WO2019038418A1 - Enzyme assisted crude palm oil extraction - Google Patents

Abstract

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

Description

Enzyme assisted crude palm oil extraction

Reference to a Sequence Listing

This application contains a Sequence Listing in computer readable form, which is incorporated herein by reference.

Background of the Invention

Field of the Invention

The present invention relates to a process for extraction or separation of crude palm oil, the crude palm oil obtainable by the process and an enzyme composition for use in the process.

Description of the Related Art

Palm oil is an edible vegetable oil which is obtained from the mesocarp of palm fruits. Palm fruits or fruitlets grow in large bunches. The palm fruitlets are stripped from the fruit bunches after being sterilized. The high temperature causes the enzymes naturally occurring enzymes in the palm fruits to denature and facilitates stripping of the fruits from the bunch stalks. The palm fruitlets are discharged into vessels commonly referred to as digesters, whereby a digested mash of palm fruits are produced under controlled temperature. The digested mash is then pressed, e.g. by using a screw press for subsequent recovery of palm oil. The crude palm oil may be subjected to screening, e.g. to remove coarse fibers, and then to a clarification process to separate oil from water, cell debris and any remaining fibrous material.

Palm fruit mesocarp contains large amounts of oil present as oil droplets within the mesocarp cells. Generally, the oil extraction rate (OER), which is a measure of the amount of extracted oil relative to the weight of the palm fruits is within the range of 20-24%, depending e.g. on fruit quality, and is subject to seasonal variation. In general, the palm oil milling process has been carefully optimized at each mill in order to minimize oil losses to the extent possible but there is still a strong incentive to improve the OER.

International patent application WO 2012/01 1130 (Advanced enzyme technologies Ltd.) concerns an enzyme composition (with exocellulolytic, pectinolytic, mammanolytic and glucanoloytic activity) used in a process for palm oil extraction. Summary of the Invention

The present invention relates to 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. The invention further relates to a crude palm oil obtainable by the above process.

The invention further concerns a composition comprising a GH10 xylanase, wherein the GH10 xylanase comprises or consists of the mature polypeptide of SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO:8; or the GH10 xylanase comprises or consists of an amino acid sequence having at least 70%, such as at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% sequence identity to the polypeptide of SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 8.

The invention further concerns the use of a GH10 xylanase in the process according to the invention, wherein the GH10 xylanase comprises or consists of the mature polypeptide of SEQ ID NO: 1 , SEQ ID NO: 2 SEQ ID NO: 3 or SEQ ID NO:8; or the GH10 xylanase comprises or consists of an amino acid sequence having at least 70%, such as at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% sequence identity to the polypeptide of SEQ ID NO: 1 , SEQ ID NO: 2,SEQ ID NO: 3 or SEQ ID NO: 8.

Definitions

Arabinofuranosidase: The term "arabinofuranosidase" or "GH62 arabinofuranosidase" means an alpha-L-arabinofuranoside arabinofuranohydrolase (EC 3.2.1.55) that catalyzes the hydrolysis of terminal non-reducing alpha-L-arabinofuranoside residues in alpha-L-arabinosides. The enzyme acts on alpha-L-arabinofuranosides, alpha-L-arabinans containing (1 ,3)- and/or (1 ,2)- and/or (1 ,5)-linkages, arabinoxylans, and arabinogalactans. Alpha-L-arabinofuranosidase is also known as arabinosidase, alpha-arabinosidase, alpha-L-arabinosidase, alpha- arabinofuranosidase, polysaccharide alpha-L-arabinofuranosidase, alpha-L-arabinofuranoside hydrolase, L-arabinosidase, or alpha-L-arabinanase. Arabinofuranosidase activity can be determined using 5 mg of medium viscosity wheat arabinoxylan (Megazyme International Ireland, Ltd., Bray, Co. Wicklow, Ireland) per ml of 100 mM sodium acetate pH 5 in a total volume of 200 μΙ for 30 minutes at 40°C followed by arabinose analysis by AMINEX® HPX-87H column chromatography (Bio-Rad Laboratories, Inc., Hercules, CA, USA).

Crude oil: The term "crude oil" (also called a non-degummed oil) refers to a pressed or extracted oil or a mixture thereof. In the present context, it is to be understood that the oil is palm oil, in particular un-refined palm oil. In particular, the term "crude oil" refers to the effluent from the screw press of a palm oil mill; i.e. to the mixture of oil and water pressed out of the palm fruit mash, before it has been subject to clarification and separation of oil from water. The crude palm oil is also designated CPO. Crude palm oil comprises water.

Digestion: The term "digestion" refers to a process where the substrate comprising palm oil is kept at a temperature in the range of 65-85°C for disintegrating the substrate and releasing palm oil from the mesocarp. The digestion can be carried out in a digestion tank and/or a precooker tank equipped with baffles. During the digestion, the substrate comprising palm oil e.g. the palm fruitlets are disintegrated and oil released from the mesocarp. According to the invention the substrate comprising palm oil can be contacted with the enzyme composition before or during the digestion.

Oil extraction rate (OER): For the purpose of the present invention, "Oil extraction rate (OER)" may be defined as by Chang et al., oil palm Industry economic journal, volume 3, 2003[9]. Chang et al. defines the Oil extract rate as ratio of oil recovered and Fresh fruit branch (FFB) times 100. According to this definition, the mathematical formula is:

OER = (weight of oil recovered/weight of FFB processed) x 100

Palm oil mill effluent (POME): Palm oil mill effluent (POME) is the waste water discharged e.g. from the sterilization process, crude oil clarification process.

Palm press liquid: The term "palm press liquid" refers to the liquid discharged from the pressing of the substrate comprising palm oil. Palm press liquid is not a crude palm oil and water has not been separated from the palm press liquid.

Sequence identity: The relatedness between two amino acid sequences or between two nucleotide sequences is described by the parameter "sequence identity".

For purposes of the present invention, the sequence identity between two amino acid sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277), preferably version 5.0.0 or later. The parameters used are gap open penalty of 10, gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. The output of Needle labeled "longest identity" (obtained using the -nobrief option) is used as the percent identity and is calculated as follows: (Identical Residues x 100)/(Length of Alignment - Total Number of Gaps in Alignment) For purposes of the present invention, the sequence identity between two deoxyribonucleotide sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, supra) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, supra), preferably version 5.0.0 or later. The parameters used are gap open penalty of 10, gap extension penalty of 0.5, and the EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix. The output of Needle labeled "longest identity" (obtained using the -nobrief option) is used as the percent identity and is calculated as follows:

(Identical Deoxyribonucleotides x 100)/(Length of Alignment - Total Number of Gaps in

Alignment)

Mature polypeptide: Mature polypeptide: The term "mature polypeptide" means a polypeptide in its final form following translation and any post-translational modifications, such as N terminal processing, C terminal truncation, glycosylation, phosphorylation, etc.

