Software Technology for “Recombinant production in trichoderma of glucoamylaseP”

Industrial Agricultural Biotech – Tuula Torkkeli, Vesa Joutsjoki, Helena Torkkeli, Arja Vainio, Richard Fagerstrom, Sirpa Aho, Matti Korhola, Helena Nevalainen, Roal Oy

Abstract for “Recombinant production in trichoderma of glucoamylaseP”

“The invention relates to the amino acid and DNA sequences for a unique glucoamylaseP that has high debranching activities, a Trichoderma host cells, transformed with such sequences and the expression of such Recombinant GlucoamylaseP, as well as the industrial uses of the enzyme and hosts modified therewith.

Background for “Recombinant production in trichoderma of glucoamylaseP”

“I. Host”

“A. Trichoderma reesei”

“The mesophilic filamentous fungus Trichoderma reesei has a high efficiency in secreting cellulase enzymes to the growth medium. Under optimal cultivation conditions, extracellular cellulase levels can reach 40 g/l (Durand and al., Enzyme Microb). Technol. Technol. 135-151).”

“Development and testing of transformation systems for T. reesei” (Knowles et.al., EP244,234 ; Pentila et.al., Gene 61,155-164 (1987); Berka et.al., EP215.594) has allowed genetic engineering to be applied to the fungus. Genetic engineering has allowed for the modification of cellulase enzyme production profiles, e.g. to produce strains with higher levels of endoglucanase 1. To promote endoglucanase, the strong cbh1 inducer was used (Nevalainen H., et.al., “The Molecular Biology of Trichoderma and its application for the expression of both heterologous and homologous genes” in Molecular Industrial Mycology Leong and Berka, eds. Marcel Dekker Inc. New York, pp. 129-148 (1992); Harkki, A., et al, Enzyme Microb. Technol. 13:227-233 (1991)).”

T. reesei’s production profile can be modified to produce homologous proteins. However, T. reesei’s production potential has been used to express heterologous proteins within the fungus. So far examples are few and include e.g., calf chymosin (Knowles et al., EP244,234; Berka et al., EP215,594; Harkki, A., et al., Bio/Technol. 7:596-603 (1989); Uusitalo, J. M., et al., J. Biotechnol. 17:35-50 (1991), CBH-1Fab fusion antibodies against 2-phenyloxazolone [Nyyssonen and al., WO92/01797] and a fungal-ligninolytic enzyme [Saloheimo and Niku-Paavola M. and M.-L. Bio/Technol. 9:987-990 (1991)). The desired gene was introduced to a cbh1 transcription cassette for improved expression and then transformed into T.reesei via protoplast transformation (Harkki A., et.al., Bio/Technol. 7:596-603 (1989); Nyyssonen et al., WO92/01797; Saloheimo, M. and Niku-Paavola, M. -L, Bio/Technol. 9:987-990 (1991)). 9:987-990 (1991) A fusion protein can also be produced to achieve higher yields in certain cases (Harkki, A., and et al., Bio/Technol. 7:596-603 (1989); Nyyssonen et al., WO92/01791). The yields of heterologous protein obtained from T.reesei range between 10-150 mg/l.

“II. Glucoamylases”

“Glucoamylase enzymes, (?-1.4,-glucan, glucohydrolase EC 3.2.1.3), are starch hydrolyzing exoacting carbohydratesohydrases. These enzymes are microbial enzymes that are extracellularly produced by some molds and yeasts. Starch is a heterogeneous, polysaccharide that contains between 15-30% and 70-85% amylopectin. Amylose is a linear, linked polymer that contains 500 or more?D(1-4)-linked sugar residues. Amylopectin, a branched polymer made of approximately 20-30??-D(1-4),-linked glucose units that are linked to one another through??D(1-6) linkages, is called. These branch points make up 4-5% of all glucosidic bonding in starch.

“Glucoamylases hydrolyze both the?-1.4 and??-1.6 linkages in polysaccharides like starch, releasing glucose units from the non-reducing ends of polysaccharides. They are two different activities. Hydrolyzing?-1.4 and??1.6 glucosidic bond, glucomylases release?-D?glucose units by hydrolyzing terminal nonreducing ends a glucose polymer like starch.

“III. “III.

“Pullulanases” are an endo-acting hydrolytic enzyme that is specific for breaking?-(1,6)glucosidic bonds. The inability of pullulanases to hydrolyze polymers smaller that maltosylmaltose limits their utility in industrial processes. Hormoconis resinae’s glucoamylase, P, can hydrolyze many polymeric substrates. This is in contrast to other glucoamylases like Hormoconis resinae-glucoamylase A. It has two glucose units linked by a?-(1,6) -glucosidic bond and panose.

“Glucoamylases have different abilities to hydrolyze ((1,6)-glucosidic) bonds. Hormoconis resinae enzymes have the highest debranching activity (previously known as Cladosporium Resinae). The fungus actually produces two different glucoamylases, with different molecular masses. The smaller glucoamylase, P, has a high level of debranching activity, while the larger glucoamylase, S, has almost no debranching activities as measured by pullulan (Fagerstrom et.al., J. Gen. Microbiol. 136:913-913 (1990); McCleary & Anderson Carbohydrate Research 86,77-96 (1980 )).”).

“IV. “IV.

“Several genes that code for glucoamylases were cloned, and they have been expressed in yeast or fungal expression systems.”

“A method for purifying glucoamylaseP as well as parts of its amino acid sequence has been published by Fagerstrom et.al., J. Gen. Microbiol. 136:913-920 (1990)). A restriction map of two overlapping glucoamylase cDNA segments has also been published. This data is in addition to evidence that suggests that recombinant laboratory yeast could be used to express the gene (Torkkeli et al. XIII International Specialized Symposium for Yeasts Leuven, Belgium (1989 )).”).

“The glucoamylase gene from Aspergillus Niiger (WO86/07091;WO88/09795; U.S. Patent. No. No. No. 4,794,175 EP Patent No. 12,6206) were cloned into yeast cells.

Saccharomyces cerevisiae has expressed the glucoamylase gene of a fungus belonging to the genus Rhizopus (EP Application Publication Number. 186066). A modified Rhizopus glucose gene was also introduced to create other amylolytic yeast types (Ashikari and al., App. Microbiol. Biotech. 32:129-133 (1989)).”

“A recombinant Saccharomyces was created by transmuting S. cerevisiae using a C. albicans-derived glucoamylase gene, resulting in the production of the?-(1,4) -glucosidic bond-cleaving protein (EP Patent Application Publication Number. 0362179).”

“The glucoamylase gene of Schwanniomyces Castellii has been cloned from Schwanniomyces and expressed in Saccharomyces cerevisiae, as well as other yeast forms (EP Patent Application Publications Nr. 0260404 and No. 0257115).”

“Brewing yeasts were transformed with a modified plasmid containing a gene coding to Saccharomyces diastaticus’ glucoamylase gene (Park et al.., MBAA Technical Quarterly 27, 112-116 (1990 )).”).

“An amylolytic S. Cervisiae strain that can use starch for its sole carbon source has been created and used to express the??amylase, glucoamylase genes in Schwanniomyces westernis (Hollenberg & Strasser Food Biotechnology 4 :527-534 (1990 )).”).

“Dohmen et. al., Gene 95.111-121 (1990) have expressed a Schwanniomyces westernis glucoamylase (S. cerevisiae cell line) by transforming cells with centromere plasmids containing the glucoamylase gene fused with different S. cerevisiae promotors.”

“The amino acids sequences of several microbial glycoamylases were determined. The complete sequences for Aspergillus Niiger (Svensson and colleagues, Carlsberg Res. Comm. 48:529-544 (1983), and Aspergillus anawamori (Nunberg, et al. Mol. Cell. Biol. 4:2306-2315 (1984).)Glucoamylases are identical. The glucoamylase enzymes of Rhizopus oryzae are identical (Ashikari et al. Agricultural and Biological Chem. 50:957-964 (1986)), Saccharomyces diastaticus (Yamashita et al., J. Bacteriol. 161:567-573 (1985)), Saccharomyces cerevisiae (Yamashita et al., J. Bacteriol. 169:2142-2149 (1987)); Aspergillus shirousami (Shibuya et al., Agric. Bio. Chem. 54:1905-1914 (1990)); Schwanniomyces occidentalis (Dohmen et al., Gene 95:111-121 (1987)); Clostridium sp. G0005 (Ohnishi et al., Eur. J. Biochem. 207:413-418 (1992)); and Saccharomycopsis fibuligera (Itoh et al., J. Bacteriol. Sequences for 169:4171-4176 (1987), have also been done.

“V. Industrial Uses for Glucoamylases.”

Saccharomyces cerevisiae is the most commonly used organism for commercial alcohol fermentation. Saccharomyces cerevisiae does not have the enzymes required to convert starch polymer into fermentable monomeric units. Starch must be subject to a pre-fermentation process in order to be used in large-scale ethanol production.

“Typically, starch polymers are ground and gelatinized using heating. Then, liquified with?-amylase (an endo-acting enzyme that hydrolyzes the?-(1,4) glucosidic bond), in the ethanol process. This results in the formation of?-limit-dextrins that contain?-(1,6)?glucosidic bond links. The main rate-limiting step in hydrolysis and industrial use of substrates that contain these bonds is currently the cleavage of the?-(1,6) glucosidic linkages.

“Efforts were made to improve the efficiency of this process by improving enzymatic protocols that treat starch. Aspergillus is the source of the most common glucoamylase used in ethanol production. This enzyme’s debranching activity is not enough for production so preparations should be supplemented by other enzymes, such as pullulanases. Small?-limit Dextrins are not degradable by pullulanases.

