CN101899427B - Lipase mutant with improved activity and built by orthogenesis - Google Patents
Lipase mutant with improved activity and built by orthogenesis Download PDFInfo
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- CN101899427B CN101899427B CN2009102351853A CN200910235185A CN101899427B CN 101899427 B CN101899427 B CN 101899427B CN 2009102351853 A CN2009102351853 A CN 2009102351853A CN 200910235185 A CN200910235185 A CN 200910235185A CN 101899427 B CN101899427 B CN 101899427B
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Abstract
The invention discloses a lipase mutant with improved activity and built by orthogenesis, belonging to the technical field of genetic engineering of enzyme. The invention discloses a series of lipase mutants obtained by orthogenesis of Rhizopus chinensis CCT CC M 201021 lipase genes. In the amino acid sequence of each mutant, the related amino acid is mutated into Ala129Ser, Lys161Arg, Thr195Tyr, Ala 230Thr, Val261Gly, Lys322Arg, Ser373Asn and the combination of two, three or four mutants of the amino acid. The lipase mutant is represented by a turnover number Kcat, and the enzyme activity of the mutants is improved if compared with parent strains.
Description
Technical field
The invention belongs to the gene engineering technology field of enzyme, relate in particular to the zhizopchin lipase mutant that enzyme activity improves.
Background technology
Lypase (EC 3.1.1.3) can not only catalyzing oil hydrolysis by using, also can be in nonaqueous phase reactions such as synthetic, the transesterificationization of catalysis ester, acidolysis, be widely used in chemistry, food is in pharmacy and washing composition or the bioenergy industry.Mikrobe is an important source of lypase, and head mold is the important production bacterium of microbial lipase.Nowadays, existingly surpass 30 kinds of rizolipases and realized commercialization production.Rizolipase mostly has height 1, and therefore the 3-regioselectivity is usually used in the grease processing.In addition, it is good that rizolipase also has stability, and the transformation efficiency advantages of higher is widely used in the production of aromatic ester, biofuel, chipal compounds.
So far, reported the gene order of a plurality of rizolipases both at home and abroad.Japan, Germany are to Rhizopus oryzae lypase (Rhizopus oryzae lipase; ROL) gene order has been done more deep research with expressing; And successively with intestinal bacteria, yeast saccharomyces cerevisiae and pichia pastoris phaff successful expression lipase gene (Minning S et al.J Biotechnol; 1998,66:147-156; Beer HD et al.Biochim BiophysActa, 1998,1399:173-180; Ueda M et al.J Mol Catal B:Enzym, 2002,17:113-124).The contriver successfully screens zhizopchin (the Rhizopus chinensis CCTCC M 201021) bacterial strain of a plant height yielding lipase from the distiller's yeast of brewing aroma type yeast wine in early-stage Study; And the clone obtains lipase gene sequence (Genbank accession number EF405962) from this bacterial strain; And realize high-level secretory expression (the Yu Xiao-Wei et al.J MolCatal B:Enzym of this lypase in pichia pastoris phaff (Pichia pastoris); 2009,57:304-311).
Orthogenesis belongs to irrational design; Be meant through simulation Darwin nature evolutionary process in the laboratory, to the gene of a certain protein enzyme, the gene of the enzyme of transforming through improved induced-mutation technique; According to specific transformation purpose, screen valuable natural enzyme then.Over nearly 10 years; The orthogenesis technology obtains great success at esterase and lypase character transformation field, mainly concentrates on the catalytic reaction activity that improves enzyme, improves substrate specificity; Improve thermostability; Aspects such as enantio-selectivity (Johannes TW et al.Curr.Opin.Microbiol, 2006,9:261-267).
The raising of enzyme activity can be used the kinetic parameter K of enzyme
CatValue is represented.K
CatClaim turn over number again, refer to per molecule enzyme or each enzyme active center ability catalytic substrate molecule number (TN) in the unit time, be also referred to as catalytic constant.Therefore, K
CatThe raising that on behalf of enzyme, the raising of value promptly live.
Summary of the invention
The technical problem that the present invention solves provides the zhizopchin lypase that enzyme activity improves.
