Patent ID: 12247235

DETAILED DESCRIPTION OF THE INVENTION

Milling Process

It is an object of the invention to provide improved processes of treating crop kernels to provide starch of high quality.

In one embodiment, the enzyme compositions useful in the processes of the invention provide benefits including, improving starch yield and/or purity, improving gluten quality and/or yield, improving fiber, gluten, or steep water filtration, dewatering and evaporation, easier germ separation and/or better post-saccharification filtration, and process energy savings thereof.

Moreover, the present inventors have surprisingly found that the enzymes useful according to the invention provide reduction in fiber mass and lower protein content of the fiber due to better separation of both starch and protein fractions from the fiber fraction. Separating starch and gluten from fiber is valuable to the industry because fiber is the least valuable product of the wet milling process, and higher purity starch and protein is desirable.

Surprisingly, the present inventors have discovered that replacing some of the protease activity in an enzyme composition can provide an improvement over an otherwise similar composition containing predominantly protease activity alone. This can provide a benefit to the industry, e.g., on the basis of cost and ease of use.

The kernels are milled in order to open up the structure and to allow further processing and to separate the kernels into the four main constituents: starch, germ, fiber and protein.

In one embodiment, a wet milling process is used. Wet milling gives a very good separation of germ and meal (starch granules and protein) and is often applied at locations where there is a parallel production of syrups.

The inventors of the present invention have surprisingly found that the quality of the starch final product may be improved by treating crop kernels in the processes as described herein.

The processes of the invention result in comparison to traditional processes in a higher starch quality, in that the final starch product is more pure and/or a higher yield is obtained and/or less process time is used. Another advantage may be that the amount of chemicals, such as SO2 and NaHSO3, which need to be used, may be reduced or even fully removed.

Wet Milling

Starch is formed within plant cells as tiny granules insoluble in water. When put in cold water, the starch granules may absorb a small amount of the liquid and swell. At temperatures up to about 50° C. to 75° C. the swelling may be reversible. However, with higher temperatures an irreversible swelling called “gelatinization” begins. Granular starch to be processed according to the present invention may be a crude starch-containing material comprising (e.g., milled) whole grains including non-starch fractions such as germ residues and fibers. The raw material, such as whole grains, may be reduced in particle size, e.g., by wet milling, in order to open up the structure and allowing for further processing. Wet milling gives a good separation of germ and meal (starch granules and protein) and is often applied at locations where the starch hydrolyzate is used in the production of, e.g., syrups.

In an embodiment, the particle size is reduced to between 0.05-3.0 mm, preferably 0.1-0.5 mm, or so that at least 30%, preferably at least 50%, more preferably at least 70%, even more preferably at least 90% of the starch-containing material fits through a sieve with a 0.05-3.0 mm screen, preferably 0.1-0.5 mm screen.

More particularly, degradation of the kernels of corn and other crop kernels into starch suitable for conversion of starch into mono- and oligo-saccharides, ethanol, sweeteners, etc. consists essentially of four steps:1. Steeping and germ separation,2. Fiber washing and drying,3. Starch gluten separation, and4. Starch washing.
1. Steeping and Germ Separation

Corn kernels are softened by soaking in water for between about 30 minutes to about 48 hours, preferably 30 minutes to about 15 hours, such as about 1 hour to about 6 hours at a temperature of about 50° C., such as between about 45° C. to 60° C. During steeping, the kernels absorb water, increasing their moisture levels from 15 percent to 45 percent and more than doubling in size. The optional addition of e.g. 0.1 percent sulfur dioxide (SO2) and/or NaHSO3 to the water prevents excessive bacteria growth in the warm environment. As the corn swells and softens, the mild acidity of the steepwater begins to loosen the gluten bonds within the corn and release the starch. After the corn kernels are steeped they are cracked open to release the germ. The germ contains the valuable corn oil. The germ is separated from the heavier density mixture of starch, hulls and fiber essentially by “floating” the germ segment free of the other substances under closely controlled conditions. This method serves to eliminate any adverse effect of traces of corn oil in later processing steps.

In an embodiment of the invention the kernels are soaked in water for 2-10 hours, preferably about 3-5 hours at a temperature in the range between 4° and 60° C., preferably around 50° C.

In one embodiment, 0.01-1%, preferably 0.05-0.3%, especially 0.1% SO2and/or NaHSO3may be added during soaking.

2. Fiber Washing and Drying

To get maximum starch recovery, while keeping any fiber in the final product to an absolute minimum, it is necessary to wash the free starch from the fiber during processing. The fiber is collected, slurried and screened to reclaim any residual starch or protein.

3. Starch Gluten Separation

The starch-gluten suspension from the fiber-washing step, called mill starch, is separated into starch and gluten. Gluten has a low density compared to starch. By passing mill starch through a centrifuge, the gluten is readily spun out.

4. Starch Washing

The starch slurry from the starch separation step contains some insoluble protein and much of solubles. They have to be removed before a top quality starch (high purity starch) can be made. The starch, with just one or two percent protein remaining, is diluted, washed 8 to 14 times, re-diluted and washed again in hydroclones to remove the last trace of protein and produce high quality starch, typically more than 99.5% pure.

Products

Wet milling can be used to produce, without limitation, corn steep liquor, corn gluten feed, germ, corn oil, corn gluten meal, corn starch, modified corn starch, syrups such as corn syrup, and corn ethanol.

Palm Oil Extraction

The present invention also provides a process for enzyme assisted extraction of crude palm oil from a substrate comprising palm oil. The substrate comprising palm oil can be selected from the group consisting of palm fruitlets, pressed palm fruit liquid, mashed or partly mashed palm fruitlets. The inventors have found that by using a GH10 xylanase on the substrate comprising palm oil, the oil extraction rate (OER) can be increased.

The invention concerns a process for extraction or separation of crude palm oil (CPO), comprising the steps of:i) contacting a substrate comprising palm oil with an enzyme composition,ii) extracting or separating the crude palm oil (CPO)wherein the enzyme composition comprises a GH10 xylanase and a GH62 arabinofuranosidase.