Xylanase: The term "xylanase" means a 1 ,4-beta-D-xylan-xylohydrolase (E.C. 3.2.1 .8) that catalyzes the endohydrolysis of 1 ,4-beta-D-xylosidic linkages in xylans. For purposes of the present invention, xylan degrading activity is determined by measuring the increase in hydrolysis of birchwood xylan (Sigma Chemical Co., Inc., St. Louis, MO, USA) by xylan-degrading enzyme(s) under the following typical conditions: 1 ml reactions, 5 mg/ml substrate (total solids), 5 mg of xylanolytic protein/g of substrate, 50 mM sodium acetate pH 5, 50°C, 24 hours, sugar analysis using p-hydroxybenzoic acid hydrazide (PHBAH) assay as described by Lever, 1972, A new reaction for colorimetric determination of carbohydrates, Anal. Biochem 47: 273-279. In one aspect, the polypeptide of the present invention have at least 20%, e.g., at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 100% of the xylanase activity of the mature polypeptide of SEQ ID NO: 1 , SEQ ID NO: 2 SEQ ID NO: 3 or SEQ ID NO: 8. In another aspect the polypeptide of the present invention have at least 20%, e.g. , at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 100% of the xylanase activity of the mature polypeptide of SEQ ID NO: 1 , SEQ ID NO: 2 , SEQ ID NO: 3 or SEQ I D NO: 8. In still another aspect the polypeptide of the present invention have at least 20%, e.g., at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 100% of the xylanase activity of the mature polypeptide of SEQ ID NO: 1 , SEQ ID NO: 2 , SEQ ID NO: 3 or SEQ ID NO: 8.

Xylan degrading activity or xylanolytic activity: The term "xylan degrading activity" or "xylanolytic activity" means a biological activity that hydrolyzes xylan-containing material. The two basic approaches for measuring xylanolytic activity include: (1 ) measuring the total xylanolytic activity, and (2) measuring the individual xylanolytic activities (e.g. , endoxylanases, beta- xylosidases, arabinofuranosidases, alpha-glucuronidases, acetylxylan esterases, feruloyl esterases, and alpha-glucuronyl esterases). Recent progress in assays of xylanolytic enzymes was summarized in several publications including Biely and Puchart, 2006, Recent progress in the assays of xylanolytic enzymes, Journal of the Science of Food and Agriculture 86(1 1 ): 1636- 1647; Spanikova and Biely, 2006, Glucuronoyl esterase - Novel carbohydrate esterase produced by Schizophyllum commune, FEBS Letters 580(19): 4597-4601 ; Herrmann etai, 1997, The beta- D-xylosidase of Trichoderma reesei \s a multifunctional beta-D-xylan xylohydrolase, Biochemical Journal 321 : 375-381 .

Total xylan degrading activity can be measured by determining the reducing sugars formed from various types of xylan, including, for example, oat spelt, beechwood, and larchwood xylans, or by photometric determination of dyed xylan fragments released from various covalently dyed xylans. The most common total xylanolytic activity assay is based on production of reducing sugars from polymeric 4-O-methyl glucuronoxylan as described in Bailey et a/., 1992, Interlaboratory testing of methods for assay of xylanase activity, Journal of Biotechnology 23(3): 257-270. Xylanase activity can also be determined with 0.2% AZCL-arabinoxylan as substrate in 0.01 % TRITON® X-100 (4-(1 ,1 ,3,3-tetramethylbutyl)phenyl-polyethylene glycol) and 200 mM sodium phosphate buffer pH 6 at 37°C. One unit of xylanase activity is defined as 1 .0 μηιοΐβ of azurine produced per minute at 37°C, pH 6 from 0.2% AZCL-arabinoxylan as substrate in 200 mM sodium phosphate pH 6 buffer.

For purposes of the present invention, xylan degrading activity is determined by measuring the increase in hydrolysis of birchwood xylan (Sigma Chemical Co., Inc., St. Louis, MO, USA) by xylan-degrading enzyme(s) under the following typical conditions: 1 ml reactions, 5 mg/ml substrate (total solids), 5 mg of xylanolytic protein/g of substrate, 50 mM sodium acetate pH 5, 50°C, 24 hours, sugar analysis using p-hydroxybenzoic acid hydrazide (PHBAH) assay as described by Lever, 1972, A new reaction for colorimetric determination of carbohydrates, Anal. Biochem 47: 273-279.

The abbreviation TL GH10 is used herein to refer to GH10 xylanase derived from Talaromyces leycettanus. The abbreviation RB GH10 is used herein to refer to GH 10 xylanase derived from Rasamsonia byssochlamydoides. The abbreviation AF GH10 is used herein to refer to GH10 xylanase derived from Aspergillus fumigatus.

Overview of sequences

SEQ ID NO: 1 is a GH10 xylanase derived from Talaromyces leycettanus

SEQ ID NO: 2 is a GH10 xylanase derived from Talaromyces leycettanus, mature polypeptide

SEQ ID NO: 3 is a GH10 xylanase derived from Rasamsonia byssochlamydoides SEQ ID NO: 4 is a GH62 arabinofuranosidase derived from Talaromyces pinophilus

SEQ ID NO: 5 is a GH62 arabinofuranosidase derived from Penicillium capsulatum

SEQ ID NO: 6 is a GH62 arabinofuranosidase derived from Penicillium oxalicum

SEQ ID NO: 7 is a GH62 arabinofuranosidase derived from Aspergillus niger

SEQ ID NO: 8 is a GH10 xylanase derived from Aspergillus niger

SEQ ID NO: 9 is a GH10 xylanase derived from Aspergillus fumigatus.

SEQ ID NO 10 is a GH3 beta-xylosidase derived from Talaromyces emersonii

Brief description of figures

Figure 1 shows oil yield (see Example 9)

Figure 2 shows Residual activity of enzyme (see Example 10)

Figure 3 shows comparison of enzyme activity (% xylose conversion) at pH 4.0 at varying temperatures. □ Aspergillus fumigatus (SEQ ID NO. 9), X- Rasamsonia byssochlamydoides GH10 (SEQ ID NO. 3); I- Talaromyces Leycettanus GH10 (SEQ ID NO. 2). See also Example 1 1 .

Figure 4 shows comparison of enzyme activity (% xylose conversion) at temperature 65 °C at varying pH.□ Aspergillus fumigatus (SEQ ID NO. 9), X- Rasamsonia byssochlamydoides GH10 (SEQ ID NO. 3); Talaromyces Leycettanus GH 10 (SEQ ID NO. 2). See also Example 11 .

Detailed Description of the Invention

The present invention concerns an enzyme composition and 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.

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 mesocarp 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 pre- cooker. 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.

In one embodiment of the invention, the substrate comprising palm oil is contacted with the enzyme composition at a temperature of above 50°C and the crude palm oil is then extracted.