“U.S. Pat. No. No. No. 4,234,686 describes a method of obtaining starch degrading enzymes form Cladosporium resinae (formerly Hormoconis resinae) and a process for isolating high-debranching glucoamylase from the mixture. U.S. Pat. No. No. 4.318,927 recommends the production of a low-calorie alcoholic beverage by using starch-degrading enzymes from Cladosporium resinae culture medium (ATCC No. 20495). Other authors have also described beer production, particularly low-calorie (light beer), where the unfermentable carbohydrate glucose dextrin is converted into fermentable sugars using glucoamylase enzymes from different origins (U.S. Pat. No. No. 7, 1986, p.121). U.S. Pat. No. 4,863,864 (EP 185,327) describe alcohol production from starch using glucoseamylase. U.S. Pat. No. No. 4,898,738 is about sake production using Aspergillus.

“Glucoamylases can be used in many applications, including those that are directed at the production of alcohols and alcoholic beverages. Starch analysis is one example of such an application (Rickard J. E. et.al, J. Sc. Food and Agricul. 41:373-379 (1987); manufacture of glucose syrups [Illanes A., Alimentos 8.22-29 (1983),] high-DE glucose syrups & high-maltose syrups EP 405,283, Shen G. J. et.al., Appl. Microbiol. Biotech. 33:340-344 (1990), The production of isomaltose. (U.S. Patent. No. No. Biochem. Biotechnol. 27:164-171 (1991); preparation of high purity glucose (EP452,238, JP3,139,289); high-maltotetraose starch hydrolysates with high maltose (U.S. Patent. No. 4,971,906, U.S. Pat. No. No. No. 4,902,621); the production of polysaccharides with improved rheological properties over raw starch (AU 8826548); the production of crystalline 2-O-alpha-D-glycopyranosyl-L-ascorbic acid (EP 425,066); lubricants and gels (EP 372,184); the synthesis of branched cyclodextrin (U.S. Pat. No. No. No. No. A. et. al. Analyst 115/1289-1295 (1990); use of fragments containing starchbinding domain to prepare genetically engineered Peptide Affinity Tails for the recovery fusion proteins (Ford C. et. al. J. Cell. Biochem. (Suppl.) 14D:30 (1990), Chen, L. et al., Abst. Annu. Meet. Am. Soc. Microbiol. 90:269 (1990); wood and textile industry application such as the preparation of plywood adhesives or particle board bindings (Mukherjee S., Brazil Patent No. 400/88, Sep. 5, 1989; saccharification and lignocellulosic material preparations; the preparation high solids dextrin glues for high speed paper coating and wrinkle-free paper conversion to envelopes and poster board. (U.S. Pat. No. No. No. No. Food Chem. 37:1174-1177 (1989); bioconversion of distillery wastewaters (Perdih A. et.al, Enzyme microb. Technol. 13:848-852 (1991), or vegetable wastes, (Von Richter and G. Starch 35,113-118 (1983),) or fruit waste, (Horn C. H. et.al., Biol. Wastes 24.127-136 (1988). These can be used to make feedstuff, including feed additives. Bulk fillers. Sweetening agents. Raw materials for ethanol production. Sci. Sci. 48:1483-1490 (1979); and assays for predicting the digestibility animal feeds (Dowman M. G. et. al., J. Sci. Food Agric 33, 689-696 (1982). The recombinant bacteria biomass can be used to supplement feed for animals (SU 916.336).

“Research also focuses on the discovery, cloning, and expression of enzymes that have a higher hydrolysis efficiency. Method for preparing glucoamylase by cultivating Cladosporium resinae. U.S. Pat. No. 4,318,989.”

“Fagerstrom et al., J. Gen. Microbiol. H. resinae glycoamylase P is the most suitable enzyme for industrial use. It has an extremely high level of debranching activity. In many industrial processes, glucoamylase may be the only debranching enzyme that is needed. H. resinae does not produce glucoamylaseP in sufficient quantities (U.S. Patent. No. No. There is still a need for large-scale, economical production of glucoamylase that has a high degree of debranching.

The inventors considered the possibility of using filamentous fungi in order to express H. resinae glucoseamylase p cDNA and genome DNA. The cDNA and gene of H. resinae glucoamylase, a unique glucoamylase, have been sequenced. These efforts led to the stable transformation Trichoderma with DNA that encodes H. resinae glucoseamylase P and the large-scale expression of this protein from the recombinant hosts.

“The invention is therefore directed to nucleic acids sequences encoding H. resinae glucoseamylase P, which include the native gene sequence, biologically functional fragments, and derivatives thereof.”

“Another embodiment of the invention is directed at the amino acid sequence for glucoamylaseP, as well as biologically functional fragments or derivatives thereof.”

“Another embodiment of the invention is directed at a stable transformed Trichoderma host and methods for expression Hormoconis resinae glucose glucoamylase (P) from it.”

“In another embodiment, invention is directed at the culture medium and enzyme preparations from it, obtained through the growth or cultivation of such transformed hosts and the use thereof.”

“In another embodiment, brew methods are provided using enzyme preparations according to the invention. This includes the treatment of wort to increase its characteristics, such as increasing wort filterability and decreasing wort viscosity.

“Another embodiment of the invention is directed to mashing treatment during the mashing and/or liquefaction step. This treatment uses the enzyme preparation of the invention. It results in higher ethanol yields and better saccharification of the grain mash.

“The Trichoderma-expressed recombinant glucoamylase P is an economical source of glucoamylase P for use in a wide variety of industrial applications involving the degradation of starch-like polymers.”

“Deposits of Microorganisms.”

“An E. coli host carrying plasmid pALK305, ALKO2311, encoding a cDNA for H. resinae glucoamylaseP has been deposited at Deutsche Sammlung von Micro-organismen und Zellkulturen GMbH (“DSM”) on 17 February 1992 and assigned number DSM 6921.”

“While purified glucoamylase P proteins have been reported in the past, the inventors discovered that the glucoamylase P preparations and methods used to isolate the protein were too expensive to allow large-scale industrial use of the enzyme. The production of recombinant glucoseamylase P was therefore investigated. These investigations led to the identification of cDNA- and genomic recombinant clones of glucoamylase P and the elucidation the sequence and gene of the glucoamylase P proteins and gene. These researches have also led to the creation of Trichoderma reesei hosts that are highly efficient and can release useful amounts of recombinant glucose into the growth medium. In this application, the terms “growth medium” and “culture medium” can be interchanged.

Trichoderma was selected as a recombinant host to express glucoamylaseP because, as a comparison to Aspergillus Trichoderma (T. reesei) secretes higher amounts of?glucans and hemicellulose-decomposing enzyme activities, such as xylanases and?glucanase. These activities are particularly desirable for enzyme preparations and methods.

Trichoderma reesei, the most efficient known cellulase producer, synthesizes all the enzymes necessary for extensive hydrolysis. T. reesei’s enzymes have been well-characterized and are capable of producing high saccharification yields in hydrolysis of cellulose. Bioeng. Symp. Symp.

“Cloning of Glucoamylase P Gene Sequences”

According to the invention, the process of producing glucoamylaseP is made easier by the cloning and expression of genetic sequences capable of encoding the glucoamylaseP protein. The term “genetic sequences” as used in this invention refers to a nucleic acids molecule (preferably DNA). There are many sources of genetic sequences capable of encoding the glucoamylase protein P. There are many sources of genetic DNA: cDNA, genomic DNA, synthetic DNA and combinations thereof. A cDNA library of the filamentous fungus Hormoconis resinae is the preferred source for the glucoamylase p cDNA. A genomic library of filamentous fungus Hormoconis resinae is the preferred source for the glucoamylase p genomic DNA.

“The glucoamylase P cDNA will not contain naturally occurring introns if it was created using mature glucoamylase P mRNA (a template). Natural introns may or not be included in the glucoamylase P genomic genome DNA of this invention. This genomic DNA can also be obtained with the 5? promoter region and/or the 3′ transcriptional terminator regions of the glucoamylaseP gene sequences. These genomic DNA can also be obtained with the genetic sequences that encode the 5? non-translated glucoamylase mRNA, and/or the genetic sequences that encode the 3′ untranslated region. If a host cell is able to recognize the transcriptional or translational regulatory signals that are associated with the expressions of the mRNA/protein, then the 5? and/or the 3′ nontranscribed regions of a native gene and/or the 5′ &/or the 3′ nontranslated regions in the mRNA may be retained for transcriptional or translational regulation.

“Glucoamylase P genome DNA can be extracted from any cell, preferably a filamentous fungal cells, and purified by using methods well-known in the art. (For example, see Guide to Molecular Cloning Techniques S. L. Berger et. al., Academic Press (1987). If a cDNA clone of glucoamylase is required, it is preferred that the mRNA preparation used be enriched in mRNA coding to glucoamylaseP. This can be done either naturally from a source producing large quantities of the protein or in vitro using techniques such as sucrose gradient centrifugation. Hormoconis resinae, a filamentous fungus is preferred as a source for glucoamylase mRNA.

“To clone into a vector, the desired DNA (either genome DNA or ds-cDNA) can be randomly sheared, or enzyme cleaved. The vector will then be ligated into a suitable vector to create a recombinant (either cDNA or genomic) gene library.

“A DNA sequence encoding the glucoamylase protein P or its functional derivatives can be placed into a DNA vector according to conventional techniques. This includes restriction enzyme digestion to provide appropriate termini and filling in of cohesive end as necessary, alkalinephosphatase treatment in order to avoid unwanted joining, and ligation using appropriate ligases. These techniques are well-known in the art.

