CN111378641A - Immobilized enzyme carrier and immobilized enzyme - Google Patents
Immobilized enzyme carrier and immobilized enzyme Download PDFInfo
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- CN111378641A CN111378641A CN201811637852.6A CN201811637852A CN111378641A CN 111378641 A CN111378641 A CN 111378641A CN 201811637852 A CN201811637852 A CN 201811637852A CN 111378641 A CN111378641 A CN 111378641A
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- Prior art keywords
- rice bran
- immobilized enzyme
- lipase
- carrier
- immobilized
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- 108010093096 Immobilized Enzymes Proteins 0.000 title claims abstract description 104
- 241000209094 Oryza Species 0.000 claims abstract description 220
- 235000007164 Oryza sativa Nutrition 0.000 claims abstract description 220
- 235000009566 rice Nutrition 0.000 claims abstract description 220
- 108090001060 Lipase Proteins 0.000 claims abstract description 105
- 102000004882 Lipase Human genes 0.000 claims abstract description 105
- 239000004367 Lipase Substances 0.000 claims abstract description 100
- 235000019421 lipase Nutrition 0.000 claims abstract description 100
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 48
- 238000000034 method Methods 0.000 claims abstract description 44
- 230000000694 effects Effects 0.000 claims abstract description 35
- 229920002472 Starch Polymers 0.000 claims abstract description 29
- 239000008107 starch Substances 0.000 claims abstract description 29
- 235000019698 starch Nutrition 0.000 claims abstract description 29
- 230000007062 hydrolysis Effects 0.000 claims abstract description 27
- 238000006460 hydrolysis reaction Methods 0.000 claims abstract description 27
- 238000005886 esterification reaction Methods 0.000 claims abstract description 16
- 230000032050 esterification Effects 0.000 claims abstract description 15
- 239000003225 biodiesel Substances 0.000 claims abstract description 10
- 230000003197 catalytic effect Effects 0.000 claims abstract description 10
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 6
- 238000002360 preparation method Methods 0.000 claims abstract 3
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 39
- 239000007853 buffer solution Substances 0.000 claims description 22
- 238000006243 chemical reaction Methods 0.000 claims description 21
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims description 20
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 claims description 18
- 108010065511 Amylases Proteins 0.000 claims description 16
- 102000013142 Amylases Human genes 0.000 claims description 16
- 235000019418 amylase Nutrition 0.000 claims description 16
- 102000004190 Enzymes Human genes 0.000 claims description 15
- 108090000790 Enzymes Proteins 0.000 claims description 15
- 229940088598 enzyme Drugs 0.000 claims description 15
- 239000004382 Amylase Substances 0.000 claims description 12
- 239000002245 particle Substances 0.000 claims description 12
- 108090000637 alpha-Amylases Proteins 0.000 claims description 11
- 102000004139 alpha-Amylases Human genes 0.000 claims description 11
- 229940024171 alpha-amylase Drugs 0.000 claims description 11
- 239000006229 carbon black Substances 0.000 claims description 11
- 239000011347 resin Substances 0.000 claims description 11
- 229920005989 resin Polymers 0.000 claims description 11
- 159000000007 calcium salts Chemical class 0.000 claims description 10
- HCWCAKKEBCNQJP-UHFFFAOYSA-N magnesium orthosilicate Chemical compound [Mg+2].[Mg+2].[O-][Si]([O-])([O-])[O-] HCWCAKKEBCNQJP-UHFFFAOYSA-N 0.000 claims description 10
- 239000000391 magnesium silicate Substances 0.000 claims description 10
- 229910052919 magnesium silicate Inorganic materials 0.000 claims description 10
- 235000019792 magnesium silicate Nutrition 0.000 claims description 10
- 238000002156 mixing Methods 0.000 claims description 10
- 239000000741 silica gel Substances 0.000 claims description 10
- 229910002027 silica gel Inorganic materials 0.000 claims description 10
- 238000004132 cross linking Methods 0.000 claims description 9
- 238000001035 drying Methods 0.000 claims description 9
- 238000004108 freeze drying Methods 0.000 claims description 9
- 108010048733 Lipozyme Proteins 0.000 claims description 8
- 238000000227 grinding Methods 0.000 claims description 8
- 239000008055 phosphate buffer solution Substances 0.000 claims description 8
- 238000002791 soaking Methods 0.000 claims description 8
- 239000001110 calcium chloride Substances 0.000 claims description 7
- 229910001628 calcium chloride Inorganic materials 0.000 claims description 7
- 239000001913 cellulose Substances 0.000 claims description 6
- 229920002678 cellulose Polymers 0.000 claims description 6
- FCCDDURTIIUXBY-UHFFFAOYSA-N lipoamide Chemical compound NC(=O)CCCCC1CCSS1 FCCDDURTIIUXBY-UHFFFAOYSA-N 0.000 claims description 6
- 239000012266 salt solution Substances 0.000 claims description 6
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 claims description 5
- 241000223258 Thermomyces lanuginosus Species 0.000 claims description 5
- 238000000855 fermentation Methods 0.000 claims description 5
- 230000004151 fermentation Effects 0.000 claims description 5
- 238000001179 sorption measurement Methods 0.000 claims description 5
- 102100021851 Calbindin Human genes 0.000 claims description 4
- 101000898082 Homo sapiens Calbindin Proteins 0.000 claims description 4
- 108091005804 Peptidases Proteins 0.000 claims description 4
- 102000015439 Phospholipases Human genes 0.000 claims description 4
- 108010064785 Phospholipases Proteins 0.000 claims description 4
- 239000004365 Protease Substances 0.000 claims description 4
- 101001021643 Pseudozyma antarctica Lipase B Proteins 0.000 claims description 4
- 241000223257 Thermomyces Species 0.000 claims description 4
- 229940025131 amylases Drugs 0.000 claims description 4
- ZCCIPPOKBCJFDN-UHFFFAOYSA-N calcium nitrate Chemical compound [Ca+2].[O-][N+]([O-])=O.[O-][N+]([O-])=O ZCCIPPOKBCJFDN-UHFFFAOYSA-N 0.000 claims description 4
- 239000000969 carrier Substances 0.000 claims description 4
- 238000010353 genetic engineering Methods 0.000 claims description 4
- 238000004519 manufacturing process Methods 0.000 claims description 4
- 102000004169 proteins and genes Human genes 0.000 claims description 4
- 108090000623 proteins and genes Proteins 0.000 claims description 4
- 238000005809 transesterification reaction Methods 0.000 claims description 4
- 238000001291 vacuum drying Methods 0.000 claims description 4
- 102000035195 Peptidases Human genes 0.000 claims description 3
- 238000006136 alcoholysis reaction Methods 0.000 claims description 3
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 claims description 2
- 108010073178 Glucan 1,4-alpha-Glucosidase Proteins 0.000 claims description 2
- 102100022624 Glucoamylase Human genes 0.000 claims description 2
- 108010028688 Isoamylase Proteins 0.000 claims description 2
- 238000005903 acid hydrolysis reaction Methods 0.000 claims description 2
- 108010019077 beta-Amylase Proteins 0.000 claims description 2
- NKWPZUCBCARRDP-UHFFFAOYSA-L calcium bicarbonate Chemical compound [Ca+2].OC([O-])=O.OC([O-])=O NKWPZUCBCARRDP-UHFFFAOYSA-L 0.000 claims description 2
- 229910000020 calcium bicarbonate Inorganic materials 0.000 claims description 2
- JXRVKYBCWUJJBP-UHFFFAOYSA-L calcium;hydrogen sulfate Chemical compound [Ca+2].OS([O-])(=O)=O.OS([O-])(=O)=O JXRVKYBCWUJJBP-UHFFFAOYSA-L 0.000 claims description 2
- LJSOLTRJEQZSHV-UHFFFAOYSA-L potassium;sodium;hydron;hydroxide;phosphate Chemical compound [OH-].[Na+].[K+].OP(O)([O-])=O LJSOLTRJEQZSHV-UHFFFAOYSA-L 0.000 claims description 2
- 238000007598 dipping method Methods 0.000 claims 2
- ZIIUUSVHCHPIQD-UHFFFAOYSA-N 2,4,6-trimethyl-N-[3-(trifluoromethyl)phenyl]benzenesulfonamide Chemical compound CC1=CC(C)=CC(C)=C1S(=O)(=O)NC1=CC=CC(C(F)(F)F)=C1 ZIIUUSVHCHPIQD-UHFFFAOYSA-N 0.000 claims 1
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 claims 1
