CN101407819B - Method for constructing ester bond enzyme engineering bacteria for tendency hydrolysis of TAG sn-2 and corresponding recombinant lipase - Google Patents
Method for constructing ester bond enzyme engineering bacteria for tendency hydrolysis of TAG sn-2 and corresponding recombinant lipase Download PDFInfo
- Publication number
- CN101407819B CN101407819B CN2008101222973A CN200810122297A CN101407819B CN 101407819 B CN101407819 B CN 101407819B CN 2008101222973 A CN2008101222973 A CN 2008101222973A CN 200810122297 A CN200810122297 A CN 200810122297A CN 101407819 B CN101407819 B CN 101407819B
- Authority
- CN
- China
- Prior art keywords
- lipase
- engineering bacteria
- recombinant
- enzyme
- tag
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
- 108090001060 Lipase Proteins 0.000 title claims abstract description 66
- 102000004882 Lipase Human genes 0.000 title claims abstract description 66
- 239000004367 Lipase Substances 0.000 title claims abstract description 66
- 235000019421 lipase Nutrition 0.000 title claims abstract description 66
- 108090000790 Enzymes Proteins 0.000 title claims abstract description 51
- 102000004190 Enzymes Human genes 0.000 title claims abstract description 51
- 241000894006 Bacteria Species 0.000 title claims abstract description 45
- 238000000034 method Methods 0.000 title claims abstract description 44
- 230000007062 hydrolysis Effects 0.000 title claims description 21
- 238000006460 hydrolysis reaction Methods 0.000 title claims description 21
- 239000013598 vector Substances 0.000 claims abstract description 27
- 108091008146 restriction endonucleases Proteins 0.000 claims abstract description 13
- 101150006771 CAL-A gene Proteins 0.000 claims abstract description 5
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 21
- 238000006243 chemical reaction Methods 0.000 claims description 19
- 230000008569 process Effects 0.000 claims description 18
- 239000007788 liquid Substances 0.000 claims description 17
- 238000010276 construction Methods 0.000 claims description 12
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 claims description 6
- 238000012408 PCR amplification Methods 0.000 claims description 6
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims description 6
- 230000005611 electricity Effects 0.000 claims description 6
- 230000006698 induction Effects 0.000 claims description 6
- 238000011144 upstream manufacturing Methods 0.000 claims description 6
- 238000004255 ion exchange chromatography Methods 0.000 claims description 5
- 229920002134 Carboxymethyl cellulose Polymers 0.000 claims description 4
- 241000235648 Pichia Species 0.000 claims description 4
- 239000001768 carboxy methyl cellulose Substances 0.000 claims description 4
- 235000010948 carboxy methyl cellulose Nutrition 0.000 claims description 4
- 239000008112 carboxymethyl-cellulose Substances 0.000 claims description 4
- 230000029087 digestion Effects 0.000 claims description 4
- 230000000968 intestinal effect Effects 0.000 claims description 3
- 238000000746 purification Methods 0.000 claims description 3
- 241000222120 Candida <Saccharomycetales> Species 0.000 claims description 2
- 230000001186 cumulative effect Effects 0.000 claims description 2
- UFTFJSFQGQCHQW-UHFFFAOYSA-N triformin Chemical compound O=COCC(OC=O)COC=O UFTFJSFQGQCHQW-UHFFFAOYSA-N 0.000 abstract description 9
- 239000008157 edible vegetable oil Substances 0.000 abstract description 4
- 230000003301 hydrolyzing effect Effects 0.000 abstract description 4
- 238000001976 enzyme digestion Methods 0.000 abstract description 2
- 238000012216 screening Methods 0.000 abstract 2
- 241000193830 Bacillus <bacterium> Species 0.000 abstract 1
- 238000010367 cloning Methods 0.000 abstract 1
- 210000001072 colon Anatomy 0.000 abstract 1
- 150000003626 triacylglycerols Chemical class 0.000 description 22
- 239000003921 oil Substances 0.000 description 18
- 235000019198 oils Nutrition 0.000 description 18
- 239000000243 solution Substances 0.000 description 14
- 230000000694 effects Effects 0.000 description 12
- 239000000047 product Substances 0.000 description 12
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 10
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 10
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 9
- 235000019626 lipase activity Nutrition 0.000 description 9
- 239000000126 substance Substances 0.000 description 8
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 8
- 125000002252 acyl group Chemical group 0.000 description 7
- 239000000872 buffer Substances 0.000 description 7
- 239000003925 fat Substances 0.000 description 7
- 235000019197 fats Nutrition 0.000 description 7
- 108090000623 proteins and genes Proteins 0.000 description 7
- 239000004372 Polyvinyl alcohol Substances 0.000 description 6
- 235000009508 confectionery Nutrition 0.000 description 6
- 230000006872 improvement Effects 0.000 description 6
- 229920002451 polyvinyl alcohol Polymers 0.000 description 6
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 5
- 235000011187 glycerol Nutrition 0.000 description 5
- 239000000463 material Substances 0.000 description 5
- 238000010521 absorption reaction Methods 0.000 description 4
- 230000032050 esterification Effects 0.000 description 4
- 238000005886 esterification reaction Methods 0.000 description 4
- 150000002148 esters Chemical class 0.000 description 4
- 239000012528 membrane Substances 0.000 description 4
- 238000002360 preparation method Methods 0.000 description 4
- 239000006228 supernatant Substances 0.000 description 4
- 101000702488 Rattus norvegicus High affinity cationic amino acid transporter 1 Proteins 0.000 description 3
- 241001123227 Saccharomyces pastorianus Species 0.000 description 3
- 230000003321 amplification Effects 0.000 description 3
- 239000010775 animal oil Substances 0.000 description 3
- 230000008901 benefit Effects 0.000 description 3
- 210000004369 blood Anatomy 0.000 description 3
- 239000008280 blood Substances 0.000 description 3
- 239000000839 emulsion Substances 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 238000004519 manufacturing process Methods 0.000 description 3