Technical scheme of the present invention: the lipase mutant that the vigor that makes up through orthogenesis improves; By zhizopchin (Rhizopus chinensis) CCTCC M 201021 lipase genes (Genbank accession number EF405962); Utilization fallibility PCR method; Through many wheel reorganization and rite-directed mutagenesis; The lipase mutant that obtains through orthogenesis in the aminoacid sequence of two mutants, comprises the combination of amino acid mutation Ala129Ser, Lys161Arg, Thr195Tyr, Ala230Thr, Val261Gly, Lys322Arg, Ser373Asn and two, three or four sudden changes of above-mentioned amino acid; With turnover number K
CatExpression, the enzyme work of lipase mutant is improved than starting strain; The sequencing result of two mutants and K thereof
CatThe multiple that improves is:
Lipase mutant aminoacid replacement position K
CatThe K of (/min)
CatThe multiple that improves
Starting strain lypase-1,138 1.0
Two mutants 1-1 Ala129Ser 1,252 1.1
Two mutants 1-2 Lys161Arg 1,479 1.3
Two mutants 1-3 Thr195Tyr 1,479 1.3
Two mutants 1-4 Ala230Thr 1,821 1.6
Two mutants 1-5 Val261Gly 1,935 1.7
Two mutants 1-6 Lys322Arg 1,366 1.2
Two mutants 1-7 Ser373Asn 1,707 1.5
Two mutants 2-1 Ala129Ser/Lys161Arg 2,278 2
Two mutants 2-2 Ala129Ser/Ala230Thr 1,365 1.2
Two mutants 2-3 Ala129Ser/Val261Gly 1,593 1.4
Two mutants 2-4 Lys161Arg/Thr195Tyr 2,845 2.5
Two mutants 2-5 Lys161Arg/Lys322Arg 1,707 1.5
Two mutants 2-6 Lys161Arg/Ser373Asn 2,048 1.8
Two mutants 2-7 Ala230Thr/Ser373Asn 1,707 1.5
Two mutants 2-8 Val261Gly/Ser373Asn 3,726 3.3
Two mutants 3-1 Ala129Ser/Ala230Thr/Val261Gly 3,186 2.8
Two mutants 3-2 Ala129Ser/Ala230Thr/Lys322Arg 2,278 2
Two mutants 3-3 Lys161Arg/Thr195Tyr/Ser373Asn 2,617 2.3
Two mutants 3-4 Lys161Arg/Ala230Thr/Ser373Asn 2,278 2
Two mutants 3-5 Lys161Arg/Val261Gly/Lys322Arg 2,731 2.4
Two mutants 3-6 Ala230Thr/Lys322Arg/Ser373Asn 2,845 2.5
Two mutants 4-1 Ala129Ser/Lys161Arg/Ala230Thr/ 2,073 1.8
Lys322Arg
Zhizopchin lypase amino acid original series is: SEQ ID NO:1; Zhizopchin lypase amino acid mutation sequence is: SEQ ID NO:2,7 mutating acids show with the background color mark.
Beneficial effect of the present invention: the present invention uses fallibility PCR method rizolipase gene to China (Genbank accession number EF405962) to carry out orthogenesis; Through many wheel reorganization and rite-directed mutagenesis; Obtain the zhizopchin lipase mutant, these two mutants comprise the combination of amino acid mutation Ala129Ser, Lys161Arg, Thr195Tyr, Ala230Thr, Val261Gly, Lys322Arg, Ser373Asn and above-mentioned amino acid mutation.With turnover number K
CatExpression, the enzyme work of lipase mutant is improved.
Embodiment
The culture medium prescription that relates among the embodiment is following:
The LB liquid nutrient medium: peptone 1%, yeast extract 0.5%, NaCl 1%, pH7.0.
YPD (Yeast Extract Peptone Dextrose Medium): Yeast Extract 1%, Trypton 2%, and Dextrose 2%, adds Agar 2% when making flat board.121 ℃ of autoclaving 20min.Adding G418 when being used to screen the G418 resistance is 0.25mg/mL-1.0mg/mL to final concentration, and promptly YPD-G418 is dull and stereotyped.
MD(Minimal?Dextrose?Medium)：YNB?1.34％，Biotin?4×10
-5％，Dextrose?2％，Agar?2％。
MM(Minimal?Methanol?Medium)：YNB1.34％，Biotin4×10
-5％，Methanol?0.5％，Agar2％。
BMGY (Buffered Glycerol-complex Medium): Yeast Extract 1%, Trypton 2%, and YNB 1.34%, and Biotin 4 * 10
-5%, Glycerol 1%, potassium phosphate solution 100mmol/L.