- 239000000872 buffer Substances 0.000 claims 1
- 238000009837 dry grinding Methods 0.000 claims 1
- 238000001238 wet grinding Methods 0.000 claims 1
- 239000003921 oil Substances 0.000 description 56
- 235000019198 oils Nutrition 0.000 description 54
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 48
- 238000001914 filtration Methods 0.000 description 18
- 238000003756 stirring Methods 0.000 description 18
- 239000000243 solution Substances 0.000 description 15
- WRIDQFICGBMAFQ-UHFFFAOYSA-N (E)-8-Octadecenoic acid Natural products CCCCCCCCCC=CCCCCCCC(O)=O WRIDQFICGBMAFQ-UHFFFAOYSA-N 0.000 description 14
- LQJBNNIYVWPHFW-UHFFFAOYSA-N 20:1omega9c fatty acid Natural products CCCCCCCCCCC=CCCCCCCCC(O)=O LQJBNNIYVWPHFW-UHFFFAOYSA-N 0.000 description 14
- QSBYPNXLFMSGKH-UHFFFAOYSA-N 9-Heptadecensaeure Natural products CCCCCCCC=CCCCCCCCC(O)=O QSBYPNXLFMSGKH-UHFFFAOYSA-N 0.000 description 14
- 239000005642 Oleic acid Substances 0.000 description 14
- ZQPPMHVWECSIRJ-UHFFFAOYSA-N Oleic acid Natural products CCCCCCCCC=CCCCCCCCC(O)=O ZQPPMHVWECSIRJ-UHFFFAOYSA-N 0.000 description 14
- QXJSBBXBKPUZAA-UHFFFAOYSA-N isooleic acid Natural products CCCCCCCC=CCCCCCCCCC(O)=O QXJSBBXBKPUZAA-UHFFFAOYSA-N 0.000 description 14
- ZQPPMHVWECSIRJ-KTKRTIGZSA-N oleic acid Chemical compound CCCCCCCC\C=C/CCCCCCCC(O)=O ZQPPMHVWECSIRJ-KTKRTIGZSA-N 0.000 description 14
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 12
- 238000009835 boiling Methods 0.000 description 12
- 239000002253 acid Substances 0.000 description 10
- 239000007788 liquid Substances 0.000 description 10
- 239000012071 phase Substances 0.000 description 9
- 230000000052 comparative effect Effects 0.000 description 7
- 238000005303 weighing Methods 0.000 description 7
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 description 6
- 238000007710 freezing Methods 0.000 description 6
- 230000008014 freezing Effects 0.000 description 6
- 239000000203 mixture Substances 0.000 description 6
- 229910052757 nitrogen Inorganic materials 0.000 description 6
- XPFJYKARVSSRHE-UHFFFAOYSA-K trisodium;2-hydroxypropane-1,2,3-tricarboxylate;2-hydroxypropane-1,2,3-tricarboxylic acid Chemical compound [Na+].[Na+].[Na+].OC(=O)CC(O)(C(O)=O)CC(O)=O.[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O XPFJYKARVSSRHE-UHFFFAOYSA-K 0.000 description 6
- LEAHFJQFYSDGGP-UHFFFAOYSA-K trisodium;dihydrogen phosphate;hydrogen phosphate Chemical compound [Na+].[Na+].[Na+].OP(O)([O-])=O.OP([O-])([O-])=O LEAHFJQFYSDGGP-UHFFFAOYSA-K 0.000 description 6
- 239000003814 drug Substances 0.000 description 4
- 239000000047 product Substances 0.000 description 4
- 238000010298 pulverizing process Methods 0.000 description 4
- 239000002994 raw material Substances 0.000 description 4
- 239000000377 silicon dioxide Substances 0.000 description 4
- 101710084376 Lipase 3 Proteins 0.000 description 3
- 150000001298 alcohols Chemical class 0.000 description 3
- 230000002779 inactivation Effects 0.000 description 3
- 235000012424 soybean oil Nutrition 0.000 description 3
- 239000003549 soybean oil Substances 0.000 description 3
- 239000000758 substrate Substances 0.000 description 3
- 239000005909 Kieselgur Substances 0.000 description 2
- 239000003957 anion exchange resin Substances 0.000 description 2
- 238000001816 cooling Methods 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 239000010432 diamond Substances 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 235000019197 fats Nutrition 0.000 description 2
- 239000004519 grease Substances 0.000 description 2
- 230000003100 immobilizing effect Effects 0.000 description 2
- 238000005470 impregnation Methods 0.000 description 2
- 238000011068 loading method Methods 0.000 description 2
- 239000012047 saturated solution Substances 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- NWUYHJFMYQTDRP-UHFFFAOYSA-N 1,2-bis(ethenyl)benzene;1-ethenyl-2-ethylbenzene;styrene Chemical compound C=CC1=CC=CC=C1.CCC1=CC=CC=C1C=C.C=CC1=CC=CC=C1C=C NWUYHJFMYQTDRP-UHFFFAOYSA-N 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- 101710084373 Lipase 1 Proteins 0.000 description 1
- 101710084378 Lipase 2 Proteins 0.000 description 1
- 101710084369 Lipase 4 Proteins 0.000 description 1
- 101710084366 Lipase 5 Proteins 0.000 description 1
- 101710084370 Lipase 6 Proteins 0.000 description 1
- 239000004793 Polystyrene Substances 0.000 description 1
- 101000984201 Thermomyces lanuginosus Lipase Proteins 0.000 description 1
- 229910021536 Zeolite Inorganic materials 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 239000003463 adsorbent Substances 0.000 description 1
- 108010050181 aleurone Proteins 0.000 description 1
- 239000008346 aqueous phase Substances 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 239000012752 auxiliary agent Substances 0.000 description 1
- 239000000440 bentonite Substances 0.000 description 1
- 229910000278 bentonite Inorganic materials 0.000 description 1
- SVPXDRXYRYOSEX-UHFFFAOYSA-N bentoquatam Chemical compound O.O=[Si]=O.O=[Al]O[Al]=O SVPXDRXYRYOSEX-UHFFFAOYSA-N 0.000 description 1
- 239000011942 biocatalyst Substances 0.000 description 1
- 235000021329 brown rice Nutrition 0.000 description 1
- 239000006227 byproduct Substances 0.000 description 1
- 239000007979 citrate buffer Substances 0.000 description 1
- 208000012839 conversion disease Diseases 0.000 description 1
- 239000003431 cross linking reagent Substances 0.000 description 1
- 235000019784 crude fat Nutrition 0.000 description 1
- 230000008021 deposition Effects 0.000 description 1
- HNPSIPDUKPIQMN-UHFFFAOYSA-N dioxosilane;oxo(oxoalumanyloxy)alumane Chemical compound O=[Si]=O.O=[Al]O[Al]=O HNPSIPDUKPIQMN-UHFFFAOYSA-N 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- 125000004185 ester group Chemical group 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000004744 fabric Substances 0.000 description 1
- 230000002349 favourable effect Effects 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 125000000524 functional group Chemical group 0.000 description 1
- 239000010903 husk Substances 0.000 description 1
- 239000003456 ion exchange resin Substances 0.000 description 1
- 229920003303 ion-exchange polymer Polymers 0.000 description 1
- 239000010985 leather Substances 0.000 description 1
- 235000019626 lipase activity Nutrition 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 229920000620 organic polymer Polymers 0.000 description 1
- 239000003960 organic solvent Substances 0.000 description 1
- 229920002223 polystyrene Polymers 0.000 description 1
- 239000011148 porous material Substances 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 108010072641 thermostable lipase Proteins 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- 230000002087 whitening effect Effects 0.000 description 1
- 239000010457 zeolite Substances 0.000 description 1
Images
Classifications
-
- 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
-
- 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
- C12P7/649—Biodiesel, i.e. fatty acid alkyl esters
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
- Y02E50/00—Technologies for the production of fuel of non-fossil origin
- Y02E50/10—Biofuels, e.g. bio-diesel
Abstract
The invention provides a rice bran immobilized carrier, which has at least one of the following characteristics that a) the hydrolysis activity of natural lipase is 50-600U/Kg; b) the oil content is 5-15 wt%; c) the starch content is 8-25%; d) the water holding capacity is 220-320ml/100 g; e) the oil holding capacity is 90-150ml/100 g; meanwhile, the invention also provides a preparation method of the rice bran immobilized carrier and an immobilized enzyme prepared by the rice bran immobilized carrier; in addition, the invention also provides a method for preparing biodiesel by catalyzing the immobilized enzyme prepared by the rice bran immobilized carrier; also provided is a method for improving lipase catalytic esterification activity and/or short-chain alcohol tolerance.
