CN115074351A - Compound immobilized enzyme and preparation method thereof - Google Patents
Compound immobilized enzyme and preparation method thereof Download PDFInfo
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- CN115074351A CN115074351A CN202110223516.2A CN202110223516A CN115074351A CN 115074351 A CN115074351 A CN 115074351A CN 202110223516 A CN202110223516 A CN 202110223516A CN 115074351 A CN115074351 A CN 115074351A
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- immobilized enzyme
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- 108010093096 Immobilized Enzymes Proteins 0.000 title claims abstract description 86
- 150000001875 compounds Chemical class 0.000 title claims abstract description 32
- 238000002360 preparation method Methods 0.000 title claims abstract description 18
- 239000007788 liquid Substances 0.000 claims abstract description 93
- 229920005989 resin Polymers 0.000 claims abstract description 63
- 102000004190 Enzymes Human genes 0.000 claims abstract description 62
- 108090000790 Enzymes Proteins 0.000 claims abstract description 62
- 239000011347 resin Substances 0.000 claims abstract description 62
- 238000001179 sorption measurement Methods 0.000 claims abstract description 44
- 108090001060 Lipase Proteins 0.000 claims abstract description 40
- 102000004882 Lipase Human genes 0.000 claims abstract description 40
- 239000004367 Lipase Substances 0.000 claims abstract description 40
- 235000019421 lipase Nutrition 0.000 claims abstract description 40
- 238000000034 method Methods 0.000 claims abstract description 35
- 239000007787 solid Substances 0.000 claims abstract description 26
- 108010031186 Glycoside Hydrolases Proteins 0.000 claims abstract description 21
- 102000005744 Glycoside Hydrolases Human genes 0.000 claims abstract description 21
- 238000001035 drying Methods 0.000 claims abstract description 20
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 15
- 238000006243 chemical reaction Methods 0.000 claims abstract description 14
- 235000020256 human milk Nutrition 0.000 claims abstract description 10
- 210000004251 human milk Anatomy 0.000 claims abstract description 10
- 239000004519 grease Substances 0.000 claims abstract description 9
- 238000000926 separation method Methods 0.000 claims abstract description 9
- 229940088598 enzyme Drugs 0.000 claims description 49
- 239000000203 mixture Substances 0.000 claims description 31
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims description 20
- 229930006000 Sucrose Natural products 0.000 claims description 20
- 239000005720 sucrose Substances 0.000 claims description 20
- 108010042889 Inulosucrase Proteins 0.000 claims description 15
- 239000000758 substrate Substances 0.000 claims description 15
- DCXXMTOCNZCJGO-UHFFFAOYSA-N tristearoylglycerol Chemical compound CCCCCCCCCCCCCCCCCC(=O)OCC(OC(=O)CCCCCCCCCCCCCCCCC)COC(=O)CCCCCCCCCCCCCCCCC DCXXMTOCNZCJGO-UHFFFAOYSA-N 0.000 claims description 14
- 239000002253 acid Substances 0.000 claims description 13
- 240000005384 Rhizopus oryzae Species 0.000 claims description 9
- 235000013752 Rhizopus oryzae Nutrition 0.000 claims description 9
- 239000003921 oil Substances 0.000 claims description 9
- PPBRXRYQALVLMV-UHFFFAOYSA-N Styrene Chemical compound C=CC1=CC=CC=C1 PPBRXRYQALVLMV-UHFFFAOYSA-N 0.000 claims description 8
- 239000003513 alkali Substances 0.000 claims description 8
- 150000001412 amines Chemical group 0.000 claims description 8
- 238000002156 mixing Methods 0.000 claims description 8
- WRIDQFICGBMAFQ-UHFFFAOYSA-N (E)-8-Octadecenoic acid Natural products CCCCCCCCCC=CCCCCCCC(O)=O WRIDQFICGBMAFQ-UHFFFAOYSA-N 0.000 claims description 7
- LQJBNNIYVWPHFW-UHFFFAOYSA-N 20:1omega9c fatty acid Natural products CCCCCCCCCCC=CCCCCCCCC(O)=O LQJBNNIYVWPHFW-UHFFFAOYSA-N 0.000 claims description 7
- QSBYPNXLFMSGKH-UHFFFAOYSA-N 9-Heptadecensaeure Natural products CCCCCCCC=CCCCCCCCC(O)=O QSBYPNXLFMSGKH-UHFFFAOYSA-N 0.000 claims description 7
- 239000005642 Oleic acid Substances 0.000 claims description 7
- ZQPPMHVWECSIRJ-UHFFFAOYSA-N Oleic acid Natural products CCCCCCCCC=CCCCCCCCC(O)=O ZQPPMHVWECSIRJ-UHFFFAOYSA-N 0.000 claims description 7
- QXJSBBXBKPUZAA-UHFFFAOYSA-N isooleic acid Natural products CCCCCCCC=CCCCCCCCCC(O)=O QXJSBBXBKPUZAA-UHFFFAOYSA-N 0.000 claims description 7
- 238000004519 manufacturing process Methods 0.000 claims description 7
- ZQPPMHVWECSIRJ-KTKRTIGZSA-N oleic acid Chemical compound CCCCCCCC\C=C/CCCCCCCC(O)=O ZQPPMHVWECSIRJ-KTKRTIGZSA-N 0.000 claims description 7
- 241000228245 Aspergillus niger Species 0.000 claims description 6
- 241000589540 Pseudomonas fluorescens Species 0.000 claims description 6
- 235000013341 fat substitute Nutrition 0.000 claims description 6
- 239000003778 fat substitute Substances 0.000 claims description 6
- 230000003534 oscillatory effect Effects 0.000 claims description 5
- GUBGYTABKSRVRQ-XLOQQCSPSA-N Alpha-Lactose Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](CO)O[C@H](O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-XLOQQCSPSA-N 0.000 claims description 4
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 4
- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 claims description 4
- NIXOWILDQLNWCW-UHFFFAOYSA-N acrylic acid group Chemical group C(C=C)(=O)O NIXOWILDQLNWCW-UHFFFAOYSA-N 0.000 claims description 4
- 125000000217 alkyl group Chemical group 0.000 claims description 4
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 claims description 4
- 239000003054 catalyst Substances 0.000 claims description 4
- 239000008103 glucose Substances 0.000 claims description 4
- 239000008101 lactose Substances 0.000 claims description 4
- 239000002861 polymer material Substances 0.000 claims description 4
- 150000003141 primary amines Chemical group 0.000 claims description 4
- 150000003335 secondary amines Chemical group 0.000 claims description 4
- 238000002791 soaking Methods 0.000 claims description 4
- 150000003512 tertiary amines Chemical group 0.000 claims description 4
- 102100026189 Beta-galactosidase Human genes 0.000 claims description 3
- 108010093031 Galactosidases Proteins 0.000 claims description 3
- 102000002464 Galactosidases Human genes 0.000 claims description 3
- 108010059881 Lactase Proteins 0.000 claims description 3
- 241001465754 Metazoa Species 0.000 claims description 3
- 101710184309 Probable sucrose-6-phosphate hydrolase Proteins 0.000 claims description 3
- 241000235403 Rhizomucor miehei Species 0.000 claims description 3
- 241000235527 Rhizopus Species 0.000 claims description 3
- 102400000472 Sucrase Human genes 0.000 claims description 3
- 101710112652 Sucrose-6-phosphate hydrolase Proteins 0.000 claims description 3