In one embodiment of the invention, the substrate comprising palm oil is contacted with the enzyme composition at a temperature within the range of 55-90°C, such as a temperature within the range of 55-85°C, 55-80°C, 60-90°C, 60-85°C, 60-80°C, 66-90°C, 67-90°C, 68-90°C, 69-90°C, 70-90°C, 66-85°C, 66-80°C, 67-80°C, 66-79°C, 66-78°C, 66-77°C, 66-76°C, 66-75°C, 66- 74°C, 66-73X, 66-72°C, 66-71 °C, 67-80X, 67-79°C, 67-78°C, 67-77°C, 67-76°C, 67-75°C,

67- 74°C, 67-73X, 67-72°C, 67-71 °C, 68-79X, 68-78°C, 68-77°C, 68-76°C, 68-75°C, 68-74°C,

68- 73X, 68-72X, 68-71 X, 69-79X, 69-78X, 69-77X, 69-76X, 69-75X, 69-74X, 69-73X,

69- 72X, 69-71 X, 70-90X, 70-89X, 70-88X, 70-87X, 70-86X, or 70-85X.

In particular embodiments, the substrate comprising palm oil is contacted with the enzyme composition at a temperature within the range of 60-90X, such as 60 - 85 90X.

The substrate comprising palm oil is contacted with the enzyme composition for a period of 5-120 minutes, such as a period of 20-120 minutes, 25-120 minutes 5-60 minutes, 20-60 minutes, 25-60 minutes, 30-60 minutes, 15-50 minutes, 20-50 minutes, 25-50 minutes, 30-50 minutes, 15-40 minutes, 20-40 minutes, 25-40 minutes, 30-40 minutes, 15-30 minutes, 20-30 minutes, 25-28 minutes, 25-30 minutes, 25-35 minutes, 15-25 minutes, 20-25 minutes, 20-28 minutes, 15-20 minutes, 10-15 minutes or 5-10 minutes.

In one embodiment of the invention, the substrate comprising palm oil is contacted with the enzyme composition at a temperature of above 50X, such as a temperature within the range of 55-90X for a period of 5-120 minutes and the crude palm oil is then extracted.

The digested substrate is passed into a press, e.g. a screw press, from which the palm press liquid is discharged. The palm press liquid is a mixture of oil, water, press cake/fibre and nuts. The palm press liquid is delivered from the press to a clarification tank forfurther processing.

In one embodiment of the invention, the digestion comprises retaining the substrate comprising palm oil at temperatures above 65X and up to 85X for 10-30 minutes, such as for 10-28 minutes, 15-28 minutes, 12-30 minutes, 12-28 minutes or 12-25 minutes.

It is to be understood that the enzyme composition used according to the invention may be applied at any point in the crude palm oil extraction and/separation process, after the palm fruit bunches have been sterilized and until the oil is separated from water the water and from cell debris and fibrous material, which is also present in the palm press liquid. In particular, the substrate may be selected from the group consisting palm fruitlets, palm press liquid, mashed or partly mashed palm fruitlets.

It will be well within the capacity of the skilled person to optimize the dosing of the enzyme composition in view of other process parameters. In particular embodiments of the invention, the enzyme composition is dosed in amounts corresponding to 10-500 mg enzyme protein/kg FFB (fresh fruit bunch) comprising palm oil, such as 10-450 mg enzyme protein/kg FFB comprising palm oil, 10-400 mg enzyme protein/kg FFB comprising palm oil, 10-350 mg enzyme protein/kg FFB comprising palm oil, 10-300 mg enzyme protein/kg FFB comprising palm oil, 10-250 mg enzyme protein/kg FFB comprising palm oil, 10-200 mg enzyme protein/kg FFB comprising palm oil, 10-150 mg enzyme protein/kg FFB comprising palm oil, 10-100 mg enzyme protein/kg FFB comprising palm oil, 10-75 mg enzyme protein/kg FFB comprising palm oil, 10- 50 mg enzyme protein/kg FFB comprising palm oil, 10-500 mg enzyme protein/kg FFB comprising palm oil, 10-500 mg enzyme protein/kg FFB comprising palm oil, 10-500 mg enzyme protein/kg FFB comprising palm oil, 75-500 mg enzyme protein/kg FFB comprising palm oil, 100- 500 mg enzyme protein/kg FFB comprising palm oil, 150-500 mg enzyme protein/kg FFB comprising palm oil, 200-500 mg enzyme protein/kg FFB comprising palm oil, 250-500 mg enzyme protein/kg FFB comprising palm oil, 300-500 mg enzyme protein/kg FFB comprising palm oil, 350-500 mg enzyme protein/kg FFB comprising palm oil, 400-500 mg enzyme protein/kg FFB comprising palm oil, 30-400 mg enzyme protein/kg FFB comprising palm oil, 30-300 mg enzyme protein/kg FFB comprising palm oil, 30-200 mg enzyme protein/kg FFB comprising palm oil, 30-150 mg enzyme protein/kg FFB comprising palm oil, 30-100 mg enzyme protein/kg FFB comprising palm oil, 30-75 mg enzyme protein/kg FFB comprising palm oil, or such as 30-50 mg enzyme protein/kg FFB comprising palm oil.

According to some embodiments of the invention, the enzyme(s) are dosed at amounts corresponding to 10-1000 ppm, such as 20-1000 ppm, 30-1000 ppm, 40-1000ppm, 50-1000ppm, 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.

In one aspect, the contacting of the substrate comprising palm oil with an enzyme composition is done when the substrate is conveyed towards the digester. In one aspect, the substrate comprising palm oil is contacted with the enzyme during conveyance from threshing to the digester, such as during transport of the fruitlets on a conveyer belt, in a screw conveyor or auger conveyor. In the process according to the invention, the enzyme may alternatively be dosed directly into the digester, such that it is first contacted with the palm fruitlets in the upper one third of the digester.

In one aspect, the process comprises steps of: sterilizing and threshing fresh palm fruit bunches to provide palm fruitlets; and conveying the palm fruitlets into a digester.

In one aspect, the palm fruitlets are threshed and conveyed from threshing to a digester without being subject to disintegration other than the disintegration, which occurs during threshing and conveyance, such as without being subject to maceration/pre-cooking/mashing.

In one aspect, the substrate comprising palm oil is contacted with the enzyme composition by distributing the enzyme composition onto the surface of the palm fruitlets, such as by sprinkling or spraying the enzyme onto the fruitlets, during conveyance.

In a particular embodiment of the present invention, enzymes are sprinkled or sprayed onto the palm fruitlets during conveyance to the digester, which leads to improved exposure of the palm fruitlets to enzyme and a more homogenous mixture as compared to mixing within the digester. The skilled person would by default add the enzyme composition in the digester, without having realized that more even distribution of enzyme composition on the fruitlet surface could be obtained by sprinkling or spraying enzyme onto the fruitlet prior to entry into the digester. A further advantage of applying the enzyme composition during conveyance of substrate comprising palm oil towards the digester is early penetration of the enzyme composition into the mesocarp through scratches or bruises on the exocarp. This helps in "positioning" the enzyme in the mesocarp and further reduces the reaction time needed when appropriate temperatures are reached in the digester.