Description
Technical Field
The invention relates to the field of immobilized enzymes, and particularly relates to an immobilized carrier and application thereof.
Background
The lipase as a biocatalyst can catalyze the hydrolysis of grease, and can catalyze the reactions such as esterification, ester exchange, acidolysis and alcoholysis under the condition of a non-aqueous phase. The lipase has the advantages of mild catalytic conditions, strong catalytic diversity, strong substrate specificity and the like, and is widely applied to the industries of food, oiling, papermaking, leather, washing, pharmacy and the like.
Thermomyces lanuginosus is a fungus with wide distribution and high growth upper limit temperature, and can generate thermostable lipase with important industrial value, namely Thermomyces lanuginosus lipase (TL lipase for short). The TL lipase has a molecular weight of about 31.7kDa and a molecular size of
The isoelectric point is 4.4. Compared with other lipase types, the TL lipase has mature fermentation technology, commercial production, low price and good immobilization stability, and has good application prospect in the process of preparing biodiesel by an enzyme method. In patent CN107012136A, a crosslinking agent and macroporous adsorption resin are used for oscillating and crosslinking immobilized Lipozyme TL 100L enzyme liquid, the obtained immobilized TL lipase catalyzes methyl esterification of soybean oil deodorized distillate, the soybean oil deodorized distillate is reused for 20 batches, and the reaction conversion rate is over 90 percent. In patent CN100552003C, the commercial enzyme Lipozyme TL IM is used for catalyzing acid-containing oil and fat in a solvent-free or organic solvent to produce biodiesel, and the esterification rate is about 70 percent. Patent CN102839166B provides a method for preparing immobilized TL lipase, which uses non-polar macroporous adsorbent resin of polystyrene as carrier to immobilize TI lipase, and the obtained immobilized TL lipase has high activity and stability in catalytic transesterification.
Generally speaking, TL lipase exhibits more catalytic transesterification capability after immobilization operation, and the catalytic esterification capability is weaker, especially immobilized on some cheap carriers, such as silica, white carbon black, zeolite, cellulose, bentonite and the like; when some organic polymers such as macroporous resin, ion exchange resin and the like are used as carriers, the immobilized TL lipase can obtain better esterification activity by modulating carrier functional groups, an immobilization method and the like, but the cost price of the resin carrier is expensive, and the immobilization cost is increased. Obviously, the technical methods are not suitable for applying TL lipase to the reaction of catalyzing high-acid-value grease raw materials to prepare biodiesel. The prior art method still lacks an immobilized TL lipase product which has low cost and higher catalytic esterification activity.
The rice bran refers to a by-product generated in the brown rice whitening step in the rice processing process, and the main components of the rice bran are pericarp, rice husk, germ, aleurone layer and the like. Patent CN104651341B provides a method for immobilizing bio-enzyme by rice bran as carrier, but only as an auxiliary agent used in water treatment process, it does not relate to improvement of short-chain alcohol tolerance of enzyme in the process of immobilizing lipase.
Disclosure of Invention
The first purpose of the invention is to provide an immobilized enzyme carrier, which contains or consists of rice bran.
In a specific embodiment the immobilized enzyme support comprises rice bran.
In a particular embodiment the immobilized enzyme support consists of rice bran.
In a specific embodiment, the natural lipase hydrolysis activity of the rice bran is 50-600U/Kg.
In a specific embodiment, the hydrolysis activity of the natural lipase in the rice bran is 55-580U/Kg.
In a specific embodiment, the natural lipase hydrolysis activity of the rice bran is 55-420U/Kg.
In a specific embodiment, the natural lipase hydrolysis activity of the rice bran is 130-280U/Kg.
In a specific embodiment, the oil content of the rice bran is 5-15 wt% based on the mass of the rice bran.
In a specific embodiment, the oil content in the rice bran is 6-15 wt% based on the mass of the rice bran.
In a specific embodiment, the oil content in the rice bran is 6-14 wt% based on the mass of the rice bran.
In a specific embodiment, the oil content in the rice bran is 9-11 wt% based on the mass of the rice bran.
In a specific embodiment, the rice bran has a starch content of 8-25 wt% based on the mass of the rice bran.
In a specific embodiment, the rice bran has a starch content of 8-24 wt% based on the mass of the rice bran.
In a specific embodiment, the rice bran has a starch content of 8-17 wt% based on the mass of the rice bran.
In a specific embodiment, the content of starch in the rice bran is 10-13 wt% based on the mass of the rice bran.
In one embodiment, the rice bran has a water holding capacity of 220-320ml/100 g.
In a specific embodiment, the rice bran has a water holding capacity of 240-317 ml/100 g.
In a specific embodiment, the rice bran has a water holding capacity of 260 to 290ml/100 g.
In one embodiment the rice bran has an oil holding capacity of 90 to 150ml/100 g.
In one embodiment the rice bran has an oil holding capacity of 91 to 150ml/100 g.
In one embodiment the rice bran has an oil holding capacity of 91 to 130ml/100 g.
In one embodiment the rice bran has an oil holding capacity of 100-.
In a specific embodiment, the hydrolysis activity of the natural lipase in the immobilized enzyme carrier is 50-600U/Kg.
In a specific embodiment, the hydrolysis activity of the natural lipase in the immobilized enzyme carrier is 55-580U/Kg.
In a specific embodiment, the hydrolysis activity of the natural lipase in the immobilized enzyme carrier is 55-420U/Kg.
