Description of drawings
Confirm 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 two mutants is following.
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 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 C autoclaving 20 min.Adding G418 when being used to screen the G418 resistance is 0.25 mg/mL-1.0 mg/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 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-NMF) mix with 160 μ L Fast blue RR (80mg Fast blue RR is dissolved in the 1mL methyl-sulphoxide) 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 to zhizopchin lipase gene proRCL external.The reaction conditions of fallibility PCR is following:
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 with
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 LB (Amp that 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 following:
Extract
Be prone toThe genome in the pichia spp library of the expression lipase mutant that wrong PCR method makes up is with DNase I digestion 30min, with being dissolved in the 30 μ L sterilized waters behind the digestion product phenol chloroform extrct deproteinize.With this genome is that template is operated as follows:
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 lypase as the contrast bacterium.
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 is evenly toppled over about 15 mL of Fast-blue RR staining agent on flat board, 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, as the contrast bacterium, 30 ℃ of 250 r/min shaking culture is to OD with the pichia spp of expressing parent's zhizopchin lypase
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 lypase 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 lypase nucleotide sequence (by Shanghai Sangon Biological Engineering Technology And Service Co., Ltd's order-checking); Utilize triplet codon to infer the aminoacid sequence of lypase, the aminoacid replacement and the transformation period of lipase mutant are as shown in table 3.
Table 3 lipase mutant and transformation period thereof
Embodiment 4 lipase mutants
t 50Measure
For measuring lypase
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, whenever add the methanol induction expression of 0.5 %, behind the cultivation 3-4 d, collect fermented supernatant fluid at a distance from 24 h.
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 HC 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 is defined as pH8.0,40 ℃ down the reaction PM enzyme amount that produces 1 μ mol p-NP be the lipase hydrolysis enzyme iu of living.Lypase 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 lypase remnant enzyme activity per-cent (%).Log value with remnant enzyme activity per-cent is mapped to time T (min), and the collinear slope is the inactivation constant
k InactBy
t 50=ln2/
k InactObtain lypase under this temperature
t 50
The gene mutation site of embodiment 5 rite-directed mutagenesis combined lipase two mutants
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 influence of the combination of each sudden change in order to investigate wherein some sudden changes to the lipase mutant vigor; 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 such as embodiment 1 are said.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 two mutants lypase and
t 50The multiple that improves
Each lypase title |
Mutational site and combination thereof |
Under 65 ℃
t 50 (min)
|
t 50The multiple that improves
|
Parent lipase |
- |
16.5 |
1.0 |
Two mutants 12-1 |
Ala230Phe/Leu180His/Thr218Ser/Val261Gly/ Lys161Arg |
755 |
45.76 |
Two mutants 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 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?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 lypase amino acid mutation bodies
Lypase 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'。