As the skilled person will realize, the palm fruitlets must be retained within the digester for sufficient time to allow the enzymes to act e.g. on the cellulosic material of the palm fruitlets. The exact retention time needed in the digester will depend on the exact conditions, and whether the enzyme composition is dosed directly in the digester or onto the palm fruitlets while being conveyed to the digester. Dosing the enzymes upstream of the digester onto the palm fruitlets while they are transported to the digester will generally lower the minimum retention time needed in the digester. In a preferred embodiment, the palm fruitlets are contacted with the enzyme composition in the digester for 15-60 minutes, such as for 20-60 minutes, 25-60 minutes, 30-60 minutes, 40-60 minutes, 50-60 minutes, 15-50 minutes, 20-50 minutes, 25-50 minutes, 30-50 minutes, 40-50 minutes, 15-40 minutes, 20-40 minutes, 25-40 minutes, 30-40 minutes, 15-30 minutes, 20-30 minutes, 25-30 minutes, 15-25 minutes or 15-20 minutes.

In addition to slightly lowering the temperature in the digester as compared to the temperature used in conventional palm oil milling processes, it may also be desirable to slightly increase the retention time in the digester. This may be advantageous even when applying the enzyme composition onto the palm fruitlets while being conveyed to the digester.

The temperature in the upper one third of digester may in particular be in the range of 45-55°C, e.g. in the range of 55-65°C; in the middle one third of the digester it may be about 65°C, such as in the range of 55-65°C, in the range of 60-70°C or in the range of 65-70°C; and in the lower one third of the digester it may be about 85°C, such as in the range of 70-85°C, typically about 80°C. According to these and other embodiments contacting or incubation of the palm fruitlet with an enzyme composition is done for a period of 5-60 minutes, such as for a period of 20-60 minutes, 25-60 minutes, 30-60 minutes, 15-50 minutes, 20-50 minutes, 25-50 minutes, 30-50 minutes, 15-40 minutes, 20-40 minutes, 25-40 minutes, 30-40 minutes, 15-30 minutes, 20- 30 minutes, 25-30 minutes, 25-35 minutes, 15-25 minutes, 20-25 minutes, 15-20 minutes, 10-15 minutes or 5-10 minutes; the time period being calculated as the time from which the enzyme composition is applied onto the palm fruitlets and until the digested fruit is discharged into the press. The contacting or incubation of the palm fruitlet with an enzyme composition may in particular be done for a period of 25-35 minutes, more preferably a period of 25-30 minutes, most preferably a period of 25-28 minutes. It is further to be understood that the enzymes used in the process according to the Invention, may be inactivated, or at least substantially inactivated, when the digested fruit is pressed, due to the high temperatures reached in the screw press.

Preferably, the incubation time or retention time of the palm fruit mash at temperatures above 65°C and up to 85°C is from 10-30 minutes, such as from 10-28 minutes, 15-28 minutes, 12-30 minutes, 12-28 minutes or 12-25 minutes.

Retention time at temperatures above 65°C and up to 85°C may be controlled according to need; e.g. by increasing the digester volume, or by slowing down the screw press. Also, throughout the milling process retention time at temperatures close to 65°C may be increased by the use of a predigester, and/or by use of a slow conveyor method.

In one aspect, contacting with an enzyme composition is done at one or more contact points.

In the present invention, the palm fruitlets are conveyed into the digester by means such as but not limited to screw-conveyer or auger conveyor or belt conveyor or roller conveyor or skate-wheel conveyor or chain conveyor or bucket elevator.

In one aspect, the palm fruitlets are subject to temperatures during passage through the digester, which increase from 45-85°C. such as from 45-90°C, from 50-85°C or such as from 50- 90°C.

In one aspect, the invention concerns a crude palm oil, which is obtainable by the process according to any of the process of the invention.

In one embodiment of the invention, the substrate comprising palm oil is contacted with the enzyme composition comprising a GH 10 xylanase.

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 a Talaromyces polypeptide, an Aspergillus polypeptide, or for example a Rasamsonia polypeptide.

In another aspect, the polypeptide is a Talaromyces leycettanus polypeptide, e.g., a polypeptide obtained from Talaromyces leycettanus Strain CBS398.68.

In another aspect, the polypeptide is a Rasamsonia byssochlamydoides polypeptide, e.g., a polypeptide obtained from Rasamsonia byssochlamydoides. In another aspect, the polypeptide is an Aspergillus niger polypeptide, e.g., a polypeptide obtained from Aspergillus 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).

Preferred embodiments of the aspect of the invention relating to the GH10 polypeptide having xyalanse activity are disclosed herein below. Additional details of preferred GH10 polypeptides having xyalanse activity are found in PCT/CN2016/107281 filed 25 November 2016, hereby incorporated by reference.

In one embodiment of the invention, the GH10 xylanase comprises or consists of the mature polypeptide of SEQ ID NO: 1 , or the GH10 xylanase comprises or consists of an amino acid sequence having at least 70%, such as at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% sequence identity to the polypeptide of SEQ ID NO: 1.

In one embodiment of the invention, the GH10 xylanase comprises or consists of the mature polypeptide of SEQ ID NO: 2, or the GH10 xylanase comprises or consists of an amino acid sequence having at least 70%, such as at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% sequence identity to the polypeptide of SEQ ID NO: 2.

In one embodiment of the invention, the GH10 xylanase comprises or consists of the mature polypeptide of SEQ ID NO: 3, or the GH10 xylanase comprises or consists of an amino acid sequence having at least 70%, such as at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% sequence identity to the polypeptide of SEQ ID NO: 3.

In one embodiment of the invention, the GH10 xylanase comprises or consists of the mature polypeptide of SEQ ID NO: 8, or the GH10 xylanase comprises or consists of an amino acid sequence having at least 70%, such as at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% sequence identity to the polypeptide of SEQ ID NO: 8.

According to the invention, the enzyme composition may further comprise one or more enzymes obtained from Trichoderma reesei. Said enzymes may in some embodiments be substantially inactive at a temperature of about 70°C. Said enzymes can be glycoside hydrolases, such as cellulases. In one embodiment of the invention, the enzymes which are obtained from Trichoderma reesei comprise all enzyme activities necessary for complete degradation of cellulose.

In one embodiment of the invention, the enzyme composition comprises a GH62 arabinofuranosidase.

Preferred embodiments of the aspect of the invention relating to the GH62 polypeptide having arabinofuranosidase activity are disclosed herein below. Additional details of preferred GH62 polypeptides having arabinofuranosidase activity are found in PCT/CN2015/071015 filed 19 January 2015., which is hereby incorporated by reference.

In an embodiment, the GH62 polypeptide having arabinofuranosidase activity has a sequence identity to the mature polypeptide of SEQ ID NO: 4 of at least 80%, e.g., at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%.

In an embodiment, the GH62 polypeptide having arabinofuranosidase activity has a sequence identity to the mature polypeptide of SEQ ID NO: 5 of at least 80%, e.g., at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%.

In an embodiment, the GH62 polypeptide having arabinofuranosidase activity has a sequence identity to SEQ ID NO: 6 of at least 80%, e.g., at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%.