- 238000003199 nucleic acid amplification method Methods 0.000 description 3
- 102000004169 proteins and genes Human genes 0.000 description 3
- 230000008521 reorganization Effects 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- 238000000108 ultra-filtration Methods 0.000 description 3
- GVJHHUAWPYXKBD-UHFFFAOYSA-N (±)-α-Tocopherol Chemical compound OC1=C(C)C(C)=C2OC(CCCC(C)CCCC(C)CCCC(C)C)(C)CCC2=C1C GVJHHUAWPYXKBD-UHFFFAOYSA-N 0.000 description 2
- 241000588724 Escherichia coli Species 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- 108090000604 Hydrolases Proteins 0.000 description 2
- 102000004157 Hydrolases Human genes 0.000 description 2
- 241000235058 Komagataella pastoris Species 0.000 description 2
- 101710098556 Lipase A Proteins 0.000 description 2
- 101710099648 Lysosomal acid lipase/cholesteryl ester hydrolase Proteins 0.000 description 2
- 102100026001 Lysosomal acid lipase/cholesteryl ester hydrolase Human genes 0.000 description 2
- 241001661345 Moesziomyces antarcticus Species 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 235000009470 Theobroma cacao Nutrition 0.000 description 2
- 244000299461 Theobroma cacao Species 0.000 description 2
- 238000000246 agarose gel electrophoresis Methods 0.000 description 2
- 239000003054 catalyst Substances 0.000 description 2
- 238000006555 catalytic reaction Methods 0.000 description 2
- HVYWMOMLDIMFJA-DPAQBDIFSA-N cholesterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2 HVYWMOMLDIMFJA-DPAQBDIFSA-N 0.000 description 2
- 230000007547 defect Effects 0.000 description 2
- 238000013461 design Methods 0.000 description 2
- 206010012601 diabetes mellitus Diseases 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 239000000284 extract Substances 0.000 description 2
- 239000012634 fragment Substances 0.000 description 2
- 239000004519 grease Substances 0.000 description 2
- 230000036541 health Effects 0.000 description 2
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 description 2
- NOESYZHRGYRDHS-UHFFFAOYSA-N insulin Chemical compound N1C(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(NC(=O)CN)C(C)CC)CSSCC(C(NC(CO)C(=O)NC(CC(C)C)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CCC(N)=O)C(=O)NC(CC(C)C)C(=O)NC(CCC(O)=O)C(=O)NC(CC(N)=O)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CSSCC(NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2C=CC(O)=CC=2)NC(=O)C(CC(C)C)NC(=O)C(C)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2NC=NC=2)NC(=O)C(CO)NC(=O)CNC2=O)C(=O)NCC(=O)NC(CCC(O)=O)C(=O)NC(CCCNC(N)=N)C(=O)NCC(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC(O)=CC=3)C(=O)NC(C(C)O)C(=O)N3C(CCC3)C(=O)NC(CCCCN)C(=O)NC(C)C(O)=O)C(=O)NC(CC(N)=O)C(O)=O)=O)NC(=O)C(C(C)CC)NC(=O)C(CO)NC(=O)C(C(C)O)NC(=O)C1CSSCC2NC(=O)C(CC(C)C)NC(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CC(N)=O)NC(=O)C(NC(=O)C(N)CC=1C=CC=CC=1)C(C)C)CC1=CN=CN1 NOESYZHRGYRDHS-UHFFFAOYSA-N 0.000 description 2
- 230000004060 metabolic process Effects 0.000 description 2
- 230000000813 microbial effect Effects 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 235000015097 nutrients Nutrition 0.000 description 2
- KJFMBFZCATUALV-UHFFFAOYSA-N phenolphthalein Chemical compound C1=CC(O)=CC=C1C1(C=2C=CC(O)=CC=2)C2=CC=CC=C2C(=O)O1 KJFMBFZCATUALV-UHFFFAOYSA-N 0.000 description 2
- 230000009257 reactivity Effects 0.000 description 2
- 238000007670 refining Methods 0.000 description 2
- 230000028327 secretion Effects 0.000 description 2
- 210000000582 semen Anatomy 0.000 description 2
- 210000002966 serum Anatomy 0.000 description 2
- 239000000758 substrate Substances 0.000 description 2
- 238000001308 synthesis method Methods 0.000 description 2
- 238000004448 titration Methods 0.000 description 2
- 235000013311 vegetables Nutrition 0.000 description 2
- 101710134784 Agnoprotein Proteins 0.000 description 1
- 201000001320 Atherosclerosis Diseases 0.000 description 1
- 108010031797 Candida antarctica lipase B Proteins 0.000 description 1
- 241000321538 Candidia Species 0.000 description 1
- FBPFZTCFMRRESA-FSIIMWSLSA-N D-Glucitol Natural products OC[C@H](O)[C@H](O)[C@@H](O)[C@H](O)CO FBPFZTCFMRRESA-FSIIMWSLSA-N 0.000 description 1
- GUBGYTABKSRVRQ-WFVLMXAXSA-N DEAE-cellulose Chemical compound OC1C(O)C(O)C(CO)O[C@H]1O[C@@H]1C(CO)OC(O)C(O)C1O GUBGYTABKSRVRQ-WFVLMXAXSA-N 0.000 description 1
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 1
- 101000925662 Enterobacteria phage PRD1 Endolysin Proteins 0.000 description 1
- 206010020772 Hypertension Diseases 0.000 description 1
- 102000004877 Insulin Human genes 0.000 description 1
- 108090001061 Insulin Proteins 0.000 description 1
- 102000016267 Leptin Human genes 0.000 description 1
- 108010092277 Leptin Proteins 0.000 description 1
- 102000003960 Ligases Human genes 0.000 description 1
- 108090000364 Ligases Proteins 0.000 description 1
- 208000001145 Metabolic Syndrome Diseases 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 208000008589 Obesity Diseases 0.000 description 1
- 240000007594 Oryza sativa Species 0.000 description 1
- 235000007164 Oryza sativa Nutrition 0.000 description 1
- 235000019483 Peanut oil Nutrition 0.000 description 1
- 241001494479 Pecora Species 0.000 description 1
- 235000019484 Rapeseed oil Nutrition 0.000 description 1
- 101000968489 Rhizomucor miehei Lipase Proteins 0.000 description 1
- 235000019485 Safflower oil Nutrition 0.000 description 1
- 241000269319 Squalius cephalus Species 0.000 description 1
- 229930003427 Vitamin E Natural products 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 210000000577 adipose tissue Anatomy 0.000 description 1
- 238000013019 agitation Methods 0.000 description 1
- 230000003115 biocidal effect Effects 0.000 description 1
- 230000037396 body weight Effects 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 235000014121 butter Nutrition 0.000 description 1
- 239000010495 camellia oil Substances 0.000 description 1
- 239000000969 carrier Substances 0.000 description 1