- 241000223258 Thermomyces lanuginosus Species 0.000 claims description 3
- 108010005774 beta-Galactosidase Proteins 0.000 claims description 3
- 239000002131 composite material Substances 0.000 claims description 3
- 238000000354 decomposition reaction Methods 0.000 claims description 3
- 108010090785 inulinase Proteins 0.000 claims description 3
- 235000011073 invertase Nutrition 0.000 claims description 3
- 229940116108 lactase Drugs 0.000 claims description 3
- 244000005700 microbiome Species 0.000 claims description 3
- 241000894007 species Species 0.000 claims description 3
- 238000002203 pretreatment Methods 0.000 claims description 2
- 108010079522 solysime Proteins 0.000 claims description 2
- -1 a-glucosidase Proteins 0.000 claims 1
- 239000003925 fat Substances 0.000 claims 1
- 238000005406 washing Methods 0.000 claims 1
- 230000000694 effects Effects 0.000 abstract description 29
- 102000004169 proteins and genes Human genes 0.000 abstract description 12
- 108090000623 proteins and genes Proteins 0.000 abstract description 12
- 230000003197 catalytic effect Effects 0.000 abstract description 5
- 230000001351 cycling effect Effects 0.000 abstract description 4
- 238000003860 storage Methods 0.000 abstract description 2
- 238000003786 synthesis reaction Methods 0.000 abstract description 2
- 241000209094 Oryza Species 0.000 description 22
- 235000007164 Oryza sativa Nutrition 0.000 description 22
- 235000009566 rice Nutrition 0.000 description 22
- 238000009010 Bradford assay Methods 0.000 description 15
- 230000008859 change Effects 0.000 description 15
- 230000000052 comparative effect Effects 0.000 description 14
- 238000005303 weighing Methods 0.000 description 14
- 239000002585 base Substances 0.000 description 12
- 239000000047 product Substances 0.000 description 12
- 238000003828 vacuum filtration Methods 0.000 description 12
- 238000001914 filtration Methods 0.000 description 8
- 235000019198 oils Nutrition 0.000 description 8
- 238000001291 vacuum drying Methods 0.000 description 8
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 6
- 238000000855 fermentation Methods 0.000 description 6
- 230000004151 fermentation Effects 0.000 description 6
- 239000003223 protective agent Substances 0.000 description 5
- 238000005516 engineering process Methods 0.000 description 4
- 150000002148 esters Chemical group 0.000 description 4
- RPNUMPOLZDHAAY-UHFFFAOYSA-N Diethylenetriamine Chemical compound NCCNCCN RPNUMPOLZDHAAY-UHFFFAOYSA-N 0.000 description 3
- 230000018044 dehydration Effects 0.000 description 3
- 238000006297 dehydration reaction Methods 0.000 description 3
- 150000002632 lipids Chemical group 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- 241000196324 Embryophyta Species 0.000 description 2
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 description 2
- 238000003795 desorption Methods 0.000 description 2
- 230000009144 enzymatic modification Effects 0.000 description 2
- 238000006911 enzymatic reaction Methods 0.000 description 2
- 239000004744 fabric Substances 0.000 description 2
- 238000004108 freeze drying Methods 0.000 description 2
- 230000003993 interaction Effects 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000007935 neutral effect Effects 0.000 description 2
- 230000010355 oscillation Effects 0.000 description 2
- 238000000643 oven drying Methods 0.000 description 2
- 238000004806 packaging method and process Methods 0.000 description 2
- 229920005862 polyol Polymers 0.000 description 2
- 150000003077 polyols Chemical class 0.000 description 2
- 239000011148 porous material Substances 0.000 description 2
- 238000012545 processing Methods 0.000 description 2
- 239000003549 soybean oil Substances 0.000 description 2
- 235000012424 soybean oil Nutrition 0.000 description 2
- 238000005809 transesterification reaction Methods 0.000 description 2
- UFTFJSFQGQCHQW-UHFFFAOYSA-N triformin Chemical compound O=COCC(OC=O)COC=O UFTFJSFQGQCHQW-UHFFFAOYSA-N 0.000 description 2
- 240000006439 Aspergillus oryzae Species 0.000 description 1
- 235000002247 Aspergillus oryzae Nutrition 0.000 description 1
- 108010048733 Lipozyme Proteins 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- 238000010364 biochemical engineering Methods 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 239000007795 chemical reaction product Substances 0.000 description 1
- 239000003431 cross linking reagent Substances 0.000 description 1
- 235000014113 dietary fatty acids Nutrition 0.000 description 1
- 229940079919 digestives enzyme preparation Drugs 0.000 description 1
- 230000032050 esterification Effects 0.000 description 1
- 238000005886 esterification reaction Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 229930195729 fatty acid Natural products 0.000 description 1
- 239000000194 fatty acid Substances 0.000 description 1
- 150000004665 fatty acids Chemical class 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 239000008173 hydrogenated soybean oil Substances 0.000 description 1
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 1
- 230000003100 immobilizing effect Effects 0.000 description 1
- 230000002779 inactivation Effects 0.000 description 1
- 239000003262 industrial enzyme Substances 0.000 description 1
- FCCDDURTIIUXBY-UHFFFAOYSA-N lipoamide Chemical compound NC(=O)CCCCC1CCSS1 FCCDDURTIIUXBY-UHFFFAOYSA-N 0.000 description 1
- 238000012423 maintenance Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 230000000050 nutritive effect Effects 0.000 description 1
- 229920001542 oligosaccharide Polymers 0.000 description 1
- 150000002482 oligosaccharides Chemical class 0.000 description 1
- 230000000737 periodic effect Effects 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 230000003595 spectral effect Effects 0.000 description 1
- 238000000967 suction filtration Methods 0.000 description 1
- 239000002699 waste material Substances 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
- C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
- C12N11/08—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a synthetic polymer
- C12N11/082—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a synthetic polymer obtained by reactions only involving carbon-to-carbon unsaturated bonds
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
- C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
- C12N11/08—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a synthetic polymer
- C12N11/082—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a synthetic polymer obtained by reactions only involving carbon-to-carbon unsaturated bonds
- C12N11/087—Acrylic polymers
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
- C12N9/18—Carboxylic ester hydrolases (3.1.1)
