CN102653743B - Thermal stability improved lipase mutant constructed through orthogenesis - Google Patents

Thermal stability improved lipase mutant constructed through orthogenesis Download PDF

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CN102653743B
CN102653743B CN 201210145503 CN201210145503A CN102653743B CN 102653743 B CN102653743 B CN 102653743B CN 201210145503 CN201210145503 CN 201210145503 CN 201210145503 A CN201210145503 A CN 201210145503A CN 102653743 B CN102653743 B CN 102653743B
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CN102653743A (en
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喻晓蔚
徐岩
王睿
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Jinhu agricultural and sideline products Marketing Association
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Jiangnan University
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Abstract

The invention relates to a thermal stability improved lipase mutant constructed through orthogenesis, belonging to the technical field of enzymatic genetic engineering. The invention discloses a thermal stability improved lipase mutant obtained through a molecular biological technology by using a Rhizopuschinensis CCTCC M201021 lipase as a parent. In amino acid sequences of each mutant, the related amino acid mutant is one or more of Met101Thr, Glu107Gly, Ala129Ser, Ser151Asn, Cys160Leu, Lys161Arg, Pro168Leu, Pro168His, Leu180His, Asp182Tyr, Thr183Ala, Thr218Ser, Lys219Asp, Ala230Phe, Ser234Phe, Val261Gly, His317Pro, Val329Ala, Glu363Arg, Asn366Asp and Ser373Cys. The mutants are represented by a half-life period of t50 at 65 DEG C, and the thermal stability of the mutants are improved compared with the parent of Rhizopuschinensis lipase. The invention also discloses a DNA (Deoxyribonucleic Acid) sequence, an expression vector and a host cell for encoding the lipase mutant.

Description

The lipase mutant that the thermostability that makes up by orthogenesis improves
Technical field
The lipase mutant that the thermostability that the present invention relates to make up by orthogenesis improves relates in particular to and utilizes Protocols in Molecular Biology to obtain the lipase mutant that thermostability improves, and belongs to the gene engineering technology field of enzyme.
Background technology
Lipase (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.Microorganism is an important source of lipase, 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.But grease processing need be carried out under comparatively high temps usually, and rizolipase belong in warm lipase, only have about 15 min in the transformation period under 65 ℃, poor heat stability has not only limited its range of application, and is easy to inactivation, has increased production cost.
Orthogenesis belongs to irrational design, refer to by simulation Darwin nature evolutionary process in the laboratory, at the gene of a certain protein enzyme, the gene of the enzyme of transforming by improved induced-mutation technique, according to the specific purpose of transforming, screen valuable natural enzyme then.Over nearly 10 years, the orthogenesis technology obtains great success at esterase and lipase 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 contriver in early-stage Study, successfully from the distiller's yeast of brewing aroma type yeast wine, screen a plant height yielding lipase zhizopchin ( Rhizopus chinensis) CCTCC M 201021 bacterial strains, and from this bacterial strain first clone obtain lipase gene sequence, and realize this lipase pichia pastoris phaff ( Pichia pastoris) in high-level secretory expression (Yu Xiao-Wei et al. J Mol Catal B:Enzym, 2009,57:304-311).The present invention is template with the zhizopchin lipase gene, utilizes the orthogenesis technology to obtain the lipase mutant that thermostability significantly improves.
The thermostability of enzyme molecule improves can be with its transformation period (t 50) embody.The vigor that is enzyme is reduced to the original needed time of vigor one half.Long half time, then enzyme stability height.Otherwise, poor stability then.Therefore, t 50Raising represented the raising of enzyme molecular heat stability.
Definition:
The nomenclature of amino acid and DNA nucleotide sequence
Use the generally acknowledged IUPAC nomenclature of amino-acid residue, use the trigram code form.The DNA nucleotide sequence adopts generally acknowledges the IUPAC nomenclature.
The sign of lipase mutant
Adopt " amino acid that original amino acid position is replaced " to represent the amino acid that suddenlys change in the lipase mutant.As Glu107Gly, the amino acid of expression position 107 replaces to Gly by the Glu of parent lipase, and the numbering of position is corresponding to the aminoacid sequence numbering of parent's zhizopchin lipase RCL in the annex sequence table 1.