In a specific embodiment, the hydrolysis activity of the natural lipase in the immobilized enzyme carrier is 130-280U/Kg.
In a specific embodiment, the oil content in the immobilized enzyme carrier is 5-15 wt% based on the mass of the immobilized enzyme carrier.
In a specific embodiment, the oil content in the immobilized enzyme carrier is 6-15 wt% based on the mass of the immobilized enzyme carrier.
In a specific embodiment, the oil content in the immobilized enzyme carrier is 6-14 wt% based on the mass of the immobilized enzyme carrier.
In a specific embodiment, the oil content in the immobilized enzyme carrier is 9-11 wt% based on the mass of the immobilized enzyme carrier.
In a specific embodiment, the starch content of the immobilized enzyme carrier is 8-25 wt% based on the mass of the immobilized enzyme carrier.
In a specific embodiment, the starch content of the immobilized enzyme carrier is 8-24 wt% based on the mass of the immobilized enzyme carrier.
In a specific embodiment, the starch content of the immobilized enzyme carrier is 8-17 wt% based on the mass of the immobilized enzyme carrier.
In a specific embodiment, the starch content of the immobilized enzyme carrier is 10-13 wt% based on the mass of the immobilized enzyme carrier.
In a specific embodiment, the water holding capacity of the immobilized enzyme carrier is 220-320ml/100 g.
In a specific embodiment, the water holding capacity of the immobilized enzyme carrier is 240-317 ml/100 g.
In a specific embodiment, the water holding capacity of the immobilized enzyme carrier is 260-290 ml/100 g.
In a specific embodiment, the oil holding capacity of the immobilized enzyme carrier is 90-150ml/100 g.
In a specific embodiment, the oil holding capacity of the immobilized enzyme carrier is 91-150ml/100 g.
In a specific embodiment, the oil holding capacity of the immobilized enzyme carrier is 91-130ml/100 g.
In a specific embodiment, the oil holding capacity of the immobilized enzyme carrier is 100-115ml/100 g.
In a specific embodiment, the immobilized enzyme carrier further comprises at least one of white carbon black, silica gel, activated carbon, diatomite, magnesium silicate, cellulose and resin.
In a specific embodiment, the immobilized enzyme carrier further comprises at least one of white carbon black, silica gel, activated carbon, diatomite and magnesium silicate.
In a specific embodiment, the carrier other than rice bran in the immobilized enzyme carrier accounts for 10% by mass or less, preferably 6% by mass or less of the total mass of the immobilized carrier.
In a particular embodiment the immobilized enzyme support consists of rice bran.
A second aspect of the present invention is directed to an immobilized enzyme comprising rice bran.
In a specific embodiment, the oil content in the immobilized enzyme is 5-15 wt% based on the mass of the immobilized enzyme.
In a specific embodiment, the oil content in the immobilized enzyme is 6-15 wt% based on the mass of the immobilized enzyme.
In a specific embodiment, the oil content in the immobilized enzyme is 6-14 wt% based on the mass of the immobilized enzyme.
In a specific embodiment, the oil content in the immobilized enzyme is 9-11 wt% based on the mass of the immobilized enzyme.
In a specific embodiment, the starch content of the immobilized enzyme is 8-25 wt% based on the mass of the immobilized enzyme.
In a specific embodiment, the starch content of the immobilized enzyme is 8-24 wt% based on the mass of the immobilized enzyme.
In a specific embodiment, the starch content of the immobilized enzyme is 8-17 wt% based on the mass of the immobilized enzyme.
In a specific embodiment, the starch content of the immobilized enzyme is 10-13 wt% based on the mass of the immobilized enzyme.
In a specific embodiment, the water holding capacity of the immobilized enzyme is 220-320ml/100 g.
In a specific embodiment, the water holding capacity of the immobilized enzyme is 240-317 ml/100 g.
In a specific embodiment, the water holding capacity of the immobilized enzyme is 260-290 ml/100 g.
In a specific embodiment, the immobilized enzyme has an oil-holding capacity of 90-150ml/100 g.
In a specific embodiment, the immobilized enzyme has an oil-holding capacity of 91-150ml/100 g.
In a specific embodiment, the immobilized enzyme has an oil-holding capacity of 91-130ml/100 g.
In a specific embodiment, the immobilized enzyme has an oil holding capacity of 100-115ml/100 g.
In a specific embodiment, the natural lipase hydrolysis activity of the rice bran is 50-600U/Kg.
In a specific embodiment, the hydrolysis activity of the natural lipase in the rice bran is 55-580U/Kg.
In a specific embodiment, the natural lipase hydrolysis activity of the rice bran is 55-420U/Kg.
In a specific embodiment, the natural lipase hydrolysis activity of the rice bran is 130-280U/Kg.
In a specific embodiment, the oil content of the rice bran is 5-15 wt% based on the mass of the rice bran.
In a specific embodiment, the oil content in the rice bran is 6-15 wt% based on the mass of the rice bran.
In a specific embodiment, the oil content in the rice bran is 6-14 wt% based on the mass of the rice bran.
In a specific embodiment, the oil content in the rice bran is 9-11 wt% based on the mass of the rice bran.
In a specific embodiment, the rice bran has a starch content of 8-25 wt% based on the mass of the rice bran.
In a specific embodiment, the rice bran has a starch content of 8-24 wt% based on the mass of the rice bran.
In a specific embodiment, the rice bran has a starch content of 8-17 wt% based on the mass of the rice bran.
In a specific embodiment, the content of starch in the rice bran is 10-13 wt% based on the mass of the rice bran.
In one embodiment, the rice bran has a water holding capacity of 220-320ml/100 g.
In a specific embodiment, the rice bran has a water holding capacity of 240-317 ml/100 g.
In a specific embodiment, the rice bran has a water holding capacity of 260 to 290ml/100 g.
In one embodiment the rice bran has an oil holding capacity of 90 to 150ml/100 g.
In one embodiment the rice bran has an oil holding capacity of 91 to 150ml/100 g.
In one embodiment the rice bran has an oil holding capacity of 91 to 130ml/100 g.
In one embodiment the rice bran has an oil holding capacity of 100-.
In a specific embodiment, the immobilized enzyme carrier further comprises at least one of white carbon black, silica gel, activated carbon, diatomite, magnesium silicate, cellulose and resin.
In a specific embodiment, the immobilized enzyme carrier further comprises at least one of white carbon black, silica gel, activated carbon, diatomite and magnesium silicate.
In a specific embodiment, the carrier other than rice bran in the immobilized enzyme accounts for 10% by mass or less, preferably 6% by mass or less of the total mass of the immobilized carrier.
In a particular embodiment the lipase-immobilized carrier is selected from the carriers described above for the first aspect.
In a particular embodiment the enzyme is selected from lipases, phospholipases, proteases and/or amylases.
In a particular embodiment the enzyme is selected from lipases.
In a particular embodiment the lipase is selected from at least one of TL lipase, CALB lipase, RM lipase or ROL lipase.
In a specific embodiment the TL lipase is selected from: fermentation products of thermomyces (thermomyces lanuginosus) and/or TL lipase obtained by genetic engineering or protein engineering and/or commercial TL lipase.
In a specific embodiment the commercially available TL lipase is selected from lipozyme TL 100L.
It is an object of a third aspect of the present invention to provide rice bran or a process for the production of rice bran according to the first or second aspect of the present invention:
a) treating the defatted rice bran A with amylase to obtain defatted rice bran B;
b) mixing the defatted rice bran B with fresh whole-fat rice bran to obtain mixed rice bran C; and
c) contacting the mixed rice bran C with a soluble calcium salt solution, and separating to obtain the rice bran;
in a particular embodiment the amylase in step a) is selected from the group consisting of α -amylase, β -amylase, glucoamylase and isoamylase.