BMMY (Buffered Methanol-complex Medium): Yeast Extract 1%, Trypton 2%, and YNB 1.34%, and Biotin 4 * 10
-5%, Methanol 0.5%, potassium phosphate solution 100mmol/L.
Unit in the substratum is % (W/V).
Embodiment 1, utilize the fallibility PCR method to make up zhizopchin lypase sudden change library
Utilize the fallibility round pcr to introduce coding mutation to zhizopchin lipase gene proRCL external.
The reaction conditions of fallibility PCR is following:
dCTP(25mmol/L) 2μL
dTTP(25mmol/L) 2μL
dGTP(10mmol/L) 1μL
dATP(10mmol/L) 1μL
F(20pmol/μL) 1μL
R(20pmol/μL) 1μL
Mg
2+(25mM) 14μL
Mn
2+(5mM) 1.5μL
pPIC9K-proRCL
0.5μL
(carrying the plasmid of zhizopchin lipase gene proRCL)
Taq archaeal dna polymerase (2.5U) 1 μ L
10 * PCR damping fluid (does not contain MgCl
2) 5 μ L
ddH
2O 20μL
Wherein, primers F and R sequence are:
Upstream primer F:5 '-TCAAGATCCCTAGGGTTCCTGTTGGTCATAAAGGTTC-3 ':
Downstream primer R:5 '-AATTCCAGTGCGGCCGCTTACAAACAGCTTCCTTCG-3 '.
Pcr amplification condition: 94 ℃ of 3min; 94 ℃ of 1min, 59 ℃ of 1min, 72 ℃ of 2min, 30 circulations; 72 ℃ of 10min.
The fallibility pcr amplification product is behind DNA purification kit purifying, and restriction enzyme A vr II and NotI digest fallibility pcr amplification product and plasmid pPIC9K respectively, connects, and is converted into E.coli JM109 competent cell.Coat LB (Amp that contains 100 μ g/ μ L) flat board.Behind the growth 12h, transformant is transferred in the LB liquid nutrient medium cultivates, obtain mutant plasmid.
After restriction enzyme SalI linearizing, electricity transforms pichia spp GS115 competent cell with mutant plasmid.Conversion fluid is coated on the MD flat board, cultivated 2 days for 30 ℃, constitute the sudden change library.
Utilizing above-mentioned same method, is that masterplate carries out many wheel fallibility PCR with the mutant gene group, makes up the sudden change library.
The screening of embodiment 2, high enzyme lipase mutant alive
Use the sterilization toothpick with the His that grows on the MD flat board
+Transformant copies to YPD and the dull and stereotyped same position of BMMY, will contrast bacterium GS 115/pPIC9K-proRCL simultaneously and be seeded on the BMMY flat board.Cultivated 2 days for 30 ℃.
Dull and stereotyped primary dcreening operation: preserve the YPD flat board that growth finishes.Every 12h covers to the BMMY plate and adds the expression of 200 μ L methanol induction recombinant lipases.Induced 2-3 days.In enzyme activity determination substrate pNPP, add 0.6% agar, after mixing, fall in the dull and stereotyped top-agar that forms of BMMY.Obvious xanchromatic bacterial strain occurring in the 2min is primary dcreening operation purpose mutant strain.Under the same terms, contrast bacterium GS115/pPIC9K-proRCL can not demonstrate obvious yellow.
96 orifice plates sieve again: in 96 orifice plates of 1.8mL/ hole (flat), add 300 μ L BMGY substratum, 121 ℃ of sterilization 20min.To wherein inserting the primary dcreening operation purpose bacterial strain (inserting GS115/pPIC9K-proRCL simultaneously) be preserved on the YPD flat board as contrast, 30 ℃ of 250r/min shaking culture to OD600 be 2-6 (about 16-18h).Centrifugal, abandon supernatant, with the resuspended thalline of 900 μ L BMMY substratum, and add the expression of 1% (V/V) methanol induction lypase.After this every 24h adds 100 μ L BMMY substratum and 1% (V/V) methyl alcohol, induces 4 days.With the centrifugal 10min of 96 orifice plate fermented liquid 3000r/min of abduction delivering 96h, collect supernatant.After getting 500 times of 1 μ L supernatant dilutions, get 5 μ L in another 96 orifice plate, add substrate with the volley of rifle fire, the vibration mixing.Show the bacterial strain of obvious xanchromatic bacterial strain in the 2min rapidly for multiple sieve mesh.Under the same terms, the fermented supernatant fluid of contrast bacterium GS115/pPIC9K-proRCL can not show obvious yellow.