- 230000003197 catalytic effect Effects 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 230000004087 circulation Effects 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 239000012141 concentrate Substances 0.000 description 1
- 238000013016 damping Methods 0.000 description 1
- 238000000354 decomposition reaction Methods 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 235000013367 dietary fats Nutrition 0.000 description 1
- 235000014113 dietary fatty acids Nutrition 0.000 description 1
- 230000003203 everyday effect Effects 0.000 description 1
- 230000029142 excretion Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000000194 fatty acid Substances 0.000 description 1
- 229930195729 fatty acid Natural products 0.000 description 1
- 150000004665 fatty acids Chemical class 0.000 description 1
- 239000000835 fiber Substances 0.000 description 1
- 239000012467 final product Substances 0.000 description 1
- 235000021323 fish oil Nutrition 0.000 description 1
- 239000000796 flavoring agent Substances 0.000 description 1
- 235000019634 flavors Nutrition 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 235000021588 free fatty acids Nutrition 0.000 description 1
- WIGCFUFOHFEKBI-UHFFFAOYSA-N gamma-tocopherol Natural products CC(C)CCCC(C)CCCC(C)CCCC1CCC2C(C)C(O)C(C)C(C)C2O1 WIGCFUFOHFEKBI-UHFFFAOYSA-N 0.000 description 1
- 125000005908 glyceryl ester group Chemical group 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 229910001385 heavy metal Inorganic materials 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 238000009413 insulation Methods 0.000 description 1
- 229940125396 insulin Drugs 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 238000005342 ion exchange Methods 0.000 description 1
- NRYBAZVQPHGZNS-ZSOCWYAHSA-N leptin Chemical compound O=C([C@H](CO)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CC=1C2=CC=CC=C2NC=1)NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CO)NC(=O)CNC(=O)[C@H](CCC(N)=O)NC(=O)[C@@H](N)CC(C)C)CCSC)N1CCC[C@H]1C(=O)NCC(=O)N[C@@H](CS)C(O)=O NRYBAZVQPHGZNS-ZSOCWYAHSA-N 0.000 description 1
- 229940039781 leptin Drugs 0.000 description 1
- 235000021388 linseed oil Nutrition 0.000 description 1
- 239000000944 linseed oil Substances 0.000 description 1
- 235000012054 meals Nutrition 0.000 description 1
- 229910021645 metal ion Inorganic materials 0.000 description 1
- 239000011259 mixed solution Substances 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 230000000050 nutritive effect Effects 0.000 description 1
- 235000020824 obesity Nutrition 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 230000037361 pathway Effects 0.000 description 1
- 239000000312 peanut oil Substances 0.000 description 1
- 235000002378 plant sterols Nutrition 0.000 description 1
- 230000006798 recombination Effects 0.000 description 1
- 238000005215 recombination Methods 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000000452 restraining effect Effects 0.000 description 1
- 235000009566 rice Nutrition 0.000 description 1
- 230000000630 rising effect Effects 0.000 description 1
- 235000005713 safflower oil Nutrition 0.000 description 1
- 239000003813 safflower oil Substances 0.000 description 1
- 230000035939 shock Effects 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 1
- 239000000600 sorbitol Substances 0.000 description 1
- 235000012424 soybean oil Nutrition 0.000 description 1
- 239000003549 soybean oil Substances 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 210000002784 stomach Anatomy 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 235000020238 sunflower seed Nutrition 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 238000004809 thin layer chromatography Methods 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 229910021642 ultra pure water Inorganic materials 0.000 description 1
- 239000012498 ultrapure water Substances 0.000 description 1
- 235000015112 vegetable and seed oil Nutrition 0.000 description 1
- 239000008158 vegetable oil Substances 0.000 description 1
- 229940046009 vitamin E Drugs 0.000 description 1
- 235000019165 vitamin E Nutrition 0.000 description 1
- 239000011709 vitamin E Substances 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
Images
Landscapes
- Enzymes And Modification Thereof (AREA)
Abstract
The invention discloses a building method of enzyme engineering bacteria which inclines to a hydrated TAG sn-2 bit ester bond and includes the following steps of: 1) the cloning of a CAL-A gene; 2) the building of a recombinant vector: double enzyme digestion and connection are carried out on a PCR product and the vector by utilizing a restriction enzyme for building the recombinant vector; converting the recombinant vector into colon bacillus and screening a positive recombinant vector through PCR authentication; and 3) converting: carrying out linearization on the positive recombinant vector by adopting the restriction enzyme and electrically transferring the positive recombinant vector into a host strain, and then screening the positive engineering bacteria by adopting PCR authentication. The invention simultaneously provides a method for preparing a recombinant lipase by utilizing the engineering bacteria obtained through the method. The recombinant lipase can be used for hydrolyzing triglyceride edible oil.
Description
Technical field
The present invention relates to the construction process of a kind of proneness hydrolyzing triglyceride sn-2 position ester bond lipase engineering bacteria and corresponding recombinant lipase.
Background technology
Along with the raising of world economy level, the absorption of animal food increases, and then causes the intake of dietary fat significantly to rise.CDC (CDC) report of survey shows that Chinese residents grease every day intake was about 44g in 2002, and contained energy accounts for 35% of meals total energy, is higher than the upper limit 30% of world health organisation recommendations.The fat intake is too high, causes body to suffer from diseases such as obesity, hypertension, diabetes, atherosclerosis.The treatment of these diseases and complication thereof brings heavy economical load not only for patient family, also brings heavy social burden.