- C12N9/20—Triglyceride splitting, e.g. by means of lipase
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
- C12P7/6436—Fatty acid esters
- C12P7/6445—Glycerides
- C12P7/6472—Glycerides containing polyunsaturated fatty acid [PUFA] residues, i.e. having two or more double bonds in their backbone
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y301/00—Hydrolases acting on ester bonds (3.1)
- C12Y301/01—Carboxylic ester hydrolases (3.1.1)
- C12Y301/01003—Triacylglycerol lipase (3.1.1.3)
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- Chemical & Material Sciences (AREA)
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- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
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- Health & Medical Sciences (AREA)
- Wood Science & Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Genetics & Genomics (AREA)
- General Health & Medical Sciences (AREA)
- General Engineering & Computer Science (AREA)
- Biochemistry (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- Biomedical Technology (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Oil, Petroleum & Natural Gas (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Immobilizing And Processing Of Enzymes And Microorganisms (AREA)
Abstract
The invention provides a compound immobilized enzyme and a preparation method thereof. The compound immobilized enzyme comprises lipase, glycosidase and a resin carrier. The preparation method comprises contacting lipase liquid with resin carrier, adding glycosidase after adsorption balance of lipase is achieved; and (3) after the mixed system is fully contacted, carrying out solid-liquid separation and drying the solid matter to obtain the compound immobilized enzyme. When the compound immobilized enzyme prepared by the invention is used for modifying the grease, the product quality is greatly improved. The immobilized enzyme prepared by the method can effectively maintain the protein catalytic structure and reduce protein shedding in the grease reaction, and can be recycled for more than 50 times without adding water in the synthesis reaction of the structural grease of the human milk substitute by the enzyme method. Aiming at the liquid enzyme source with reduced enzyme activity caused by long storage time, the preparation method provided by the invention can obviously improve the enzyme activity and the cycling stability of the immobilized enzyme.
Description
Technical Field
The invention relates to the technical field of biochemical engineering, in particular to a compound immobilized enzyme and a preparation method thereof.
Background
The modification of oil by enzyme method is one of the important means for modulating the structure and composition of oil and improving the nutritive value and applicability of the oil, and the main catalyst used is lipase. Compared with a chemical method, the enzymatic modification has the advantages of mild conditions, simple steps, high product quality and the like, but the large-scale application of the enzymatic modification in the field of oil processing is limited by high cost. The lipase is immobilized to improve the stability, and is convenient to separate from a reaction system and reuse, so that the method is an effective method for reducing the production cost.
In an actual oil modification experiment by an enzyme method, gradual desorption and dehydration of an immobilized enzyme along with reaction are main reasons for reducing enzyme activity. In order to avoid desorption of the immobilized enzyme, glutaraldehyde is usually selected as a cross-linking agent for treatment in the preparation process of the immobilized enzyme. In the preparation of enzyme preparations for the food industry, glutaraldehyde is prohibited. In order to avoid the gradual dehydration of the immobilized enzyme, a polyol having water-holding capacity is often added as a protective agent to the liquid enzyme or the immobilized enzyme, for example, the immobilized enzyme mentioned in patent WO 2009/010561 a1 mainly comprises a carrier, a polyol and a lipase. In addition, periodic rehydration of the immobilized enzyme also contributes to the maintenance or recovery of enzyme activity.
There is still a need in the art to develop a new method for preparing immobilized enzyme, which can make the oil industrial enzyme preparation have good catalytic activity and cycling stability.
Disclosure of Invention
The first aspect of the invention provides a preparation method of a compound immobilized enzyme, which specifically comprises the following steps:
1) the lipase liquid is contacted with a resin carrier for mixing, and is vibrated and adsorbed to obtain a mixture A; the lipase liquid contains polyhydroxy compounds with the concentration of 5-50%, preferably sucrose, glucose or lactose; the resin carrier is selected from a porous high polymer material with a styrene type or acrylic type skeleton, and the surface of the resin carrier can be distributed with basic groups containing alkyl long chains and terminal quaternary amines, tertiary amines, secondary amines or primary amines; preferably a basic group with a quaternary amine at the end;
2) adding glycosidase into the mixture A in the step 1) to obtain a mixture B; preferably, glycosidase is added after the lipase in the mixture A reaches adsorption equilibrium;
3) carrying out solid-liquid separation on the mixture B in the step 2) and drying the solid matter to obtain the compound immobilized enzyme; preferably, the mixture B is stirred for more than 30min at the temperature ranging from 4 ℃ to 30 ℃ and then subjected to solid-liquid separation.
Alternatively, the method comprises the following steps:
1) the lipase liquid is contacted with a resin carrier for mixing, and is vibrated and adsorbed to obtain a mixture A; the lipase liquid contains polyhydroxy compounds with the concentration of 5-50%, preferably sucrose, glucose or lactose; the resin carrier is selected from a porous high polymer material with a styrene type or acrylic type skeleton, and the surface of the resin carrier can be distributed with basic groups containing alkyl long chains and terminal quaternary amines, tertiary amines, secondary amines or primary amines; preferably a basic group with a quaternary amine at the end;
2) after the lipase liquid and the resin carrier are oscillated and adsorbed for a period of time, adding glycosidase to form a mixture B; preferably, glycosidase is added after the lipase in the mixture A reaches adsorption equilibrium;
3) and after the mixture B is fully contacted, carrying out solid-liquid separation and drying the solid matter to obtain the compound immobilized enzyme.