As Leu180His/ Thr218Ser, sudden change has all taken place in the amino acid of expression position 180 and position 218.
Summary of the invention
The object of the present invention is to provide the lipase mutant of the thermostability raising that makes up by orthogenesis.
Technical scheme of the present invention: the lipase mutant that the thermostability that makes up by orthogenesis improves, by parent's zhizopchin ( Rhizopus chinensis) CCTCC M 201021 lipase carry out orthogenesis, by fallibility PCR, DNA Shuffling and rite-directed mutagenesis, obtain lipase mutant, undergo mutation in the aminoacid replacement position among parent's zhizopchin aminoacid sequence SEQ ID NO:1, adopt " amino acid of original amino acid-position-replacement " to represent the amino acid that suddenlys change in the lipase mutant, described lipase mutant is:
Table 1
Figure 340786DEST_PATH_IMAGE001
The aminoacid sequence of zhizopchin lipase amino acid mutation body is SEQ ID NO:2, and 22 mutating acids are arranged, and the background color mark shows.
The expression vector that is used for the described lipase mutant of expression is: pPIC9K, pPIC3.5K, pPICZ α, pPICZ;
Being used for described expression vector microorganism transformed host cell is pichia spp.
Compare with parent's zhizopchin lipase, the thermostability of described lipase mutant has obtained raising, with the transformation period under 65 ℃ t 50Represent the raising of thermostability, the transformation period of described lipase mutant and the multiple of raising are as follows:
Table 2
Figure 943412DEST_PATH_IMAGE002
The transformation period of parent's zhizopchin lipase under 65 ℃ t 50Be 16.5 min.
Beneficial effect of the present invention: use molecular biology method that parent's zhizopchin lipase is carried out orthogenesis, by fallibility PCR, DNA Shuffling and rite-directed mutagenesis, obtain lipase mutant, with transformation period t 50Expression, the thermostability of lipase mutant is improved, and is as shown in table 2.
Description of drawings
Determine mutating acid according to Shanghai Sangon Biological Engineering Technology And Service Co., Ltd's sequencing result, the amino acid mutation site sequencing result of each mutant is as follows.
Fig. 1 lipase mutant 1:Ser234Phe peak figure that checks order.
Fig. 2 lipase mutant 2:a:Pro168Leu, the b:Val329Ala peak figure that checks order.
Fig. 3 lipase mutant 3:Cys160Leu peak figure that checks order.
Fig. 4 lipase mutant 4:a:His317Pro, the b:Met101Thr peak figure that checks order.
Fig. 5 lipase mutant 5:a:Ser151Asn, the b:Leu180His peak figure that checks order.
Fig. 6 lipase mutant 6:a:Val261Gly, the b:Ser373Cys peak figure that checks order.
Fig. 7 lipase mutant 7:a:Asp182Tyr, the b:Ala230Phe peak figure that checks order.
Fig. 8 lipase mutant 8:a:Leu180His, the b:Thr218Ser peak figure that checks order.
Fig. 9 lipase mutant 9:a:Asn366Asp, b:Leu180His, the c:Val329Ala peak figure that checks order.
Figure 10 lipase mutant 10:a:Ser234Phe, b:Leu180His, the c:Thr218Ser peak figure that checks order.
Figure 11 lipase mutant 11:a:Ser234Phe, b:Pro168 His, c:Val329Ala, d:Cys160Leu, e:His317Pro, the f:Met101Thr peak figure that checks order.
Figure 12 lipase mutant 12:a:Leu180His, b:Thr218Ser, c:Val261Gly, the d:Lys161Arg peak figure that checks order.
Figure 13 lipase mutant 13:a:Glu107Gly, b:Ala129Ser, C:Glu363Arg, d:Lys161Arg, e:Val261Gly, the f:Leu180His peak figure that checks order.
Figure 14 lipase mutant 14:a:Lys161Arg, b:Leu180His, c:Thr218Ser, d:Met101Thr, e:Thr183Ala, f:Glu107Gly, g:Ser151Asn, the h:Glu363Arg peak figure that checks order.