In a particular embodiment the amylase is selected from the group consisting of α -amylase.
In a specific embodiment the α -amylase is selected from high temperature amylases.
In a particular embodiment the contacting temperature of the amylase with the defatted rice bran A in step a) is from 80 to 90 ℃.
In one embodiment the contact time is 30 min.
In one embodiment the contact pH is from 5.0 to 6.6.
In a specific embodiment, the particle size of the obtained mixed rice bran C in the step b) is adjusted to 140-700 mesh.
In a particular embodiment the particle size of the defatted rice bran B and/or fresh whole rice bran is adjusted to 140-700 mesh prior to the mixing step of step B).
In a specific embodiment, the particle size adjusting method employs at least one of dry pulverization, wet pulverization, ultra-fine pulverization, and low-temperature pulverization.
In a specific embodiment, the mass ratio of the defatted rice bran B to the fresh whole rice bran is 1: 4-4: 1
In a particular embodiment said soluble calcium salt in step c) is selected from at least one of calcium chloride, calcium nitrate, calcium hydrogen sulphate, calcium bicarbonate.
In a particular embodiment the soluble calcium salt is selected from calcium chloride.
In a particular embodiment the soluble calcium salt solution has a concentration of 10% to 80% (w/v).
In a specific embodiment, the volume of the soluble calcium salt solution is at least 1mL/g based on the mass of the mixed rice bran C.
In one embodiment, the soaking time is 1-2 hr.
In one embodiment the impregnation temperature is from 25 to 35 ℃.
In a particular embodiment the impregnation is followed by drying to a moisture content of 5-10%.
In a specific embodiment, the drying method adopts at least one of freeze drying, low-temperature vacuum drying, natural airing and low-temperature fluidized drying.
In a specific embodiment, in step a), the method further comprises: a1) a step of contacting defatted rice bran A with water at a pH of 5.0 to 6.6.
In a particular embodiment the defatted rice bran a is contacted with water prior to treatment with amylase.
In a specific embodiment the defatted rice bran A is contacted with the buffer solution at a temperature of from 80 to 120 ℃.
In a specific embodiment, the buffer solution is selected from at least one of a citric acid buffer solution, a phosphate buffer solution, a potassium dihydrogen phosphate-sodium hydroxide buffer solution, a tris-hydrochloric acid buffer solution, and a PBS buffer solution.
In a particular embodiment the buffer solution is selected from a citrate buffer.
In one embodiment the contact time is 20-40 min.
In a specific embodiment, the concentration of the citric acid buffer solution is 0.01-0.1 mol/L.
In a fourth aspect, the present invention provides rice bran produced by the process according to the third aspect, the rice bran having at least one of the following characteristics;
1) the hydrolysis activity of natural lipase in the rice bran is 50-600U/Kg;
2) the oil content of the rice bran is 5-15 wt%;
3) the starch content in the rice bran is 8-25%;
4) the water holding capacity of the rice bran is 220-320ml/100 g;
5) the oil holding capacity of the rice bran is 90-150ml/100 g.
In a specific embodiment, the natural lipase hydrolysis activity of the rice bran is 50-600U/Kg.
In a specific embodiment, the hydrolysis activity of the natural lipase in the rice bran is 55-580U/Kg.
In a specific embodiment, the natural lipase hydrolysis activity of the rice bran is 55-420U/Kg.
In a specific embodiment, the natural lipase hydrolysis activity of the rice bran is 130-280U/Kg.
In a specific embodiment, the rice bran contains 5-15 wt% of oil.
In a specific embodiment, the rice bran has an oil content of 6 to 15 wt%.
In a specific embodiment, the rice bran has an oil content of 6 to 14 wt%.
In a specific embodiment, the rice bran has an oil content of 9 to 11 wt%.
In a specific embodiment, the rice bran has a starch content of 8 to 25 wt%.
In a specific embodiment, the rice bran has a starch content of 8-24 wt%.
In a specific embodiment, the rice bran has a starch content of 8-17 wt%.
In a specific embodiment, the rice bran contains 10-13 wt% of starch.
In one embodiment, the rice bran has a water holding capacity of 220-320ml/100 g.
In a specific embodiment, the rice bran has a water holding capacity of 240-317 ml/100 g.
In a specific embodiment, the rice bran has a water holding capacity of 260 to 290ml/100 g.
In one embodiment the rice bran has an oil holding capacity of 90 to 150ml/100 g.
In one embodiment the rice bran has an oil holding capacity of 91 to 150ml/100 g.
In one embodiment the rice bran has an oil holding capacity of 91 to 130ml/100 g.
In one embodiment the rice bran has an oil holding capacity of 100-.
An object of the fifth aspect of the present invention is to provide an immobilized enzyme comprising the immobilized enzyme carrier object of the first aspect of the present invention,
in a particular embodiment the enzyme is selected from lipases, phospholipases, proteases and/or amylases.
In a particular embodiment the enzyme is selected from lipases.
In a particular embodiment the lipase is selected from at least one of TL lipase, CALB lipase, RM lipase or ROL lipase.
In a specific embodiment the TL lipase is selected from: fermentation products of thermomyces (thermomyces lanuginosus) and/or TL lipase obtained by genetic engineering or protein engineering and/or commercial TL lipase.
In a specific embodiment the commercially available TL lipase is selected from lipozyme TL 100L.
In a specific embodiment, the immobilized enzyme further comprises at least one carrier selected from the group consisting of silica, silica gel, activated carbon, diatomaceous earth, magnesium silicate, cellulose and resin.
In a specific embodiment, the immobilized enzyme further comprises at least one carrier selected from the group consisting of silica, silica gel, activated carbon, diatomaceous earth and magnesium silicate.
In a specific embodiment, the carrier other than rice bran in the immobilized enzyme accounts for 10% by mass or less, preferably 6% by mass or less of the total mass of the immobilized carrier.
An object of a sixth aspect of the present invention is to provide a process for preparing an immobilized enzyme according to the object of the fifth aspect of the present invention, characterized by contacting an immobilized enzyme carrier according to the object of the first aspect of the present invention with an enzyme.
In a specific embodiment, the immobilized enzyme is prepared by at least one of an adsorption method, a covalent bonding method, an embedding method and a crosslinking method.
An object of the seventh aspect of the present invention is to provide a use of the immobilized enzyme according to the object of the second aspect of the present invention or the object of the fifth aspect of the present invention in at least one reaction selected from catalytic esterification, transesterification, alcoholysis, acid hydrolysis and/or hydrolysis.
The object of the eighth aspect of the invention is to provide a use of the immobilized enzyme according to the object of the second aspect of the invention or the object of the fifth aspect of the invention for preparing biodiesel.
The ninth aspect of the present invention is to provide a method for improving lipase catalytic esterification activity and/or short-chain alcohol tolerance, which is characterized by contacting the immobilized carrier of the first aspect of the present invention with enzyme.
Drawings
FIG. 1 is a test result of the tolerance of immobilized TL lipase to short-chain alcohols such as methanol in the process of preparing biodiesel by catalyzing oleic acid and methanol esterification with immobilized TL lipase, wherein a curve marked by diamonds is immobilized TL lipase 3 prepared by the method of the present invention; the curve of the square mark is the immobilized TL lipase of the weak-base anion exchange resin prepared by other methods; the curve obtained by operating 9 batches was repeated with a molar ratio of oleic acid to methanol of 1: 2.