“Libraries containing sequences coding for glucoamylase P may be screened and a sequence coding for glucoamylase P identified by any means which specifically selects for such sequence, such as, for example, a) by hybridization with an appropriate nucleic acid probe(s) containing a sequence specific for the DNA of this protein, or b) by hybridization-selected translational analysis in which native mRNA which hybridizes to the clone in question is translated in vitro and the translation products are further characterized, c) if the cloned genetic sequences are themselves capable of expressing mRNA, by immunoprecipitation of a translated glucoamylase P product produced by the host containing the clone, or d) by assay of glucoamylase P activity in the growth medium of the transformed hosts.”

“Oligonucleotide probes that are specific for glucoamylaseP can be used to identify clones of this protein. They can be constructed from knowledge about the amino acid sequence. The amino acid sequence is presented horizontally. Unless otherwise noted, the amino terminus and carboxy terminus are to be listed at the left and right ends, respectively.

“Because the gene code is degenerate, multiple codons may be used to encode a particular amino acid (Watson, J. D., in: Molecular Biology of the Gene, Third Ed., W. A. Benjamin, Inc., Menlo Park, Calif. (1977), pp. 356-357). Analyzing the amino acid sequence can help identify sequences that may be encoded using oligonucleotides with the lowest degree degeneracy. It is best to identify sequences that only contain one codon of amino acids.

“Although an amino acid sequence can sometimes be encoded only by one oligonucleotide sequence at a time, it is possible for the sequence to be encoded using any number of similar oligonucleotides. It is important to note that, while all members of the set have oligonucleotide sequencings capable of encoding the exact same peptide sequence and thus may contain the same sequence of oligonucleotides as the gene which encodes it, only one member contains the nucleotide that matches the exon coding sequence. This member of the set is capable of hybridizing with DNA, even when there are other members in the set. It is therefore possible to use the unfractionated set oligonucleotides in a similar way as one would to clone a single gene that encodes the protein.

“Using the genetic code (Watson, J. D., et al., in: Molecular Biology of the Gene, Third Ed., W. A. Benjamin, Inc., Menlo Park, Calif. (1977),), one or more oligonucleotides are identified. This can be done by looking at abnormal base pairing relationships and the frequency that a specific codon is used to encode a particular amino acids in eukaryotic cells. These “codon usage guidelines” were disclosed by Lathe, R. and colleagues, J. Molec. Biol. 183:1-12 (1985). The “codon use rules” of Lathe allow us to identify a single or set of oligonucleotide sequencings that have a theoretical’most probable’ nucleotide sequence capable encoding the glucoamylase P sequences.

“The appropriate oligonucleotide or set of oligonucleotides that is capable of encoding a glucoamylase fragment (or which is complementary to such an oligonucleotide or set of oligonucleotides), may be synthesized using methods well known in art (see, for instance, Synthesis and Application of DNA and RNA by S. A. Narang 1987, Academic Press San Diego, Calif.). It can then used as cloned glucoamylase gene to isolate glucoamylase cloned glucoamylase glucoamylase gene to identify and to cloned to oligonucleotide or set of oligonucleotides or set of oligonucleotide oligonucleotide oligonucleotide -to otide or set of oligonotide and es, es, es, es, IRL Press, Washington, D. Maniatis, T., and colleagues have described techniques for nucleic acid hybridization, and clone identification. Hames, B. D., and others, have published Nucleic Acid Hybridization, a Practical Approach, IRL Press, Washington, D.C. (1985). The members of the gene library described above that are capable of hybridization are then examined to determine the nature and extent of glucoamylase-P encoding sequences they contain.

The above-described DNA probe has been labeled with a detectable number to facilitate detection of the desired glucoamylase DNA encoding sequence. Any material with a detectable chemical or physical property can be considered a detectable group. These materials are well-developed in the field nucleic acid hybridization. In general, any label that is useful in such methods can also be used in the present invention. Radio-active labels such as 32P, 3H, 14C, 35S, and the like are particularly useful. Any radioactive label that provides sufficient signal strength and has a long half-life can be used. Radioactive labeling may be used to radioactively label single-stranded oligonucleotides using kinase reaction. Polynucleotides, which are equally useful, can also be radioactively labelled using kinase reactions to be used as nucleic acids hybridization probes. See, for example, Leary, J. J. et al., Proc. Natl. Acad. Sci. USA 80:4045 (1983); Renz, M. et al., Nucl. Acids Res. 12:3435 (84); Renz, M. EMBO J. 6:817 (1983).”

In summary, the actual identification and sequencing of glucoamylase P proteins allows for the identification of a hypothetical “most likely” DNA sequence or a set thereof capable of encoding such a peptide. One can construct an oligonucleotide that is complementary to this hypothetical sequence, or a set oligonucleotides that are complementary to the set “most likely” oligonucleotides. This will give one a DNA molecule (or a number of DNA molecules) capable of serving as a probe for the isolation and identification of clones containing the glucoamylase p gene.

“An alternative method of cloning a glucoamylase P genes is to prepare a library using an expression vector. This involves cloning DNA, or more preferably cDNA, from a cell that can express glucoamylase P into a vector. After screening the library for members that express glucoamylase P (e.g., screening it with antibodies or assaying growth medium to determine glucoamylase P activity), the library is analyzed for any such members.

“The methods discussed above are therefore capable of identifying genetic sequences capable of encoding either glucoamylaseP or its fragments. It is important to express these proteins in order to further identify such genetic sequences and to create recombinant glucoseamylase P. This includes full-length, antigenic, or enzymatically activated fragments. This expression identifies clones that express proteins with glucoamylase P characteristics. It can be used to identify clones that have the ability of binding glucoamylase P antibodies, to produce antibodies capable of binding to glucoamylase P enzyme activity, or to provide a glucoamylase P function to a recipient.

Site-directed mutation may be used to prepare mutant glucoamylase P activities. This is as per the art. Site-directed mutation may be used to modify the thermal stability of the invention, as described in Itoh, T. and colleagues, Agric. Biol. Chem. 53:3159-3167 (1989), for the glucoamylase of Saccharomycopsis Fibuligera. Ala 81, Asp 89 and Asp 89 have been shown to be critical for the enzyme’s thermal stability.

“Expression of GlucoamylaseP and its Functional Derivatives.”

“To express glucoamylaseP and/or its enzymatically activate derivatives, transcriptional or translational signals that are recognizable by a suitable host are required. Cloned glucoamylase protein encoding sequences may be linked to sequences controlling transcriptional activity in an expression vector. These sequences can then be introduced into prokaryote and eukaryote cells to create recombinant glucoseamylaseP or a functional derivative. Depending on which strand of glucoamylase P encoding is operably linked with the sequences controlling transcriptional activity, it may be possible to express glucoamylase P antisense RNA and a functional derivative thereof.

“Expressions of the glucoamylase in different hosts can result in different post-translational modification which could alter the properties of this protein. Glucoamylase or a functional derivative thereof can be expressed in eukaryotic and especially fungal cells. The present invention preferably includes the expression of glucoamylase or a functional derivative thereof in transformed Trichoderma Reesei.

“A nucleic acid molecule such as DNA is considered to be capable of expressing a polypeptide” if it contains expression control sequencings that contain transcriptional regulatory information. Such sequences can be operatably linked to the nucleotide sequence that encodes the polypeptide.

An operable linkage refers to a connection in which a sequence connects to a regulatory sequence or sequences in such a manner that it places expression of the sequence under control or influence of the regulatory sequence. If two DNA sequences, such as a glucoamylase P encoding and a promoter sequence linked to 5′ of the protein encoding, are induction of promoter functions, and if the nature and function of the linkage between them does not (1) result or interfere with the ability to regulate the expression of glucoamylase P or antisense RNA or protein or (3) affect the ability to transcribe the glucoamylase P template by the promoter sequence, then they are considered operable links. A promoter region is considered operable if it can effect transcription of a particular DNA sequence.

The exact nature of regulatory regions required for gene expression can vary depending on species and cell types. However, they will generally include 5′ nontranscribing (non-coding), and 5′ translating sequences that are involved in initiation of transcription or translation, such as the TATA Box, CAAT sequence, Capping sequence, and the capping sequence. These 5′ non-transcribing sequences will contain a region that contains a promoter to control transcription of the operably linked genes. These transcriptional control sequences can also contain enhancer sequences and upstream activator sequences, if desired.

“Expression of glucoamylaseP in recombinant host requires the use regulatory regions that are functional in such hosts. Depending on the host’s nature, there are many transcriptional and/or translational regulatory sequences that can be used. You can also derive transcriptional and/or translational regulatory signals from the genome sequences of viruses that infect host cells. A desired regulatory signal should be associated with a specific gene that is capable of high expression in the host cells.

“In eukaryotes where transcription is not linked with translation, such control areas may or may not contain an initiator methionine codon (AUG), depending on whether the cloned sequencing contains such a methionine. These regions generally contain a promoter area that can direct the initiation and maintenance of RNA synthesis within the host cell. Promoters from heterologous fungal gene promoters that encode a mRNA product capable to translation are preferred. In particular, promoters for T.reesei cellobiohydrolase I, cellobiohydrolase II, cellobiohydrolase III (CBHII), and endoglucanase I, (EGII), may be used, particularly in the T.reesei host. The yeast GAL4 gene promor or the glycolytic gene promoter (such as the glycerol-3 phosphate dehydrogenase enhancer) may be used in laboratory yeast hosts.

“As it is well known, the codon that encodes the first methionine is where translation of the eukaryotic transcript begins. It is important to ensure that the linkage between an eukaryotic promoter, a DNA sequence that encodes the glucoamylaseP, or a functional derivative thereof does not include any codons capable of encoding methionine. If such codons are present, they can lead to a formation of either a fusion protein (if AUG codons are in the same frame as the glucoamylaseP encoding DNA sequence sequence) or a frameshift mutation (if AUG codons are not in the same frame as the glucoamylaseP encoding sequence).