Figure 15 lipase mutant 15:a:Glu107Gly, b:Ala129Ser, c:Ser151Asn, d:Lys161Arg, e:Leu180His, f:Ser234Phe, g:Glu363Arg, the h:Val329Ala peak figure that checks order.
Figure 16 lipase mutant 12-1:a:Ala230Phe, b:Leu180His, c:Thr218Ser, d:Val261Gly, the e:Lys161Arg peak figure that checks order.
Figure 17 lipase mutant 13-1:a:Glu107Gly, b:Ala129Ser, c:Ser151Asn, d:Lys219Asp, e:Leu180His, f:Glu363Arg, the g:Ala230Phe peak figure that checks order.
Embodiment
The substratum and the agent prescription that relate among the embodiment are as follows:
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 C autoclaving 20 min.Add when being used for screening G418 resistance G418 to final concentration be 0.25 mg/mL-1.0 mg/mL, i.e. YPD-G418 flat board.
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 pH 6.0,100 mmol/L.
BMMY (Buffered Methanol-complex Medium): Yeast Extract 1%, Trypton 2%, YNB1.34%, Biotin 4 * 10 -5%, Methanol 0.5%, potassium phosphate solution 100 mmol/L.
Unit in the substratum is %(W/V)
Fast-blue RR staining agent: 360 μ L naphthalene esters (20mg naphthalene ester is dissolved in 1mL 2-methylformamide) are dissolved in the 1mL methyl-sulphoxide with 160 μ L Fast blue RR(80mg Fast blue RR) mix mutually.
Embodiment 1, utilize the pichia spp library of fallibility PCR method construction expression lipase mutant
Utilize the fallibility round pcr to introduce coding mutation external to zhizopchin lipase gene proRCL.The reaction conditions of fallibility PCR is as follows:
Figure 723150DEST_PATH_IMAGE003
Wherein, upstream primer F and downstream primer R sequence are:
F: 5'-TCAAGATCCCTAGGGTTCCTGTTGGTCATAAAGGTTC-3';
R: 5'-AATTCCAGTGCGGCCGCTTACAAACAGCTTCCTTCG-3'。
Pcr amplification condition: 94 ℃ of 3min; 94 ℃ of 1 min, 59 ℃ of 1 min, 72 ℃ of 2 min, 30 circulations; 72 ℃ of 10 min.
The fallibility pcr amplification product behind DNA purification kit purifying, restriction enzyme AvrII and NotI digests fallibility pcr amplification product and plasmid pPIC9K respectively, connects, and obtains to comprise the expression plasmid of mutant gene, is converted into E .coliThe JM109 competent cell.Coat the Amp that LB(contains 100 μ g/ μ L) flat board.Grow behind 12 h, transformant is transferred in the LB liquid nutrient medium cultivates, obtain expression plasmid.
With expression plasmid through restriction enzyme SalAfter the I linearizing, electricity transforms pichia spp GS115 competent cell.Conversion fluid is coated on the MD flat board, and 30 ° of C cultivate 2 d, constitute the pichia spp library of expressing lipase mutant.
Embodiment 2, utilize the pichia spp library of DNA Shuffling method construction expression lipase mutant
Utilize the method for DNA Shuffling that the mutational site in the lipase mutant of fallibility PCR structure is made up at random.The condition of DNA Shuffling is as follows:
Extract EasilyThe genome in the pichia spp library of the expression lipase mutant that wrong PCR method makes up with DNase I digestion 30min, will be dissolved in the 30 μ L sterilized waters behind the digestion product phenol chloroform extrct deproteinize.Be that template is carried out following operation with this genome:
Step 1: PCR reaction system:
Taq (2.5U) 0.5 μL
5×buffer（Mg 2+ plus） 10 μL
Genome 0.5 μL
dNTP(25mmol/L) 4 μL
dd H 2O 34.5 μL
Pcr amplification condition: 94 ℃ of 3min; 94 ℃ of 1 min, 59 ℃ of 1 min, 72 ℃ of 2 min, 10 circulations; 72 ℃ of 10 min.