FIG. 2 is a test result of the tolerance of immobilized TL lipase to short-chain alcohols such as methanol in the process of preparing biodiesel by catalyzing oleic acid and methanol esterification with immobilized TL lipase, wherein the curve marked by diamonds is immobilized TL lipase 3 prepared by the method of the present invention; the curve of the square mark is the immobilized TL lipase of the weak-base anion exchange resin prepared by other methods; the curve obtained for 9 batches was repeated with an oleic acid to methanol molar ratio of 1: 4.
Detailed Description
The embodiments and technical effects of the present invention will be specifically described below with reference to examples, but the present invention is not limited to the following examples.
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Raw materials:
sources of defatted rice bran: yihaijiali group Jiamusi factory
Sources of whole rice bran: yihaijiali group Jiamusi factory
High temperature amylase sources: from HebeiPengyu Biotech Co., Ltd
The lipase source is as follows: lipozyme TL 1OOL available from Novoxin
Calcium chloride: purchased from national medicine group
The citric acid source is as follows: purchased from national medicine group
Crushing equipment: tester high speed universal pulverizer FW100
A freeze dryer: shanghai Guansen Biotech Co Ltd
Oil sources: fengyi oil & fat science & technology (Shanghai) Co., Ltd
Sources of methanol: purchased from national medicine group
Ethanol source: purchased from national medicine group
The biodiesel detection method comprises the following steps:
the oleic acid conversion rate is (oil phase initial acid value-oil phase acid value after reaction)/oil phase initial acid value × 100%, wherein the oil phase acid value determination method refers to the determination of the acid value and acidity of GB/T5530- "
The rice bran detection method comprises the following steps:
reference is made to the literature "Antita V.Prabhu, ShreehasP.Tambe, et al.Rice bran lipase: extract, activity and activity on biotechnol.Prog.1999,15, 1083-1089".
The method for measuring the oil content in the rice bran refers to the measurement of crude fat in the feed of GB/T6433-.
The method for measuring the starch content in the rice bran refers to an optical rotation method for measuring the starch content in animal feed GB/T20194 and 2018.
The method for measuring the water holding capacity and the oil holding capacity of the rice bran comprises the following steps: weighing 100g of rice bran sample in a 1L beaker, pouring 500mL of distilled water (or soybean oil), placing the beaker in a water bath kettle at 20 ℃ to absorb water (or oil) for 2h at a constant temperature, then placing the rice bran absorbing water (or oil) on a filter cloth upside down until no continuous liquid drops drip, measuring the volume V of flowing liquid, and taking the water holding capacity (or oil holding capacity) as 500-V.
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Example 1
Weighing 50g of defatted rice bran in a 1L beaker, adding 250mL of 0.01moL/L citric acid-sodium citrate buffer solution with the pH value of 5.0, boiling in boiling water for 30min, reducing the temperature to 80 ℃, adding 0.2 wt% (based on the weight of the defatted rice bran) of high-temperature α -amylase, stirring for 30min, filtering, uniformly mixing with 200g of fresh rice bran according to the ratio of 1:4 (based on the weight of the defatted rice bran), grinding to the particle size of 140 meshes and 700 meshes, adding 500mL of CaCl2Stirring and soaking the saturated solution at 25 deg.C for 2hr, filtering, pre-freezing with liquid nitrogen, and freeze-drying to water content of 5%; adding 125mL of TL lipase enzyme solution into the pretreated rice bran carrier, adding 250mL of 0.01mol/L disodium hydrogen phosphate-sodium dihydrogen phosphate buffer solution with the pH value of 7.0, stirring for 2h, and then adding 10mL of 0.5% glutaraldehyde solution for crosslinking reaction for 1 h; and filtering and separating rice bran, and naturally airing at room temperature until the moisture content is 10% to obtain the immobilized TL lipase 1.
Example 2
Weighing 50g defatted rice bran in a 1L beaker, adding 250mL 0.03moL/L citric acid-sodium citrate buffer solution with pH of 5.4, boiling in boiling water for 30min, cooling to 90 deg.C, andadding 0.3 wt% (based on the weight of the defatted rice bran) of high-temperature α -amylase, stirring for 30min, filtering, mixing with 100g of fresh rice bran at a ratio of 1:2, grinding to particle size of 140 meshes and 700 meshes, adding 300mL of 10% CaCl by mass2Stirring and soaking the solution at 30 ℃ for 1h, filtering, pre-freezing with liquid nitrogen, and freeze-drying to water content of 7%; adding 150mL of TL lipase enzyme solution into the pretreated rice bran carrier, adding 150mL of 0.01mol/L disodium hydrogen phosphate-sodium dihydrogen phosphate buffer solution with the pH value of 7.0, stirring for 3h, and then adding 8mL of 0.5% glutaraldehyde solution for crosslinking reaction for 0.5 h; filtering to separate rice bran, and vacuum drying at 40 deg.C until water content is 8% to obtain immobilized TL lipase 2.
Example 3
Weighing 50g of defatted rice bran into a 1L beaker, adding 250mL of 0.06moL/L citric acid-sodium citrate buffer solution with the pH value of 5.8, boiling the mixture in boiling water for 30min, reducing the temperature to 85 ℃, adding 0.4 wt% (based on the weight of the defatted rice bran) of high-temperature α -amylase, stirring the mixture for 30min, filtering the mixture, uniformly mixing the mixture with 50g of fresh rice bran according to the ratio of 1:1 (based on the weight of the defatted rice bran), grinding the mixture to the particle size of 140 meshes and 700 meshes, adding 100mL of CaCl with the mass concentration of 40 percent2Stirring and soaking the solution at 35 deg.C for 1.5h, filtering, pre-freezing with liquid nitrogen, and freeze-drying to water content of 10%; adding 200mL of TL lipase enzyme solution into the pretreated rice bran carrier, adding 100mL of 0.01mol/L disodium hydrogen phosphate-sodium dihydrogen phosphate buffer solution with the pH value of 7.0, stirring for 4 hours, and then adding 6mL of 0.5% glutaraldehyde solution for crosslinking reaction for 0.5 hour; filtering to separate rice bran, and freeze drying to water content of 5% to obtain immobilized TL lipase 3.
Example 4
Weighing 50g defatted rice bran in a 1L beaker, adding 250mL of 0.1moL/L citric acid-sodium citrate buffer solution with pH of 6.0, boiling in boiling water for 30min, cooling to 85 deg.C, adding 0.3 wt% (based on the weight of defatted rice bran) of high temperature α -amylase, stirring for 30min, filtering, mixing with 25g fresh rice bran at a ratio of 2:1 (based on the weight of defatted rice bran), and grinding to particle size of 140-700 mesh, 75mL of CaCl with the mass concentration of 60 percent is added2Stirring and soaking the solution at 30 deg.C for 1.5h, filtering, pre-freezing with liquid nitrogen, and freeze-drying to water content of 8%; adding 225mL TL lipase enzyme solution into the pretreated rice bran carrier, adding 75mL of 0.01mol/L disodium hydrogen phosphate-sodium dihydrogen phosphate buffer solution with pH of 7.0, stirring for 3h, and then adding 3mL of 0.5% glutaraldehyde solution for crosslinking reaction for 1 h; filtering to separate rice bran, vacuum drying at 35 deg.C to water content of 7% to obtain immobilized TL lipase 4.
