CN102604911B - Highly active Rhizopus chinensis lipase mutant - Google Patents

Abstract

The high-activity Rhizopus chinensis lipase mutant belongs to the technical field of enzyme genetic engineering. The invention discloses a series of lipase mutants obtained from Rhizopus chinensis CCTCC M 201021 lipase gene through directed evolution. In the amino acid sequence of each mutant, the amino acid mutations involved are Ala129Ser, Lys161Arg, Thr195Tyr, Ala230Thr, Val261Gly, Lys322Arg, Ser373Asn and combinations of two, three or four mutations of the above amino acids. The enzyme activity of these mutants was improved compared with the starting strain, expressed by the turnover number K _cat .

Description

Highly active Rhizopus chinensis lipase mutant

This application is a divisional application of the original application number 200910235185.3, the filing date is 2009.11.11, and the invention title is "a lipase mutant with improved activity constructed by directed evolution".

technical field

The invention belongs to the technical field of gene engineering of enzymes, and in particular relates to a lipase mutant of Rhizopus sinensis with improved enzyme activity.

Background technique

Lipase (EC 3.1.1.3) can not only catalyze the hydrolysis of oil, but also catalyze ester synthesis, transesterification, acid hydrolysis and other reactions in non-aqueous phase. It is widely used in chemistry, food, pharmaceutical and detergent or bioenergy in industry. Microorganisms are an important source of lipase, and Rhizopus is an important producer of microbial lipase. Today, more than 30 Rhizopus lipases have been commercially produced. Most Rhizopus lipases have high 1,3-position selectivity, so they are often used in oil processing. In addition, Rhizopus lipase has the advantages of good stability and high conversion efficiency, and is widely used in the production of aromatic esters, biodiesel, and chiral compounds.

So far, many gene sequences of Rhizopus lipase have been reported at home and abroad. Japan and Germany have done in-depth research on the gene sequence and expression of Rhizopus oryzae lipase ( Rhizopus oryzae lipase, ROL ), and have successfully expressed the lipase gene with Escherichia coli, Saccharomyces cerevisiae and Pichia pastoris successively ( Minning S et al. J Biotechnol , 1998, 66 : 147-156; Beer HD et al. Biochim Biophys Acta , 1998, 1399 : 173-180; Ueda M et al. J Mol Catal B: Enzym , 2002, 17 : 113 -124). In the previous research, the inventor successfully screened a strain of Rhizopus chinensis CCTCC M 201021 with high lipase production from the koji for brewing Luzhou-flavor Daqu liquor, and cloned the lipase gene sequence from the strain (Genbank accession number EF405962), and achieved high-level secretory expression of the lipase in Pichia pastoris (Yu Xiao-Wei et al. J Mol Catal B: Enzym , 2009, 57:304-311) .

Directed evolution is an irrational design, which means that by simulating Darwin's natural evolution process in the laboratory, the gene of a certain protein enzyme is modified by an improved mutagenesis technology, and then screened for the specific purpose of transformation. of natural enzymes. In the past 10 years, directed evolution technology has achieved great success in the field of esterase and lipase property modification, mainly focusing on improving the catalytic activity of enzymes, improving substrate specificity, improving thermal stability, enantioselectivity, etc. (Johannes TW et al. Curr. Opin. Microbiol , 2006, 9: 261-267).

The improvement of enzyme activity can be expressed by the kinetic parameter K _cat value of the enzyme. K _cat , also known as the conversion number, refers to the number of substrate molecules (TN) that can be catalyzed per molecule of enzyme or each enzyme active center per unit time, also known as the catalytic constant. Therefore, an increase in K _cat value represents an increase in enzyme activity.

Contents of the invention

The technical problem solved by the invention is to provide rhizopus sinorius lipase with improved enzyme activity.

The technical solution of the present invention: the lipase mutant with improved activity constructed by directed evolution is composed of Rhizopus chinensis CCTCC M 201021 lipase gene (Genbank accession number EF405962), using error-prone PCR method, through multiple rounds of recombination And site-directed mutation, the lipase mutant obtained by directed evolution, in the amino acid sequence of the mutant, contains amino acid mutations Ala129Ser, Lys161Arg, Thr195Tyr, Ala230Thr, Val261Gly, Lys322Arg, Ser373Asn and two, three or four of the above amino acids The combination of mutations; represented by the conversion number K _cat , the enzyme activity of the lipase mutant has been improved compared with the starting strain; the sequencing results of the mutant and the multiples of its K _cat improvement are:

lipase mutant amino acid substitution position K _cat (/min) The multiple of K _cat improvement Starting strain lipase - 1138 1.0 Mutant 1-1 Ala129Ser 1252 1.1 Mutant 1-2 Lys161Arg 1479 1.3 Mutant 1-3 Thr195Tyr 1479 1.3 Mutant 1-4 Ala230Thr 1821 1.6 Mutant 1-5 Val261Gly 1935 1.7 Mutant 1-6 Lys322Arg 1366 1.2 Mutant 1-7 Ser373Asn 1707 1.5 Mutant 2-1 Ala129Ser/Lys161Arg 2278 2 Mutant 2-2 Ala129Ser / Ala230Thr 1365 1.2 Mutant 2-3 Ala129Ser/Val261Gly 1593 1.4 Mutant 2-4 Lys161Arg/Thr195Tyr 2845 2.5 Mutant 2-5 Lys161Arg / Lys322Arg 1707 1.5 Mutant 2-6 Lys161Arg/Ser373Asn 2048 1.8 Mutant 2-7 Ala230Thr /Ser373Asn 1707 1.5 Mutant 2-8 Val261Gly/ Ser373Asn 3726 3.3 Mutant 3-1 Ala129Ser/Ala230Thr/Val261Gly 3186 2.8 Mutant 3-2 Ala129Ser/Ala230Thr/Lys322Arg 2278 2 Mutant 3-3 Lys161Arg/Thr195Tyr/Ser373Asn 2617 2.3 Mutant 3-4 Lys161Arg/Ala230Thr/Ser373Asn 2278 2 Mutant 3-5 Lys161Arg/Val261Gly/Lys322Arg 2731 2.4 Mutant 3-6 Ala230Thr/Lys322Arg/Ser373Asn 2845 2.5 Mutant 4-1 Ala129Ser/Lys161Arg/Ala230Thr/Lys322Arg 2073 1.8

The original amino acid sequence of Rhizopus sinensis lipase is: SEQ ID NO: 1; the amino acid mutant sequence of Rhizopus sinensis lipase is: SEQ ID NO: 2, and the 7 mutated amino acids are marked with background color.