“A glucoamylase fusion product P can be made if desired. Full-length glucoamylaseP encodes a signal sequence of 29 amino acids that allows secretion from fungal hosts such as Hormoconis resinae Saccharomyces cerevisiae, Trichoderma reesei.

“The sequence coding mature glucoamylase may be linked with a heterologous signal sequence that will allow secretion or compartmentalization of the protein within a specific host. These signal sequences can be constructed with or without specific protease site so that the signal sequence can be removed later. T. reesei hosts can use signal sequences from cellulase genes and, in particular, cellobiohydrolase II and cellobiohydrolase I signal sequences.

“Transcriptional initiating regulatory signals can also be selected that allow activation or repression of genes, so that they can be controlled.”

The non-transcribed/or nontranslated regions 3′ to P sequence coding can be obtained if desired. The 3′-nontranscribed region can be retained to its transcriptional termination regulatory elements. The 3′ non-transcribed area may be retained to its translational regulatory sequence elements or elements that direct polyadenylation within eukaryotic cell. If the native expression control sequences fail to function in the host cells, the sequences that work in the host cells may be replaced.

“To transform a fungal cells with the DNA constructs, many vector systems are possible depending on whether the glucoamylase DNA construct is to be inserted into the host cell’s chromosomal DNA or allowed to exist in an extrachromosomal version. EP 244,234 describes the stable transformation of Trichoderma reesei as well as the expression of heterologous gene in such transformants. The preferred plasmid system is described in the same as in U.S. Application Ser. No. No. 19, 1990 or in U.S. Application Ser. 07/524.308 was filed May 16, 1990. It is incorporated by reference.”

The transient expression may occur if the glucoamylase-P encoding sequence is introduced into a recipient eukaryotic cells as a nonreplicating DNA or RNA molecule. This non-replicating DNA or RNA molecule can be either a linear molecule, or more preferably, a closed covalent circle molecule that is ineligible for autonomous replication.

“Preferable embodiments may include genetically stable transformants that are constructed using vector systems or transformation systems where glucoamylase-P DNA is integrated into the host genome. This integration can occur naturally within the cell, or it may be assisted by a vector inserting the vector into the host chromosome. The vector can integrate the desired sequences of gene into the fungal host cell’s chromosome. Such vectors can be used to integrate a T. reesei gene into a fungal host cell chromosome. This is called the cellobiohydrolase 1 gene. It promotes homologous recombination on a particular site of the host chromosome.

“Cells that have successfully integrated the DNA into their chromosomes can be selected by adding one or more markers. This allows for the selection of host cells that contain the expression vector. The marker might provide biocide resistance, such as resistance to antibiotics or heavy metals like copper. You can link the selectable marker gene directly to the DNA sequences to be expressed or introduce the marker into the same cell via co-transfection. U.S. patent Ser. No. No. 07/496,155 filed Mar. 19, 1990; and in Finkelstein, D. B., Chapter 6 in Biotechnology of Filamentous Fungi, D. B. Finkelstein et al., eds., Butterworth-Heinemann, Boston, 1992, pp. 113-156 are both included herein completely by reference.

“Factors that are important in choosing a vector include the ability of recipient cells to recognize the vector and select them from others; the number and quality of copies of vectors desired by a host; and the possibility of being able to shuttle the vector between different host cells.

Once the vector or DNA sequence containing construct(s), is ready for expression, the DNA construct(s), can be introduced into the appropriate host cell using any of a number of suitable methods. Recipient cells are then grown in a selective media (if required), to allow for the growth vector-containing cells. The cloned gene sequences can be expressed to produce the glucoamylase (or a portion) of this protein. The expression can occur in continuous fashion in the transformed cells or it can be controlled by a stimulus that induces the host cells to produce the cloned glucoseamylase P.

“The expressed recombinant glucoseamylase P protein can also be isolated and purified if desired. This is done according to conventional conditions such as extraction, precipitation or affinity chromatography.

“Industrial Uses for Glucoamylase P.”

“Distilled alcohol is produced by a series, which includes the conversion of raw materials to sugars that can then be used by yeast and the fermentation process, followed by distillation. The residue usually contains proteins and minerals and is useful for animal feed. An integrated process, in which barley is converted to starch, carbon dioxide and other feed fractions, can be used.

For alcohol manufacturing, the smaller starch grains, known as “B starch”, are used with a diameter of approximately 5 microns. A-starch granules with a diameter of 15 microns or more are used primarily for coating paper.

“The key advantages of integrating ethanol and starch production are in the process and the product economics. The water from starch manufacturing can also be used in alcohol production, material flows can be flexible, and the emphasis can be put on the production end products that are most in demand.

“Processe such as the invention which increase the efficiency of the enzymes that convert starch to sugars are highly desirable. The T. reesei-glucoamylase P strains are particularly useful in brewing, including the production of beer and the manufacturing of grain alcohol. The strains of the invention secrete a mixture of enzymes (?-1,4-glucoamylase, ?-1,6-glucoamylase P, ?-glucanase, hydroxyethylcellulose degrading activity (HEC), filter paper degrading activity (FPU), xylanase (hemicellulose degrading activity), acid protease (protein degrading activity at pH 4.7) and ?-glucosidase (cellobiose degrading activity)) into their growth medium. The medium can be used immediately after separation of the solids. This allows the medium to efficiently and quickly break down macromolecular substances found in ground grain slurry into substrates that are utilizable for yeast. The growth medium can be diluted or concentrated to adjust the activity of the desired enzyme(s), using techniques such as lyophilization and dialysis.

The enzyme preparations of this invention are active on the 1,6 linkages. They degrade isomaltose and panose 5-30 times faster than preparations that contain Aspergillus glucoamylase or Rhizopus. Hydrolysis of starch to its entirety increases the production of simple sugars, which in turn increases the overall alcohol yield.

A cascade fermentation is more economical and efficient than a batch method of fermentation. A continuous volumetric productivity can be achieved with higher cell concentrations (either by cell recycling, or immobilization), which is 5-20 times greater than that of the batch mode.

“Cascading fermentation has the advantage that there is no lag for starting fermentation. Also, the inhibition of fermentation caused ethanol can be reduced over the entire range of fermentation. This is done by limiting the severity of the inhibition to the last vessel of the series. Cascade fermentations do not last forever, as the process is stopped every 2 to 3 weeks and the tanks are emptied for cleaning.

“All enzymes secreted in Trichoderma culture growth medium are contained.” This medium contains enzymes that have been reasonably well characterised (see U.S. Appl. Ser. No. No. Ser. No. No. The medium can be used either as a source or mixture of these enzymes.

“The Hormoconis resinae glucose containing growth medium is produced by any Trichoderma stain that has been transformed using cDNA or genomic sequences that encode glucoamylase. It is a useful preparation and contains the desired mix of enzyme activities. Trichoderma alko 2743 (ALKO?GA) is an example of one such strain. Starch-based biological processing and specifically ethanol production are some examples of possible uses. T. reesei strains 2743 and 233 have approximately the same activity levels as their respective cellulases and?-glucanase activities. Strain 233 has about 1/3 of the xylanase activity as strain 2743.

“Generally, there are three steps to mashing grain for ethanol production.

“1) Heating (also known as gelatinization);

“2) Addition of?-amylase enzyme

“3) Addition of amylolytic enzymes like pullulanase and glucoamylase (also known as saccharification).

The invention’s glucoamylase-containing medium (or enzyme preparation), may be added at any stage of mashing. The heating will inactivate the enzyme if it is added prior to or during gelatinization. Additional enzyme must be added after heating.

“The addition of growth medium of invention (that is, growth media taken from the culture Trichoderma and transformed with sequences encoded the glucoamylaseP of the invention), at the beginning mashing or fermentation increases the recovery and alters beer’s characteristics. Pilot and laboratory scale experiments show identical results. These results also differ between the ALKO?GA and commercial glucoamylase concentrate NOVOAMG 300 L, which are produced by Aspergillus.

“The enzyme activity tests from enzyme concentrates showed that ALKO-GA has starch-decomposing side activities such as cellulases, xylanases, and?glucosidase in amounts greater than those currently available like NOVO AMG 300L.”

“The amount of ALKOGA enzyme addition to the wort affected the characteristics of the wort when it was mixed in laboratory scale mashing. This dose effect gives you maximum flexibility to alter the characteristics of your wort. The enzyme effects were similar with ALKO/GA and NOVOAMG 300L except that the concentrations of?-glucans found in the treated wort were very low when ALKO/GA was used. However, the concentrations of NOVOAMG 300L and ALKO-GA were comparable to the control group. ALKO-GA worts have a lower viscosity than the control group and a higher filtration rate, which are both benefits in an industrial setting. The ALKO-GA treated mashing is significantly more efficient than the NOVO AMG 300L treated mashing. This is why the volume of the wort is so much higher. This is another advantage of the invention.

The following are the reasons why ALKO-GA has a better response (even at 0.1 g/100g mash): The?-glucanase xylanase, cellulase and cellulase activity in ALKO-GA are higher than those in NOVO AMG300 L. ALKO-GA’s?-glucanase seems to have a higher temperature stability than the Aspergillus Niger??glucanase. However, it seems that the fermentation mixture does not lose its ability to maintain temperature. Although the extract yield is the same in all conditions, the extract volume was significantly higher when ALKOGA was added to mashing than other commercial preparations such as the NOVO AMG300 L. This was even more so when compared with the control experiment.