In one embodiment of the invention, the substrate comprising palm oil is palm fruitlets, which comprise oil in the mesocarp of the fruit. The palm fruitlets are contacted with the enzyme composition. In one embodiment, the substrate is palm fruitlets, which are mashed or partly mashed and contacted with the enzyme composition. This increases availability of mesocarp cells and thereby enhances enzyme activity on the mesocarp cells. In one embodiment, the substrate comprising palm oil is crude palm oil which is contacted with the enzyme composition. In the various aspects and embodiments of the invention the substrate, which comprises palm oil may be a substrate which also comprises fiber, in particular fiber from the mescocarp of palm fruitlets.

In one embodiment of the invention the substrate comprising palm oil is sterilized before being contacted with the enzyme composition. Palm fruits grow in large bunches and needs to be stripped from the bunch stalks before being contacted with the enzyme composition. Steam sterilization of the fresh fruit bunches facilitates fruits being stripped from bunches to give the palm fruitlet. The sterilization step has several advantages one being that it softens the fruit mesocarp for subsequent digestion. A further advantage is that the quality of the final palm oil product is better if the palm fruits are stripped from the bunch stalks.

The sterilization can be a batch sterilization or a continuous sterilization. The sterilization process can be carried out at a temperature of 100° C.-150° C. In one embodiment of the invention, the pressure is reduced during the sterilization procedure.

After the sterilization, the palm fruitlets are stripped from the bunch stalks. Stripping or threshing can be carried out in a mechanized system having a rotating drum or fixed drum equipped with rotary beater bars which detach the fruit from the bunch and leaves the spikelets on the stem. The stripped palm fruitlets can be contacted with the enzyme composition according to the invention.

In one embodiment of the invention, the substrate comprising palm oil is subjected to digestion before extracting the crude palm oil. The stripped palm fruitlets can be transported into a digester by one or more transportation means, e.g. a conveyor belt. In the digester, the fruitlets are further heated in order to loosen the pericarp. The digester is typically a steam heated vessel, which has rotating shafts to which stirring arms are attached or is equipped with baffles. The fruitlets are rotated, causing the loosening of the pericarps from the nuts and degradation of the mesocarp. The digester is a continuous process where the digester is kept full and as the digested fruit is drawn out, freshly stripped fruits are brought in.

In one embodiment of the invention, the first part of the digestion is carried out in a precooker. The substrate may be held at a temperature within the range of 65-85° C. for some time and then transferred to the digester tank.

Polypeptides Having GH62 Arabinofuranosidase Activity

Preferred embodiments of the aspect of the invention relating to the GH62 polypeptide having arabinofuranosidase activity are disclosed herein below.

In an embodiment, the polypeptide having GH62 arabinofuranosidase activity of the present invention, is selected from the group consisting of:(a) a polypeptide having at least 85%, e.g., at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the mature polypeptide of SEQ ID NO: 1;(b) a variant of the mature polypeptide of SEQ ID NO: 1 comprising a substitution, deletion, and/or insertion at one or more (several) positions;(c) a polypeptide encoded by a polynucleotide having at least 85%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the mature polypeptide coding sequence of SEQ ID NO: 2 or the cDNA sequence thereof.

In an embodiment, the polypeptide having GH62 arabinofuranosidase activity of the present invention, is selected from the group consisting of:(a) a polypeptide having at least 85%, e.g., at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the mature polypeptide of SEQ ID NO: 3;(b) a variant of the mature polypeptide of SEQ ID NO: 3 comprising a substitution, deletion, and/or insertion at one or more (several) positions.

In one aspect, the polypeptide differs by up to 10 amino acids, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, from the mature polypeptide. In another embodiment, the present invention relates to variants of the mature polypeptide comprising a substitution, deletion, and/or insertion at one or more (e.g., several) positions. In an embodiment, the number of amino acid substitutions, deletions and/or insertions introduced into the mature polypeptide is up to 10, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. The amino acid changes may be of a minor nature, that is conservative amino acid substitutions or insertions that do not significantly affect the folding and/or activity of the protein; small deletions, typically of 1-30 amino acids; small amino- or carboxyl-terminal extensions, such as an amino-terminal methionine residue; a small linker peptide of up to 20-25 residues; or a small extension that facilitates purification by changing net charge or another function.

A polypeptide having arabinofuranosidase activity may be obtained from microorganisms of any genus. For purposes of the present invention, the term “obtained from” as used herein in connection with a given source shall mean that the polypeptide encoded by a polynucleotide is produced by the source or by a strain in which the polynucleotide from the source has been inserted. In one aspect, the polypeptide obtained from a given source is secreted extracellularly.

The polypeptide may be a fungal polypeptide. In one embodiment, the polypeptide is from a fungus of the order Eurotiales, or from the family Aspergillaceae, or from the genusAspergillusor from the speciesAspergillus clavatusorAspergillus wentiiorAspergillus niger.

In one embodiment, the GH62 arabinofuranosidase is derived from a strain of the genusAspergillus, such as a strain ofAspergillus niger.

In one embodiment, the polypeptide is from a fungus of the order Eurotiales, or from the family Aspergillaceae, or from the genus Neosartorya or from the species Neosartoryafischeri.

In one embodiment, the polypeptide is from a fungus of the order Eurotiales, or from the family Trichocomaceae, or from the genusTalaromycesor from the speciesTalaromyces pinophilus.

The polypeptide may be a bacterial polypeptide. In one embodiment, the polypeptide is from a bacterium of the order Actinomycetales, or from the family Streptomycetaceae, or from the genusStreptomycesor from the speciesStreptomyces nitrosporeusorStreptomyces beijiangensis.

In one embodiment, the polypeptide is from a bacterium of the order Actinomycetales, or from the family Streptosporangiaceae, or from the genusStreptosporangiumor from the speciesStreptosporangiumsp-60756.

It will be understood that for the aforementioned species, the invention encompasses both the perfect and imperfect states, and other taxonomic equivalents, e.g., anamorphs, regardless of the species name by which they are known. Those skilled in the art will readily recognize the identity of appropriate equivalents.

Strains of these species are readily accessible to the public in a number of culture collections, such as the American Type Culture Collection (ATCC), Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ), Centraalbureau Voor Schimmelcultures (CBS), and Agricultural Research Service Patent Culture Collection, Northern Regional Research Center (NRRL).