1,3-triglyceride (DAG) is the micro-moiety in the natural fats and oils.Numerous scientific researches are found, 1, the absorption of 3-DAG can reduce obese patient's body weight and body fat and form, reduce diabetes B patient blood sugar metabolism index of correlation, for example reduce its serum insulin level and leptin level etc., also can reduce serum total cholesterol and the always concentration of triglyceride level (TAG) on an empty stomach.Therefore, 1, the absorption of 3-DAG can significantly improve all kinds of metabolic syndromes that Yin Putong grease Excessive Intake is caused.Simultaneously, 1,3-DAG is at aspect and TAG basically identicals such as local flavor, color and lusters, and has energy density identical with TAG and bioavailability, and without any side effects to body.
In view of 1, the health efficacy that 3-DAG had, it has prepared the extensive concern of Chinese scholars.Existing multinomial both at home and abroad at present about 1, the patent and the article of 3-DAG preparation, involved method mainly contains direct esterification method, glycerine solution, TAG and decomposes back synthesis method and portion water solution etc. earlier.
The direct esterification method be glycerine and FA 1, under the catalysis of 3-specific lipase (rhizomucor miehei lipase), esterification directly takes place generates 1,3-DAG.The advantage of this method is in the product 1, and the content of 3-DAG is very high, but that shortcoming is required substrate cost is very high, also needing constantly to remove the water that generates in the reaction system is carried out reaction, this has increased the difficulty of patent working, as Chinese patent CN1560020A, CN1615365.
The glycerine solution is meant 1, under the effect of 3-specific lipase, the acyl group transfer takes place between TAG and the glycerine and generate 1,3-DAG.The required cost of this method is higher, has limited its application equally, as Chinese patent CN1438308A, CN1544412A.
Triglyceride level decomposes the back synthesis method earlier and is meant that triglyceride level generates glycerine and FA thorough decomposition the under the effect of steam or non-selectivity lipase (as candida antarctica lipase B), and both are 1, and esterification generates 1,3-DAG under the effect of 3-specificity lytic enzyme.The required substrate cost of this method is lower, but needs constantly to remove moisture in the reaction process, and technical process is comparatively loaded down with trivial details.If adopt steam that TAG is hydrolyzed, other nutritive ingredient in the natural fats and oils such as vitamin-E, plant sterol etc. suffer brokenly words, also need to add this type of material again in final product, and the corresponding increase of cost is as Chinese patent CN1267322A.
The direct hydrolysis method is meant that under chemical catalyst or action of lipase TAG sn-2 position acyl group is generated 1,3-DAG under the direct hydrolysis.If adopt chemical catalyst to carry out catalysis, 1,3-DAG content is lower in the hydrolysis products therefrom; and in order to prevent Fatty Acid Oxidation under the high temperature; reaction needed is carried out under the protection of vacuum or rare gas element, has increased cost, as Chinese patent CN1635068A, CN1585814.If adopt lipase that TAG is carried out catalytic hydrolysis, owing to do not find the lipase of sn-2 position acyl group specificity hydrolysis at present as yet, causing the DAG in the product is 1,3-DAG and 1, the mixture of 2-DAG.From pathways metabolism, 1,2-DAG is the same with TAG, all changes into the MAG and the FA of sn-2 position after enzymic digestion, and the two enters after the blood, all is used for synthetic TAG.Therefore, excessive absorption also can cause problems such as blood fat rising.
Antarctic candidia lipase A (CAL-A) is the lipase of at present known tool TAG sn-2 position acyl hydrolase tendency.Patent ZL200610049242.5 has groped CAL-A hydrolysis TAG preparation 1, the condition of 3-DAG, and successfully prepare high by 1, the edible oil of 3-DAG content.But, limited the industrial applications of this method greatly because CAL-A output in its original strain is very low.
Pichia pastoris phaff expression system (Pichia pastoris) has the expression amount height, can modify and advantage such as regulating and expressing foreign protein, more and more be subjected to the attention of molecular biosciences educational circles, the expression of succeeding in this system of more than 300 kind of foreign protein has been arranged at present.
Summary of the invention
The technical problem to be solved in the present invention provides the construction process of a kind of tendency hydrolysis TAG sn-2 position ester bond enzyme engineering bacteria, and utilizes this project bacterium to prepare the method for recombinant lipase.
In order to solve the problems of the technologies described above, the invention provides the construction process of a kind of tendency hydrolysis TAG sn-2 position ester bond enzyme engineering bacteria, may further comprise the steps:
1), the clone of CAL-A gene:
With the antarctic candida genome is template, utilizes a pair of Auele Specific Primer to carry out pcr amplification;
The upstream primer of described Auele Specific Primer and downstream primer are as follows respectively:
Upstream primer (LPU): AATAAGGAATTCGCGGCGCTGCCCAACCCCTACGAC is SEQID NO:1;
Downstream primer (LPD): AAGGAAAAAAGCGGCCGCCATCGACCGTGGGAACTG; Be SEQ ID NO:2;
2), the structure of recombinant vectors:
Utilize restriction enzyme that the PCR product is carried out double digestion, is connected with carrier, make up recombinant vectors; Then above-mentioned recombinant vectors conversion is entered in the intestinal bacteria, identify, filter out positive recombinant vectors by PCR;
3), transform:
With above-mentioned positive recombinant vectors, adopt restriction enzyme to carry out linearizing, and its electricity commentaries on classics is entered in the host bacterium; Adopt PCR to identify then, filter out positive engineering bacteria.
Improvement as the construction process of tendency hydrolysis TAG sn-2 of the present invention position ester bond enzyme engineering bacteria: the carrier step 2) is pPIC9K or pPICZ α, and the host bacterium in the step 3) is Pichia yeast GS115 or X33.
Further improvement as the construction process of tendency hydrolysis TAG sn-2 of the present invention position ester bond enzyme engineering bacteria: the PCR primers designed step 2) is Auele Specific Primer in the step 1) and the standard primer of this carrier self.
Further improvement as the construction process of tendency hydrolysis TAG sn-2 of the present invention position ester bond enzyme engineering bacteria: the restriction enzyme in the step 3) is Sal I or Sac I.
Further improvement as the construction process of tendency hydrolysis TAG sn-2 of the present invention position ester bond enzyme engineering bacteria: the PCR primers designed in the step 3) is the combination of standard primer of standard primer, the Auele Specific Primer in the step 1) and this carrier self of Auele Specific Primer, this carrier self in the step 1).