The apparent limit of attenuation increased from 84.7% up to 89% when the ALKOGA dose was 3g/100g mash. Although the higher dose did not have any effect, there was an increase in fermentable sugars (glucose and maltose etc.). These correlate with an increase in enzyme dosage. If NOVO-AMG 300L was used in mashing, the result is identical. This result shows that the ALKO-GA dosage 3 g/100g mash is preferable for Pilot scale (100L) brewing experiments.

Summary for “Recombinant production in trichoderma of glucoamylaseP”

“I. Host”

“A. Trichoderma reesei”

“The mesophilic filamentous fungus Trichoderma reesei has a high efficiency in secreting cellulase enzymes to the growth medium. Under optimal cultivation conditions, extracellular cellulase levels can reach 40 g/l (Durand and al., Enzyme Microb). Technol. Technol. 135-151).”

“Development and testing of transformation systems for T. reesei” (Knowles et.al., EP244,234 ; Pentila et.al., Gene 61,155-164 (1987); Berka et.al., EP215.594) has allowed genetic engineering to be applied to the fungus. Genetic engineering has allowed for the modification of cellulase enzyme production profiles, e.g. to produce strains with higher levels of endoglucanase 1. To promote endoglucanase, the strong cbh1 inducer was used (Nevalainen H., et.al., “The Molecular Biology of Trichoderma and its application for the expression of both heterologous and homologous genes” in Molecular Industrial Mycology Leong and Berka, eds. Marcel Dekker Inc. New York, pp. 129-148 (1992); Harkki, A., et al, Enzyme Microb. Technol. 13:227-233 (1991)).”

T. reesei’s production profile can be modified to produce homologous proteins. However, T. reesei’s production potential has been used to express heterologous proteins within the fungus. So far examples are few and include e.g., calf chymosin (Knowles et al., EP244,234; Berka et al., EP215,594; Harkki, A., et al., Bio/Technol. 7:596-603 (1989); Uusitalo, J. M., et al., J. Biotechnol. 17:35-50 (1991), CBH-1Fab fusion antibodies against 2-phenyloxazolone [Nyyssonen and al., WO92/01797] and a fungal-ligninolytic enzyme [Saloheimo and Niku-Paavola M. and M.-L. Bio/Technol. 9:987-990 (1991)). The desired gene was introduced to a cbh1 transcription cassette for improved expression and then transformed into T.reesei via protoplast transformation (Harkki A., et.al., Bio/Technol. 7:596-603 (1989); Nyyssonen et al., WO92/01797; Saloheimo, M. and Niku-Paavola, M. -L, Bio/Technol. 9:987-990 (1991)). 9:987-990 (1991) A fusion protein can also be produced to achieve higher yields in certain cases (Harkki, A., and et al., Bio/Technol. 7:596-603 (1989); Nyyssonen et al., WO92/01791). The yields of heterologous protein obtained from T.reesei range between 10-150 mg/l.

“II. Glucoamylases”

“Glucoamylase enzymes, (?-1.4,-glucan, glucohydrolase EC 3.2.1.3), are starch hydrolyzing exoacting carbohydratesohydrases. These enzymes are microbial enzymes that are extracellularly produced by some molds and yeasts. Starch is a heterogeneous, polysaccharide that contains between 15-30% and 70-85% amylopectin. Amylose is a linear, linked polymer that contains 500 or more?D(1-4)-linked sugar residues. Amylopectin, a branched polymer made of approximately 20-30??-D(1-4),-linked glucose units that are linked to one another through??D(1-6) linkages, is called. These branch points make up 4-5% of all glucosidic bonding in starch.

“Glucoamylases hydrolyze both the?-1.4 and??-1.6 linkages in polysaccharides like starch, releasing glucose units from the non-reducing ends of polysaccharides. They are two different activities. Hydrolyzing?-1.4 and??1.6 glucosidic bond, glucomylases release?-D?glucose units by hydrolyzing terminal nonreducing ends a glucose polymer like starch.

“III. “III.

“Pullulanases” are an endo-acting hydrolytic enzyme that is specific for breaking?-(1,6)glucosidic bonds. The inability of pullulanases to hydrolyze polymers smaller that maltosylmaltose limits their utility in industrial processes. Hormoconis resinae’s glucoamylase, P, can hydrolyze many polymeric substrates. This is in contrast to other glucoamylases like Hormoconis resinae-glucoamylase A. It has two glucose units linked by a?-(1,6) -glucosidic bond and panose.

“Glucoamylases have different abilities to hydrolyze ((1,6)-glucosidic) bonds. Hormoconis resinae enzymes have the highest debranching activity (previously known as Cladosporium Resinae). The fungus actually produces two different glucoamylases, with different molecular masses. The smaller glucoamylase, P, has a high level of debranching activity, while the larger glucoamylase, S, has almost no debranching activities as measured by pullulan (Fagerstrom et.al., J. Gen. Microbiol. 136:913-913 (1990); McCleary & Anderson Carbohydrate Research 86,77-96 (1980 )).”).

“IV. “IV.

“Several genes that code for glucoamylases were cloned, and they have been expressed in yeast or fungal expression systems.”

“A method for purifying glucoamylaseP as well as parts of its amino acid sequence has been published by Fagerstrom et.al., J. Gen. Microbiol. 136:913-920 (1990)). A restriction map of two overlapping glucoamylase cDNA segments has also been published. This data is in addition to evidence that suggests that recombinant laboratory yeast could be used to express the gene (Torkkeli et al. XIII International Specialized Symposium for Yeasts Leuven, Belgium (1989 )).”).

“The glucoamylase gene from Aspergillus Niiger (WO86/07091;WO88/09795; U.S. Patent. No. No. No. 4,794,175 EP Patent No. 12,6206) were cloned into yeast cells.

Saccharomyces cerevisiae has expressed the glucoamylase gene of a fungus belonging to the genus Rhizopus (EP Application Publication Number. 186066). A modified Rhizopus glucose gene was also introduced to create other amylolytic yeast types (Ashikari and al., App. Microbiol. Biotech. 32:129-133 (1989)).”

“A recombinant Saccharomyces was created by transmuting S. cerevisiae using a C. albicans-derived glucoamylase gene, resulting in the production of the?-(1,4) -glucosidic bond-cleaving protein (EP Patent Application Publication Number. 0362179).”

“The glucoamylase gene of Schwanniomyces Castellii has been cloned from Schwanniomyces and expressed in Saccharomyces cerevisiae, as well as other yeast forms (EP Patent Application Publications Nr. 0260404 and No. 0257115).”

“Brewing yeasts were transformed with a modified plasmid containing a gene coding to Saccharomyces diastaticus’ glucoamylase gene (Park et al.., MBAA Technical Quarterly 27, 112-116 (1990 )).”).

“An amylolytic S. Cervisiae strain that can use starch for its sole carbon source has been created and used to express the??amylase, glucoamylase genes in Schwanniomyces westernis (Hollenberg & Strasser Food Biotechnology 4 :527-534 (1990 )).”).

“Dohmen et. al., Gene 95.111-121 (1990) have expressed a Schwanniomyces westernis glucoamylase (S. cerevisiae cell line) by transforming cells with centromere plasmids containing the glucoamylase gene fused with different S. cerevisiae promotors.”

“The amino acids sequences of several microbial glycoamylases were determined. The complete sequences for Aspergillus Niiger (Svensson and colleagues, Carlsberg Res. Comm. 48:529-544 (1983), and Aspergillus anawamori (Nunberg, et al. Mol. Cell. Biol. 4:2306-2315 (1984).)Glucoamylases are identical. The glucoamylase enzymes of Rhizopus oryzae are identical (Ashikari et al. Agricultural and Biological Chem. 50:957-964 (1986)), Saccharomyces diastaticus (Yamashita et al., J. Bacteriol. 161:567-573 (1985)), Saccharomyces cerevisiae (Yamashita et al., J. Bacteriol. 169:2142-2149 (1987)); Aspergillus shirousami (Shibuya et al., Agric. Bio. Chem. 54:1905-1914 (1990)); Schwanniomyces occidentalis (Dohmen et al., Gene 95:111-121 (1987)); Clostridium sp. G0005 (Ohnishi et al., Eur. J. Biochem. 207:413-418 (1992)); and Saccharomycopsis fibuligera (Itoh et al., J. Bacteriol. Sequences for 169:4171-4176 (1987), have also been done.

“V. Industrial Uses for Glucoamylases.”

Saccharomyces cerevisiae is the most commonly used organism for commercial alcohol fermentation. Saccharomyces cerevisiae does not have the enzymes required to convert starch polymer into fermentable monomeric units. Starch must be subject to a pre-fermentation process in order to be used in large-scale ethanol production.

“Typically, starch polymers are ground and gelatinized using heating. Then, liquified with?-amylase (an endo-acting enzyme that hydrolyzes the?-(1,4) glucosidic bond), in the ethanol process. This results in the formation of?-limit-dextrins that contain?-(1,6)?glucosidic bond links. The main rate-limiting step in hydrolysis and industrial use of substrates that contain these bonds is currently the cleavage of the?-(1,6) glucosidic linkages.

“Efforts were made to improve the efficiency of this process by improving enzymatic protocols that treat starch. Aspergillus is the source of the most common glucoamylase used in ethanol production. This enzyme’s debranching activity is not enough for production so preparations should be supplemented by other enzymes, such as pullulanases. Small?-limit Dextrins are not degradable by pullulanases.