It is specifically noted that the adsorption of the lipase liquid and the resin carrier takes a certain time, and the achievement of the adsorption equilibrium in step 2) of the above method is common knowledge in the art, and specifically means that the adsorption equilibrium is achieved without a significant change in the residual protein content of the solution. If the oscillation adsorption time of the lipase liquid and the resin carrier is short and the adsorption balance is far away, a large amount of lipase is remained in the lipase liquid and can not be fixed on the carrier, so that the final protein content is difficult to control, and resource waste is also caused.
In one or more embodiments, sufficient contacting as described in step 3) means that the mixed system is stirred at a temperature in the range of 4 to 30 ℃ for more than 30 min.
In one or more embodiments, the resin may be selected from NKX-8 resin (available from tianjin nan and science and technology ltd), or from LX1000HA, EPHA, LX1000EPN, LX1000NH, LX1000EA, LXSR-2 (available from seian dawn scientific and technology co., ltd.), or from Lewatit S4228 \4268\4328\4468\4528\5221\5228\5328\5128\5528\ 8806268 \6368\7468 (available from langerhans chemical (china) ltd.), or from ECR 4\ 6\ 1508\ 1504\ 1640 (available from laite china ltd.). The resins of the present invention include, in addition to the specific products defined above by trade names, any commercially available resin products having other trade names or resins obtained by self-preparation or processing, as long as they have the same or similar structure and adsorption or exchange properties as the specific products.
In one or more embodiments, before step 1), a step of pre-treating the resin carrier is further included, and preferably, the pre-treating method includes: respectively soaking by using acid and alkali solutions, wherein the last step needs to be the alkali solution. For example, acid, base … acid, base; base, acid, base … acid, base are also possible, but the last step must be base since the resin of the invention is a basic resin. Preferably, acid and alkali are alternately soaked for more than two times, and then the mixture is washed to be neutral by water to remove free water. More preferably, reference may be made to the pretreatment method of the resin carrier in patent CN 102839166B.
In one or more embodiments, the mass ratio of the resin carrier to the enzyme protein in the lipase liquor in step 1) is 1: 0.01-0.05.
In one or more embodiments, the lipase in step 2) is an enzyme for the decomposition of lipids, selected from lipases of animal, plant or microbial origin; preferably, the lipase is selected from Thermomyces lanuginosus, Mucor miehei, Rhizopus oryzae (Rhizopus oryzae), Pseudomonas fluorescens (Pseudomonas fluorescens), Rhizopus oryzae (Rhizopus miehei), Aspergillus niger (Aspergillus niger) or genetically modified species of the above microorganisms.
In one or more embodiments, the oscillatory adsorption in step 1) is carried out at a temperature of 4-30 ℃ at an oscillatory speed of 100-.
In one or more embodiments, the glycosidase in step 2) is a fructosyltransferase, a sucrase, an inulinase, an a-glucosidase, a lactase or a galactosidase, and the mass of the enzyme protein in the glycosidase is 1-10%, preferably 2.5-5% of the mass of the enzyme protein in the lipase liquor in step 1).
In one or more embodiments, the solid-liquid separation method described in step 3) is vacuum filtration or cloth bag centrifugation.
In one or more embodiments, the drying process described in step 3) is room temperature tray drying, vacuum oven drying, fluidized bed drying, or freeze drying.
The second aspect of the invention provides a compound immobilized enzyme or a packaging product thereof, wherein the compound immobilized enzyme is prepared by the method, and has a protein content of 3-10% and a moisture content of 3-15%.
The third aspect of the invention provides the application of the compound immobilized enzyme in the grease industry as a catalyst and/or the application in preparing breast milk substitutes, such as OPO.
In a fourth aspect, the present invention provides a method for preparing a human milk fat substitute, such as OPO, comprising adding the complex immobilized enzyme prepared by the above method to a mixed substrate of glycerol tristearate and oleic acid, preferably, the method comprises the following steps: mixing and dissolving glycerol tristearate and oleic acid according to the mass ratio of 1:2-1:4 to prepare a substrate, adding the composite immobilized enzyme prepared by the method, and reacting for 1-6h at the temperature of 60-70 ℃ in a gas bath at the speed of 150-200 rpm; the addition amount of the compound immobilized enzyme is 6-12% of the mass of the substrate.
In the present invention, glycosidases are added for the purpose of: (1) the existence structure of the polyhydroxy protective agent around the protein and the interaction form with the protein are modulated, because glycosidase can catalyze the protective agent sucrose of lipase to react to generate functional oligosaccharide, the hydroxyl number of the protective agent around the protein and the interaction form with the lipase are changed, the steric hindrance and the water holding capacity in a nanometer pore channel are increased, and the falling and dehydration inactivation of the protein in the oil reaction are further effectively reduced; (2) after lipase is adsorbed, glycosidase is adsorbed on a resin carrier, so that most glycosidase is adsorbed to the periphery and the orifice of a resin pore channel, and the steric hindrance can be increased by virtue of protein, and the falling of the protein is reduced.
Compared with the prior art, the invention has the advantages that:
(1) the compound immobilized enzyme prepared by the invention can be used for various esterification and ester exchange related applications in the grease industry, and the immobilized enzyme shows excellent catalytic activity and cycling stability.
(2) The immobilized enzyme prepared by the method can effectively maintain the protein catalytic structure and reduce protein shedding in the grease reaction, and can be recycled for more than 50 times without adding water in the synthesis reaction of the structural grease of the human milk substitute by the enzyme method.
(3) Aiming at the liquid enzyme source with reduced enzyme activity caused by long storage time, the preparation method provided by the invention can obviously improve the enzyme activity and the cycling stability of the immobilized enzyme.
Drawings
FIG. 1 shows the content of C52 in the NKX-8 carrier-series immobilized enzymes (A, B, C, F, I, J, Q, Lipozyme RM IM) in OPO production as a function of reaction batch.
FIG. 2 shows the variation of C52 content of LXSR-2 and ECR1640 carrier series immobilized enzymes (D, E, G, H) with reaction batch in OPO production.
FIG. 3 shows RML enzyme solution (K, L, M, N, O, P) obtained by immobilizing NKX-8 and LXSR-2 resin and storing the immobilized enzyme for one year, and improving the enzyme activity and the service life of the immobilized enzyme by using glycosidase treatment technology.
Detailed Description
It is to be understood that within the scope of the present invention, the above-described technical features of the present invention and the technical features described in detail below (e.g., the embodiments) may be combined with each other to constitute a preferred embodiment.