In another embodiment, the GH62 polypeptide having arabinofuranosidase activity has a sequence identity to the mature polypeptide of SEQ ID NO: 7 of at least 80%, e.g., at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%.

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 genus Penicillium or from the species Penicillium aurantiogriseum, Penicillium oxalicum or Penicillium capsulatum.

In one embodiment, the polypeptide is from a fungus of the order Eurotiales, or from the family Aspergillaceae, or from the genus Aspergillus or from the species Aspergillus clavatus or

Aspergillus wentii or Aspergillus 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 Neosartorya fischeri.

In one embodiment, the polypeptide is from a fungus of the order Eurotiales, or from the family Trichocomaceae, or from the genus Talaromyces or from the species Talaromyces pinophilus.

In one embodiment, the polypeptide is from a fungus of the order Ustilaginales, or from the family Ustilaginaceae, or from the genus Ustilago or from the species Ustilago maydis.

In one embodiment, the polypeptide is from a fungus of the phylum Ascomycota, or from the genus Acrophialophora or from the species Acrophialophora fusispora.

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 genus Streptomyces or from the species Streptomyces nitrosporeus or Streptomyces beijiangensis. In one embodiment, the polypeptide is from a bacterium of the order Actinomycetales, or from the family Streptosporangiaceae, or from the genus Streptosporangium or from the species Streptosporangium sp-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).

The invention further concerns an enzyme composition comprising a GH10 xylanase, wherein the GH10 xylanase comprises or consists of the mature polypeptide of SEQ ID NO: 1 , SEQ ID NO: 2 ,SEQ ID NO: 3 or SEQ ID NO:8; or the GH10 xylanase comprises or consists of an amino acid sequence having at least 70%, such as at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% sequence identity to the polypeptide of SEQ ID NO: 1 , SEQ ID NO: 2,SEQ ID NO: 3 or SEQ ID NO: 8.

The enzyme composition can further comprise a GH62 arabinofuranosidase, wherein the GH62 arabinofuranosidase comprises or consists of the mature polypeptide of SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6or SEQ ID NO: 7; or the GH62 arabinofuranosidase comprises or consists comprising or consisting of an amino acid sequence having at least 80%, such as at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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% or at least 99% sequence identity to the polypeptide of SEQ I D NO: 4, SEQ ID NO: 5, SEQ I D NO: 6 or SEQ I D NO: 7.

In one embodiment of the invention, the enzyme composition further comprises one or more enzymes obtained from Tric oderma reesei. These enzymes are substantially inactive at a temperature of about 70°C.

Examples

Example 1

Preparation of sterilized palm fruit mesocarp

4 Pre-incubate the mashed mesocarp by placing the containers in a water bath at 90°C for 10 minutes followed by 70°C for 5 minutes. [For control, pre-incubate only at 90°C for 10 minutes]

5 Inoculate the required enzyme composition into the substrate. For control, add 1 ml of reverse osmosis water

6 Mix the substrate and inoculum with a spatula in clockwise followed anti-clockwise

direction, 5 rounds in each direction.

7 Close the lid and incubate the substrate in a water bath at 70°C for 30 minutes. Keep mixing the substrate as mentioned in step 6 mixing after every 10 minutes. [For control, skip this step and go directly to step 8]

8 Add 30ml of boiling water to the substrate, mix it with a spatula and transfer it into a water bath at 90°C for 15 minutes for first conditioning and to stop the enzyme reaction.

9 Press the substrate using a para-press at 4 Kg/cm² for 25 seconds. (Take 25 seconds to increase the pressure to 4 Kg/cm², and maintain it for another 25 seconds before releasing the pressure).

10 Manually transfer the pressed extract into a pre-weighed 250ml conical bottom centrifuge tubes.

11 Put the pressed fiber cake in the substrate vessel and add 50ml of boiling water to it.

12 Using a spatula, try to open the compact fiber cake such that it is completely soaked in the water.

13 Put the container back at 90°C for 15 minutes for second conditioning.

14 Press the fiber cake using a para-press at 4 Kg/cm² for 25 seconds. (Take 25 seconds to increase the pressure to 4 Kg/cm², and maintain it for another 25 seconds before releasing the pressure).

15 Add the collected extract to the respective centrifuge tube (containing the extract collected from step 9).

16 Note the weight of tube containing the extract.

17 Put the tube in a water bath at 90°C for 30 minutes for clarification.

18 Centrifuge the clarified extract at 5000 r.p.m. for 10 minutes in a swinging bucket type rotor.

19 Using a glass pipette and pipette controller, pipette out the complete top oil layer into a pre-weighed petri dish. Make sure that the sludge layer is not disturbed during the process and no sludge particles come with the oil.

20 Aspirate some boiling water in the pipette and slowly dispense it into a waste beaker. A layer of oil will form on the water while dispensing. Add this oil layer to the rest of the oil collected in the petri dish.

21 Note the weight of centrifuge tube containing the sludge.

22 Note the weight of petri plate containing the oil.

The oil yield can by calculated by:

Oil yield = Weight of petri dish containing oil extracted - Weight of empty petri dish

Example 3 Oil yield in gram from a 50 gram mashed substrate was compared on a batch sterilized substrate obtained from Ribubonus mill, Malaysia.

The substrate comprising palm oil was prepared from palm fruitlets following the method described in example 1 . The substrate was then digested and pressed as described in example 2. The experiment was carried out three times for each sample and the result is given as the average of the three oil yields. For the samples, where enzyme composition was added, the enzyme addition was done in step 5 as described in example 2. Control sample was without enzyme addition.

Palmora^® OER 1 .0 is a commercial product sold by Novozymes A/S

Oil yield in gram from a 50 gram mashed substrate was compared on a continuous mill sterilized substrate obtained from AIP mill, Bengkulu, Indonesia. The mashed substrate was obtained from a continuous sterilizer at the mentioned mill and kept at 4°C until the substrate wash then digested and pressed as described in example 2. The experiment was carried out three times for each sample and the result is given as the average of the three oil yields. For the samples, where enzyme composition was added, the enzyme addition was done in step 5 as described in example 2. Control sample was without enzyme addition.

Palmora^® OER 1 .0 is a commercial product sold by Novozymes A S

As seen from the above two tables, the enzyme composition comprising SEQ I D NO: 1 and SEQ ID NO: 4 performed equally well on substrate from both batch and continuous sterilizer. And the enzyme composition was comparable to Palmora^® OER 1 .0 at a dosage of 750 ppm and also performed better than current dosage of Palmora^® OER 1.0 at 300ppm. Example 4

Oil yield in gram from a 50 gram mashed substrate was compared on a batch sterilized substrate obtained from Ribubonus mill, Malaysia.

This experiment was done to demonstrate robustness of the enzyme composition comprising SEQ ID NO: 1 and SEQ ID NO: 4 by incubating for 15 and 30 minutes. The objective was to simulate digester residence time with enzyme for lowered time vs. higher time. The mashed substrate was prepared following method listed in example 1 .