“U.S. Pat. No. No. No. 4,234,686 describes a method of obtaining starch degrading enzymes form Cladosporium resinae (formerly Hormoconis resinae) and a process for isolating high-debranching glucoamylase from the mixture. U.S. Pat. No. No. 4.318,927 recommends the production of a low-calorie alcoholic beverage by using starch-degrading enzymes from Cladosporium resinae culture medium (ATCC No. 20495). Other authors have also described beer production, particularly low-calorie (light beer), where the unfermentable carbohydrate glucose dextrin is converted into fermentable sugars using glucoamylase enzymes from different origins (U.S. Pat. No. No. 7, 1986, p.121). U.S. Pat. No. 4,863,864 (EP 185,327) describe alcohol production from starch using glucoseamylase. U.S. Pat. No. No. 4,898,738 is about sake production using Aspergillus.

“Glucoamylases can be used in many applications, including those that are directed at the production of alcohols and alcoholic beverages. Starch analysis is one example of such an application (Rickard J. E. et.al, J. Sc. Food and Agricul. 41:373-379 (1987); manufacture of glucose syrups [Illanes A., Alimentos 8.22-29 (1983),] high-DE glucose syrups & high-maltose syrups EP 405,283, Shen G. J. et.al., Appl. Microbiol. Biotech. 33:340-344 (1990), The production of isomaltose. (U.S. Patent. No. No. Biochem. Biotechnol. 27:164-171 (1991); preparation of high purity glucose (EP452,238, JP3,139,289); high-maltotetraose starch hydrolysates with high maltose (U.S. Patent. No. 4,971,906, U.S. Pat. No. No. No. 4,902,621); the production of polysaccharides with improved rheological properties over raw starch (AU 8826548); the production of crystalline 2-O-alpha-D-glycopyranosyl-L-ascorbic acid (EP 425,066); lubricants and gels (EP 372,184); the synthesis of branched cyclodextrin (U.S. Pat. No. No. No. No. A. et. al. Analyst 115/1289-1295 (1990); use of fragments containing starchbinding domain to prepare genetically engineered Peptide Affinity Tails for the recovery fusion proteins (Ford C. et. al. J. Cell. Biochem. (Suppl.) 14D:30 (1990), Chen, L. et al., Abst. Annu. Meet. Am. Soc. Microbiol. 90:269 (1990); wood and textile industry application such as the preparation of plywood adhesives or particle board bindings (Mukherjee S., Brazil Patent No. 400/88, Sep. 5, 1989; saccharification and lignocellulosic material preparations; the preparation high solids dextrin glues for high speed paper coating and wrinkle-free paper conversion to envelopes and poster board. (U.S. Pat. No. No. No. No. Food Chem. 37:1174-1177 (1989); bioconversion of distillery wastewaters (Perdih A. et.al, Enzyme microb. Technol. 13:848-852 (1991), or vegetable wastes, (Von Richter and G. Starch 35,113-118 (1983),) or fruit waste, (Horn C. H. et.al., Biol. Wastes 24.127-136 (1988). These can be used to make feedstuff, including feed additives. Bulk fillers. Sweetening agents. Raw materials for ethanol production. Sci. Sci. 48:1483-1490 (1979); and assays for predicting the digestibility animal feeds (Dowman M. G. et. al., J. Sci. Food Agric 33, 689-696 (1982). The recombinant bacteria biomass can be used to supplement feed for animals (SU 916.336).

“Research also focuses on the discovery, cloning, and expression of enzymes that have a higher hydrolysis efficiency. Method for preparing glucoamylase by cultivating Cladosporium resinae. U.S. Pat. No. 4,318,989.”

“Fagerstrom et al., J. Gen. Microbiol. H. resinae glycoamylase P is the most suitable enzyme for industrial use. It has an extremely high level of debranching activity. In many industrial processes, glucoamylase may be the only debranching enzyme that is needed. H. resinae does not produce glucoamylaseP in sufficient quantities (U.S. Patent. No. No. There is still a need for large-scale, economical production of glucoamylase that has a high degree of debranching.

The inventors considered the possibility of using filamentous fungi in order to express H. resinae glucoseamylase p cDNA and genome DNA. The cDNA and gene of H. resinae glucoamylase, a unique glucoamylase, have been sequenced. These efforts led to the stable transformation Trichoderma with DNA that encodes H. resinae glucoseamylase P and the large-scale expression of this protein from the recombinant hosts.

“The invention is therefore directed to nucleic acids sequences encoding H. resinae glucoseamylase P, which include the native gene sequence, biologically functional fragments, and derivatives thereof.”

“Another embodiment of the invention is directed at the amino acid sequence for glucoamylaseP, as well as biologically functional fragments or derivatives thereof.”

“Another embodiment of the invention is directed at a stable transformed Trichoderma host and methods for expression Hormoconis resinae glucose glucoamylase (P) from it.”

“In another embodiment, invention is directed at the culture medium and enzyme preparations from it, obtained through the growth or cultivation of such transformed hosts and the use thereof.”

“In another embodiment, brew methods are provided using enzyme preparations according to the invention. This includes the treatment of wort to increase its characteristics, such as increasing wort filterability and decreasing wort viscosity.

“Another embodiment of the invention is directed to mashing treatment during the mashing and/or liquefaction step. This treatment uses the enzyme preparation of the invention. It results in higher ethanol yields and better saccharification of the grain mash.

“The Trichoderma-expressed recombinant glucoamylase P is an economical source of glucoamylase P for use in a wide variety of industrial applications involving the degradation of starch-like polymers.”

“Deposits of Microorganisms.”

“An E. coli host carrying plasmid pALK305, ALKO2311, encoding a cDNA for H. resinae glucoamylaseP has been deposited at Deutsche Sammlung von Micro-organismen und Zellkulturen GMbH (“DSM”) on 17 February 1992 and assigned number DSM 6921.”

“While purified glucoamylase P proteins have been reported in the past, the inventors discovered that the glucoamylase P preparations and methods used to isolate the protein were too expensive to allow large-scale industrial use of the enzyme. The production of recombinant glucoseamylase P was therefore investigated. These investigations led to the identification of cDNA- and genomic recombinant clones of glucoamylase P and the elucidation the sequence and gene of the glucoamylase P proteins and gene. These researches have also led to the creation of Trichoderma reesei hosts that are highly efficient and can release useful amounts of recombinant glucose into the growth medium. In this application, the terms “growth medium” and “culture medium” can be interchanged.

Trichoderma was selected as a recombinant host to express glucoamylaseP because, as a comparison to Aspergillus Trichoderma (T. reesei) secretes higher amounts of?glucans and hemicellulose-decomposing enzyme activities, such as xylanases and?glucanase. These activities are particularly desirable for enzyme preparations and methods.

Trichoderma reesei, the most efficient known cellulase producer, synthesizes all the enzymes necessary for extensive hydrolysis. T. reesei’s enzymes have been well-characterized and are capable of producing high saccharification yields in hydrolysis of cellulose. Bioeng. Symp. Symp.

“Cloning of Glucoamylase P Gene Sequences”

According to the invention, the process of producing glucoamylaseP is made easier by the cloning and expression of genetic sequences capable of encoding the glucoamylaseP protein. The term “genetic sequences” as used in this invention refers to a nucleic acids molecule (preferably DNA). There are many sources of genetic sequences capable of encoding the glucoamylase protein P. There are many sources of genetic DNA: cDNA, genomic DNA, synthetic DNA and combinations thereof. A cDNA library of the filamentous fungus Hormoconis resinae is the preferred source for the glucoamylase p cDNA. A genomic library of filamentous fungus Hormoconis resinae is the preferred source for the glucoamylase p genomic DNA.

“The glucoamylase P cDNA will not contain naturally occurring introns if it was created using mature glucoamylase P mRNA (a template). Natural introns may or not be included in the glucoamylase P genomic genome DNA of this invention. This genomic DNA can also be obtained with the 5? promoter region and/or the 3′ transcriptional terminator regions of the glucoamylaseP gene sequences. These genomic DNA can also be obtained with the genetic sequences that encode the 5? non-translated glucoamylase mRNA, and/or the genetic sequences that encode the 3′ untranslated region. If a host cell is able to recognize the transcriptional or translational regulatory signals that are associated with the expressions of the mRNA/protein, then the 5? and/or the 3′ nontranscribed regions of a native gene and/or the 5′ &/or the 3′ nontranslated regions in the mRNA may be retained for transcriptional or translational regulation.

“Glucoamylase P genome DNA can be extracted from any cell, preferably a filamentous fungal cells, and purified by using methods well-known in the art. (For example, see Guide to Molecular Cloning Techniques S. L. Berger et. al., Academic Press (1987). If a cDNA clone of glucoamylase is required, it is preferred that the mRNA preparation used be enriched in mRNA coding to glucoamylaseP. This can be done either naturally from a source producing large quantities of the protein or in vitro using techniques such as sucrose gradient centrifugation. Hormoconis resinae, a filamentous fungus is preferred as a source for glucoamylase mRNA.

“To clone into a vector, the desired DNA (either genome DNA or ds-cDNA) can be randomly sheared, or enzyme cleaved. The vector will then be ligated into a suitable vector to create a recombinant (either cDNA or genomic) gene library.

“A DNA sequence encoding the glucoamylase protein P or its functional derivatives can be placed into a DNA vector according to conventional techniques. This includes restriction enzyme digestion to provide appropriate termini and filling in of cohesive end as necessary, alkalinephosphatase treatment in order to avoid unwanted joining, and ligation using appropriate ligases. These techniques are well-known in the art.

“Libraries containing sequences coding for glucoamylase P may be screened and a sequence coding for glucoamylase P identified by any means which specifically selects for such sequence, such as, for example, a) by hybridization with an appropriate nucleic acid probe(s) containing a sequence specific for the DNA of this protein, or b) by hybridization-selected translational analysis in which native mRNA which hybridizes to the clone in question is translated in vitro and the translation products are further characterized, c) if the cloned genetic sequences are themselves capable of expressing mRNA, by immunoprecipitation of a translated glucoamylase P product produced by the host containing the clone, or d) by assay of glucoamylase P activity in the growth medium of the transformed hosts.”