The invention provides a preparation method of a compound immobilized enzyme, which specifically comprises the following steps:
1) the lipase liquid is contacted with a resin carrier for mixing, and is oscillated for adsorption; the lipase liquid contains polyhydroxy compounds with the concentration of 5-50%, preferably sucrose, glucose or lactose; the resin carrier is selected from a porous high polymer material with a styrene type or acrylic type skeleton, and the surface of the resin carrier can be distributed with basic groups containing alkyl long chains and terminal quaternary amines, tertiary amines, secondary amines or primary amines; preferably a basic group with a quaternary amine at the end;
2) after the lipase liquid and the resin carrier are oscillated and adsorbed for a period of time, adding glycosidase to form a mixed system; preferably, glycosidase is added after the lipase reaches adsorption equilibrium;
3) and (3) after the mixed system is fully contacted, carrying out solid-liquid separation and drying the solid matter to obtain the compound immobilized enzyme.
In one or more embodiments, the resin may be selected from NKX-8 resin (available from tianjin nan and science and technology ltd), or from LX1000HA, EPHA, LX1000EPN, LX1000NH, LX1000EA, LXSR-2 (available from seian dawn scientific and technology co., ltd.), or from Lewatit S4228 \4268\4328\4468\4528\5221\5228\5328\5128\5528\ 8806268 \6368\7468 (available from langerhans chemical (china) ltd.), or from ECR 4\ 6\ 1508\ 1504\ 1640 (available from laite china ltd.).
In one or more embodiments, before step 1), a step of pre-treating the resin carrier is further included, and preferably, the pre-treating method includes: respectively soaking by using acid and alkali solutions, wherein the last step needs to be the alkali solution. For example, acid, base … acid, base; base, acid, base … acid, base are also possible, but the last step must be base since the resin of the invention is a basic resin. Preferably, acid and alkali are alternately soaked for more than two times, and then the mixture is washed by water to be neutral, and free water is removed.
In one or more embodiments, the mass ratio of the resin carrier to the enzyme protein in the lipase liquor in step 1) is 1: 0.01-0.05.
In one or more embodiments, the lipase in step 2) is an enzyme for the decomposition of lipids, selected from animal, plant or microbial lipases; preferably, the lipase is selected from Thermomyces lanuginosus, Mucor miehei, Rhizopus oryzae (Rhizopus oryzae), Pseudomonas fluorescens (Pseudomonas fluorescens), Rhizopus oryzae (Rhizopus miehei), Aspergillus niger (Aspergillus niger) or genetically modified species of the above microorganisms.
In one or more embodiments, said oscillatory adsorption in step 1) is carried out at a temperature of between 4 and 30 ℃ and at an oscillation speed of between 100 and 200rpm, preferably at a speed of between 120 and 150 rpm.
In one or more embodiments, the glycosidase in step 2) is a fructosyltransferase, a sucrase, an inulinase, an a-glucosidase, a lactase or a galactosidase, and the mass of the enzyme protein in the glycosidase is 1-10%, preferably 2.5-5% of the mass of the enzyme protein in the lipase liquor in step 1).
In one or more embodiments, sufficient contacting as described in step 3) means that the mixed system is stirred at a temperature in the range of 4 to 30 ℃ for more than 30 min.
In one or more embodiments, the solid-liquid separation method in step 3) is vacuum filtration or cloth bag centrifugal filtration.
In one or more embodiments, the drying process described in step 3) is room temperature tray drying, vacuum oven drying, fluidized bed drying, or freeze drying.
The second aspect of the invention provides a compound immobilized enzyme or a packaging product thereof, wherein the immobilized compound immobilized enzyme is prepared by the method, the protein content is 3-10%, and the moisture content is 3-15%.
The third aspect of the invention provides the application of the compound immobilized enzyme in the grease industry as a catalyst and/or the application in preparing breast milk fat substitutes, such as OPO.
In a fourth aspect, the present invention provides a method for preparing a human milk fat substitute, such as OPO, comprising adding the complex immobilized enzyme prepared by the above method to a mixed substrate of glycerol tristearate and oleic acid, preferably, the method comprises the following steps: mixing and dissolving glycerol tristearate and oleic acid according to the mass ratio of 1:2-1:4 to prepare a substrate, adding the compound immobilized enzyme prepared by the method, carrying out air bath at 60-70 ℃, and reacting at 150-; the addition amount of the compound immobilized enzyme is 6-12% of the mass of the substrate.
Examples
Example (1)
NKX-8 resin (purchased from Tianjin Nankai and science and technology Co., Ltd.) was treated with 1M HCl solution and 1M NaOH solution twice (and then treated with NaOH solution) before use, and the treatment was carried out for two hours each time, and finally washed with clear water to neutrality, and free water was removed by suction filtration for further use. Weighing 50g of treated NKX-8, adding 100ml of self-produced RML liquid (strain BC4-RML (CGMCC No:19263) to ferment by using aspergillus oryzae at 10mg/ml, containing 30% of sucrose solution, fresh, and having enzyme activity of 8000U/mg), oscillating at room temperature of 150rpm for 24h, and determining that the protein content in the solution is below 0.5mg/ml by using a Bradford method. And the content of the residual protein in the solution does not change obviously along with the prolonging of the adsorption time, which indicates that the adsorption balance is achieved. Then adding 25ml fructosyl transferase liquid (purchased from Weifang kang Dien Biotechnology Co., Ltd., 1mg/ml) into the solid-liquid mixture, continuing oscillating for 8h, removing the liquid by vacuum filtration, and drying the resin solid for 24h at room temperature to obtain the immobilized enzyme A.
Example (2)
Weighing 50g of treated NKX-8 (same as example 1), adding 250ml of self-produced RML liquid (strain BC4-RML (CGMCC No:19263) to perform rice koji enzyme fermentation, wherein the concentration of the liquid is 10mg/ml, the liquid contains 30% of sucrose, and the liquid is fresh and has enzyme activity of 8000U/mg), oscillating the liquid at room temperature of 150rpm for 24 hours, determining that the protein content in the solution is below 0.5mg/ml by a Bradford method, and ensuring that the residual protein content in the solution does not change obviously along with the extension of adsorption time, thereby indicating that the adsorption balance is achieved. Then adding 25ml fructosyl transferase liquid (purchased from Weifang Kangdian Biotechnology Co., Ltd., 1mg/ml) into the solid-liquid mixture, continuing oscillating for 1h, removing the liquid by vacuum filtration, and drying the resin solid for 24h at room temperature to obtain the immobilized enzyme B.