The sudden change library that makes up with fallibility PCR is that screen on the basis, obtains the bacterial strain that 6 strain enzymes are lived and obviously improved, and measures the lypase nucleotide sequence, utilizes triplet codon to infer the aminoacid sequence of lypase, the aminoacid replacement of lipase mutant and K
CatIt is as shown in table 1 that value improves multiple.Measure the K of lipase mutant according to the method for embodiment 3
CatValue.
The sequencing result of table 1 two mutants
| Lipase mutant | The aminoacid replacement position | K cat(/min) | K catThe multiple that improves |
| Starting strain lypase | - | 1138 | 1.0 |
| Two mutants 2-1 | Ala129Ser/Lys161Arg | 2278 | 2 |
| Two mutants 2-4 | Lys161Arg/Thr19STyr | 2845 | 2.5 |
| Two mutants 2-7 | Ala230Thr/Ser373Asn | 1707 | 1.5 |
| Two mutants 2-8 | Val261Gly/Ser373Asn | 3726 | 3.3 |
| Two mutants 3-2 | Ala129Ser/Ala230Thr/Lys322Arg | 2278 | 2 |
| Two mutants 3-4 | Lys161Arg/Ala230Thr/Ser373Asn | 2278 | 2 |
The K of embodiment 3 lipase mutants
CatPH-value determination pH
For measuring the K of lypase
CatValue need be carried out separation and purification to enzyme.
Shake flask fermentation:, be seeded in the 25mL BMGY substratum 30 ℃ of shaking culture 16～20h to OD with starting strain and each lipase mutant
600Be 2～6, centrifugal collection thalline is diluted to OD with the BMMY substratum
600Be 1, every methanol induction at a distance from 24h interpolation 0.5% is expressed, and cultivates after 3-4 days, collects fermented supernatant fluid.
Separation and purification: the fermented supernatant fluid of mutant strain is concentrated through the 10KD ultra-filtration membrane, obtain the sudden change lipase activity component of purifying behind SP-SepharoseFF strong cation exchange chromatography and the Phenyl-Sepharose 6FF HC column chromatography.Concrete operations reference Yu Xiao-Wei et al.J Mol Catal B:Enzym, 2009,57:304-311.
Measuring method:
The measuring method of lipase activity be the pNPP method (Pencreach G et al.Enzyme and MicrobialTechnol.1996,18:417-422.).Enzyme is lived, and to be defined as enzyme amount that PM under certain reaction conditions produces 1 μ mol p-NP be the lipase hydrolysis enzyme iu of living.In substrate p-NP cetylate concentration is in 0～25mmol/L scope, measures enzyme activity, calculates the kinetic parameter K that obtains lypase
Cat
The gene mutation site of embodiment 4 rite-directed mutagenesis combined lipase two mutants
Through the too much wheel fallibility PCR library construction that suddenlys change, obtain including 7 mutant strains of amino acid mutation site Ala129Ser, Lys161Arg, Thr195Tyr, Ala230Thr, Val261Gly, Lys322Arg, Ser373Asn.Reach the influence of the combination of each sudden change in order to investigate wherein some sudden changes, the rite-directed mutagenesis combination is carried out in the mutational site of finding, obtain a plurality of lipase mutants lypase mutant strain vigor.(rite-directed mutagenesis can utilize commercially available test kit to carry out.)
The gene that will contain the said mutation Sites Combination is connected with carrier pPIC9K, and electricity transforms pichia spp GS115, to obtain the efficient secretory expression of lypase.Measure the K of lipase mutant with the method that is equal to embodiment 3
CatValue.The combination site and the K thereof of each mutant enzyme
CatThe multiple that improves is as shown in table 2.