The polypeptide may be identified and obtained from other sources including microorganisms isolated from nature (e.g., soil, composts, water, etc.) or DNA samples obtained directly from natural materials (e.g., soil, composts, water, etc.) using the above-mentioned probes. Techniques for isolating microorganisms and DNA directly from natural habitats are well known in the art. A polynucleotide encoding the polypeptide may then be obtained by similarly screening a genomic DNA or cDNA library of another microorganism or mixed DNA sample. Once a polynucleotide encoding a polypeptide has been detected with the probe(s), the polynucleotide can be isolated or cloned by utilizing techniques that are known to those of ordinary skill in the art (see, e.g., Sambrook et al., 1989, supra).

Polypeptides Having GH10 Xylanase Activity

Exemplary embodiments relating to the GH10 polypeptide having xylanase activity are disclosed herein below.

In an embodiment, the polypeptide having GH10 xylanase activity, selected from the group consisting of:(a) a polypeptide having at least 85%, e.g., at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the mature polypeptide of SEQ ID NO: 4;(b) a variant of the mature polypeptide of SEQ ID NO: 4 comprising a substitution, deletion, and/or insertion at one or more (several) positions;(c) a polypeptide encoded by a polynucleotide having at least 85%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the mature polypeptide coding sequence of SEQ ID NO: 5 or the cDNA sequence thereof.

In one aspect, the polypeptide differs by up to 10 amino acids, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, from the mature polypeptide. In another embodiment, the present invention relates to variants of the mature polypeptide comprising a substitution, deletion, and/or insertion at one or more (e.g., several) positions. In an embodiment, the number of amino acid substitutions, deletions and/or insertions introduced into the mature polypeptide is up to 10, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. The amino acid changes may be of a minor nature, that is conservative amino acid substitutions or insertions that do not significantly affect the folding and/or activity of the protein; small deletions, typically of 1-30 amino acids; small amino- or carboxyl-terminal extensions, such as an amino-terminal methionine residue; a small linker peptide of up to 20-25 residues; or a small extension that facilitates purification by changing net charge or another function.

A polypeptide having xylanase activity of the present invention (GH10 xylanase) may be obtained from microorganisms of any genus. For purposes of the present invention, the term “obtained from” as used herein in connection with a given source shall mean that the polypeptide encoded by a polynucleotide is produced by the source or by a strain in which the polynucleotide from the source has been inserted. In one aspect, the polypeptide obtained from a given source is secreted extracellularly.

The polypeptide may be aTalaromycespolypeptide.

In another embodiment, the polypeptide is aTalaromyces leycettanuspolypeptide, e.g., a polypeptide obtained fromTalaromyces leycettanusStrain CBS398.68.

The polypeptide may be anAspergilluspolypeptide. In another embodiment, the polypeptide is anAspergillus nigerpolypeptide,

In one embodiment, the GH10 xylanase is derived from a strain of the genusAspergillus, such as a strain ofAspergillus niger.

It will be understood that for the aforementioned species, the invention encompasses both the perfect and imperfect states, and other taxonomic equivalents, e.g., anamorphs, regardless of the species name by which they are known. Those skilled in the art will readily recognize the identity of appropriate equivalents.

Strains of these species are readily accessible to the public in a number of culture collections, such as the American Type Culture Collection (ATCC), Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ), Centraalbureau Voor Schimmelcultures (CBS), and Agricultural Research Service Patent Culture Collection, Northern Regional Research Center (NRRL).

The polypeptide may be identified and obtained from other sources including microorganisms isolated from nature (e.g., soil, composts, water, etc.) or DNA samples obtained directly from natural materials (e.g., soil, composts, water, etc.) using the above-mentioned probes. Techniques for isolating microorganisms and DNA directly from natural habitats are well known in the art. A polynucleotide encoding the polypeptide may then be obtained by similarly screening a genomic DNA or cDNA library of another microorganism or mixed DNA sample. Once a polynucleotide encoding a polypeptide has been detected with the probe(s), the polynucleotide can be isolated or cloned by utilizing techniques that are known to those of ordinary skill in the art (see, e.g., Sambrook et al., 1989, supra).

Polypeptides Having GH30 Xylanase Activity

GH30 polypeptide refers to a polypeptide with enzyme activity, the polypeptide being classified as member of the Glycoside hydrolase family 30 in the database of Carbohydrate-Active enZYmes (CAZymes) (http://www.cazy.org/).

In one embodiment, the polypeptide having GH30 xylanase activity is selected from the group wherein the polypeptide having GH30 xylanase activity is selected from the group consisting of:(a) a polypeptide having at least 85%, e.g., at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the mature polypeptide of SEQ ID NO: 6;(b) a variant of the mature polypeptide of SEQ ID NO: 6 comprising a substitution, deletion, and/or insertion at one or more (several) positions.

In one aspect, the polypeptide differs by up to 10 amino acids, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, from the mature polypeptide. In another embodiment, the present invention relates to variants of the mature polypeptide comprising a substitution, deletion, and/or insertion at one or more (e.g., several) positions. In an embodiment, the number of amino acid substitutions, deletions and/or insertions introduced into the mature polypeptide is up to 10, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. The amino acid changes may be of a minor nature, that is conservative amino acid substitutions or insertions that do not significantly affect the folding and/or activity of the protein; small deletions, typically of 1-30 amino acids; small amino- or carboxyl-terminal extensions, such as an amino-terminal methionine residue; a small linker peptide of up to 20-25 residues; or a small extension that facilitates purification by changing net charge or another function.

A polypeptide having xylanase activity of the present invention (GH30 xylanase) may be obtained from microorganisms of any genus. For purposes of the present invention, the term “obtained from” as used herein in connection with a given source shall mean that the polypeptide encoded by a polynucleotide is produced by the source or by a strain in which the polynucleotide from the source has been inserted. In one aspect, the polypeptide obtained from a given source is secreted extracellularly.

In one embodiment, the polypeptide having GH30 xylanase activity is derived from a strain of the genusBacillus, such as a strain ofBacillus subtilis.

The polypeptide may be a bacterial polypeptide. In one embodiment, the polypeptide may be aBacilluspolypeptide. In another embodiment, the polypeptide is aBacillus subtilispolypeptide.

It will be understood that for the aforementioned species, the invention encompasses both the perfect and imperfect states, and other taxonomic equivalents, e.g., anamorphs, regardless of the species name by which they are known. Those skilled in the art will readily recognize the identity of appropriate equivalents.