The experiment was carried out three times for each sample and the result is given as the average of the three oil yields. For the samples, where enzyme composition was added, the enzyme addition was done in step 5 as described in example 2. Control sample was without enzyme addition. For experiments with an incubation time of 15 minutes, incubation was carried out for 15 minutes instead of the 30 minutes stated in step 7 of example 2.

A comparison of oil yield of the mashed substrate was done as against Palmora^® OER 1 .0 for 15 and 30 minutes respectively.

Example 5

Contributing enzyme activities enzyme composition

Oil yield in gram from a 50 gram mashed substrate was compared on a batch sterilized substrate obtained from Ribubonus mill, Malaysia. The substrate comprising palm oil was prepared from palm fruitlets following the method described in example 1 . The substrate was then digested and pressed as described in example 2. The experiment was carried out three times for each sample and the result is given as the average of the three oil yields. For the samples, where enzyme composition was added, the enzyme addition was done in step 5 as described in example 2. Control sample was without enzyme addition.

Example 6

Determination of Td by Differential Scanning Calorimetry.

The thermostability of SEQ ID NO: 1 was determined by Differential Scanning Calorimetry (DSC) using a VP-Capillary Differential Scanning Calorimeter (MicroCal Inc., Piscataway, NJ, USA). The thermal denaturation temperature, Td (°C), was taken as the top of denaturation peak (major endothermic peak) in thermograms (Cp vs. T) obtained after heating enzyme solutions (approx. 0.5 mg/ml) in buffer (50 mM acetate buffer pH 5.0) at a constant programmed heating rate of 200 K/hr.

Sample- and reference-solutions (approx. 0.2 ml) were loaded into the calorimeter

(reference: buffer without enzyme) from storage conditions at 10°C and thermally pre-equilibrated for 20 minutes at 20°C prior to DSC scan from 20°C to 105°C. Denaturation temperatures were determined at an accuracy of approximately +/- 1 °C. Td obtained under these conditions for SEQ ID NO: 1 was approx. 89°C.

Example 7

Preparation of substrate.

The sterilized palm fruit mesocarp is pressed.

Manually mix the mashed mesocarp until it is uniformly mixed

Example 8:

Enzymes:

GH62 Arabinofuranosidase A: GH62 arabinofuranosidase derived from Aspergillus niger (SEQ ID NO: 7).

GH10 Xylanase A: GH10 xylanase derived from Aspergillus niger (SEQ ID NO: 8).

10 qms assay protocol for palm substrate

1. 10g of prepared mash is aliquoted into 50ml 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 500μΙ_ of water in case of Control and 500μΙ_ 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 / 30mins) with intermittent mixing at every 15th minute of incubation with spatula, as specified in Step 6;

8. At the end of incubation, add 20ml 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;

1 1. 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.

As shown in table below, the addition of GH10 Xylanase A ( a GH10 Xylanase from Aspergillus niger, SEQ ID NO: 8) and GH62 Arabinofuranosidase A (GH62

Arabinofuranosidase from Aspergillus niger, SEQ ID NO 7), lead to an increase the oil yield from 10 gms of palm mesocarp. Samples Mg Enzyme for 10 g substrate Oil yield in grams

Control No enzyme 2.88 ±0.2

GH10 Xylanase A + 0.12 + 0.04 3.18 ± 0.1

GH62 Arabinofuranosidase A

Example 9: Oil yield

Experimental procedure:

50g mesocarp tissue is used as substrate. The mesocarp is generated after separating the nuts from the pressure-cooked oil palm fruits. This separated mesocarp part is then mashed for 2-3min to make a uniform mash. The mash substrate is warmed to 70DC in the pre-set water bath followed by addition of required enzyme dilution. For control, equivalent amount of water is added. The enzyme is manually mixed with the substrate and kept for incubation at 70DC for 30min to digest the substrate. Intermittent manual mixing is carried out every 10min interval during incubation period. Then 30ml of boiling water is added to the enzyme substrate mix and transferred it into a water bath at 90DC for 15 minutes to stop the enzyme reaction. The digested mash is then pressed using a mechanical press at 4 Kg/cm2 for 25 seconds and collected the extract in a pre-weighed vessel. The pressed-fiber is washed with 50ml of hot water and further pressed after conditioning the material at 90DC for 15 minutes under same condition and mixed the extract with the respective previous extract. The entire amount of extract was clarified at 90DC for 60 min in a water bath under static condition. The clarified material is then cen-trifuged at 5000 rpm at - 30DC for 10min in a swing bucket centrifuge. The free oil is pipetted out and weighed in a fine balance. This is assessed for the oil yield.

Table 1. Oil ield

^*Levels not connected by same letter are significantly different.

See also figure 1 .

The data shows that Talaromyces Leycettanus GH10 (SEQ ID NO. 2) leads to significantly higher oil yields, than both control and Aspergillus fumigatus GH10 (SEQ ID NO. 9).

Example 10: Residual activity of enzyme

Set up:

Sample stock: 10 mg/ml enzyme> 0.25mg/ml (5ml) in 50mM Na acetate pH 5 buffer Stress temperature: 50, 60, 70, 80, 90 °C

Unstress temperature: 4°C

Stress duration: 30 min

Sample volume for stress : 100μΙ ( 0.25 mg/ml) in a PCR Plate

Assay

Substrate: 0.2% Arabinoxylan in diluted buffer.

4X dilution after stress (120μΙ buffer +40μΙ sample)

dilution buffer: 50Mm Na acetate +0.01% TRITON X

Assay :20min 25DC @ 800 RPM (20μΙ sample+ 180μΙ substrate)

settling time: 10 min

Absorbance read at 590nm using 50ul sample in 384 well plate (50μΙ)

Note: Sample diluted on volume basis.

Results: Table 2- Residual activity (see also Figure 1)

The results are also shown in Figure 2.

The results show that the Talaromyces leycettanus GH10 (SEQ ID NO. 2) displays a significantly higher residual activity at higher temperatures as compared to Aspergillus fumigatus GH 10 (SEQ ID NO. 9).

Example 11 : Xylose conversion

The enzymes Talaromyces leycettanus GH10 and Rasamsonia byssochlamydoides GH10 behave similarly in temperature and pH sensitivity testing, while differing from Aspergillus fumigatus GH10. This is shown below

Materials and method:

Pretreated corn cobs hydrolysis assay

Corn cobs were pretreated with NaOH (0.08 g/g dry weight cobs) at 120 DC for 60 minutes at 15% total dry weight solids (TS). The resulting material was washed with water until it was pH 8.2, resulting in washed alkaline pretreated corn cobs (APCC). Ground Sieved Alkaline Pretreated Corn Cobs (GS-APCC) was prepared by adjusting the pH of APCC to 5.0 by addition of 6 M HCI and water with extensive mixing, milling APCC in a Cosmos ICMG 40 wet multi-utility grinder (EssEmm Corporation, Tamil Nadu, India), and autoclaving for 45 minutes at 121 DC, with a final TS of 3.33%. The hydrolysis of GS-APCC was conducted using 2.2 ml deep-well plates (Axygen, Union City, CA, USA) in a total reaction volume of 1.0 ml.