The present invention also provides the engineering bacteria that utilizes above-mentioned any one method to obtain to prepare the method for recombinant lipase simultaneously: the positive engineering bacteria of gained is seeded in the BMGY substratum, and 30 ℃ of incubated overnight are to OD
600Be 2～6; Centrifugal then collection thalline is diluted to OD with the BMMY substratum again
600Be 1, after 12h adds the methanol induction expression that accounts for cumulative volume 0.5%, induces 120 hours,, get the crude enzyme liquid of recombinant lipase medium centrifugal.
As the improvement for preparing the method for recombinant lipase of the present invention: the crude enzyme liquid of recombinant lipase is concentrated and purifying, recombinant lipase.
Further improvements in methods as preparation recombinant lipase of the present invention: enrichment step adopts hollow carboxymethyl cellulose film, and purification step adopts ion exchange chromatography.
The present invention is by CAL-A gene and Yeast expression carrier are made up recombinant vectors, and its conversion is entered the histidine defect type finishes in the red saccharomyces pastorianus, makes up engineering bacteria; Adopt methyl alcohol to carry out abduction delivering then, prepared recombinant C AL-A in a large number.
The constructing plan of engineering bacteria of the present invention can be summarized in following steps:
1. from ncbi database, search out the gene order of coding CAL-A;
2. design a pair of Auele Specific Primer, with the gene order of this lipase of amplification coding;
3. select suitable carriers and host bacterium;
4. utilize restriction enzyme that the PCR product is carried out double digestion, is connected with carrier, make up recombinant vectors;
5. the recombinant vectors conversion is entered in the intestinal bacteria, identify, filter out positive recombinant vectors by PCR;
6. extract positive recombinant vectors, adopt suitable restriction enzyme to carry out linearizing, and its electricity commentaries on classics is entered in the pichia spp;
7. adopt PCR to identify, screen positive engineering bacteria.
The contriver has utilized the engineering bacteria of gained to adopt the methanol induction recombination yeast, has investigated the methyl alcohol addition, adds batch, induction time is to the influence of recombinant lipase yield of enzyme, thereby optimized the condition of producing enzyme.The contriver adopts hollow carboxymethyl cellulose film that the crude enzyme liquid of recombinant lipase is concentrated, adopt ion exchange chromatography that crude enzyme liquid is carried out purifying again, and studied the influence of the optimal reactive temperature of recombinant lipase and pH, thermostability and pH stability and different chemical material the reorganization lipase activity.
The recombinant lipase of gained of the present invention can be used for hydrolyzing triglyceride edible oil (TAG), thereby prepares 1,3-DAG; Last available bar-shaped thin-layer chromatography and high performance liquid chromatography come the proportion of composing of various glyceryl ester in the detection reaction system.Triglyceride edible oil is at least a in animal oil, vegetables oil, microbial oil, animal oil refining fat, vegetable oil refining fat and the concise fat of microbial oil; Vegetables oil can be selected rapeseed oil, soybean oil, Oleum Gossypii semen, Semen Maydis oil, linseed oil, safflower oil, plam oil, Oleum Cocois, sweet oil, Rice pollard oil, sunflower seed oil, tea-seed oil, peanut oil, cocoa ester or class cocoa ester for use; Animal oil can be selected butter, sheep oil, lard, fish oil or newborn ester for use.
In sum, method of the present invention is a kind of method of CAL-A being expressed at complete red saccharomyces pastorianus expression system.Compare with patent with existing technology, the present invention has the following advantages:
1. adopting yeast secretion type carrier PPIC9K etc. be carrier, makes up recombinant vectors with target gene fragment and transform to enter in the complete red saccharomyces pastorianus of histidine defect type.Induce in the process, recombinant lipase secretion enters in the substratum, can direct centrifugal collection, avoided the bigger breaking yeast cellule membrane process of the required difficulty of intracellular enzyme.
2. excretion vector PPIC9K self excretory foreign protein is less, helps the purifying of gained recombinant lipase.The supernatant that experiment showed, abduction delivering only needs hollow carboxymethyl cellulose membrane ultrafiltration to concentrate and the ion exchange chromatography two-step purifying, can obtain electrophoretically pure recombinant lipase.
3. gained recombinant lipase optimal reactive temperature increases than original lipase.The optimal reactive temperature of original lipase is about the 50-70 degree, and the optimal reactive temperature of recombinant lipase is 75 degree.
4. the gained recombinant lipase has certain resistivity to the toxic action of heavy metal ion.In the metal ion of all tests, has only Hg
+The activity of ion pair recombinant lipase has stronger restraining effect.
5. the gained recombinant lipase is slightly higher than original lipase to the proneness of triglyceride level sn-2 position acyl hydrolase.
6, the gained engineering bacteria induces process simple, only can carry out abduction delivering with cheap methyl alcohol, and production cost is lower.
Description of drawings
Below in conjunction with accompanying drawing the specific embodiment of the present invention is described in further detail.
Fig. 1 is a recombinant lipase abduction delivering graphic representation of the present invention.
Embodiment
The construction process of embodiment 1, a kind of proneness hydrolysis TAG sn-2 position ester bond enzyme engineering bacteria, carry out following steps successively:
1), the clone of CAL-A gene
Selecting EcoR I and Not I on the pPPIC9K for use is restriction enzyme site, designs a pair of Auele Specific Primer.
Upstream primer (LPU): AATAAGGAATTCGCGGCGCTGCCCAACCCCTACGAC
Downstream primer (LPD): AAGGAAAAAAGCGGCCGCCATCGACCGTGGGAACTG
With Candida antarctica genome is template, carries out pcr amplification.The PCR system is as follows: Buffer (no Mg
2+) 5 μ L, Mg
2+Solution 5 μ L, dNTP solution 4 μ L, upstream primer 1 μ L, downstream primer 1 μ L, template 1 μ L, Taq enzyme 0.5 μ L add water and supply 50 μ L.Amplification condition is as follows: 95 ℃ of 5min; 94 ℃ of 1min, 65 ℃ of 1min, 72 ℃ of 1min, 30 circulations; 72 ℃ of insulation 10min.After amplification finishes, adopt the test kit (TaKaRa DNAFragment Purification Kit Ver.2.0) of Takara company to carry out purifying.