The sequencing result of table 2 two mutants and K thereof
CatThe multiple that improves
| Lipase mutant | The aminoacid replacement position | K cat(/min) | K catThe multiple that improves |
| Starting strain lypase | - | 1138 | 1.0 |
| Two mutants 1-1 | Ala129Ser | 1252 | 1.1 |
| Two mutants 1-2 | Lys161Arg | 1479 | 1.3 |
| Two mutants 1-3 | Thr195Tyr | 1479 | 1.3 |
| Two mutants 1-4 | Ala230Thr | 1821 | 1.6 |
| Two mutants 1-5 | Val261Gly | 1935 | 1.7 |
| Two mutants 1-6 | Lys322Arg | 1366 | 1.2 |
| Two mutants 1-7 | Ser373Asn | 1707 | 1.5 |
| Two mutants 2-2 | Ala129Ser/Ala230Thr | 1365 | 1.2 |
| Two mutants 2-3 | Ala129Ser/Val261Gly | 1593 | 1.4 |
| Two mutants 2-5 | Lys161Arg/Lys322Arg | 1707 | 1.5 |
| Two mutants 2-6 | Lys161Arg/Ser373Asn | 2048 | 1.8 |
| Two mutants 3-1 | Ala129Ser/Ala230Thr/Val261Gly | 3186 | 2.8 |
| Two mutants 3-3 | Lys161Arg/Thr195Tyr/Ser373Asn | 2617 | 2.3 |
| Two mutants 3-5 | Lys161Arg/Val261Gly/Lys322Arg | 2731 | 2.4 |
| Two mutants 3-6 | Ala230Thr/Lys322Arg/Ser373Asn | 2845 | 2.5 |
| Two mutants 4-1 | Ala129Ser/Lys161Arg/Ala230Thr/ Lys322Arg | 2073 | 1.8 |
Sequence table
<210>SEQ?ID?NO：1
<211>389
<212>PRT
< 213>zhizopchin (Rhizopus chinensis) CCTCC M 201021 lypase amino acid
<400>1
Met?Val?Ser?Phe?Ile Ser?Ile?Ser?Gln?Gly Val?Ser?Leu?Cys?Leu
5 10 15
Leu?Val?Ser?Ser?Met Met?Leu?Gly?Ser?Ser Ala?Val?Pro?Val?Ala
20 25 30
Gly?His?Lys?Gly?Ser Val?Lys?Ala?Thr?Asn Gly?Thr?Asp?Phe?Gln
35 40 45
Leu?Pro?Pro?Leu?Ile Ser?Ser?Arg?Cys?Thr Pro?Pro?Ser?His?Pro
50 55 60
Glu?Thr?Thr?Gly?Asp Pro?Asp?Ala?Glu?Ala Tyr?Tyr?Ile?Asn?Lys
65 70 75
Ser?Val?Gln?Trp?Tyr Gln?Ala?His?Gly?Gly Asn?Tyr?Thr?Ala?Leu
80 85 90
Ile?Lys?Arg?Asp?Thr Glu?Thr?Val?Gly?Gly Met?Thr?Leu?Asp?Leu
95 100 105
Pro?Glu?Asn?Pro?Pro Pro?Ile?Pro?Ala?Thr Ser?Thr?Ala?Pro?Ser
110 115 120
Ser?Asp?Ser?Gly?Glu Val?Val?Thr?Ala?Thr Ala?Ala?Gln?Ile?Lys
125 130 135
Glu?Leu?Thr?Asn?Tyr Ala?Gly?Val?Ala?Ala Thr?Ala?Tyr?Cys?Arg
140 145 150
Ser?Val?Val?Pro?Gly Thr?Lys?Trp?Asp?Cys Lys?Gln?Cys?Leu?Lys
155 160 165
Tyr?Val?Pro?Asp?Gly Lys?Leu?Ile?Lys?Thr Phe?Thr?Ser?Leu?Leu
170 175 180
Thr?Asp?Thr?Asn?Gly Phe?Ile?Leu?Arg?Ser Asp?Ala?Gln?Lys?Thr
185 190 195
Ile?Tyr?Val?Thr?Phe Arg?Gly?Thr?Asn?Ser Phe?Arg?Ser?Ala?Ile
200 205 210
Thr?Asp?MET?Val?Phe Thr?Phe?Thr?Asp?Tyr Ser?Pro?Val?Lys?Gly
215 220 225
Ala?Lys?Val?His?Ala Gly?Phe?Leu?Ser?Ser Tyr?Asn?Gln?Val?Val
230 235 240
Lys?Asp?Tyr?Phe?Pro Val?Val?Gln?Asp?Gln Leu?Thr?Ala?Tyr?Pro
245 250 255
Asp?Tyr?Lys?Val?Ile Val?Thr?Gly?His?Ser Leu?Gly?Gly?Ala?Gln
260 265 270
Ala?Leu?Leu?Ala?Gly Met?Asp?Leu?Tyr?Gln Arg?Glu?Lys?Arg?Leu
275 280 285