Step 2: in above-mentioned system, add primers F and each 1 μ L of R, pcr amplification condition: 94 ℃ of 3min; 94 ℃ of 1 min, 59 ℃ of 1 min, 72 ℃ of 2 min, 30 circulations; 72 ℃ of 10 min.
Amplified production is after glue reclaims the purification kit purifying, with the same method of embodiment 1, the pichia spp library of construction expression lipase mutant.
The screening of embodiment 3, high enzyme lipase mutant alive
Xerox and to copy on the MM flat board being stored in pichia spp library on the MD flat board, cultivated 2 days for 30 ℃, with the pichia spp of expressing parent's zhizopchin lipase bacterium in contrast.
Dull and stereotyped primary dcreening operation: per 12 h cover to the MM plate and add the expression of 200 μ L methanol induction recombinant lipases, after inducing 2-3 days, flat board is placed 65 ℃ of thermal treatment 60 min, cool to room temperature, on flat board, evenly topple over about 15 mL of Fast-blue RR staining agent, in 2 min mono-clonal the apparent chocolate bacterium colony that is deeper than contrast be primary dcreening operation purpose bacterial strain.
96 orifice plate screening methods: in 96 orifice plates in 1.8 mL/ holes (flat), add 300 μ L BMGY substratum, 121 ℃ of sterilization 20 min.To wherein inserting the bacterial strain that primary dcreening operation obtains, with the pichia spp of expressing parent's zhizopchin lipase bacterium in contrast, 30 ℃ of 250 r/min shaking culture is to OD 600Be 2-6.Centrifugal, abandon supernatant, with the resuspended thalline of 900 μ L BMMY substratum, per 24 h add 1 % (V/V) methanol induction lipase to express, and induces 4 days, and centrifugal collection supernatant is measured the transformation period of lipase mutant under 65 ℃ according to the method for embodiment 4 t 50
The pichia spp library of the expression lipase mutant that screening fallibility PCR and DNA Shuffling make up, obtain the bacterial strain that 14 strain thermostabilitys obviously improve respectively, measure lipase nucleotide sequence (by Shanghai Sangon Biological Engineering Technology And Service Co., Ltd's order-checking), utilize triplet codon to infer the aminoacid sequence of lipase, aminoacid replacement and the transformation period of lipase mutant are as shown in table 3.
Table 3 lipase mutant and transformation period thereof
Figure 56042DEST_PATH_IMAGE005
Embodiment 4 lipase mutants t 50Measure
For measuring lipase t 50Value need be carried out separation and purification to enzyme.
Shake flask fermentation: inoculum size 10%(V/V), in the 25 mL BMGY substratum, 30 ℃ of shaking culture 16～20 h to OD 600Be 2～6, centrifugal collection thalline is diluted to OD with the BMMY substratum 600Be 1, add the methanol induction of 0.5 % every 24 h and express, behind the cultivation 3-4 d, collect fermented supernatant fluid.
Separation and purification: the fermented supernatant fluid of mutant strain is concentrated through 10 KD ultra-filtration membranes, obtain the sudden change lipase activity component of purifying behind SP-Sepharose FF strong cation exchange chromatography and the Phenyl-Sepharose 6 FF hydrophobic chromatography column chromatographies.Concrete operations reference Yu Xiao-Wei et al. J Mol Catal B:Enzym, 2009,57:304-311.