Example (3)
Weighing 50g of treated NKX-8 (same as example 1), adding 50ml of self-produced RML liquid (strain BC4-RML (CGMCC No:19263) to perform rice koji enzyme fermentation, wherein the concentration of the liquid is 10mg/ml, the liquid contains 30% of sucrose, and the liquid is fresh and has enzyme activity of 8000U/mg), oscillating the liquid at room temperature of 150rpm for 12h, determining that the protein content in the solution is below 0.5mg/ml by a Bradford method, and ensuring that the residual protein content in the solution does not change obviously along with the extension of adsorption time, thereby indicating that the adsorption balance is achieved. Then adding 25ml fructosyl transferase liquid (purchased from Weifang Kangdian Biotechnology Co., Ltd., 1mg/ml) into the solid-liquid mixture, continuing to oscillate for 24h, vacuum-filtering to remove the liquid, and drying the resin solid for 24h at room temperature to obtain the immobilized enzyme C.
Example (4)
LXSR-2 resin (available from New science and technology materials, Inc., Xian blue, as described in EXAMPLE 1) was processed in the same manner as described in example 1.
Weighing 50g of treated LXSR-2, adding 100ml of self-produced RML liquid (strain BC4-RML (CGMCC No:19263) to perform rice koji enzyme fermentation at 10mg/ml, containing 30% of sucrose solution, fresh, and having enzyme activity of 8000U/mg), oscillating at room temperature of 150rpm for 24h, determining that the protein content in the solution is below 0.5mg/ml by a Bradford method, and ensuring that the residual protein content in the solution does not change obviously along with the extension of adsorption time, thereby indicating that the adsorption balance is achieved. Adding 100ml fructosyl transferase solution (purchased from Weifang kang Di En Biotechnology Co., Ltd., 1mg/ml) into the solid-liquid mixture, continuing to oscillate for 24h, vacuum-filtering to remove the liquid, and drying the resin solid at room temperature for 24h to obtain the immobilized enzyme D.
Example (5)
ECR1604 resin (available from Brand resin Co., Ltd.) was processed in the same manner as in example (1).
Weighing 50g of the treated ECR1604 resin, adding 100ml of self-produced RML liquid (strain BC4-RML (CGMCC No:19263) to perform rice koji enzyme fermentation at 10mg/ml, containing 30% of sucrose solution, fresh, and having enzyme activity of 8000U/mg), oscillating at room temperature of 150rpm for 16h, determining that the protein content in the solution is below 0.5mg/ml by a Bradford method, and ensuring that the residual protein content in the solution does not change obviously along with the extension of adsorption time, thereby indicating that the adsorption balance is achieved. Adding 10ml fructosyl transferase solution (purchased from Weifang kang Di En Biotechnology Co., Ltd., 1mg/ml) into the solid-liquid mixture, continuing to oscillate for 8h, vacuum-filtering to remove the liquid, and drying the resin solid at room temperature for 24h to obtain the immobilized enzyme E.
COMPARATIVE EXAMPLE (1)
Weighing 50g of treated NKX-8 (same as example 1), adding 100ml of RML liquid (strain BC4-RML (CGMCC No: 19263)) from rice koji, fermenting with rice koji enzyme, 10mg/ml, 30% sucrose solution, fresh, 8000U/mg enzyme activity, oscillating at room temperature of 150rpm for 16h, determining that the protein content in the solution is below 0.2mg/ml by Bradford method, and the residual protein content in the solution does not change obviously along with the extension of adsorption time, which indicates that the adsorption balance is reached. And (3) removing the liquid by vacuum filtration, and drying the resin solid at room temperature for 24h to obtain the immobilized enzyme F.
COMPARATIVE EXAMPLE (2)
50g of the treated LXSR-2 resin (same as the example 4) is weighed, 100ml of RML liquid (strain BC4-RML (CGMCC No: 19263)) from the rice koji is added to be fermented by the rice koji enzyme, 10mg/ml of the RML liquid contains 30% of sucrose solution, the RML liquid is fresh, the enzyme activity is 8000U/mg, after the RML liquid is shaken for 16 hours at the room temperature of 150rpm, the protein content in the solution is determined to be below 0.2mg/ml by a Bradford method, and the residual protein content in the solution does not change obviously along with the extension of the adsorption time, which indicates that the adsorption balance is reached. And (4) removing the liquid by vacuum filtration, and drying the resin solid at room temperature for 24h to obtain the immobilized enzyme G.
COMPARATIVE EXAMPLE (3)
Weighing 50g of treated ECR1604 resin (same as example 5), adding 100ml of RML liquid (strain BC4-RML (CGMCC No:19263) from rice koji, fermenting with rice koji enzyme at 10mg/ml, adding 30% sucrose solution, keeping fresh, and keeping enzyme activity at 8000U/mg), oscillating at 150rpm at room temperature for 16h, measuring the protein content in the solution by Bradford method to be below 0.2mg/ml, and ensuring that the residual protein content in the solution does not change obviously along with the extension of adsorption time, thus indicating that the adsorption balance is reached. And (3) removing the liquid by vacuum filtration, and drying the resin solid at room temperature for 24H to obtain the immobilized enzyme H.
COMPARATIVE EXAMPLE (4)
Weighing 50g of treated NKX-8 (same as example 1), adding 100ml of self-produced RML liquid (strain BC4-RML (CGMCC No:19263) to perform rice koji enzyme fermentation at 10mg/ml, without polyhydroxy protective agent, fresh, enzyme activity of 8000U/mg), oscillating at room temperature of 150rpm for 8h, determining that the protein content in the solution is below 0.5mg/ml by Bradford method, and the residual protein content in the solution does not change obviously along with the extension of adsorption time, which indicates that the adsorption balance is achieved. Adding 25ml fructosyl transferase liquid (purchased from Weifang kang dien biotechnology Limited company, 1mg/ml) into the solid-liquid mixture, continuing to oscillate for 8h, then carrying out vacuum filtration to remove the liquid, and drying the resin solid for 24h at room temperature to obtain the immobilized enzyme I.