Ser?Pro?Lys?Asn?Leu Ser?Ile?Tyr?Thr?Val Gly?Cys?Pro?Arg?Val
290 295 300
Gly?Asn?Asn?Ala?Phe Ala?Tyr?Tyr?Val?Asp Ser?Thr?Gly?Ile?Pro
305 310 315
Phe?His?Arg?Thr?Val His?Lys?Arg?Asp?Ile Val?Pro?His?Val?Pro
320 325 330
Pro?Gln?Ala?Phe?Gly Tyr?Leu?His?Pro?Gly Val?Glu?Ser?Trp?Ile
335 340 345
Lys?Glu?Asp?Pro?Ala Asp?Val?Gln?Ile?Cys Thr?Ser?Asn?Ile?Glu
350 355 360
Thr?Lys?Gln?Cys?Ser Asn?Ser?Ile?Val?Pro Phe?Thr?Ser?Ile?Ala
365 370 375
Asp?His?Leu?Thr?Tyr Phe?Gly?Ile?Asn?Glu Gly?Ser?Cys?Leu
380 385 389
<210>SEQ?ID?NO：2
<211>389
<212>PRT
< 213>zhizopchin (Rhizopus chinensis) CCTCC M 201021 lypase amino acid mutation bodies
<400>2
Met?Val?Ser?Phe?Ile Ser?Ile?Ser?Gln?Gly Val?Ser?Leu?Cys?Leu
5 10 15
Leu?Val?Ser?Ser?Met Met?Leu?Gly?Ser?Ser Ala?Val?Pro?Val?Ala
20 25 30
Gly?His?Lys?Gly?Ser Val?Lys?Ala?Thr?Asn Gly?Thr?Asp?Phe?Gln
35 40 45
Leu?Pro?Pro?Leu?Ile Ser?Ser?Arg?Cys?Thr Pro?Pro?Ser?His?Pro
50 55 60
Glu?Thr?Thr?Gly?Asp Pro?Asp?Ala?Glu?Ala Tyr?Tyr?Ile?Asn?Lys
65 70 75
Ser?Val?Gln?Trp?Tyr Gln?Ala?His?Gly?Gly Asn?Tyr?Thr?Ala?Leu
80 85 90
Ile?Lys?Arg?Asp?Thr Glu?Thr?Val?Gly?Gly Met?Thr?Leu?Asp?Leu
95 100 105
Pro?Glu?Asn?Pro?Pro Pro?Ile?Pro?Ala?Thr Ser?Thr?Ala?Pro?Ser
110 115 120
Ser?Asp?Ser?Gly?Glu Val?Val?Thr?Ser?Thr Ala?Ala?Gln?Ile?Lys
125 130 135
Glu?Leu?Thr?Asn?Tyr Ala?Gly?Val?Ala?Ala Thr?Ala?Tyr?Cys?Arg
140 145 150
Ser?Val?Val?Pro?Gly Thr?Lys?Trp?Asp?Cys Arg?Gln?Cys?Leu?Lys
155 160 165
Tyr?Val?Pro?Asp?Gly Lys?Leu?Ile?Lys?Thr Phe?Thr?Ser?Leu?Leu
170 175 180
Thr?Asp?Thr?Asn?Gly Phe?Ile?Leu?Arg?Ser Asp?Ala?Gln?Lys?Tyr
185 190 195
Ile?Tyr?Val?Thr?Phe Arg?Gly?Thr?Asn?Ser Phe?Arg?Ser?Ala?Ile
200 205 210
Thr?Asp?MET?Val?Phe Thr?Phe?Thr?Asp?Tyr Ser?Pro?Val?Lys?Gly
215 220 225
Ala?Lys?Val?His?Thr Gly?Phe?Leu?Ser?Ser Tyr?Asn?Gln?Val?Val
230 235 240
Lys?Asp?Tyr?Phe?Pro Val?Val?Gln?Asp?Gln Leu?Thr?Ala?Tyr?Pro
245 250 255
Asp?Tyr?Lys?Val?Ile Gly?Thr?Gly?His?Ser Leu?Gly?Gly?Ala?Gln
260 265 270
Ala?Leu?Leu?Ala?Gly Met?Asp?Leu?Tyr?Gln Arg?Glu?Lys?Arg?Leu
275 280 285
Ser?Pro?Lys?Asn?Leu Ser?Ile?Tyr?Thr?Val Gly?Cys?Pro?Arg?Val
290 295 300
Gly?Asn?Asn?Ala?Phe Ala?Tyr?Tyr?Val?Asp Ser?Thr?Gly?Ile?Pro
305 310 315
Phe?His?Arg?Thr?Val His?Arg?Arg?Asp?Ile Val?Pro?His?Val?Pro
320 325 330
Pro?Gln?Ala?Phe?Gly Tyr?Leu?His?Pro?Gly Val?Glu?Ser?Trp?Ile
335 340 345
Lys?Glu?Asp?Pro?Ala Asp?Val?Gln?Ile?Cys Thr?Ser?Asn?Ile?Glu
350 355 360
Thr?Lys?Gln?Cys?Ser Asn?Ser?Ile?Val?Pro Phe?Thr?Asn?Ile?Ala
365 370 375
Asp?His?Leu?Thr?Tyr Phe?Gly?Ile?Asn?Glu Gly?Ser?Cys?Leu
380 385 389
<210>SEQ?ID?NO：3
<400>3
F：5′-TCAAGATCCCTAGGGTTCCTGTTGGTCATAAAGGTTC-3′；
R：5′-AATTCCAGTGCGGCCGCTTACAAACAGCTTCCTTCG-3′。