Strains of these species are readily accessible to the public in a number of culture collections, such as the American Type Culture Collection (ATCC), Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ), Centraalbureau Voor Schimmelcultures (CBS), and Agricultural Research Service Patent Culture Collection, Northern Regional Research Center (NRRL).

The polypeptide may be identified and obtained from other sources including microorganisms isolated from nature (e.g., soil, composts, water, etc.) or DNA samples obtained directly from natural materials (e.g., soil, composts, water, etc.) using the above-mentioned probes. Techniques for isolating microorganisms and DNA directly from natural habitats are well known in the art. A polynucleotide encoding the polypeptide may then be obtained by similarly screening a genomic DNA or cDNA library of another microorganism or mixed DNA sample. Once a polynucleotide encoding a polypeptide has been detected with the probe(s), the polynucleotide can be isolated or cloned by utilizing techniques that are known to those of ordinary skill in the art (see, e.g., Sambrook et al., 1989, supra).

Cellulolytic Composition

Exemplary cellulolytic compositions are as described in e.g., WO 2015/081139 and PCT/US2015/034179.

In an embodiment, the cellulolytic composition is derived from a strain ofTrichoderma, such as a strain ofTrichoderma reesei; a strain ofHumicola, such as a strain ofHumicola insolens, and/or a strain ofChrysosporium, such as a strain ofChrysosporium lucknowense.

In a preferred embodiment, the cellulolytic composition is derived from a strain ofTrichoderma reesei.

In a preferred embodiment, the cellulolytic composition is aTrichoderma reeseicellulase preparation.

In an embodiment, the cellulolytic composition comprises aTrichoderma reeseicellulase preparation containingAspergillus oryzaebeta-glucosidase fusion protein (WO 2008/057637) andThermoascus aurantiacusGH61A polypeptide (WO 2005/074656).

In an embodiment, the cellulolytic composition comprises aTrichoderma reeseicellulolytic enzyme composition, further comprisingThermoascus aurantiacusGH61A polypeptide having cellulolytic enhancing activity (WO 2005/074656) andAspergillus oryzaebeta-glucosidase fusion protein (WO 2008/057637).

In another embodiment, the cellulolytic composition comprises aTrichoderma reeseicellulolytic enzyme composition, further comprisingThermoascus aurantiacusGH61A polypeptide having cellulolytic enhancing activity (Sequence Number 2 in WO 2005/074656) andAspergillus fumigatusbeta-glucosidase (Sequence Number 2 of WO 2005/047499).

In another embodiment, the cellulolytic composition comprises aTrichoderma reeseicellulolytic enzyme composition, further comprisingPenicillium emersoniiGH61A polypeptide having cellulolytic enhancing activity disclosed in WO 2011/041397,Aspergillus fumigatusbeta-glucosidase (Sequence Number 2 of WO 2005/047499) or a variant thereof with the following substitutions: F100D, S283G, N456E, F512Y.

In an embodiment, the cellulolytic composition is derived fromTrichoderma reeseiRutC30.

In an embodiment, the cellulolytic composition comprises aTrichoderma reeseicellulase preparation containingTrichophaea saccataGH10 xylanase (WO 2011/057083) andTalaromyces emersoniibeta-xylosidase.

Enzyme Composition

The present invention also provides an enzyme composition comprising of a GH62 arabinofuranosidase, a GH10 xylanase, and/or a GH30 xylanase.

In an embodiment, the enzyme composition of the present invention further comprises one or more hydrolytic enzymes, preferably one or more cellulolytic enzyme, preferably, the one or more cellulolytic enzymes is expressed in an organism, such asTrichoderma reesei.

In an embodiment, the enzyme composition of the present invention further comprises a cellulolytic composition.

Preferably, the compositions are enriched in the polypeptides useful according to the invention. The term “enriched” indicates that the enzymatic activity of the composition has been increased, e.g., with an enrichment factor of at least 1.1, such as at least 1.2, at least 1.3, at least 1.4, at least 1.5, at least 2.0, at least 3.0, at least 4.0, at least 5.0, at least 10. In an embodiment, the composition comprises the polypeptides of the first aspect of the invention and one or more formulating agents, as described in the ‘formulating agent’ section below.

The compositions may comprise a polypeptide of the present invention as the major enzymatic component, e.g., a mono-component composition. Such a composition may further comprise a formulating agent, as described in the ‘formulating agent’ section below. Alternatively, the compositions may comprise multiple enzymatic activities, such as one or more (e.g., several) enzymes selected from the group consisting of phytase, xylanase, galactanase, alpha-galactosidase, protease, phospholipase, glucoronidase, lysophospholipase, amylase, beta-glucanase, arabinofuranosidase, beta-xylosidase, endo-1,4-beta-xylanase acetyl xylan esterase, feruloyl esterase, cellulase, cellobiohydrolase, beta-glycosidase, pullulanase, or any mixture thereof. Additional cellulolytic activities are particularly contemplated, as further outlined below.

Where arabinofuranosidase and xylanase activity are contemplated, it is at present contemplated that the xylanase is used in one or more of the following amounts (dosage ranges): 0.01-200; 0.05-100; 0.1-50; 0.2-20; 0.1-1; 0.2-2; 0.5-5; or 1-10 wherein all these ranges are mg xylanase protein per kg substrate (ppm). It is at present contemplated that the arabinofuranosidase is administered in one or more of the following amounts (dosage ranges): 0.01-200; 0.05-100; 0.1-50; 0.2-20; 0.1-1; 0.2-2; 0.5-5; or 1-10 wherein all these ranges are mg arabinofuranosidase protein per kg substrate (ppm). It is further contemplated that the ratio of the GH10 xylanase to GH62 arabinofuranosidase is in the range of 100:1 to 1:100 xylanase:arabinofuranosidase such as the ranges 50:1 to 1:50, 50:1 to 1:10, 25:1 to 1:5, 10:1 to 1:2 or such as 10:1 to 1:50, 5:1 to 1:25, 2:1 to 1:10 xylanase:arabinofuranosidase.