2), the structure of recombinant vectors
Use restriction enzyme EcoR I and Not I respectively pPIC9K and above-mentioned PCR product to be carried out enzyme and cut, it is as follows that enzyme is cut system: pPIC9K or PCR product 30 μ L, 10 * Buffer, 10 μ L, EcoR I 2 μ L, Nde I 2 μ L add water and supply 100 μ L.After 30 ℃ of enzymes are cut 3h, adopt the test kit in the step 1) to carry out purifying.
The pPIC9K of double digestion is connected with the PCR product, and linked system is as follows: PCR product 1 μ L, pPIC9K1 μ L, 10 * Buffer1 μ L, dna ligase 0.5 μ L add water and supply 10 μ L.PCR product water consumption substitution in the contrast linked system.16 ℃ connect 12h, will connect the product conversion then and enter bacillus coli DH 5 alpha.Step of converting is as follows:
1. in 100 μ L bacillus coli DH 5 alpha competent cells, add 5 μ L linked systems, mixing and ice bath 30min;
2. competent cell is positioned in 42 ℃ the water-bath, accurately is incubated 45s.
3. with competent cell ice bath 1min, add 600 μ L LB substratum then, put into 37 ℃ of water-baths immediately and be incubated 5min, put it in 37 ℃ of shaking tables again, thermal agitation is cultivated 1h;
4. draw 200 μ L, coat and contain the antibiotic LB flat board of Kan, cultivate for 37 ℃, up to growing single bacterium colony.
Adopt bacterium colony PCR method that the single bacterium colony that screens on the flat board is identified.Before PCR identified, the single bacterium colony of picking contained in the 1.5ml centrifuge tube of microbiotic substratum in 0.3ml, and it is little muddy that 37 ℃ of shaking tables are cultured to substratum, was that template is carried out PCR and identified the same step 1) of PCR system and flow process with this nutrient solution then.(this is in order to ensure exactness to adopt the standard primer of a pair of Auele Specific Primer in the step 1), pPIC9K carrier to increase respectively, therefore only use above-mentioned any one primer also to be fine), and utilize agarose gel electrophoresis that amplified production is detected.If pcr amplification goes out the gene fragment band of the same size (being the size of the PCR band in the step 1)) with coding lipase, then show the positive transformant of this bacterium colony.This positive recombinant of picking is cultivated in the LB substratum, and extracts recombinant vectors in a large number.
3), the conversion of pichia spp
Adopt restriction enzyme Sal I that above-mentioned recombinant vectors is carried out linearization for enzyme restriction, enzyme is cut system with step 2), and it is mixed with 80 μ L GS115 competent cells, carry out electricity commentaries on classics behind the ice bath 5min, electric commentaries on classics condition is the 1.5KV 5ms that descends to shock by electricity.After electricity change to finish, add the Sorbitol Solution USP of 1ml ice precooling immediately and, be transferred in the centrifuge tube of 1.5mL, then the thalline suspension is coated on the MD flat board the thalline mixing, per 200 μ L coating one flat plate, 30 ℃ of cultivations are until growing single bacterium colony.
Picking list bacterium colony is put respectively on MM and the MD flat board, to identify the phenotype of recombinant conversion.On the MD flat board growth normal and poky on the MM flat board be Mut
sType, the normal transformant of all growing on MD and MM flat board is Mut
+Type.Picking Mut
+The type recon is cultivated in the YPD substratum, and is that template is carried out pcr amplification and identified with its genome, identifies the same step 1) of flow process.(this is in order to ensure exactness in order to be combined into the performing PCR amplification to adopt a pair of Auele Specific Primer in the step 1), the standard primer of pPIC9K carrier, an Auele Specific Primer and a standard primer respectively, therefore only use above-mentioned any one primer also to be fine), and amplified production is carried out agarose gel electrophoresis detect.The transformant that amplifies purpose clip size band (being PCR product size in the step 1)) is positive transformant.
The engineering bacteria that embodiment 2, a kind of embodiment of utilization 1 are obtained prepares the method for recombinant lipase, adopts saccharomycetic abduction delivering, and concrete steps are as follows:
The positive transformant of embodiment 1 gained is seeded in the BMGY substratum, and 30 ℃ of incubated overnight are to OD
600Be 2 to 6, centrifugal then collection thalline.The thalline of gained is diluted to OD with the BMMY substratum
600Be 1, add the methanol induction that accounts for total nutrient solution volume 0.5% every 12h and express.Got the 1.5ml medium centrifugal every 24 hours, supernatant is the crude enzyme liquid of recombinant lipase.Inducing sustainedly stopped to the 5th day.
The preparation of polyvinyl alcohol sweet oil emulsion: add the 4g polyvinyl alcohol in the 100mL ultrapure water, the microwave heating dissolving becomes polyvinyl alcohol solution.With polyvinyl alcohol solution and the sweet oil mixed with 4:1 (w:w), high-speed homogenizer stirs 3 times then, each 1min, 3min at interval; Get polyvinyl alcohol sweet oil emulsion.
The lipase activity determination step is as follows: add 0.025mol/L in Erlenmeyer flask, the phosphoric acid buffer 5mL of pH7.5 and polyvinyl alcohol sweet oil emulsion 4mL, 40 ℃ of water-bath 5min.The 1mL crude enzyme liquid that adds embodiment 2 gained then, reaction 15min adds 95% ethanol 15mL termination reaction, adds 3 of phenolphthalein indicators again.Become pink with 0.05mol/L sodium hydroxide titration to solution, deduct the blank sodium hydroxide that consumes.Per minute is decomposed sweet oil to be discharged the required enzyme amount of 1 μ mol free fatty acids and is defined as 1 lipase activity unit (U) [136].The enzyme of enzyme liquid available following formula alive calculates:
Particular content as shown in Figure 1.Found that with the prolongation of induction time, the activity of recombinant lipase raises gradually in the fermented liquid supernatant, during by the 5th day, the enzyme work in the fermented liquid supernatant reaches 10.68U/mL, and among the original Candida antarctica lipase A almost detect less than.