The hydrolysis was performed with 10 mg of GS-APCC total solids per ml of 50 mM sodium acetate (pH 4.0 to 5.5) or 50 mM Tris (pH 6.0 to 7.0) buffer containing 1 mM manganese sulfate and various protein loadings of various enzyme compositions (expressed as mg protein per gram of cellulose). Enzyme compositions were prepared and then added simultaneously to all wells in a volume ranging from 50 μΙ to 200 μΙ, for a final volume of 1 ml in each reaction. The plate was then sealed using an ALPS-300™ plate heat sealer (Abgene, Epsom, United Kingdom), mixed thoroughly, and incubated at a specific temperature for 72 hours. All experiments reported were performed in triplicate.

Following hydrolysis, samples were filtered using a 0.45 μιη MULTISCREEN® 96-well filter plate (Millipore, Bedford, MA, USA) and filtrates were analyzed for sugar content as described below. When not used immediately, filtered aliquots were frozen at -20°C. The sugar concentrations of samples diluted in 0.005 M H2S04 were measured using a 4.6 x 250 mm AMINEX® HPX-87H column (Bio-Rad Laboratories, Inc., Hercules, CA, USA) by elution with 0.05% w/w benzoic acid-0.005 M H2S04 at 65°C at a flow rate of 0.6 ml per minute, and quantitation by integration of the glucose, cellobiose, and xylose signals from refractive index detection (CHEMSTATION®, AGILENT® 1100 HPLC, Agilent Technologies, Santa Clara, CA, USA) calibrated by pure sugar samples. The resultant glucose equivalents were used to calculate the percentage of cellulose conversion for each reaction. The resultant xylose equivalents were used to calculate the percentage of xylo-oligosaccharide conversion for each reaction.

Glucose, cellobiose, and xylose were measured individually. Measured sugar concentrations were adjusted for the appropriate dilution factor. All HPLC data processing was performed using MICROSOFT EXCEL™ software (Microsoft, Richland, WA, USA).

The degree of xylo-oligosaccharide conversion to xylose was calculated using the following equation: % xylose conversion = xylose concentration/xylose concentration in a limit digest. In order to calculate % conversion, a 100% conversion point was set based on a cellulase control (100 mg of Trichoderma reesei cellulase supplemented with P. emersonii GH61A polypeptide (WO 201 1/041397), A. fumigatus GH10 xylanase (xyn3) (WO 2006/078256), and T. emersonii GH3 beta-xylosidase (WO 2003/070956) per gram cellulose), and all values were divided by this number and then multiplied by 100. The % relative activity for each temperature was calculated using the following equation: % relative activity = (% xylose conversion of a xylanase at a certain pH and temperature - % xylose conversion of beta-xylosidase at that certain pH and temperature) / (% xylose conversion of the xylanase for the pH and temperature containing the highest % xylose conversion - % xylose conversion of beta-xylosidase for the pH and temperature containing the highest % xylose conversion) X 100

Results

Table 3: % xylose conversion at ph 4.0 and varied temperatures (see also fig 3)

Aspergillus 55 49 42 41

fumigatus GH10

(SEQ ID NO 9)

Talaromyces 76 76 76 78

Lecyttanus GH10

(SEQ ID NO. 2)

Rasamsonia 70 72 78 83

byssochlamydoides

GH10

(SEQ ID NO. 3)

It can be seen that while both Talaromyces Leycettanus GH10 (SEQ ID NO. 1 ) and Rasamsonia byssochlamydoides GH10 (SEQ ID NO. 3) display maintained higher levels of xylose conversion, the xylose conversion by Aspergillus fumigatus GH10 (SEQ ID NO 9) decreases as the temperature increases.

Table 4: % xylose conversion at Temp and varied pH (see also figure 4)

These experiments demonstrate the similar activity profile of Talaromyces Leycettanus GH10 (SEQ ID NO. 2) and Rasamsonia byssochlamydoides GH10 (SEQ ID NO. 3); and that this activity profile is different from that of Aspergillus fumigatus (SEQ ID NO. 9). Example 12: Preparation of Talaromyces leycettanus GH10 xylanase (SEQ ID NO 1 )

The Talaromyces leycettanus GH10 xylanase (SEQ ID NO 1 ) was prepared

recombinantly and purified according to WO 2013/019827 using Aspergillus oryzae as a host.

Example 13 : Preparation of Rasamsonia byssochlamydoides GH10 xylanase (SEQ ID NO 3)

The Rasamsonia byssochlamydoides GH10 xylanase (SEQ ID NO. 3) was prepared recombinantly and purified according to WO 2014/182990 using Aspergillus oryzae as a host.

Example 14: Preparation of Aspergillus fumigatus GH10 xylanase (SEQ ID NO. 9)

Aspergillus fumigatus NN055679 GH10 xylanase (xyn3) (GENESEQP:AEC74753) was prepared recombinantly according to WO 2006/078256 using Aspergillus oryzae BECh2 (WO 2000/39322) as a host.

The filtered broth was desalted and buffer-exchanged into 50 mM sodium acetate pH 5.0 using a HIPREP® 26/10 Desalting Column (GE Healthcare, Piscataway, NJ, USA) according to the manufacturer's instructions. Protein concentration was determined using a Microplate BCA™ Protein Assay Kit with bovine serum albumin as a protein standard.

Example 15: Preparation of Talaromyces emersonii CBS 393.64 GH3 beta-xylosidase (SEDQ ID NO. 10)

A Talaromyces emersonii CBS 393.64 beta-xylosidase (GENESEQP:AZI 104896) was prepared recombinantly according to Rasmussen et al., 2006, Biotechnology and Bioengineering 94: 869-876 using Aspergillus oryzae JaL355 as a host (WO 2003/070956). The filtered broth was concentrated and desalted with 50 mM sodium acetate pH 5.0 using a tangential flow concentrator equipped with a 10 kDa polyethersulfone membrane. Protein concentration was determined using a Microplate BCA™ Protein Assay Kit (Thermo Fischer Scientific, Waltham, MA, USA) in which bovine serum albumin was used as a protein standard

Claims

Claims

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

wherein the enzyme composition comprises a GH10 xylanase.

A process according to claim 1 , wherein the GH10 xylanase comprises or consists of the mature polypeptide of SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 8, or the GH10 xylanase comprises or consists of an amino acid sequence having at least 70%, such as at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% sequence identity to the polypeptide of SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO:8.

A process according to any of claims 1 -2, wherein the GH10 xylanase is obtained from Talaromyces leycettanus.

A process according to any of claims 1 -2, wherein the GH10 xylanase is obtained from Rasamsonia byssochlamydoides.

A process according to any of claims 1 -2, wherein the GH10 xylanase is obtained from Aspergillus niger.

A process according to any of the preceding claims, wherein the enzyme composition further comprises one or more enzymes, such as one or more glycoside hydrolysases, obtained from Trichoderma reesei.

A process according to claim 6 wherein the one or more enzymes are substantially inactive at a temperature of about 70°C.

