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WO2019038418A1 - Extraction d'huile de palme brute assistée par des enzymes - Google Patents

Extraction d'huile de palme brute assistée par des enzymes Download PDF

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Publication number
WO2019038418A1
WO2019038418A1 PCT/EP2018/072873 EP2018072873W WO2019038418A1 WO 2019038418 A1 WO2019038418 A1 WO 2019038418A1 EP 2018072873 W EP2018072873 W EP 2018072873W WO 2019038418 A1 WO2019038418 A1 WO 2019038418A1
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Prior art keywords
seq
minutes
palm oil
ffb
enzyme
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English (en)
Inventor
Purna Venkatesh
Kim Borch
Aindrila Dasgupta
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Novozymes AS
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Novozymes AS
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Priority to MYPI2020000914A priority Critical patent/MY208100A/en
Priority to AU2018319349A priority patent/AU2018319349B2/en
Publication of WO2019038418A1 publication Critical patent/WO2019038418A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11B—PRODUCING, e.g. BY PRESSING RAW MATERIALS OR BY EXTRACTION FROM WASTE MATERIALS, REFINING OR PRESERVING FATS, FATTY SUBSTANCES, e.g. LANOLIN, FATTY OILS OR WAXES; ESSENTIAL OILS; PERFUMES
    • C11B1/00—Production of fats or fatty oils from raw materials
    • C11B1/02—Pretreatment
    • C11B1/025—Pretreatment by enzymes or microorganisms, living or dead
    • C—CHEMISTRY; METALLURGY
    • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11B—PRODUCING, e.g. BY PRESSING RAW MATERIALS OR BY EXTRACTION FROM WASTE MATERIALS, REFINING OR PRESERVING FATS, FATTY SUBSTANCES, e.g. LANOLIN, FATTY OILS OR WAXES; ESSENTIAL OILS; PERFUMES
    • C11B1/00—Production of fats or fatty oils from raw materials
    • C11B1/10—Production of fats or fatty oils from raw materials by extracting