Formulating Agent

The enzyme of the invention may be formulated as a liquid or a solid. For a liquid formulation, the formulating agent may comprise a polyol (such as e.g. glycerol, ethylene glycol or propylene glycol), a salt (such as e.g. sodium chloride, sodium benzoate, potassium sorbate) or a sugar or sugar derivative (such as e.g. dextrin, glucose, sucrose, and sorbitol). Thus in one embodiment, the composition is a liquid composition comprising the polypeptide of the invention and one or more formulating agents selected from the list consisting of glycerol, ethylene glycol, 1,2-propylene glycol, 1,3-propylene glycol, sodium chloride, sodium benzoate, potassium sorbate, dextrin, glucose, sucrose, and sorbitol.

For a solid formulation, the formulation may be for example as a granule, spray dried powder or agglomerate. The formulating agent may comprise a salt (organic or inorganic zinc, sodium, potassium or calcium salts such as e.g. such as calcium acetate, calcium benzoate, calcium carbonate, calcium chloride, calcium citrate, calcium sorbate, calcium sulfate, potassium acetate, potassium benzoate, potassium carbonate, potassium chloride, potassium citrate, potassium sorbate, potassium sulfate, sodium acetate, sodium benzoate, sodium carbonate, sodium chloride, sodium citrate, sodium sulfate, zinc acetate, zinc benzoate, zinc carbonate, zinc chloride, zinc citrate, zinc sorbate, zinc sulfate), starch or a sugar or sugar derivative (such as e.g. sucrose, dextrin, glucose, lactose, sorbitol).

In an embodiment, the solid composition is in granulated form. The granule may have a matrix structure where the components are mixed homogeneously. However, the granule typically comprises a core particle and one or more coatings, which typically are salt and/or wax coatings. The core particle can either be a homogeneous blend of xylanase of the invention optionally combined with one or more additional enzymes and optionally together with one or more salts or an inert particle with the xylanase of the invention optionally combined with one or more additional enzymes applied onto it.

In an embodiment, the material of the core particles are selected from the group consisting of inorganic salts (such as calcium acetate, calcium benzoate, calcium carbonate, calcium chloride, calcium citrate, calcium sorbate, calcium sulfate, potassium acetate, potassium benzoate, potassium carbonate, potassium chloride, potassium citrate, potassium sorbate, potassium sulfate, sodium acetate, sodium benzoate, sodium carbonate, sodium chloride, sodium citrate, sodium sulfate, zinc acetate, zinc benzoate, zinc carbonate, zinc chloride, zinc citrate, zinc sorbate, zinc sulfate), starch or a sugar or sugar derivative (such as e.g. sucrose, dextrin, glucose, lactose, sorbitol), sugar or sugar derivative (such as e.g. sucrose, dextrin, glucose, lactose, sorbitol), small organic molecules, starch, flour, cellulose and minerals.

The salt coating is typically at least 1 μm thick and can either be one particular salt or a mixture of salts, such as Na2SO4, K2SO4, MgSO4and/or sodium citrate. Other examples are those described in e.g. WO 2008/017659, WO 2006/034710, WO 1997/05245, WO 1998/54980, WO 1998/55599, WO 2000/70034 or polymer coating such as described in WO 2001/00042.

Enzymatic Amount

Enzymes may be added in an effective amount during wet milling process, which can be adjusted according to the practitioner and particular process needs. In general, enzyme may be present in an amount of 0.0001-1 mg enzyme protein per g dry solids (DS) kernels, such as 0.001-0.1 mg enzyme protein per g DS kernels. In particular embodiments, the enzyme may be present in an amount of, e.g., 1 μg, 2.5 μg, 5 μg, 10 μg, 20 μg, 25 μg, 30 μg, 35 μg, 40 μg, 45 μg, 50 μg, 75 μg, 100 μg, 125 μg, 150 μg, 175 μg, 200 μg, 225 μg, 250 μg, 275 μg, 300 μg, 325 μg, 350 μg, 375 μg, 400 μg, 450 μg, 500 μg, 550 μg, 600 μg, 650 μg, 700 μg, 750 μg, 800 μg, 850 μg, 900 μg, 950 μg, 1000 μg enzyme protein per g DS kernels.

In some embodiments of palm oil extraction, the enzyme(s) are dosed at amounts corresponding to 10-1000 ppm, such as 20-1000 ppm, 30-1000 ppm, 40-1000 ppm, 50-1000 ppm, 100-1000 ppm, 200-1000 ppm, 100-500 ppm, such as 200-500 ppm, 250-400 ppm or 350-1000 ppm relative to the amount of substrate comprising palm oil.

The present invention is further described by the following examples that should not be construed as limiting the scope of the invention.

EXAMPLES

Materials and Methods

Enzymes

GH62 Arabinofuranosidase A: GH62 arabinofuranosidase derived fromAspergillus niger(SEQ ID NO: 1).

GH62 Arabinofuranosidase B: GH62 arabinofuranosidase derived fromAspergillus niger(SEQ ID NO: 3).

GH10 Xylanase A: GH10 xylanase derived fromAspergillus niger(SEQ ID NO: 4).

GH30 Xylanase A: GH30 xylanase derived fromBacillus subtilis(SEQ ID NO: 6)

Celluclast 1.5 L: a commercial product comprising cellulase and available at Novozymes A/S.

Example 1: Cloning and Recombinant Expression of a GH62 Arabinofuranosidase fromAspergillus niger

The arabinofuranosidase encoding gene with SEQ ID NO: 2 was PCR amplified from genomic DNA isolated from anAspergillus nigerstrain, which was isolated from Ireland, using gene-specific primers that also included a Kozak translation initiation sequence, “CACC”, immediately 5′ of the start codon. The PCR amplified product was cloned into theAspergillusexpression vector pMStr57 (WO 04/032648) that had been digested with the restriction enzymes BamHI and XhoI.

The sequence of the GH62 arabinofuranosidase encoding gene cloned in the expression vector was confirmed, and the expression construct was transformed into theAspergillus oryzaestrain MT3568 by the methods described in Christensen et al., 1988, Biotechnology 6, 1419-1422 and WO 04/032648. Transformants were selected during regeneration from protoplasts based on the ability to utilize acetamide as a nitrogen source conferred by a selectable marker in the expression vector. Production of the recombinant arabinofuranosidase was evaluated by culturing the transformants in 96-well deep-well microtiter plates for 4 days at 30° C. in YPG medium (WO 05/066338) and monitoring arabinofuranosidase expression by SDS-PAGE. The transformant showing the highest level of expression in microtiter plate culture was selected and re-isolated twice under selection.

