Invented by Badal C. Saha, Rodney J. Bothast, US Department of Agriculture USDA

Thermostable ?-L-arabinofuranosidase from Aureobasidium pullulans is an enzyme that is gaining popularity in the market due to its unique properties and potential applications. This enzyme is a type of arabinofuranosidase that is capable of breaking down arabinose, a type of sugar found in plant cell walls. The thermostable nature of this enzyme makes it ideal for use in a variety of industrial processes, including biofuel production, food processing, and animal feed production. The market for thermostable ?-L-arabinofuranosidase from Aureobasidium pullulans is expected to grow significantly in the coming years. This is due to the increasing demand for sustainable and eco-friendly products in various industries. The use of this enzyme in biofuel production is particularly promising, as it can help to increase the efficiency of the process and reduce the overall cost of production. In addition to biofuel production, thermostable ?-L-arabinofuranosidase from Aureobasidium pullulans has potential applications in the food industry. This enzyme can be used to break down arabinose in plant cell walls, making it easier to extract nutrients and flavors from plant-based ingredients. This can lead to the development of new and innovative food products that are both nutritious and flavorful. Animal feed production is another area where thermostable ?-L-arabinofuranosidase from Aureobasidium pullulans can be used. This enzyme can help to break down the cell walls of plant-based feed ingredients, making them more digestible for animals. This can lead to improved animal health and productivity, as well as reduced feed costs for farmers. Overall, the market for thermostable ?-L-arabinofuranosidase from Aureobasidium pullulans is expected to continue to grow in the coming years. This enzyme has unique properties that make it ideal for use in a variety of industrial processes, and its potential applications are vast. As more industries look for sustainable and eco-friendly solutions, the demand for this enzyme is likely to increase, making it a promising investment for businesses in the biotech and industrial sectors.

The US Department of Agriculture USDA invention works as follows

An ?-L-arabinofuranosidase enzyme which is highly thermostable, and is effective for the hydrolysis of arabinofuranosyl residues from L-arabinose containing polysaccharides and hemicelluloses is disclosed. The enzyme is produced from color variant Aureobasidium Pullulans strain NRRL-Y-21792. This ?-L-arabinofuranosidase may be used in conjunction with xylanolytic enzymes for the treatment of hemicellulosic materials to produce fermentable sugars, particularly xylose and L-arabinose.

Background for Thermostable ?-L-arabinofuranosidase from Aureobasidium pullulans

1. “1.

This invention relates to an ?-L-arabinofuranosidase effective for hydrolyzing arabinosyl residues from L-arabinose containing polysaccharides and hemicelluloses, and which is stable at high temperatures.

2. “2.

More that one billion gallons ethanol is produced each year in the United States. Approximately 95% of this ethanol comes from the fermentation of corn starch. (Bothast 1994, Genetically modified microorganisms used to convert multiple substrates into ethanol, Proceedings of the National Corn Growers Assoc., St. Louis, Mo.). Corn Utilization Conf., National Corn Growers Assoc., St. Louis, Mo.). As well as being abundant and low-value, lignocellulosic agricultural wastes like corn fiber, corn stover and straw, bagasse and corn fiber can be used to produce fuel alcohol. The use of lignocellulosic biomass to produce fuel alcohol is a significant challenge, both economically and technically. Its success will depend on the development and application of cost-effective and highly efficient biocatalysts to convert pretreated biomass into fermentable sugars.

Bioresourc., 50:3-16. Technol., 50:3-16). L-Arabinosyls are found in many hemicelluloses. They are monomeric or oligomeric side-chains on the?-1?4-linked xylose and galactose xylans (xylans), arabinoxylans (arabinogalactans) and arabinans (forming?-1?5)-linkages. J. 302:443-449. Ward and Moo Young, 1989, CRC Critical. Rev. Biotechnol., 8:237-274). These side chains inhibit the enzymatic hydrolysis by xylanases of hemicelluloses (Bachmann & McCarthy, 1991, Appl. Environ. Microbiol., 57:2121-2130). ?-L-Arabinofuranosidases (?-L-arabinofuranoside arabinofuranohydrolase, EC 3.2.1.55, ?-L-AFase) are exo-type enzymes which hydrolyze terminal non-reducing ?-L-arabinofuranosyl groups from L-arabinose containing polysaccharides. These enzymes can hydrolyze (1?3)- or (1?5) -?-L-arabinofuranosyl linkages of arabinan or both. These?-L AFases are essential for the breakdown of heteroxylans in microbial xylanolytic system (Bachmann & McCarthy, 1991; ibid. ; Greve et al. 1984, Appl. Environ. Microbiol. 47:1135-1141; Lee and Forsberg 1987, Can. J. Microbiol., 33:1011-1016; Poutanen, 1988, J. Biotechnol., 113:15-22).