“Oligonucleotide probes that are specific for glucoamylaseP can be used to identify clones of this protein. They can be constructed from knowledge about the amino acid sequence. The amino acid sequence is presented horizontally. Unless otherwise noted, the amino terminus and carboxy terminus are to be listed at the left and right ends, respectively.

“Because the gene code is degenerate, multiple codons may be used to encode a particular amino acid (Watson, J. D., in: Molecular Biology of the Gene, Third Ed., W. A. Benjamin, Inc., Menlo Park, Calif. (1977), pp. 356-357). Analyzing the amino acid sequence can help identify sequences that may be encoded using oligonucleotides with the lowest degree degeneracy. It is best to identify sequences that only contain one codon of amino acids.

“Although an amino acid sequence can sometimes be encoded only by one oligonucleotide sequence at a time, it is possible for the sequence to be encoded using any number of similar oligonucleotides. It is important to note that, while all members of the set have oligonucleotide sequencings capable of encoding the exact same peptide sequence and thus may contain the same sequence of oligonucleotides as the gene which encodes it, only one member contains the nucleotide that matches the exon coding sequence. This member of the set is capable of hybridizing with DNA, even when there are other members in the set. It is therefore possible to use the unfractionated set oligonucleotides in a similar way as one would to clone a single gene that encodes the protein.

“Using the genetic code (Watson, J. D., et al., in: Molecular Biology of the Gene, Third Ed., W. A. Benjamin, Inc., Menlo Park, Calif. (1977),), one or more oligonucleotides are identified. This can be done by looking at abnormal base pairing relationships and the frequency that a specific codon is used to encode a particular amino acids in eukaryotic cells. These “codon usage guidelines” were disclosed by Lathe, R. and colleagues, J. Molec. Biol. 183:1-12 (1985). The “codon use rules” of Lathe allow us to identify a single or set of oligonucleotide sequencings that have a theoretical’most probable’ nucleotide sequence capable encoding the glucoamylase P sequences.

“The appropriate oligonucleotide or set of oligonucleotides that is capable of encoding a glucoamylase fragment (or which is complementary to such an oligonucleotide or set of oligonucleotides), may be synthesized using methods well known in art (see, for instance, Synthesis and Application of DNA and RNA by S. A. Narang 1987, Academic Press San Diego, Calif.). It can then used as cloned glucoamylase gene to isolate glucoamylase cloned glucoamylase glucoamylase gene to identify and to cloned to oligonucleotide or set of oligonucleotides or set of oligonucleotide oligonucleotide oligonucleotide -to otide or set of oligonotide and es, es, es, es, IRL Press, Washington, D. Maniatis, T., and colleagues have described techniques for nucleic acid hybridization, and clone identification. Hames, B. D., and others, have published Nucleic Acid Hybridization, a Practical Approach, IRL Press, Washington, D.C. (1985). The members of the gene library described above that are capable of hybridization are then examined to determine the nature and extent of glucoamylase-P encoding sequences they contain.

The above-described DNA probe has been labeled with a detectable number to facilitate detection of the desired glucoamylase DNA encoding sequence. Any material with a detectable chemical or physical property can be considered a detectable group. These materials are well-developed in the field nucleic acid hybridization. In general, any label that is useful in such methods can also be used in the present invention. Radio-active labels such as 32P, 3H, 14C, 35S, and the like are particularly useful. Any radioactive label that provides sufficient signal strength and has a long half-life can be used. Radioactive labeling may be used to radioactively label single-stranded oligonucleotides using kinase reaction. Polynucleotides, which are equally useful, can also be radioactively labelled using kinase reactions to be used as nucleic acids hybridization probes. See, for example, Leary, J. J. et al., Proc. Natl. Acad. Sci. USA 80:4045 (1983); Renz, M. et al., Nucl. Acids Res. 12:3435 (84); Renz, M. EMBO J. 6:817 (1983).”

In summary, the actual identification and sequencing of glucoamylase P proteins allows for the identification of a hypothetical “most likely” DNA sequence or a set thereof capable of encoding such a peptide. One can construct an oligonucleotide that is complementary to this hypothetical sequence, or a set oligonucleotides that are complementary to the set “most likely” oligonucleotides. This will give one a DNA molecule (or a number of DNA molecules) capable of serving as a probe for the isolation and identification of clones containing the glucoamylase p gene.

“An alternative method of cloning a glucoamylase P genes is to prepare a library using an expression vector. This involves cloning DNA, or more preferably cDNA, from a cell that can express glucoamylase P into a vector. After screening the library for members that express glucoamylase P (e.g., screening it with antibodies or assaying growth medium to determine glucoamylase P activity), the library is analyzed for any such members.

“The methods discussed above are therefore capable of identifying genetic sequences capable of encoding either glucoamylaseP or its fragments. It is important to express these proteins in order to further identify such genetic sequences and to create recombinant glucoseamylase P. This includes full-length, antigenic, or enzymatically activated fragments. This expression identifies clones that express proteins with glucoamylase P characteristics. It can be used to identify clones that have the ability of binding glucoamylase P antibodies, to produce antibodies capable of binding to glucoamylase P enzyme activity, or to provide a glucoamylase P function to a recipient.

Site-directed mutation may be used to prepare mutant glucoamylase P activities. This is as per the art. Site-directed mutation may be used to modify the thermal stability of the invention, as described in Itoh, T. and colleagues, Agric. Biol. Chem. 53:3159-3167 (1989), for the glucoamylase of Saccharomycopsis Fibuligera. Ala 81, Asp 89 and Asp 89 have been shown to be critical for the enzyme’s thermal stability.

“Expression of GlucoamylaseP and its Functional Derivatives.”

“To express glucoamylaseP and/or its enzymatically activate derivatives, transcriptional or translational signals that are recognizable by a suitable host are required. Cloned glucoamylase protein encoding sequences may be linked to sequences controlling transcriptional activity in an expression vector. These sequences can then be introduced into prokaryote and eukaryote cells to create recombinant glucoseamylaseP or a functional derivative. Depending on which strand of glucoamylase P encoding is operably linked with the sequences controlling transcriptional activity, it may be possible to express glucoamylase P antisense RNA and a functional derivative thereof.

“Expressions of the glucoamylase in different hosts can result in different post-translational modification which could alter the properties of this protein. Glucoamylase or a functional derivative thereof can be expressed in eukaryotic and especially fungal cells. The present invention preferably includes the expression of glucoamylase or a functional derivative thereof in transformed Trichoderma Reesei.

“A nucleic acid molecule such as DNA is considered to be capable of expressing a polypeptide” if it contains expression control sequencings that contain transcriptional regulatory information. Such sequences can be operatably linked to the nucleotide sequence that encodes the polypeptide.

An operable linkage refers to a connection in which a sequence connects to a regulatory sequence or sequences in such a manner that it places expression of the sequence under control or influence of the regulatory sequence. If two DNA sequences, such as a glucoamylase P encoding and a promoter sequence linked to 5′ of the protein encoding, are induction of promoter functions, and if the nature and function of the linkage between them does not (1) result or interfere with the ability to regulate the expression of glucoamylase P or antisense RNA or protein or (3) affect the ability to transcribe the glucoamylase P template by the promoter sequence, then they are considered operable links. A promoter region is considered operable if it can effect transcription of a particular DNA sequence.

The exact nature of regulatory regions required for gene expression can vary depending on species and cell types. However, they will generally include 5′ nontranscribing (non-coding), and 5′ translating sequences that are involved in initiation of transcription or translation, such as the TATA Box, CAAT sequence, Capping sequence, and the capping sequence. These 5′ non-transcribing sequences will contain a region that contains a promoter to control transcription of the operably linked genes. These transcriptional control sequences can also contain enhancer sequences and upstream activator sequences, if desired.

“Expression of glucoamylaseP in recombinant host requires the use regulatory regions that are functional in such hosts. Depending on the host’s nature, there are many transcriptional and/or translational regulatory sequences that can be used. You can also derive transcriptional and/or translational regulatory signals from the genome sequences of viruses that infect host cells. A desired regulatory signal should be associated with a specific gene that is capable of high expression in the host cells.

“In eukaryotes where transcription is not linked with translation, such control areas may or may not contain an initiator methionine codon (AUG), depending on whether the cloned sequencing contains such a methionine. These regions generally contain a promoter area that can direct the initiation and maintenance of RNA synthesis within the host cell. Promoters from heterologous fungal gene promoters that encode a mRNA product capable to translation are preferred. In particular, promoters for T.reesei cellobiohydrolase I, cellobiohydrolase II, cellobiohydrolase III (CBHII), and endoglucanase I, (EGII), may be used, particularly in the T.reesei host. The yeast GAL4 gene promor or the glycolytic gene promoter (such as the glycerol-3 phosphate dehydrogenase enhancer) may be used in laboratory yeast hosts.

“As it is well known, the codon that encodes the first methionine is where translation of the eukaryotic transcript begins. It is important to ensure that the linkage between an eukaryotic promoter, a DNA sequence that encodes the glucoamylaseP, or a functional derivative thereof does not include any codons capable of encoding methionine. If such codons are present, they can lead to a formation of either a fusion protein (if AUG codons are in the same frame as the glucoamylaseP encoding DNA sequence sequence) or a frameshift mutation (if AUG codons are not in the same frame as the glucoamylaseP encoding sequence).

“A glucoamylase fusion product P can be made if desired. Full-length glucoamylaseP encodes a signal sequence of 29 amino acids that allows secretion from fungal hosts such as Hormoconis resinae Saccharomyces cerevisiae, Trichoderma reesei.