For larger-scale production of the recombinant arabinofuranosidase, the selected transformant was cultured in 500 ml baffled flasks containing 150 ml of DAP-4C-1 medium (WO 12/103350). The cultures were shaken on a rotary table at 150 RPM at for 4 days. The culture broth was subsequently separated from cellular material by passage through a 0.22 μm filtration unit.

Example 2: Chromatographic Purification of the Recombinant Arabinofuranosidase fromAspergillus niger

pH of the filtered sample was adjusted to around pH 7.5 and 1.8M ammonium sulfate was added. The sample was applied to a 5 ml HiTrap™ Phenyl (HS) column on an Åkta Explorer. Prior to loading, the column had been equilibrated in 5 column volumes (CV) of 50 mM HEPES+1.8M AMS (ammonium sulfate) pH 7. In order to remove unbound material, the column was washed with 5 CV of 50 mM HEPES+1.8M AMS pH 7. The target protein was eluted from the column into a 10 ml loop using 50 mM HEPES+20% isopropanol pH 7. From the loop, the sample was loaded onto a desalting column (HiPrep™ 26/10 Desalting), which had been equilibrated with 3 CV of 50 mM HEPES+100 mM NaCl pH 7.0. The target protein was eluted with 50 mM HEPES+100 mM NaCl pH 7.0 and relevant fractions were selected and pooled based on the chromatogram. The flow rate was 5 ml/min.

The GH62 arabinofuranosidase coding sequence and the full-length amino acid sequence of the GH62 arabinofuranosidase are shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively. Determination of the N-terminal sequence was: KCSLPSS, which was determined by N-terminal Edman degradation Sequencing.

Example 3: Genomic DNA Extraction fromAspergillus nigerNN053297

TheAspergillus nigerstrain NN053297 was isolated from hot spring soil samples collected from Yunnan province in 2010.

Aspergillus nigerstrain NN053297 was inoculated on PDA plate and incubated for 37 C for 4 days. Mycelia were collected and frozen in liquid nitrogen in a sterilized mortar and grounded with pestle to fine powders. Then the genomic DNA was extracted with Biospin Fungus Genomic DNA Extraction Kit (Bioer Technology Co. Ltd., Hangzhou, China) following the manufacturer's instruction.

Example 4: Cloning ofAspergillus nigerGH10 Xylanase Gene into anAspergillus oryzaeExpression Vector

Based on the DNA information from public database, oligonucleotide primers, shown below, were designed to amplify the coding sequence of theAspergillus nigerGH10 xylanase. The GH10 xylanase coding sequence and the full-length amino acid sequence are shown as SEQ ID NO: 5 & SEQ ID NO: 4. The primers were synthesized by Invitrogen, Beijing, China.

primer1ACACAACTGGGGATCCACCatggttcagatcaaggtagctgcacprimer2CCCTCTAGATCTCGAGctagagagcatttgcgatagcagtgta

Lowercase characters of primer1 and primer2 represent the coding region the gene. While bold characters represent a region homologous to insertion sites ofAspergillus oryzaeexpression vector pCaHj505 as described in WO2013029496. The 4 underlined letters in primer1 represent the Kozark sequence as the initiation of translation process.

The genomic DNA was prepared in Example 1. A Phusion™ High-Fidelity DNA Polymerase (Finnzymes Oy, Espoo, Finland) was used for the PCR amplification. An In-fusion CF Dry-down PCR Cloning Kit (BD Biosciences, Palo Alto, CA, USA) was used to clone the fragment into the expression vector pCaHj505. The expression vector pCaHj505 contained the TAKA-amylase promoter derived fromAspergillus oryzaeand theAspergillus nigerglucoamylase terminator elements. Furthermore pCaHj505 had pUC19 derived sequences for selection and propagation inE. coli, and an amdS gene, which encoded an acetoamidase gene derived fromAspergillus nidulansfor selection of an amds+Aspergillustransformant. Plasmid pCaHj505 was linearized by digestion with Bam I and Xho I, isolated by 1.0% agarose gel electrophoresis using TBE buffer, and purified using an illustra GFX PCR DNA and Gel Band Purification Kit (GE Healthcare, Buckinghamshire, UK) following the manufacturer's instructions.

For the gene amplification, the PCR reaction was performed which contained the primer pair, primer 1 & 2, and the genomic DNA ofAspergillus nigerNN053297 as the template. In brief, 20 picomoles of each of the primer pair were used in a PCR reaction composed of 2 μl of genomic DNA, 10 μl of 5× Phusion® GC Buffer (Finnzymes Oy, Espoo, Finland), 1 μl of 2.5 mM each of dATP, dTTP, dGTP, and dCTP, and 0.6 unit of PHUSION™ High-Fidelity DNA Polymerase (Finnzymes Oy, Espoo, Finland) in a final volume of 50 μl with deionized water. The amplification was performed using a Peltier Thermal Cycler (MJ Research Inc., South San Francisco, CA, USA) programmed for denaturing at 98° C. for 1 minute; 10 cycles each of denaturing at 98° C. for 15 seconds, annealing at 68° C. for 30 seconds, with a 1° C. decrease per cycle and elongation at 72° C. for 3 minutes; 25 cycles each at 98° C. for 15 seconds, 58° C. for 30 seconds, and 72° C. for 3 minutes; and a final extension at 72° C. for 7 minutes. The heat block then went to a 4° C. soak cycle.

The PCR product, ˜1.5 kb, was purified from solution by using an ILLUSTRA™ GFX™ PCR DNA and Gel Band Purification Kit.

The purified PCR product was then ligated to the linearized vector pCaHj505 by using In-Fusion™ Dry-down PCR Cloning Kit, resulting in p505-GH10_AnNz, in which the transcription of the ofAspergillus nigerGH10 xylanase gene was under the control of a promoter from the gene forAspergillus oryzaealpha-amylase. Briefly, for the ligation reaction, the pellet of In-Fusion Dry Down mix was suspended in 2 μl of double distilled H2O and 1 μl was taken to add to 0.3 μl of the linearized vector pCaHj505 and 3.7 μl PCR product in the tube. The ligation reaction was incubated at 50° C. for 15 min.