Definitions

  • 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.
  • 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.
  • 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.
  • OER oil extraction rate
  • 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.
  • the present invention relates to a process for extraction or separation of crude palm oil (CPO), comprising the steps of:
  • 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
  • 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
  • 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
  • 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.
  • Crude oil refers to a pressed or extracted oil or a mixture thereof.
  • the oil is palm oil, in particular un-refined palm oil.
  • 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 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.
  • the substrate comprising palm oil e.g. the palm fruitlets are disintegrated and oil released from the mesocarp.
  • the substrate comprising palm oil can be contacted with the enzyme composition before or during the digestion.
  • Oil extraction rate (OER):
  • 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 is the waste water discharged e.g. from the sterilization process, crude oil clarification process.
  • 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”.
  • 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.
  • 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)
  • 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:
  • Mature polypeptide 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 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.
  • xylan degrading activity is determined by measuring the increase in hydrolysis of birchwood xylan (Sigma Chemical Co., Inc., St.
  • 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.
  • PBAH p-hydroxybenzoic acid hydrazide
  • 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.
  • the polypeptide of the present invention have at least 20%, e.g.
  • 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.
  • 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 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).
  • 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.
  • TRITON® X-100 (4-(1 ,1 ,3,3-tetramethylbutyl)phenyl-polyethylene glycol)
  • 200 mM sodium phosphate buffer pH 6 at 37°C One unit of xylanase activity is defined as 1 .0 ⁇ 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.
  • 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.
  • PBAH p-hydroxybenzoic acid hydrazide
  • 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.
  • 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
  • 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 .
  • 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:
  • the enzyme composition comprises a GH10 xylanase.
  • 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.
  • 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.
  • the substrate comprising palm oil is crude palm oil which is contacted with the enzyme composition.
  • the substrate, which comprises palm oil may be a substrate which also comprises fiber, in particular fiber from the mesocarp of palm fruitlets.
  • 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.
  • the pressure is reduced during the sterilization procedure.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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, 66-75°C, 66- 74°C, 66-73X, 66-72°C,
  • 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.
  • 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.
  • 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.
  • 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.
  • the substrate may be selected from the group consisting palm fruitlets, palm press liquid, mashed or partly mashed palm fruitlets.
  • 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, 10-500 mg enzyme protein/kg FFB comprising palm oil, 75-500 mg enzyme protein/kg FFB comprising palm oil, 75-500 mg enzyme protein/kg FFB compris
  • 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.
  • the contacting of the substrate comprising palm oil with an enzyme composition is done when the substrate is conveyed towards the digester.
  • 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.
  • 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.
  • the process comprises steps of: sterilizing and threshing fresh palm fruit bunches to provide palm fruitlets; and conveying the palm fruitlets into a digester.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • contacting with an enzyme composition is done at one or more contact points.
  • 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.
  • 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.
  • the invention concerns a crude palm oil, which is obtainable by the process according to any of the process 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 may be obtained from microorganisms of any genus.
  • 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.
  • 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.
  • the polypeptide is a Talaromyces leycettanus polypeptide, e.g., a polypeptide obtained from Talaromyces leycettanus Strain CBS398.68.
  • the polypeptide is a Rasamsonia byssochlamydoides polypeptide, e.g., a polypeptide obtained from Rasamsonia byssochlamydoides.
  • the polypeptide is an Aspergillus niger polypeptide, e.g., a polypeptide obtained from Aspergillus niger.
  • 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.
  • ATCC American Type Culture Collection
  • DSMZ Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH
  • CBS Centraalbureau Voor Schimmelcultures
  • NRRL Northern Regional Research Center
  • 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.
  • 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 GH10 xylanase comprises or consists of the mature polypeptide of SEQ ID NO: 1
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the enzymes which are obtained from Trichoderma reesei comprise all enzyme activities necessary for complete degradation of cellulose.
  • the enzyme composition comprises a GH62 arabinofuranosidase.
  • 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%.
  • 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%.
  • 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%.
  • 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.
  • 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.
  • the polypeptide obtained from a given source is secreted extracellularly.
  • the polypeptide may be a fungal polypeptide.
  • 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.
  • 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
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • ATCC American Type Culture Collection
  • DSMZ Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH
  • CBS Centraalbureau Voor Schimmelcultures
  • NRRL Northern Regional Research Center
  • 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.
  • 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
  • a GH10 xylanase 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
  • 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.
  • 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.
  • step 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]
  • 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.
  • 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.
  • 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
  • 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.
  • 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.
  • the enzyme addition was done in step 5 as described in example 2. Control sample was without enzyme addition.
  • 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.
  • the sterilized palm fruit mesocarp is pressed.
  • 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).
  • GH10 Xylanase A a GH10 Xylanase from Aspergillus niger, SEQ ID NO: 8
  • GH62 Arabinofuranosidase A GH62
  • 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.
  • 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
  • Substrate 0.2% Arabinoxylan in diluted buffer.
  • dilution buffer 50Mm Na acetate +0.01% TRITON X
  • 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
  • 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 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 ⁇ to 200 ⁇ , for a final volume of 1 ml in each reaction. The plate was then sealed using an ALPS-300TM 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.
  • 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 EXCELTM software (Microsoft, Richland, WA, USA).
  • % xylose conversion xylose concentration/xylose concentration in a limit digest.
  • % 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.
  • % 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
  • the Talaromyces leycettanus GH10 xylanase (SEQ ID NO 1 ) was prepared.
  • 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) 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 BCATM 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 BCATM Protein Assay Kit (Thermo Fischer Scientific, Waltham, MA, USA) in which bovine serum albumin was used as a protein standard

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Abstract

La présente invention concerne un procédé d'extraction ou de séparation d'huile de palme brute (CPO), comprenant les étapes consistant à : mettre en contact un substrat comprenant de l'huile de palme avec une composition enzymatique comprenant une xylanase GH10 et extraire ou séparer l'huile de palme brute.
PCT/EP2018/072873 2017-08-25 2018-08-24 Extraction d'huile de palme brute assistée par des enzymes Ceased WO2019038418A1 (fr)

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WO2025207659A1 (fr) * 2024-03-26 2025-10-02 Danisco Us Inc. Procédés et systèmes de production d'huile de palme brute

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WO2021198077A1 (fr) 2020-04-02 2021-10-07 Novozymes A/S Procédé pour récupérer de l'huile de palme brute

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