Example 5
Weighing 100g of defatted rice bran in a 1L beaker, adding 500mL of 0.01moL/L citric acid-sodium citrate buffer solution with pH of 6.6, boiling in boiling water for 30min, reducing the temperature to 80 ℃, adding 0.3 wt% (based on the weight of the defatted rice bran) of high-temperature α -amylase, stirring for 30min, filtering, uniformly mixing with 25g of fresh rice bran according to the ratio of 4:1 (based on the weight of the defatted rice bran), grinding to the particle size of 140 meshes and 700 meshes, adding 125mL of CaCl with the mass concentration of 80%2Stirring and soaking the solution at 35 deg.C for 2h, filtering, pre-freezing with liquid nitrogen, and freeze-drying to water content of 5%; adding 375mL of TL lipase enzyme solution into the pretreated rice bran carrier, adding 125mL of 0.01mol/L disodium hydrogen phosphate-sodium dihydrogen phosphate buffer solution with pH of 7.0, stirring for 3h, and then adding 5mL of 0.5% glutaraldehyde solution for crosslinking reaction for 1 h; and filtering to separate a rice bran part, and naturally airing at room temperature until the moisture content is 9% to obtain the immobilized TL lipase 5.
Example 6
Weighing 70g of defatted rice bran in a 1L beaker, adding 350mL of 0.01moL/L citric acid-sodium citrate buffer solution with pH of 6.6, boiling in boiling water for 30min, reducing the temperature to 80 ℃, adding 0.3 wt% (based on the weight of the defatted rice bran) of high-temperature α -amylase, stirring for 30min, filtering, adding 30g of white carbon black, uniformly mixing with 400g of fresh rice bran according to the ratio of 1:4 (based on the total weight of the defatted rice bran and the white carbon black), grinding to the particle size of 140 meshes and 700 meshes, adding 500mL of CaCl2Stirring and soaking the saturated solution at 25 deg.C for 2hr, filtering, pre-freezing with liquid nitrogen, and freeze-drying to water content of 5%; then, the product is processedAdding 125mL of TL lipase liquid into the pretreated carrier, adding 250mL of 0.01mol/L disodium hydrogen phosphate-sodium dihydrogen phosphate buffer solution with the pH value of 7.0, stirring for 2h, and then adding 10mL of 0.5% glutaraldehyde solution for crosslinking reaction for 1 h; and filtering and separating rice bran, and naturally airing at room temperature until the moisture content is 10% to obtain the immobilized TL lipase 6.
Comparative example 1
Fresh whole rice bran was used as the immobilization carrier directly, and the conditions for immobilization and drying were exactly the same as in example 1.
Comparative example 2
The stabilized defatted rice bran was used as an immobilization carrier directly, and the conditions for immobilization and drying were exactly the same as in example 1.
Comparative example 3
Defatted and de-starched rice bran was used as the immobilization carrier, and the conditions for immobilization and drying were exactly the same as in example 1.
And (3) rice bran carrier related index determination:
results of the experiment
1. Determination of esterification Activity
20g of oleic acid (oleic acid purity is 75%) is weighed into a 50mL round-bottom flask, 3% of immobilized TL lipase (based on the mass of an oil phase substrate) is added into the flask in 2h, 6mL of methanol is added in three times, the mixture is reacted for 4h at the temperature of 40 ℃, and the conversion rate of the oleic acid is determined.
Oleic acid conversion (oil phase starting acid value-oil phase acid value after reaction)/oil phase starting acid value × 100%
From experimental results, the rice bran immobilized TL lipase disclosed by the invention has the advantage that in the process of catalyzing esterification reaction of oleic acid and methanol, the conversion rate of the oleic acid is more than 93%, and is more than 20% higher than that of commercial enzyme Lipozyme TL IM (TL is immobilized on a silica carrier). Comparative example 1 immobilized TL lipase uses fresh rice bran, which is itself a relatively dense structure lacking pores, which is not conducive to deposition and adsorption of TL lipase, thus TL has a low loading on fresh rice bran and minimal esterification activity; comparative example 2 and comparative example 3 immobilized TL lipase using defatted rice bran or defatted, de-starched rice bran, although favorable for loading and adsorption of TL lipase, comparative example 2 immobilized TL lipase activity was not significantly improved due to lack of natural lipase of rice bran and prior activation of TL lipase by lipid hydrolysis of rice bran.
2. Methanol tolerance assay
20g of oleic acid (oleic acid purity of 75%) was weighed into a 50mL round-bottom flask, and 3% of immobilized TL lipase shown in FIG. 1 or FIG. 2 (based on the mass of the oil phase substrate) and 6mL and 12mL of methanol (molar ratio of oleic acid to methanol is 1:2 and 1:4, respectively) were added in one portion and reacted for 4 hours. After the reaction is finished, the immobilized TL lipase is filtered out and is continuously used for the next batch of reaction, and the reaction is carried out according to the batch times shown in figure 1 or figure 2.
Because short-chain alcohols such as methanol and the like have strong inactivation effect on lipase, methanol is often added in batches or fed-batch manner in the process of preparing biodiesel. As shown in fig. 2 or fig. 1, the above-mentioned method can reduce the inactivation of enzyme to some extent, but also greatly reduces the reaction efficiency. Compared with resin immobilized TL lipase, the rice bran immobilized TL lipase provided by the invention has better methanol tolerance, and particularly in a reaction system with high methanol concentration, the inactivation speed of the rice bran immobilized TL lipase is obviously slower than that of the resin immobilized lipase.
Claims (10)
1. An immobilized enzyme carrier, which is characterized in that the immobilized enzyme carrier contains rice bran or consists of the rice bran;
preferably, the hydrolysis activity of the natural lipase in the rice bran is 50-600U/Kg, preferably 55-580U/Kg, preferably 55-420U/Kg, preferably 130-280U/Kg;
preferably, the oil content of the rice bran is 5-15 wt%, preferably 6-14 wt%, preferably 9-11 wt%, based on the mass of the rice bran;
preferably, the content of starch in the rice bran is 8-25 wt%, preferably 8-24 wt%, preferably 8-17 wt%, preferably 10-13 wt%, based on the mass of the rice bran;
preferably, the water holding capacity of the rice bran is 220-320ml/100g, preferably 240-317 ml/100g, preferably 260-290 ml/100 g;
preferably, the oil holding capacity of the rice bran is 90-150ml/100g, preferably 91-130ml/100g, preferably 100-115ml/100 g;
preferably, the hydrolysis activity of the natural lipase in the immobilized enzyme carrier is 50-600U/Kg, preferably 55-580U/Kg, preferably 55-420U/Kg, preferably 130-280U/Kg;
preferably, the oil content in the immobilized enzyme carrier is 5-15 wt%, preferably 6-14 wt%, preferably 9-11 wt%, based on the mass of the immobilized enzyme carrier;
preferably, the starch content of the immobilized enzyme carrier is 8-25 wt%, preferably 8-24 wt%, preferably 8-17 wt%, preferably 10-13 wt%;
preferably, the water holding capacity of the immobilized enzyme carrier is 220-320ml/100g, preferably 240-317 ml/100g, preferably 260-290 ml/100 g;
preferably, the oil holding capacity of the immobilized enzyme carrier is 90-150ml/100g, preferably 91-130ml/100g, preferably 100-115ml/100 g;
preferably, the immobilized enzyme carrier further contains at least one of white carbon black, silica gel, activated carbon, diatomite, magnesium silicate, cellulose and resin, preferably at least one of white carbon black, silica gel, activated carbon, diatomite and magnesium silicate; or
Preferably, the immobilized enzyme carrier consists of rice bran.