All 5 μl of the ligation solution were used for transformation ofE. coliTOP10 competent cell (TIANGEN Biotech (Beijing) Co. Ltd., Beijing, China). The ligation solution was added to 50 μl of frozen-thaw competent cells and kept on ice for 30 minutes. Then the cells were heat-shocked at 42° C. for 1 min, and placed on ice for 2 min. Next, 200 μl of LB medium were added to the cells and incubated at 37° C. for 60 min shaking at 350 rpm in a thermomixer. Finally, all the cells were spread on LB plate containing 100 ug/ml of ampicillin and incubated at 37° C. overnight.

2 colonies were picked up for sequencing using 3730XL DNA Analyzers (Applied Biosystems Inc, Foster City, CA, USA). After the sequences were confirmed, the colony with correct insertion was inoculated for plasmid DNA extraction with a QIAPREP® Spin Miniprep Kit (QIAGEN GmbH, Hilden, Germany) by following the manufacturer's instruction. Thus the plasmid DNA of p505-GH10_AnNz was prepared forA. oryzaetransformations in Example 5.

Example 5: Expression of theAspergillus nigerGH10 Xylanase Gene inAspergillus oryzae MT3568

Aspergillus oryzaeMT3568 protoplasts were prepared according to the method of Christensen et al., 1988, Bio/Technology 6: 1419-1422. For the transformations, 3 μg of plasmid DNA of p505-GH10_AnNz were used to transformAspergillus oryzaeMT3568. The transformation yielded several transformants. Four transformants were isolated and inoculated into 3 ml of Dap4C medium in 24-well plate and incubated at 30° C., 150 rpm. After 3 days incubation, 20 μl of supernatant from each culture were analyzed on NuPAGE Novex 4-12% Bis-Tris Gel w/MES (Invitrogen Corporation, Carlsbad, CA, USA) according to the manufacturer's instructions. The resulting gel was stained with Instant Blue (Expedeon Ltd., Babraham Cambridge, UK). SDS-PAGE profiles of the cultures showed that all 4 transformants had a protein band at 35 kDa. The transformant with the highest expression level of each gene was designated as 034EQ8.

Example 6: Preparation of anAspergillus nigerGH10 Xylanase

2 slants of the expression strain 034EQ8 used for inoculation of 12 flasks of 2-liter each containing 400 ml of Dap4C medium. In detail, each slant was washed with 10 ml of Dap4C medium and inoculated into 6 flasks, shaking at 30 C, 80 rpm. The culture was harvested on day 4 and filtered using a 0.22 μm DURAPORE Membrane (Millipore, Bedford, MA, USA). After purification theAspergillus nigerGH10 xylanase was obtained.

Example 7: Use of Enzyme in Wet Milling Process

The amount of starch and gluten separated from fiber, after incubation with and without enzyme, is measured by 10-g fiber assay. The 10-g fiber assay is generally described that it incubates wet fiber samples obtained from wet-milling plant after fiber pressing and re-suspended in lactic acid buffer to 200-g slurry containing 5% dry solids of fiber, which equals to the fiber content from 100 gram dry substance of corn, in the presence of enzymes, at conditions relevant to the process (pH 3.5 to 4, temperature around 52° C.) and over a time period of between 1 to 4 hr. After incubation the slurry is transferred and pressed over a sieve (typically 100 micron or smaller), while collecting the filtrate passing through. The fiber that retained over the sieve is pressed using a spatula to recover as much filtrate as possible. The pressed fiber is then transferred to a beaker containing 200-ml of water and stirred. The slurry is passed through the 75-micron sieve and the collected filtrate is combined with the first. The pressing, washing and filtering steps above is repeated once more, so that a final filtrate is recovered and combined with the first two. The combined filtrate is then vacuum filtered, this time through a glass micro filter paper (Whatman) which retains the insoluble solids that are released from the fiber and passed through the 75-micron screen. After passing 200 ml water over the filter paper to remove any trace of solubles, the total insoluble solids retained on the filter paper is dried and weighed. The dry weight is reported as Starch+Gluten released as percentage (w/w) of fiber dry matter of starting substrate.

Example 8: Use of GH10 Xylanase and/or GH62 Arabinofuranosidase

10-g fiber assay is performed at pH 3.8, incubating at 52° C. for 1 hour at dose of 35 ug enzyme protein per gram corn, using a blend including Celluclast and GH10 Xylanase A, in combination with either GH62 Arabinofuranosidase A or GH62 Arabinofuranosidase B. Blend consists of 5% GH62 Arabinofuranosidase A or GH62 Arabinofuranosidase B, 15% of GH10 Xylanase A, and the remaining 80% from Celluclast. For comparison, blend containing Celluclast and GH10 Xylanase A only (no GH62 Arabinofuranosidase) was included. The corn fiber with 13.63% residual starch and 10.44% residual protein was used as substrate in the fiber assay. Release of starch+gluten (dry substance) from corn fiber at the specified doses below was measured.

TABLE 1Dose (ug enzymeStarch + GlutenTreatmentsprotein/g corn)RecoveredNo Enzyme06.55%Celluclast + GH10 Xylanase A358.90%Celluclast + GH10 Xylanase A +3510.57%GH62 Arabinofuranosidase ACelluclast + GH10 Xylanase A +3510.73%GH62 Arabinofuranosidase B

As shown in table 1 the addition of GH62 Arabinofuranosidase A and GH62 Arabinofuranosidase B on top of Celluclast+GH10 Xylanase A can significantly increase the yield of starch+gluten in corn wet-milling process.

Example 9: Use of GH10 Xylanase and/or GH62 Arabinofuranosidase

10-g fiber assay is performed at pH 3.8, incubating at 52° C. for 1 hour at dose of 30 ug enzyme protein per gram corn, using a blend including Celluclast and GH10 Xylanase A, in combination with either GH62 Arabinofuranosidase A or GH62 Arabinofuranosidase B. Blend consists of 5% GH62 Arabinofuranosidase A or GH62 Arabinofuranosidase B, 15% of GH10 Xylanase A, and the remaining 80% from Celluclast. For comparison, blend containing Celluclast and GH10 Xylanase A only (no GH62) was included. The corn fiber with 16.67% residual starch and 10.77% residual protein was used as substrate in the fiber assay. Release of starch+gluten (dry substance) as well as individual starch and protein from corn fiber at the specified doses below was measured.