COMPARATIVE EXAMPLE (5)
Weighing 50g of treated NKX-8 (same as example 1), adding 100ml of self-produced RML liquid (strain BC4-RML (CGMCC No: 19263)) to perform rice koji enzyme fermentation, wherein the concentration of the liquid is 10mg/ml, the liquid contains 30% of sucrose, and the activity of the liquid is 8000U/mg, oscillating the liquid at room temperature of 150rpm for 8 hours, determining that the protein content in the solution is below 0.5mg/ml by a Bradford method, and ensuring that the residual protein content in the solution does not change obviously along with the extension of adsorption time, thereby indicating that the adsorption balance is achieved. Adding 25ml fructosyl transferase solution (purchased from Weifang Kangdian Biotechnology Ltd., 1mg/ml) into the solid-liquid mixture, oscillating for 10min, vacuum filtering to remove liquid, and drying the resin solid at room temperature for 24h to obtain immobilized enzyme J.
Example (4)
Weighing 50g of treated NKX-8 (same as example 1), adding 100ml of RML liquid (strain BC4-RML (CGMCC No:19263) from rice koji, fermenting with rice koji enzyme, 10mg/ml, containing 30% of sucrose solution, storing for one year, and keeping the enzyme activity at 5000U/mg), oscillating at room temperature of 150rpm for 6h, measuring the protein content in the solution to be below 0.2mg/ml by Bradford method, and ensuring that the residual protein content in the solution does not change obviously along with the extension of the adsorption time, thus indicating that the adsorption balance is reached. Adding 25ml fructosyl transferase liquid (purchased from Weifang kang dien biotechnology Limited company, 1mg/ml) into the solid-liquid mixture, continuing to oscillate for 10h, then carrying out vacuum filtration to remove the liquid, and drying the resin solid for 24h at room temperature to obtain the immobilized enzyme K.
COMPARATIVE EXAMPLE (6)
Weighing 50g of treated NKX-8 (same as example 1), adding 100ml of RML liquid (strain BC4-RML (CGMCC No:19263) from rice koji, fermenting with rice koji, 10mg/ml, storing for one year in 30% sucrose solution, and keeping enzyme activity at 6000U/mg), oscillating at room temperature of 150rpm for 12h, measuring the protein content in the solution to be below 0.2mg/ml by Bradford method, and ensuring that the residual protein content in the solution does not change obviously along with the extension of adsorption time, thus indicating that the adsorption balance is reached. And (3) removing the liquid by vacuum filtration, and drying the resin solid at room temperature for 24h to obtain the immobilized enzyme L.
COMPARATIVE EXAMPLE (7)
50g of treated NKX-8 (same as example 1) was weighed, and 100ml of an RML liquid derived from koji (strain BC4-RML (CGMCC No: 19263)) was added thereto, and the mixture was fermented with koji enzyme at 10mg/ml in a 30% sucrose solution and stored for one year at an enzyme activity of 5000U/mg, and 25ml of a fructosyltransferase solution (purchased from Weifang Kangdian Biotech Ltd., 1mg/ml) was added thereto. Oscillating at room temperature of 150rpm for 24h, vacuum-filtering to remove liquid, and drying the resin solid at room temperature for 24h to obtain the immobilized enzyme M.
Example (5)
Weighing 50g of treated LXSR-2 (same as example 4), adding 100ml of RML liquid (strain BC4-RML (CGMCC No:19263) from rice koji, fermenting by rice koji enzyme, 10mg/ml, containing 30% of sucrose solution, storing for one year, and keeping the enzyme activity at 5000U/mg), oscillating at room temperature of 150rpm for 8 hours, measuring the protein content in the solution to be below 0.2mg/ml by a Bradford method, and ensuring that the residual protein content in the solution does not change obviously along with the extension of the adsorption time, thereby indicating that the adsorption balance is reached. Adding 25ml fructosyl transferase solution (purchased from Weifang kang Di En Biotechnology Co., Ltd., 1mg/ml) into the solid-liquid mixture, continuing oscillating for 7h, vacuum filtering to remove liquid, and drying the resin solid at room temperature for 24h to obtain the immobilized enzyme N.
COMPARATIVE EXAMPLE (8)
Weighing 50g of treated LXSR-2 (same as example 4), adding 100ml of RML liquid (strain BC4-RML (CGMCC No:19263) from rice koji, fermenting by rice koji enzyme, 10mg/ml, containing 30% of sucrose solution, storing for one year, and keeping the enzyme activity at 5000U/mg), oscillating at room temperature of 150rpm for 18h, measuring the protein content in the solution to be below 0.2mg/ml by a Bradford method, wherein the residual protein content in the solution does not change obviously along with the extension of the adsorption time, and thus the adsorption balance is achieved. And (3) removing the liquid by vacuum filtration, and drying the resin solid at room temperature for 24h to obtain the immobilized enzyme O.
COMPARATIVE EXAMPLE (9)
Weighing 50g of treated LXSR-2 (same as example 4), simultaneously adding 100ml of RML liquid (strain BC4-RML (CGMCC No: 19263)) from rice koji, fermenting by rice koji enzyme, 10mg/ml of 10mg/ml sucrose solution containing 30 percent, storing for one year, activating enzyme by 5000U/mg, oscillating at room temperature of 150rpm for 4h (at the moment, the adsorption balance is not reached), adding 25ml of fructosyl transferase liquid (purchased from Weifang Kangdien bioscience, Inc., 1mg/ml), continuously oscillating for 10h, removing the liquid by vacuum filtration, and drying the resin solid at room temperature for 24h to obtain the immobilized enzyme P.
COMPARATIVE EXAMPLE (10)
50g of treated NKX-8 (same as example 1) was weighed, 200ml of Palatase 20000L liquid (Novozymes, 20000U/g, 5mg/ml) was added thereto, and after shaking at 150rpm at room temperature for 8 hours, the protein content in the solution was determined to be 0.5mg/ml or less by the Bradford method. Adding 25ml fructosyl transferase solution (purchased from Weifang Kangdian Biotechnology Ltd., 1mg/ml) into the solid-liquid mixture, continuing to oscillate for 8h, vacuum-filtering to remove the liquid, and drying the resin solid at room temperature for 24h to obtain the immobilized enzyme Q.
Table 1 shows the contents of the two enzymes used in the preparation of the immobilized enzymes of the above examples and comparative examples and the water content of the protein of the immobilized enzyme
Determination of ester exchange Activity Using 1 immobilized enzyme
Refined soybean oil (sold in Yuanbao brand soybean oil) and extremely hydrogenated soybean oil (purchased from special fat Co., Ltd.) are used as substrates (w/w 73:27), the mass ratio of enzyme (calculated by the total mass of the carrier and the enzyme) to the substrates is 1:20, the mixture is reacted for 30min at 70 ℃, and after the reaction is finished, the product is taken out to measure the Solid Fat (SFC) content at 40 ℃. Each time the product was removed leaving the enzyme in the reactor, and the substrate was added for reaction for 5 cycles.