The purifying of embodiment 4, recombinant lipase:
Adopt hollow-fibre membrane that the crude enzyme liquid of embodiment 2 gained is carried out ultrafiltration and concentration, the molecular weight that dams of ultra-filtration membrane is 10kD.When enzyme liquid is concentrated into smaller size smaller, constantly add the phosphoric acid buffer of 25mmol/L, to the concentrated solution washing of dialysing.
Ion exchange chromatography uses the DEAE-cellulose medium.Phosphoric acid buffer with 20mmol/L pH6.0 carries out balance earlier, will impose on the ion exchange column upper end then with the concentrated enzyme liquid that the phosphoric acid buffer of 20mmol/L pH6.0 is dialysed, and the NaCl solution with 0～0.5mol/L carries out wash-out afterwards, and flow velocity is 2mL/min.Collect all elutriants, 10 pipes are measured enzymic activity at interval, and adopt SDS-PAGE to detect, and will have only the collection tube of recombinant lipase band to merge, and get pure recombinant lipase.
The character of embodiment 5, recombinant lipase
1), the optimal reactive temperature of recombinant lipase and thermostability
In 30～80 ℃ of scopes, with 10 ℃ be gradient, the enzyme of measuring under the differing temps is lived.After recording optimum temperuture, the enzyme that adds again when surveying 5 ℃ of optimum temperuture plus-minuss is lived.Live to measure enzyme that the enzyme activity under the temperature is defined as 100% when the highest, calculate the relative reactivity of enzyme under other temperature.Under 30～80 ℃, is gradient be incubated 30min after, again in 40 ℃ time to measure its vigor with 10 ℃ with enzyme liquid.Be defined as 100% with the lipase activity of preserving down at 0 ℃, calculate the relative surplus activity of handling the back enzyme under other temperature.
Found that the optimal reactive temperature of recombinant lipase is 75 degree, and is slightly higher than original lipase (50-70 ℃); Recombinant C AL-A has good thermostability, has only when envrionment temperature surpasses 70 ℃, just can exert an influence to its activity.And original lipase has stability preferably below 60 ℃.
2), the optimal reaction pH of recombinant lipase and pH stability
In the damping fluid of pH3.0～10.0, measure this lipase activity down for 40 ℃.With measure enzyme activity when the highest enzyme work under the pH value be defined as 100%, calculate the relative reactivity under other pH value.With recombinant lipase liquid respectively in the corresponding buffer solution system of pH3.0～10.0 25 ℃ preserve 16h, measure its enzyme down in 40 ℃ of pH7.5 again and live, make the relative vigor curve of enzyme.
Found that the optimal reaction pH of recombinant lipase is 7, and is identical with original lipase; Recombinant lipase has stability preferably in the scope of pH4-8, with original lipase basically identical.
3), the different chemical material is to the influence of reorganization lipase activity
Enzyme liquid joined in the solution that contains the different chemical material in 25 ℃ and handle 30min, in the phosphoric acid buffer reaction system, measure enzyme then and live.With water is contrast, calculates the influence of other chemical substance to lipase activity.The result is as shown in table 1 below:
Table 1, different chemical material are to the influence of reorganization lipase activity
| Chemical substance | Concentration (mmol/L) | |
| CoCl | ||
| 2 | 10 | 175±22 |
| |
10 | 89±12 |
| Li 2SO 4 | 10 | 86±16 |
| |
10 | 91±7.8 |
| CuSO 4 | 10 | 86±3.9 |
| HgSO 4 | 10 | 50±7.9 |
| AgNO 3 | 10 | 84±17 |
| Pb(Ac) 2 | 10 | 84±7.9 |
| ZnSO 4 | 10 | 91±10 |
| |
10 | 93±10 |
| |
10 | 82±12 |
| Fe 2(SO 4) 3 | 10 | 93±16 |
| |
10 | 89±6.8 |
| |
10 | 265±18 |
Embodiment 6, recombinant lipase are to the mensuration of triglyceride sn-2 position ester linkage hydrolyzing ability
To water oil ratio is to add 2% recombinant lipase (oily w%) in the mixed solution of 3:1 (w/w), and behind 60 ℃ of reaction 35h, in the reaction system 1, the content of 3-DAG is about 13.60%, 1, and the content of 2 (2,3)-DAG is 4.73%.This shows that the ability of recombinant lipase hydrolysis TAG sn-2 position acyl group is 2.83 times of hydrolysis sn-1 (3) position acyl group ability, than original lipase height (original lipase is 1.84 times)
At last, it is also to be noted that what more than enumerate only is several specific embodiments of the present invention.Obviously, the invention is not restricted to above embodiment, many distortion can also be arranged.All distortion that those of ordinary skill in the art can directly derive or associate from content disclosed by the invention all should be thought protection scope of the present invention.
Sequence table
SEQ?ID?NO：1
SEQ?ID?NO：2
Claims (7)
1. the construction process of a tendency hydrolysis TAG sn-2 position ester bond enzyme engineering bacteria is characterized in that may further comprise the steps:
1), the clone of CAL-A gene:
With the antarctic candida genome is template, utilizes a pair of Auele Specific Primer to carry out pcr amplification;
The upstream primer of described Auele Specific Primer and downstream primer are as follows respectively:
Upstream primer: AATAAGGAATTCGCGGCGCTGCCCAACCCCTACGAC,
Downstream primer: AAGGAAAAAAGCGGCCGCCATCGACCGTGGGAACTG;
2), the structure of recombinant vectors:
Utilize restriction enzyme that the PCR product is carried out double digestion, is connected with carrier, make up recombinant vectors, described carrier is pPIC9K; Then above-mentioned recombinant vectors conversion is entered in the intestinal bacteria, identify, filter out positive recombinant vectors by PCR;
3), transform:
With above-mentioned positive recombinant vectors, to adopt restriction enzyme to carry out linearizing, and its electricity commentaries on classics is entered in the host bacterium, described host bacterium is Pichia yeast GS115; Adopt PCR to identify then, filter out positive engineering bacteria.
2. the construction process of tendency hydrolysis TAG sn-2 according to claim 1 position ester bond enzyme engineering bacteria, it is characterized in that: the PCR primers designed described step 2) is: the standard primer of the Auele Specific Primer in the step 1) or this carrier.
3. the construction process of tendency hydrolysis TAG sn-2 according to claim 2 position ester bond enzyme engineering bacteria, it is characterized in that: the restriction enzyme in the described step 3) is SalI or SacI.