In recent years arabinofuranosidases received much attention due to their practical applications in agro-industrial process such as efficient conversion hemicellulosic biomass into fuels and chemicals. Delignification pulp. Environ. Microbiol. 40:57-62 Gilead & Shoham 1995, Appl. Environ. Microbiol., 61:170-174; Gunata et al., 1990, J. Agric. Food Chem., 38:772-776; Utt et al., 1991, Appl. Environ. Microbiol., 57:1227-1234). It is necessary to develop a?-L -AFase that can be used to convert hemicellulose into fermentable sugars to produce fuel ethanol or other value-added chemical products. Several bacteria and fungi produce?-L -AFases, but only a small number of them have been purified and characterized. Environ. Microbiol. 58:1082-1088. Kaji and Tagawa 1970, Biochim. Biophys. Acta, 207, 456-464. Lee and Forsberg ibid. Tajana et. al. 1992, Appl. Environ. Microbiol. ; 58:1447-1450). Aureobasidium Pullulans, a yeast-like fungus, has been identified as a superior producer of xylanase, amylases and?-glucosidase. Environ. Microbiol., 52:1026-1030; Saha et al., 1993, Curr. Microbiol., 26:267-273; Saha et al., 1994, Appl. Environ. Microbiol., 60:3774-3780).

We have now discovered a new thermostable ?-L-arabinofuranosidase enzyme which is effective for the hydrolysis of arabinofuranosyl residues from L-arabinose containing polysaccharides and hemicelluloses at high temperatures. The enzyme is produced from color variant A. Pullulans strains NRRL Y-21792. This ?-L-arabinofuranosidase may be used in conjunction with xylanolytic enzymes for the treatment of hemicellulosic materials to produce fermentable sugars, particularly xylose and L-arabinose.

In accordance with this discovery, it is an object of this invention to provide a novel ?-L-arabinofuranosidase enzyme for the enzymatic hydrolysis of L-arabinose containing polysaccharides or hemicellulose, which is highly thermostable.

Another object of this invention is to provide an improved process for the enzymatic conversion of hemicellulose to L-arabinose using an ?-L-arabinofuranosidase insensitive to high temperatures.

Another object of this invention is to provide an improved process for the enzymatic conversion of hemicellulose to L-arabinose, which may be subsequently fermented to ethanol, using an ?-L-arabinofuranosidase insensitive to high temperatures.

The following description will make it clear that this invention has many other objects and benefits.

DESCRIPTION DU DRAWINGS

FIG. 2. Temperature and stability (?) Effect of temperature on stability (?) of purified ?-L-arabinofuranosidase from A. pullulans Y-21792. The residual enzyme activity was then measured after incubating the enzyme solution for 30 minutes at different temperatures in an acetate buffer of 50 mM and pH 5.0. The standard assay method was used to measure the activity of the enzyme at different temperatures. Enzyme used, 18 mU/ml.

The enzyme of this invention is an ?-L-arabinofuranosidase (?-L-arabinofuranoside arabinofuranohydrolase, EC 3.2.1.55) which is effective for the exo-hydrolysis of L-arabinose containing polysaccharides and hemicelluloses with the release of L-arabinose (?-L-arabinofuranose) therefrom. The enzyme can hydrolyze a variety of L-arabinose substrates including, but not limited, to xylans and arabinoxylans. “The enzyme has specific hydrolytic activity on both (1?3) and (1?5) linked, terminal, non-reducing -L-arabinofuranoses in any L-arabinose-containing polysaccharide, but does not act upon any internal -L-arabinosyl links.

The preferred microorganism for the production of the ?-L-arabinofuranosidase of this invention is a strain of Aureobasidium pullans, strain NRRL Y-21792. The strain was deposited in Peoria (Ill.) in the Agricultural Research Service Culture Collection on June 24, 1997, under the Budapest Treaty. 24, 1997. The Agricultural Research Service Culture Collection also has this strain of Aureobasidium Pullulans under the accession number NRRL Y 12974.

The color variant strain A. pullulans used in the present invention is NRRL strain Y-21792. This strain’s morphological patterns and carbon assimilation patterns are similar to those described by Barnett & Hunter in Illustrated Genera of Imperfect Fungi (Burgess Publishing, Minneapolis Minn. 1972). The Yeasts: A Taxonomy Study (4th Edition, Kurtzman & Fell ed. !, Elsevier Science B. V., 1997, pp. The contents of these publications are incorporated herein by reference. The strain differs from typical A. pullulans strains by its bright pink pigment, high xylanase production, and low DNA similarity (Leathers et al., 1988, J. Indust. Microbiol., 3:231-239).

Click here to view the patent on Google Patents.