Claims (1)
1. the lipase mutant that the vigor that makes up through orthogenesis improves; It is characterized in that 201021 lipase genes, its Genbank accession number EF405962, utilization fallibility PCR method by zhizopchin (Rhizopus chinensis) CCTCC M; Through many wheel reorganization and rite-directed mutagenesis; The lipase mutant that obtains through orthogenesis in the aminoacid sequence of two mutants, comprises amino acid mutation Ala129Ser; With turnover number K
CatExpression, the enzyme work of lipase mutant is improved than starting strain; The sequencing result of two mutants and K thereof
CatThe multiple that improves is:
。
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|---|---|---|---|
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| CN 201210083907 Division CN102604910B (en) | 2009-11-11 | 2009-11-11 | Lipase mutant with high catalytic activity |
| CN 201210083900 Division CN102586203B (en) | 2009-11-11 | 2009-11-11 | Determinate-evolution-constructed lipase mutant with improved catalysis activity |
| CN 201210083902 Division CN102604908B (en) | 2009-11-11 | 2009-11-11 | Lipase mutant with high catalytic activity |
| CN 201210083905 Division CN102604909B (en) | 2009-11-11 | 2009-11-11 | Directed-evolution structured lipase mutant with enhanced catalytic activity |
| CN 201210083915 Division CN102604912B (en) | 2009-11-11 | 2009-11-11 | Rhizopuschinensis lipase mutant with high activity |
| CN 201210083913 Division CN102604911B (en) | 2009-11-11 | 2009-11-11 | Rhizopuschinensis lipase mutant with high activity |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102586203A (en) * | 2009-11-11 | 2012-07-18 | 江南大学 | Determinate-evolution-constructed lipase mutant with improved catalysis activity |
| CN102604909A (en) * | 2009-11-11 | 2012-07-25 | 江南大学 | Directed-evolution structured lipase mutant with enhanced catalytic activity |
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| CN102660517B (en) * | 2011-12-08 | 2013-04-03 | 上海交通大学 | Lipase mutant with improved heat stability, and construction method thereof |
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| CN102604909A (en) * | 2009-11-11 | 2012-07-25 | 江南大学 | Directed-evolution structured lipase mutant with enhanced catalytic activity |
| CN102586203B (en) * | 2009-11-11 | 2013-04-17 | 江南大学 | Determinate-evolution-constructed lipase mutant with improved catalysis activity |
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| CN101899427A (en) | 2010-12-01 |
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