A process according to any of claims 1 -7, wherein the enzyme composition further comprises one or more glycoside hydrolases selected from GH62 arabinofuranosidases. A process according to claim 8, wherein the GH62 arabinofuranosidase comprises or consists of the mature polypeptide of SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6 or SEQ ID NO: 7; or the GH62 arabinofuranosidase comprises or consists comprising or consisting of an amino acid sequence having at least 80%, such as at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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% or at least 99% sequence identity to the polypeptide of SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6 or SEQ ID NO: 7.

10. A process according to any of the preceding claims, wherein the substrate comprising palm oil is selected from the group consisting of palm fruitlets, palm press liquid, mashed or partly mashed palm fruitlets.

1 1. The process according to any of the preceding claims, wherein the substrate comprising palm oil is subjected to sterilization before being contacted with the enzyme composition.

12. The process according to claim 1 1 , wherein the substrate is sterilization is batch sterilization or continuous sterilization.

13. The process according to any of claims 1 1 -12, wherein the sterilization temperature is in the range of 100-150°C.

14. The process according to any of claims 1 1-13, wherein the palm fruits are stripped from the fresh fruit bunches (FFB) after being sterilized.

15. The process according to any of the preceding claims, wherein the process comprises contacting the substrate with the enzyme composition at a temperature of above 50°C before extracting the crude palm oil.

16. The process according to any of the preceding claims, wherein the substrate contacted with the enzyme composition is subjected to digestion, before extracting the crude palm oil.

17. The process according to any of claims15-16, wherein the temperature is within the range of 55-85°C, 55-80°C, 60-90°C, 60-85°C, 60-80°C, 66-90°C, 67-90°C, 68-90°C, 69-90°C, 70-90°C, 66-85°C, 66-80°C, 67-80°C, 66-79°C, 66-78°C, 66-77°C, 66-76°C, 66-75°C, 66-

74°C, 66-73°C, 66-72°C, 66-71 °C, 67-80°C, 67-79°C, 67-78°C, 67-77°C, 67-76°C, 67- 75°C, 67-74°C, 67-73°C, 67-72°C, 67-71 °C, 68-79°C, 68-78°C, 68-77°C, 68-76°C, 68- 75°C, 68-74°C, 68-73°C, 68-72°C, 68-71 °C, 69-79°C, 69-78°C, 69-77°C, 69-76°C, 69- 75°C, 69-74°C, 69-73°C, 69-72°C, 69-71 °C, 70-90°C, 70-89°C, 70-88°C, 70-87°C, 70- 86°C, or 70-85°C.

18. The process according to any of the preceding claims, wherein the process comprises contacting the substrate with the enzyme composition for a period of 5-120 minutes, such as a period of 20-120 minutes, 25-120 minutes 5-60 minutes, 20-60 minutes, 25-60 minutes, 30-60 minutes, 15-50 minutes, 20-50 minutes, 25-50 minutes, 30-50 minutes, 15-40 minutes, 20-40 minutes, 25-40 minutes, 30-40 minutes, 15-30 minutes, 20-30 minutes, 25-28 minutes, 25-30 minutes, 25-35 minutes, 15-25 minutes, 20-25 minutes, 20-28 minutes, 15-20 minutes, 10-15 minutes or 5-10 minutes.

19. The process according to any of the preceding claims, wherein the substrate comprising palm oil is retained at a temperature above 65°C and below 85°C for 10-30 minutes, such as for 10-28 minutes, 15-28 minutes, 12-30 minutes, 12-28 minutes or 12-25 minutes.

20. The process according to any of the preceding claims, wherein the enzyme composition is dosed in amounts corresponding to 10-500 mg enzyme protein/kg FFB (fresh fruit bunch) comprising palm oil, such as 10-450 mg enzyme protein/kg FFB comprising palm oil, 10-400 mg enzyme protein/kg FFB comprising palm oil, 10-350 mg enzyme protein/kg FFB comprising palm oil, 10-300 mg enzyme protein/kg FFB comprising palm oil, 10-250 mg enzyme protein/kg FFB comprising palm oil, 10-200 mg enzyme protein/kg FFB comprising palm oil, 10-150 mg enzyme protein/kg FFB comprising palm oil, 10-100 mg enzyme protein/kg FFB comprising palm oil, 10-75 mg enzyme protein/kg FFB comprising palm oil, 10-50 mg enzyme protein/kg FFB comprising palm oil, 10-500 mg enzyme protein/kg FFB comprising palm oil, 10-500 mg enzyme protein/kg FFB comprising palm oil, 10-500 mg enzyme protein/kg FFB comprising palm oil, 75-500 mg enzyme protein/kg FFB comprising palm oil, 100-500 mg enzyme protein/kg FFB comprising palm oil, 150- 500 mg enzyme protein/kg FFB comprising palm oil, 200-500 mg enzyme protein/kg FFB comprising palm oil, 250-500 mg enzyme protein/kg FFB comprising palm oil, 300-500 mg enzyme protein/kg FFB comprising palm oil, 350-500 mg enzyme protein/kg FFB comprising palm oil, 400-500 mg enzyme protein/kg FFB comprising palm oil, 30-400 mg enzyme protein/kg FFB comprising palm oil, 30-300 mg enzyme protein/kg FFB comprising palm oil, 30-200 mg enzyme protein/kg FFB comprising palm oil, 30-150 mg enzyme protein/kg FFB comprising palm oil, 30-100 mg enzyme protein/kg FFB comprising palm oil, 30-75 mg enzyme protein/kg FFB comprising palm oil, or such as

30-50 mg enzyme protein/kg FFB comprising palm oil.

21. The process according to any of the preceding claims, wherein the enzyme composition is dosed such that the amount of enzyme protein corresponds to 10-1000 ppm, such as 20-1000 ppm, 30-1000 ppm, 40-1000ppm, 50-1000ppm, 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.

22. A crude palm oil, which is obtainable by the process according to any of the preceding claims.

23. A composition comprising a GH10 xylanase, wherein the GH10 xylanase comprises or consists of the mature polypeptide of SEQ ID NO: 1 , SEQ ID NO: 2 ,SEQ ID NO: 3 or

SEQ ID NO: 8; or the GH 10 xylanase comprises or consists of an amino acid sequence having at least 70%, such as at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% sequence identity to the polypeptide of SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 8.

24. Composition according to claim 23, wherein the composition further comprises a GH62 arabinofuranosidase, wherein the GH62 arabinofuranosidase comprises or consists of the mature polypeptide of SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6 or SEQ ID NO: 7; or the GH62 arabinofuranosidase comprises or consists comprising or consisting of an amino acid sequence having at least 80%, such as at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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% or at least 99% sequence identity to the polypeptide of SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6 or SEQ ID NO: 7.

25. Composition according to any of claims 23-24, wherein the enzyme composition further comprises one or more enzymes obtained from Trichoderma reesei.

26. Composition according to claim 25, wherein the one or more enzymes are substantially inactive at a temperature of about 70°C.

27. Composition according to any of claims 25-26, wherein the composition is for use in the process of claims 1-21 .