The immobilized enzymes prepared in the above examples and comparative examples were subjected to 5 rounds of reactions according to the method, respectively.
The method for detecting the SFC content at 40 ℃ comprises the following steps: the lipid-fixing tube with the sample is respectively put into an oven at 100 ℃ for 15min, a water bath at 60 ℃ for 5min, a water bath at 0 ℃ for 60min and a water bath at 40 ℃ for 30min, and then the solid lipid content (SFC) of the lipid-fixing tube is measured by a nucleic acid resonance instrument (model mq20 of Bruker spectral instruments, Germany). The formula of the ester exchange activity (IUN) is shown as formula (1). Table 2 shows the transesterification activity variation of different immobilized enzymes in 5 cycles of reaction.
Ester interchange activity (IUN) ═ SFC 0 -SFC 1 ) /30X 1260 (formula 1)
In the formula, SFC 0 The SFC value of the substrate oil at 40 ℃ without enzyme reaction is shown as 1 Refers to the SFC value of the enzyme reaction product sample at 40 ℃. SFC (solid fat content) refers to the solid fat content.
TABLE 2 transesterification Activity
Preparation of human milk fat substitute OPO by applying 2 immobilized enzyme
Mixing and dissolving glyceryl tristearate and oleic acid according to the mass ratio of 1:2 to prepare a substrate, reacting the substrate with an immobilized enzyme with the addition of 9% (w/w) at 60 ℃ and at 150rpm for 5h to prepare the breast milk fat substitute 1, 3-dioleate-2-palmitic acid triglyceride (OPO). After the reaction was completed, the fatty acids were removed, and the product was analyzed for CN52 triglyceride content and diglyceride content by GC and HPLC.
The immobilized enzymes prepared in the above examples and comparative examples were subjected to the preparation of OPO according to the methods. Table 3 gives the lifetime of different immobilized enzymes for OPO production. FIGS. 1-3 show the variation of C52 content in the product of different immobilized enzymes in the production of OPO.
And (3) judging the catalytic performance of the immobilized enzyme: (1) the content of CN52 in the OPO product is used as a comparative standard of enzyme activity, and the high content of CN52 indicates that the enzyme activity is high. (2) According to the standard that the content of CN52 triglyceride is more than 40 percent, which is mentioned in the national standard GB30604-2015, of OPO product, the reaction batch up to the standard is calculated and used as the service life data of the immobilized enzyme.
TABLE 3 Life of immobilized enzymes for OPO production
Claims (10)
1. A preparation method of a compound immobilized enzyme is characterized by comprising the following steps:
1) the lipase liquid is contacted with a resin carrier for mixing, and is vibrated and adsorbed to obtain a mixture A; the lipase liquid contains polyhydroxy compounds with the concentration of 5-50%, preferably sucrose, glucose or lactose; the resin carrier is selected from a porous high polymer material with a styrene type or acrylic type skeleton, and the surface of the resin carrier can be distributed with basic groups containing alkyl long chains and terminal quaternary amines, tertiary amines, secondary amines or primary amines; preferably a basic group with a quaternary amine at the end;
2) adding glycosidase into the mixture A in the step 1) to obtain a mixture B; preferably, after the lipase in the mixture A reaches adsorption equilibrium, adding glycosidase;
3) and (3) carrying out solid-liquid separation on the mixture B obtained in the step 2) and drying the solid matter to obtain the compound immobilized enzyme.
2. The method for preparing a composite immobilized enzyme according to claim 1), which further comprises a step of pretreating the resin carrier before step 1), preferably, the pretreatment method comprises: alternately soaking with acid and alkali for more than two times, soaking with alkali solution in the last step, washing with water to neutrality, and removing free water.
3. The method for preparing a compound immobilized enzyme according to claim 1, wherein the mass ratio of the resin carrier to the enzyme protein in the lipase liquid in step 1) is 1: 0.01-0.05.
4. The method for preparing a complex immobilized enzyme according to claim 1, wherein the lipase in step 2) is an enzyme for the decomposition of fats and oils, and is selected from animal, plant or microbial lipases; preferably, the lipase is selected from Thermomyces lanuginosus, Mucor miehei, Rhizopus oryzae (Rhizopus oryzae), Pseudomonas fluorescens (Pseudomonas fluorescens), Rhizopus oryzae (Rhizopus miehei), Aspergillus niger (Aspergillus niger) or genetically modified species of the above microorganisms.
5. The method for preparing a composite immobilized enzyme according to claim 1, wherein the oscillatory adsorption in step 1) is performed at a temperature of 4-30 ℃ and an oscillatory speed of 100-200rpm, preferably 120-150 rpm.
6. The method for preparing a compound immobilized enzyme according to claim 1, wherein the glycosidase in step 2) is fructosyltransferase, sucrase, inulase, a-glucosidase, lactase or galactosidase, and the mass of the enzyme protein in the glycosidase is 1-10%, preferably 2.5-5% of the mass of the enzyme protein in the lipase liquid in step 1).
7. The method for preparing a complex immobilized enzyme according to claim 1, wherein in step 3), the mixture B is stirred at a temperature of 4-30 ℃ for more than 30min before solid-liquid separation.
8. A complex immobilized enzyme or a packaged product thereof, wherein the complex immobilized enzyme is produced by the method according to any one of claims 1 to 7.
9. Use of the complex immobilized enzyme of claim 8 as a catalyst in the grease industry and/or for the preparation of breast milk substitutes, e.g., OPO.
10. A method for producing a human milk fat substitute, such as OPO, comprising adding the complex immobilized enzyme of claim 8 to a mixed substrate of glycerol tristearate and oleic acid, preferably, the method comprising the steps of: mixing and dissolving glycerol tristearate and oleic acid according to the mass ratio of 1:2-1:4 to prepare a substrate, adding the compound immobilized enzyme of claim 8, carrying out a reaction at 60-70 ℃ in a gas bath at 200rpm of 150-; the addition amount of the compound immobilized enzyme is 6-12% of the mass of the substrate.
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| CN117230054B (en) * | 2023-11-16 | 2024-03-19 | 广东惠尔泰生物科技有限公司 | Preparation method of immobilized lipase and method for preparing UPU type glyceride by using immobilized lipase |
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