“The sequence coding mature glucoamylase may be linked with a heterologous signal sequence that will allow secretion or compartmentalization of the protein within a specific host. These signal sequences can be constructed with or without specific protease site so that the signal sequence can be removed later. T. reesei hosts can use signal sequences from cellulase genes and, in particular, cellobiohydrolase II and cellobiohydrolase I signal sequences.

“Transcriptional initiating regulatory signals can also be selected that allow activation or repression of genes, so that they can be controlled.”

The non-transcribed/or nontranslated regions 3′ to P sequence coding can be obtained if desired. The 3′-nontranscribed region can be retained to its transcriptional termination regulatory elements. The 3′ non-transcribed area may be retained to its translational regulatory sequence elements or elements that direct polyadenylation within eukaryotic cell. If the native expression control sequences fail to function in the host cells, the sequences that work in the host cells may be replaced.

“To transform a fungal cells with the DNA constructs, many vector systems are possible depending on whether the glucoamylase DNA construct is to be inserted into the host cell’s chromosomal DNA or allowed to exist in an extrachromosomal version. EP 244,234 describes the stable transformation of Trichoderma reesei as well as the expression of heterologous gene in such transformants. The preferred plasmid system is described in the same as in U.S. Application Ser. No. No. 19, 1990 or in U.S. Application Ser. 07/524.308 was filed May 16, 1990. It is incorporated by reference.”

The transient expression may occur if the glucoamylase-P encoding sequence is introduced into a recipient eukaryotic cells as a nonreplicating DNA or RNA molecule. This non-replicating DNA or RNA molecule can be either a linear molecule, or more preferably, a closed covalent circle molecule that is ineligible for autonomous replication.

“Preferable embodiments may include genetically stable transformants that are constructed using vector systems or transformation systems where glucoamylase-P DNA is integrated into the host genome. This integration can occur naturally within the cell, or it may be assisted by a vector inserting the vector into the host chromosome. The vector can integrate the desired sequences of gene into the fungal host cell’s chromosome. Such vectors can be used to integrate a T. reesei gene into a fungal host cell chromosome. This is called the cellobiohydrolase 1 gene. It promotes homologous recombination on a particular site of the host chromosome.

“Cells that have successfully integrated the DNA into their chromosomes can be selected by adding one or more markers. This allows for the selection of host cells that contain the expression vector. The marker might provide biocide resistance, such as resistance to antibiotics or heavy metals like copper. You can link the selectable marker gene directly to the DNA sequences to be expressed or introduce the marker into the same cell via co-transfection. U.S. patent Ser. No. No. 07/496,155 filed Mar. 19, 1990; and in Finkelstein, D. B., Chapter 6 in Biotechnology of Filamentous Fungi, D. B. Finkelstein et al., eds., Butterworth-Heinemann, Boston, 1992, pp. 113-156 are both included herein completely by reference.

“Factors that are important in choosing a vector include the ability of recipient cells to recognize the vector and select them from others; the number and quality of copies of vectors desired by a host; and the possibility of being able to shuttle the vector between different host cells.

Once the vector or DNA sequence containing construct(s), is ready for expression, the DNA construct(s), can be introduced into the appropriate host cell using any of a number of suitable methods. Recipient cells are then grown in a selective media (if required), to allow for the growth vector-containing cells. The cloned gene sequences can be expressed to produce the glucoamylase (or a portion) of this protein. The expression can occur in continuous fashion in the transformed cells or it can be controlled by a stimulus that induces the host cells to produce the cloned glucoseamylase P.

“The expressed recombinant glucoseamylase P protein can also be isolated and purified if desired. This is done according to conventional conditions such as extraction, precipitation or affinity chromatography.

“Industrial Uses for Glucoamylase P.”

“Distilled alcohol is produced by a series, which includes the conversion of raw materials to sugars that can then be used by yeast and the fermentation process, followed by distillation. The residue usually contains proteins and minerals and is useful for animal feed. An integrated process, in which barley is converted to starch, carbon dioxide and other feed fractions, can be used.

For alcohol manufacturing, the smaller starch grains, known as “B starch”, are used with a diameter of approximately 5 microns. A-starch granules with a diameter of 15 microns or more are used primarily for coating paper.

“The key advantages of integrating ethanol and starch production are in the process and the product economics. The water from starch manufacturing can also be used in alcohol production, material flows can be flexible, and the emphasis can be put on the production end products that are most in demand.

“Processe such as the invention which increase the efficiency of the enzymes that convert starch to sugars are highly desirable. The T. reesei-glucoamylase P strains are particularly useful in brewing, including the production of beer and the manufacturing of grain alcohol. The strains of the invention secrete a mixture of enzymes (?-1,4-glucoamylase, ?-1,6-glucoamylase P, ?-glucanase, hydroxyethylcellulose degrading activity (HEC), filter paper degrading activity (FPU), xylanase (hemicellulose degrading activity), acid protease (protein degrading activity at pH 4.7) and ?-glucosidase (cellobiose degrading activity)) into their growth medium. The medium can be used immediately after separation of the solids. This allows the medium to efficiently and quickly break down macromolecular substances found in ground grain slurry into substrates that are utilizable for yeast. The growth medium can be diluted or concentrated to adjust the activity of the desired enzyme(s), using techniques such as lyophilization and dialysis.

The enzyme preparations of this invention are active on the 1,6 linkages. They degrade isomaltose and panose 5-30 times faster than preparations that contain Aspergillus glucoamylase or Rhizopus. Hydrolysis of starch to its entirety increases the production of simple sugars, which in turn increases the overall alcohol yield.

A cascade fermentation is more economical and efficient than a batch method of fermentation. A continuous volumetric productivity can be achieved with higher cell concentrations (either by cell recycling, or immobilization), which is 5-20 times greater than that of the batch mode.

“Cascading fermentation has the advantage that there is no lag for starting fermentation. Also, the inhibition of fermentation caused ethanol can be reduced over the entire range of fermentation. This is done by limiting the severity of the inhibition to the last vessel of the series. Cascade fermentations do not last forever, as the process is stopped every 2 to 3 weeks and the tanks are emptied for cleaning.

“All enzymes secreted in Trichoderma culture growth medium are contained.” This medium contains enzymes that have been reasonably well characterised (see U.S. Appl. Ser. No. No. Ser. No. No. The medium can be used either as a source or mixture of these enzymes.

“The Hormoconis resinae glucose containing growth medium is produced by any Trichoderma stain that has been transformed using cDNA or genomic sequences that encode glucoamylase. It is a useful preparation and contains the desired mix of enzyme activities. Trichoderma alko 2743 (ALKO?GA) is an example of one such strain. Starch-based biological processing and specifically ethanol production are some examples of possible uses. T. reesei strains 2743 and 233 have approximately the same activity levels as their respective cellulases and?-glucanase activities. Strain 233 has about 1/3 of the xylanase activity as strain 2743.

“Generally, there are three steps to mashing grain for ethanol production.

“1) Heating (also known as gelatinization);

“2) Addition of?-amylase enzyme

“3) Addition of amylolytic enzymes like pullulanase and glucoamylase (also known as saccharification).

The invention’s glucoamylase-containing medium (or enzyme preparation), may be added at any stage of mashing. The heating will inactivate the enzyme if it is added prior to or during gelatinization. Additional enzyme must be added after heating.

“The addition of growth medium of invention (that is, growth media taken from the culture Trichoderma and transformed with sequences encoded the glucoamylaseP of the invention), at the beginning mashing or fermentation increases the recovery and alters beer’s characteristics. Pilot and laboratory scale experiments show identical results. These results also differ between the ALKO?GA and commercial glucoamylase concentrate NOVOAMG 300 L, which are produced by Aspergillus.

“The enzyme activity tests from enzyme concentrates showed that ALKO-GA has starch-decomposing side activities such as cellulases, xylanases, and?glucosidase in amounts greater than those currently available like NOVO AMG 300L.”

“The amount of ALKOGA enzyme addition to the wort affected the characteristics of the wort when it was mixed in laboratory scale mashing. This dose effect gives you maximum flexibility to alter the characteristics of your wort. The enzyme effects were similar with ALKO/GA and NOVOAMG 300L except that the concentrations of?-glucans found in the treated wort were very low when ALKO/GA was used. However, the concentrations of NOVOAMG 300L and ALKO-GA were comparable to the control group. ALKO-GA worts have a lower viscosity than the control group and a higher filtration rate, which are both benefits in an industrial setting. The ALKO-GA treated mashing is significantly more efficient than the NOVO AMG 300L treated mashing. This is why the volume of the wort is so much higher. This is another advantage of the invention.

The following are the reasons why ALKO-GA has a better response (even at 0.1 g/100g mash): The?-glucanase xylanase, cellulase and cellulase activity in ALKO-GA are higher than those in NOVO AMG300 L. ALKO-GA’s?-glucanase seems to have a higher temperature stability than the Aspergillus Niger??glucanase. However, it seems that the fermentation mixture does not lose its ability to maintain temperature. Although the extract yield is the same in all conditions, the extract volume was significantly higher when ALKOGA was added to mashing than other commercial preparations such as the NOVO AMG300 L. This was even more so when compared with the control experiment.

The apparent limit of attenuation increased from 84.7% up to 89% when the ALKOGA dose was 3g/100g mash. Although the higher dose did not have any effect, there was an increase in fermentable sugars (glucose and maltose etc.). These correlate with an increase in enzyme dosage. If NOVO-AMG 300L was used in mashing, the result is identical. This result shows that the ALKO-GA dosage 3 g/100g mash is preferable for Pilot scale (100L) brewing experiments.

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