TABLE 2Dose (ugenzymeStarch +IndividualIndividualprotein/gGlutenStarchProteinTreatmentscorn)RecoveredRecoveredRecoveredNo Enzyme09.75%4.95%4.80%Celluclast + GH103013.95%8.29%5.66%Xylanase ACelluclast + GH1028.513.50%8.03%5.47%Xylanase ACelluclast + GH103015.40%9.58%5.82%Xylanase A + GH62Arabinofuranosidase ACelluclast + GH103015.20%9.46%5.74%Xylanase A + GH62Arabinofuranosidase B

As shown in table 2 the addition of GH62 Arabinofuranosidase A and GH62 Arabinofuranosidase B on top of Celluclast+GH10 Xylanase A can significantly increase the yield of starch+gluten in corn wet-milling process.

Example 10: Use of GH10 Xylanase, GH30 Xylanase and/or GH62 Arabinofuranosidase

A 10-g fiber assay was performed at pH 3.8, with incubation at 52° C. for 1 hour and a dosage of 35 ug enzyme protein per gram corn, using enzyme blends containing GH10 Xylanase A, GH30 Xylanase A, GH62 Arabinofuranosidase A, and Celluclast with the detailed ratio of 35 ug EP/g corn as below table.

TABLE 3GH10GH30GH62XylanaseXylanaseArabinofur-CelluclastAAanosidase A(ug-EP/g(ug-EP/g(ug-EP/g(ug-EP/gTreatmentscorn)corn)corn)corn)No Enzyme0000Celluclast35000Celluclast + GH10285.2501.75Xylanase A + GH62Arabinofuranosidase ACelluclast + GH10285.251.750Xylanase A + GH30Xylanase ACelluclast + GH302805.251.75Xylanase A + GH62Arabinofuranosidase ACelluclast + GH10283.51.751.75Xylanase A + GH30Xylanase A + GH62Arabinofuranosidase A

For comparison, an enzyme composition containing only Celluclast was included. A corn fiber with 15.52% residual starch and 12.00% residual protein in fiber was used as substrate in the fiber assay. Release of starch+gluten (dry substance) from the corn fiber at the specified dosage was measured; the results are provided in the table 4 below.

TABLE 4DoseStarch +(ug enzymeGlutenTreatmentsprotein/g corn)RecoveredNo Enzyme04.39%Celluclast356.68%Celluclast + GH10 Xylanase A + GH62359.40%Arabinofuranosidase ACelluclast + GH10 Xylanase A + GH30359.45%Xylanase ACelluclast + GH30 Xylanase A + GH62358.55%Arabinofuranosidase ACelluclast + GH10 Xylanase A + GH30359.70%Xylanase A + GH62 Arabinofuranosidase A

As shown in table 4, the addition of combined GH62 Arabinofuranosidase A+GH30 Xylanase A on top of Celluclast+GH10 Xylanase A can significantly increase the yield of starch+gluten in corn wet-milling process.

Example 11: Preparation of Sterilized Palm Fruit Mesocarp

StepAction1In palm oil mill, oil palm FFBs (fresh fruit bunches) are receiveddirectly from the field, subjected to sterilization in industrialautoclave (120° C. for 120 minutes) and then threshed to obtainoil palm fruitlets along with calyx leaves. Collect the sterilizedpalm fruitlets.2Separate the oil palm fruitlets and discard the rest of the biomass[calyx leaves and small pieces of fruit bunch stalk (called asempty fruit bunch or EFB)].3Pack the fruitlets in an autoclavable plastic cover and cook it ina kitchen pressure cooker (10 L Capacity; Aluminum) forbelow induction cooktop program:Program Name: Pressure CookerTime: 30 minutesWatt/Temperature: 1300 W/180° C.4Spread the cooked fruitlets in a tray and allow it to cool down toapproximately 50° C.5Peel off the mesocarp from the nut. Collect the mesocarp in apre-weighed plastic storage container and record the weight.6Record the weight of nuts and discard it.7The peeled mesocarp is stored at 4° C. until use.

Example 12: Preparation of Substrate

The sterilized palm fruit mesocarp is pressed:

StepAction1Mash required amount of mesocarp in mash bath at ~200 r.p.m.for:a. 3 minutes, if mesocarp quantity is more than 2 Kilograms.b. 2 minutes, if mesocarp quantity is 2-1 Kilograms.2Manually mix the mashed mesocarp until it is uniformly mixed

Example 13: 10 Gins Assay Protocol for Palm Substrate

1. 10 g of prepared mash is aliquoted into 50 ml Falcon tubes with intermittent mixing to ensure substrate homogeneity. Note down the exact weight of substrate weighed;2. Also, note down the empty weight of plastic pertriplates that are to be used for collecting extracted oil;3. Pre-condition the tubes with substrate, keeping them at 90° C. for 5 minutes;4. Transfer the tubes to respective incubation temperature (55° C.) water bath and pre-condition them for 10 minutes;5. Inoculate the tubes with 500 μL of water in case of Control and 500 μL of enzyme solution in case of other enzyme treatments;6. After adding enzyme/water, mix the contents with a microspatula 5 times in clock-wise and 5 times in anti-clockwise direction to ensure proper mixing;7. Incubate for specified time (15 mins/30 mins) with intermittent mixing at every 15th minute of incubation with spatula, as specified in Step 6;8. At the end of incubation, add 20 ml of water into each tube and mix well;9. For clarification, transfer the tubes to 90° C. water bath and allow it to clarify for 30 mins;10. Centrifuge the tubes in table top centrifuge at 7000 rpm, 30° C. for 10 min to get oil layer at the top;11. Pipette out the oil layer into pre-weighed petriplates. Use hot water to completely extract free oil from each tube;12. Note down the weight of petriplates with extracted oil;13. The oil yield can by calculated by: Oil yield=Weight of petri dish containing oil extracted−Weight of empty petri dish.

TABLE 5mg Enzyme forOil yield inSamples10 g substrategramsControlno enzyme2.88 ± 0.2GH10 Xylanase A + GH620.12 + 0.043.18 ± 0.1Arabinofuranosidase A

As shown in table 5 the addition of GH10 Xylanase A and GH62 Arabinofuranosidase A can increase the oil yield from 10 gms of palm mesocarp.