The application is dividing an application of original applying number 200910235185.3, applying date 2009.11.11, denomination of invention " lipase mutant that the vigor that makes up through orthogenesis improves ".
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 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 100 mmol/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 100 mmol/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:
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 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 is converted into E .coli 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 mutant plasmid.
With mutant plasmid through restriction enzyme
SalAfter the I linearizing, electricity transforms pichia spp GS115 competent cell.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 GS115/ pPIC9K-proRCL simultaneously and be seeded on the BMMY flat board.30 ° of C cultivated 2 days.
Dull and stereotyped primary dcreening operation: preserve the YPD flat board that growth finishes.Per 12 h cover to the BMMY plate and add 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 2 min 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 in 1.8 mL/ holes (flat), add 300 μ L BMGY substratum, 121 ℃ of sterilization 20 min.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 250 r/min shaking culture to OD600 be 2-6 (about 16-18 h).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 per 24 h add 100 μ L BMMY substratum and 1 % (V/V) methyl alcohol, induce 4 days.With centrifugal 10 min of 96 orifice plate fermented liquids, 3000 r/min of abduction delivering 96 h, 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 2 min 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 6 strain enzymes bacterial strain that obviously improves alive, measures the lypase nucleotide sequence, utilizes triplet codon to infer the aminoacid sequence of lypase, and the aminoacid replacement of lipase mutant reaches
K CatIt is as shown in table 1 that value improves multiple.Measure lipase mutant according to the method for embodiment 3
K 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/Thr195Tyr |
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 |
Embodiment 3 lipase mutants
K CatPH-value determination pH
For measuring lypase
K CatValue need be carried out separation and purification to enzyme.
Shake flask fermentation:, be seeded in the 25 mL BMGY substratum 30 ℃ of shaking culture 16~20 h 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, whenever add the methanol induction expression of 0.5 %, cultivate after 3-4 days, 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.
Measuring 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 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 0~25 mmol/L scope, measures enzyme activity, calculates the kinetic parameter that obtains lypase
K 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 lipase mutant with the method that is equal to embodiment 3
K CatValue.The combination site of each mutant enzyme and
K CatThe multiple that improves is as shown in table 2.
The sequencing result of table 2 two mutants and
K 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 |
<210> SEQ?ID?NO:?1
<211> 389
<212> PRT
<213>Zhizopchin (
Rhizopuschinensis) 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 (
Rhizopuschinensis) 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'。