2. An immobilized enzyme, wherein the immobilized enzyme comprises rice bran;
preferably, the oil content in the immobilized enzyme is 5-15 wt%, preferably 6-14 wt%, preferably 9-11 wt% based on the mass of the immobilized enzyme;
preferably, the starch content of the immobilized enzyme is 8-25 wt%, preferably 8-24 wt%, preferably 8-17 wt%, preferably 10-13 wt%;
preferably, the water holding capacity of the immobilized enzyme is 220-320ml/100g, preferably 240-317 ml/100g, preferably 260-290 ml/100 g;
preferably, the oil holding capacity of the immobilized enzyme is 90-150ml/100g, preferably 91-130ml/100g, preferably 100-115ml/100 g;
preferably, the hydrolysis activity of the natural lipase in the rice bran is 50-600U/Kg, preferably 55-580U/Kg, preferably 55-420U/Kg, preferably 130-280U/Kg;
preferably, the oil content of the rice bran is 5-15 wt%, preferably 6-14 wt%, preferably 9-11 wt%, based on the mass of the rice bran;
preferably, the content of starch in the rice bran is 8-25 wt%, preferably 8-24 wt%, preferably 8-17 wt%, preferably 10-13 wt%, based on the mass of the rice bran;
preferably, the water holding capacity of the rice bran is 220-320ml/100g, preferably 240-317 ml/100g, preferably 260-290 ml/100 g;
preferably, the oil holding capacity of the rice bran is 90-150ml/100g, preferably 91-130ml/100g, preferably 100-115ml/100 g;
preferably, the hydrolysis activity of the natural lipase in the rice bran is 50-600U/Kg, preferably 55-580U/Kg, preferably 55-420U/Kg, preferably 130-280U/Kg;
preferably, the immobilized enzyme further comprises at least one of white carbon black, silica gel, activated carbon, diatomite, magnesium silicate, cellulose and resin, preferably at least one of white carbon black, silica gel, activated carbon, diatomite and magnesium silicate; preferably the carrier of the immobilized enzyme is selected from the carriers of claim 1; or
Preferably, the enzyme is selected from lipases, phospholipases, proteases and/or amylases; preferably the lipase is selected from at least one of TL lipase, CALB lipase, RM lipase or ROL lipase; preferably the TL lipase is selected from: fermentation products of Thermomyces (Thermomyces lanuginosus) and/or TL lipases obtained by genetic engineering or protein engineering and/or commercially available TL lipases, preferably selected from lipozyme TL 100L.
3. A rice bran or a process for the preparation of rice bran as claimed in claim 1 or claim 2, wherein:
a) treating the defatted rice bran A with amylase to obtain defatted rice bran B;
b) mixing the defatted rice bran B with fresh whole-fat rice bran to obtain mixed rice bran C; and
c) contacting the mixed rice bran C with a soluble calcium salt solution, and separating to obtain the rice bran;
preferably, the amylase in step a) is selected from α -amylase, β -amylase, glucoamylase and isoamylase, preferably α -amylase, further preferably high temperature amylase;
preferably, the contact temperature of the amylase and the defatted rice bran A in the step a) is 80-90 ℃; preferably the contact time is 30 min; preferably the contact pH is from 5.0 to 6.6;
preferably, the particle size of the mixed rice bran C obtained in the step b) is adjusted to 140-700 meshes; preferably, the particle size of the defatted rice bran B and/or the fresh whole fat rice bran is adjusted to 140-700 mesh before the mixing step of step B); preferably, the particle size adjusting method adopts at least one of dry grinding, wet grinding, ultra-fine grinding and low-temperature grinding; preferably, the mass ratio of the defatted rice bran B to the fresh full-fat rice bran is 1: 4-4: 1; or
Preferably, the soluble calcium salt in step c) is selected from at least one of calcium chloride, calcium nitrate, calcium hydrogen sulfate and calcium bicarbonate; preferably the soluble calcium salt is selected from calcium chloride, preferably the soluble calcium salt solution is at a concentration of 10% to 80% (w/v); preferably, the volume dosage of the soluble calcium salt solution is at least 1mL/g based on the mass of the mixed rice bran C; preferably soaking for 1-2 hr; the preferred dipping temperature is 25-35 ℃; preferably drying after dipping to the moisture of 5-10%; preferably, the drying method adopts at least one of freeze drying, low-temperature vacuum drying, natural airing and low-temperature fluidized drying.
4. The method of claim 3, further comprising, in the step a):
a1) a step of contacting defatted rice bran A with water at a pH of 5.0 to 6.6;
preferably, the contacting of the defatted rice bran a with water is performed before the defatted rice bran a is treated with amylase; or
Preferably, the contact temperature of the defatted rice bran A and the buffer solution is 80-120 ℃; preferably, the buffer solution is at least one selected from citric acid buffer solution, phosphate buffer solution, potassium dihydrogen phosphate-sodium hydroxide buffer solution, tris-hydrochloric acid buffer solution and PBS buffer solution; preferably the buffer solution is selected from citric acid buffers; preferably the contact time is 20-40 min; preferably, the concentration of the citric acid buffer solution is 0.01-0.1 mol/L.
5. A rice bran produced by the process of claim 3 or claim 4, wherein the rice bran has at least one of the following characteristics;
1) the hydrolysis activity of natural lipase in the rice bran is 50-600U/Kg, preferably 55-580U/Kg, preferably 55-420U/Kg, preferably 130-280U/Kg;
2) the oil content of the rice bran is 5-15 wt%, preferably 6-14 wt%, preferably 9-11 wt%, based on the mass of the rice bran;
3) the content of starch in the rice bran is 8-25 wt%, preferably 8-24 wt%, preferably 8-17 wt%, preferably 10-13 wt%, based on the mass of the rice bran;
4) the water holding capacity of the rice bran is 220-320ml/100g, preferably 240-317 ml/100g, preferably 260-290 ml/100 g; or
5) The oil holding capacity of the rice bran is 90-150ml/100g, preferably 91-130ml/100g, preferably 100-115ml/100 g.
6. An immobilized enzyme comprising the immobilized enzyme carrier of claim 1, preferably the enzyme is selected from the group consisting of lipase, phospholipase, protease and/or amylase; preferably the lipase is selected from at least one of TL lipase, CALB lipase, RM lipase or ROL lipase; preferably the TL lipase is selected from: fermentation products of thermomyces thermophilus (thermomyces lanuginosus) and/or TL lipases obtained by genetic engineering or protein engineering and/or commercially available TL lipases, preferably selected from lipozyme TL 100L.
7. A process for preparing the immobilized enzyme of claim 6, characterized by contacting the immobilized enzyme carrier of claim 1 with an enzyme; preferably, the immobilized enzyme is prepared by at least one of an adsorption method, a covalent bonding method, an embedding method and a crosslinking method.
8. Use of an immobilized enzyme according to claim 2 or claim 6 in at least one reaction selected from catalytic esterification, transesterification, alcoholysis, acid hydrolysis and/or hydrolysis.
9. Use of an immobilized enzyme according to claim 2 or claim 6 in the preparation of biodiesel.
10. A method for improving lipase-catalyzed esterification activity and/or short-chain alcohol tolerance, which comprises contacting the immobilized carrier of claim 1 with an enzyme.
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| CN114438072A (en) * | 2022-04-08 | 2022-05-06 | 山东天力药业有限公司 | Production method of trehalose |
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| CN114480360B (en) * | 2021-12-14 | 2024-03-12 | 山东省农业科学院 | Method for preparing diglyceride by immobilized Sn-2 lipase |
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