4. the construction process of tendency hydrolysis TAG sn-2 according to claim 3 position ester bond enzyme engineering bacteria, it is characterized in that: the PCR primers designed in the described step 3) is: the combination of the standard primer of the Auele Specific Primer in the step 1), this carrier or the standard primer of the Auele Specific Primer in the step 1) and this carrier.
5. the engineering bacteria that utilizes in the claim 1～4 any one method to obtain prepares the method for recombinant lipase, and it is characterized in that: the positive engineering bacteria of gained is seeded in the BMGY substratum, and 30 ℃ of incubated overnight are to OD
600Be 2～6; Centrifugal then collection thalline is diluted to OD with the BMMY substratum again
600Be 1, after 12h adds the methanol induction expression that accounts for cumulative volume 0.5%, induces 120 hours,, get the crude enzyme liquid of recombinant lipase medium centrifugal.
6. the method for preparing recombinant lipase according to claim 5 is characterized in that: the crude enzyme liquid of recombinant lipase is concentrated and purifying, get recombinant lipase.
7. the method for preparing recombinant lipase according to claim 6 is characterized in that: described enrichment step adopts hollow carboxymethyl cellulose film, and described purification step adopts ion exchange chromatography.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2008101222973A CN101407819B (en) | 2008-11-17 | 2008-11-17 | Method for constructing ester bond enzyme engineering bacteria for tendency hydrolysis of TAG sn-2 and corresponding recombinant lipase |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2008101222973A CN101407819B (en) | 2008-11-17 | 2008-11-17 | Method for constructing ester bond enzyme engineering bacteria for tendency hydrolysis of TAG sn-2 and corresponding recombinant lipase |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN101407819A CN101407819A (en) | 2009-04-15 |
| CN101407819B true CN101407819B (en) | 2011-05-04 |
Family
ID=40571030
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2008101222973A Expired - Fee Related CN101407819B (en) | 2008-11-17 | 2008-11-17 | Method for constructing ester bond enzyme engineering bacteria for tendency hydrolysis of TAG sn-2 and corresponding recombinant lipase |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN101407819B (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104450670B (en) * | 2014-12-09 | 2019-01-25 | 云南师范大学 | A method of improving host cell inulinase-producing activity |
| CN114426993B (en) * | 2022-02-18 | 2024-01-26 | 山东省农业科学院 | Method for obtaining 1, 3-diglyceride from high oleic acid sunflower seed oil |
-
2008
- 2008-11-17 CN CN2008101222973A patent/CN101407819B/en not_active Expired - Fee Related
Also Published As
| Publication number | Publication date |
|---|---|
| CN101407819A (en) | 2009-04-15 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN102796675B (en) | Rhodotorula glutinis oil genetic engineering strain and construction method and application thereof | |
| CN110846346B (en) | Microbial oil rich in Sn-2 DHA, and preparation method and application thereof | |
| CN103820335B (en) | Mortierella alpina, M. alpina genetic engineering strain of overexpression omega 3 desaturase gene and construction method of strain | |
| AU2020277167B2 (en) | Microbial oil containing ara at sn-2 position and preparation method and uses therefor | |
| CN102834523A (en) | Process for biodiesel production from a yeast strain | |
| CN103361327A (en) | Recombinant pichia pastoris for heterogenous high level expression of lipase | |
| CN107557309A (en) | The method that microbial fermentation produces single cell protein and Unicell Oils and Fats | |
| CN104388485B (en) | A kind of production method of the glyceride type fish oil rich in polyunsaturated fatty acid | |
| CN107488615A (en) | The pseudomonad of one plant height yielding lipase and its enzymatic production method | |
| CN101407819B (en) | Method for constructing ester bond enzyme engineering bacteria for tendency hydrolysis of TAG sn-2 and corresponding recombinant lipase | |
| CN103243038B (en) | Yeast engineering strain for expressing lipase mutants of thermomyces lanuginosus | |
| CN104726477A (en) | Lipase coding gene and engineering strain thereof | |
| CN109666709A (en) | A method of diglyceride is prepared by raw material of high-acid value grease | |
| CN105567579A (en) | Recombinant strain for expressing heterogenous Omega-3 desaturase in mortierella alpina and construction method thereof | |
| CN103555600A (en) | Yeast engineered strain of high-activity-expression thermomyces lanuginosus lipase LN | |
| CN104278004B (en) | Escherichia F6, the F6 lipase of expression lipase and its production and application | |
| CN107446904A (en) | A kind of lipase and its production method and application | |
| CN107893032A (en) | Utilize the Karl Jaspers bacterial strain of vegetable fat deodorizing distillate production lipase | |
| CN102839134B (en) | Yeast strain for producing alpha-linolenic acid, culture method and application thereof | |
| CN107760657B (en) | Culture medium for sn-2 selective extracellular lipase and use method thereof | |
| CN106318984A (en) | Method for preparing fatty glyceride by using oleaginous microorganisms as well as glyceride and applications thereof | |
| CN104630079A (en) | Recombinant mortierella alpina strain for heterologous expression of linoleic acid isomerase gene and construction method thereof | |
| CN105462863A (en) | Alkaline-lipase-producing Aspergillus niger mutant strain | |
| CN102373229B (en) | Saccharomyces oleaginosus delta12 and delta15 difunctional fatty acid desaturase gene and cloning method thereof | |
| CN109355323A (en) | A kind of method that low temperature compound lipases improve glycerol ester type omega-fatty acid content in transesterification |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| EE01 | Entry into force of recordation of patent licensing contract |
Application publication date: 20090415 Assignee: Hangzhou Guan Hai Technology Co., Ltd. Assignor: Zhejiang University Contract record no.: 2014330000193 Denomination of invention: Method for constructing ester bond enzyme engineering bacteria for tendency hydrolysis of TAG sn-2 and corresponding recombinant lipase Granted publication date: 20110504 License type: Exclusive License Record date: 20140523 |
|
| LICC | Enforcement, change and cancellation of record of contracts on the licence for exploitation of a patent or utility model | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20110504 Termination date: 20201117 |
|
| CF01 | Termination of patent right due to non-payment of annual fee |