t 50Measuring method:
The measuring method of lipase activity is pNPP method (Pencreach G et al. Enzyme and Microbial Technol.1996,18:417-422.).Enzyme is lived and to be defined as pH8.0,40 ℃ down the reaction per minute enzyme amount that produces 1 μ mol p-NP be the lipase hydrolysis enzyme international unit of living.Lipase at the measuring method of 65 ℃ of half-life is: at 65 ℃ of following treat enzyme liquid, at the different treatment time sampling, the pNPP method is measured lipase remnant enzyme activity per-cent (%).Log value with remnant enzyme activity per-cent is mapped to time T (min), and the slope of straight line is the inactivation constant k InactBy t 50=ln2/ k InactObtain lipase under this temperature t 50
The gene mutation site of embodiment 5 rite-directed mutagenesis combined lipase mutant
Through the structure in above-mentioned fallibility PCR and DNA Shuffling sudden change library, screening obtains lipase mutant as shown in table 3.Reach the combination of each sudden change to the influence of lipase mutant vigor in order to investigate wherein some sudden changes, rite-directed mutagenesis combination (rite-directed mutagenesis can utilize commercially available test kit to carry out) is carried out in mutational site in the his-and-hers watches 3, the gene that will contain the said mutation Sites Combination is connected with carrier pPIC9K, obtain expression plasmid, follow-up Pichia anomala expression is as described in the embodiment 1.Measure lipase mutant under 65 ℃ with the method for embodiment 4 t 50The lipase mutant that the thermostability acquisition improves is as shown in table 4.
Table 4 mutant lipase and t 50The multiple that improves
Each lipase title Mutational site and combination thereof Under 65 ℃ t 50 (min) t 50The multiple that improves
Parent lipase - 16.5 1.0
Mutant 12-1 Ala230Phe/Leu180His/Thr218Ser/Val261Gly/ Lys161Arg 755 45.76
Mutant 13-1 Glu107Gly/Ala129Ser/Ser151Asn/Lys219Asp/Leu180His/Glu363Arg/Ala230Phe 690 41.82
At last, what note also is, above-mentioned that enumerate only is several embodiment of the present invention.Obviously, the invention is not restricted to above embodiment.
<210> SEQ ID NO: 1
<211> 389
<212> PRT
＜213〉zhizopchin ( Rhizopus chinensis) CCTCC M 201021 lipase 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 Lys 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 Glu 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 lipase amino acid mutation bodies
Lipase amino acid mutation site: 20 mutating acid background color marks show.
<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 Thr Thr Leu Asp Leu
95 100 105
Pro Gly 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
Asn Val Val Pro Gly Thr Lys Trp Asp Leu Arg Gln Cys Leu Lys
155 160 165
Tyr Val Leu or His Asp Gly Lys Leu Ile Lys Thr Phe Thr Ser Leu His
170 175 180
Thr Tyr Ala 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 Ser Asp Tyr Ser Pro Val Lys Gly
215 220 225
Ala Lys Val His Phe Tyr Phe Leu Phe 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 Pro Arg Thr Val His Arg Arg Asp Ile Val Pro His Ala 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 Arg Cys Ser Asp Ser Ile Val Pro Phe Thr Cys 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 (2)

1. the lipase mutant that improves of the thermostability that makes up by orthogenesis, it is characterized in that by parent's zhizopchin ( Rhizopus chinensis) CCTCC NO:M 201021 lipase carry out orthogenesis, by fallibility PCR, DNA Shuffling and rite-directed mutagenesis, obtain lipase mutant, undergo mutation in the aminoacid replacement position among parent's zhizopchin aminoacid sequence SEQ ID NO:1, adopt " amino acid of original amino acid-position-replacement " to represent the amino acid that suddenlys change in the lipase mutant, described lipase mutant is:
Lipase mutant aminoacid replacement position
Mutant 10 Ser234Phe/Leu180His/Thr218Ser
The aminoacid sequence of zhizopchin lipase amino acid mutation body is SEQ ID NO:2;
The expression vector that is used for the described lipase mutant of expression is: pPIC9K, pPIC3.5K, pPICZ α, pPICZ;
Being used for described expression vector microorganism transformed host cell is pichia spp.
2. lipase mutant according to claim 1 is characterized in that comparing with parent's zhizopchin lipase, and the thermostability of described lipase mutant has obtained raising, with the transformation period under 65 ℃ t 50Represent the raising of thermostability, the transformation period of described lipase mutant and the multiple of raising are as follows:
65 ℃ of t50 of the 65 ℃ of t50 in lipase mutant aminoacid replacement position (min) (min) improve multiple
Mutant 10 Ser234Phe/Leu180His/Thr218Ser 89.9 5.45
The transformation period of parent's zhizopchin lipase under 65 ℃ t 50Be 16.5 min.
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