Beneficial effects of the present invention: the present invention uses the error-prone PCR method to carry out directed evolution to Rhizopus sinensis lipase gene (Genbank accession number EF405962), and obtains Rhizopus sinensis lipase mutants through multiple rounds of recombination and site-directed mutation. These mutants Contains amino acid mutations Ala129Ser, Lys161Arg, Thr195Tyr, Ala230Thr, Val261Gly, Lys322Arg, Ser373Asn and the above amino acid mutations

changing combination. The enzyme activity of the lipase mutant was improved, expressed by the turnover number K _cat .

Detailed ways

The medium formula involved in the embodiment is as follows:

LB liquid medium: peptone 1%, yeast extract 0.5%, NaCl 1%, pH7.0.

YPD(Yeast Extract Peptone Dextrose Medium): Yeast Extract 1%, Trypton 2%, Dextrose 2%, add Agar 2% when making the tablet. Autoclave at 121 °C for 20 min. When used to screen G418 resistance, add G418 to a final concentration of 0.25 mg/mL-1.0 mg/mL, that is, YPD-G418 plate.

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%, YNB 1.34%, Biotin 4× ^10-5 %, Glycerol 1%, potassium phosphate solution 100 mmol/L.

BMMY (Buffered Methanol-complex Medium): Yeast Extract 1%, Trypton 2%, YNB 1.34%, Biotin 4× ^10-5 %, Methanol 0.5%, potassium phosphate solution 100 mmol/L.

The unit in the medium is % (W/V)

Embodiment 1, utilize the error-prone PCR method to construct Rhizopus sinica lipase mutant library

Nucleotide mutations were introduced into the lipase gene proRCL of Rhizopus sinensis in vitro by error-prone PCR. The reaction conditions for error-prone PCR are as follows:

Wherein, the sequences of primers F and R are:

Upstream primer F: 5'-TCAAGATCCCTAGGGTTCCTGTTGGTCATAAAGGTTC-3';

Downstream primer R: 5'-AATTCCAGTGCGGCCGCTTACAAACAGCTTCCTTCG-3'.

PCR amplification conditions: 94°C for 3 min; 94°C for 1 min, 59°C for 1 min, 72°C for 2 min, 30 cycles; 72°C for 10 min.

After the error-prone PCR amplification product was purified by a DNA purification kit, the restriction endonucleases Avr Ⅱ and Not I digested the error-prone PCR amplification product and plasmid pPIC9K respectively, ligated, and transformed into E.coli JM109 competent cells. Spread on LB (containing 100 μg/μL of Amp) plate. After 12 h of growth, the transformants were transferred to LB liquid medium for culture to obtain mutant plasmids.

After the mutant plasmid was linearized by the restriction endonuclease Sal I, the competent cells of Pichia pastoris GS115 were electrotransformed. Spread the transformation solution on the MD plate and incubate at 30°C for 2 days to form a mutant library.

Using the same method as above, multiple rounds of error-prone PCR were performed using the mutant genome as a template to construct a mutant library.

Embodiment 2, the screening of lipase mutant with high enzymatic activity

Use a sterilized toothpick to copy the His ⁺ transformant grown on the MD plate to the same position on the YPD and BMMY plates, and at the same time inoculate the control bacteria GS115/pPIC9K-proRCL on the BMMY plate. Incubate at 30°C for 2 days.

Plate primary screening: save the grown YPD plate. Every 12 h, 200 μL of methanol was added to the cover of the BMMY plate to induce the expression of recombinant lipase. Induce for 2-3 days. Add 0.6% agar to the enzyme activity assay substrate pNPP, mix well, and pour it on the BMMY plate to form the top layer of agar. The strains that showed obvious yellow within 2 min were the mutant strains of primary screening purpose. Under the same conditions, the control bacteria GS115/pPIC9K-proRCL could not show obvious yellow color.

96-well plate re-screening: Add 300 μL of BMGY medium to a 1.8 mL/well (flat-bottomed) 96-well plate, and sterilize at 121°C for 20 min. Insert the target strain of primary screening preserved on the YPD plate (incorporate GS115/pPIC9K-proRCL at the same time as a control), and shake at 250 r/min at 30°C until the OD600 is 2-6 (about 16-18 h). Centrifuge, discard the supernatant, resuspend the cells with 900 μL BMMY medium, and add 1% (V/V) methanol to induce lipase expression. After that, 100 μL of BMMY medium and 1% (V/V) methanol were added every 24 h for induction for 4 days. The 96-well plate fermentation broth induced for 96 h was centrifuged at 3000 r/min for 10 min, and the supernatant was collected. Take 1 μL of the supernatant and dilute 500 times, take 5 μL into another 96-well plate, add the substrate with a row gun, shake and mix. The strains that quickly show obvious yellow color within 2 minutes are the strains for re-screening. Under the same conditions, the fermentation supernatant of the control bacterium GS115/pPIC9K-proRCL could not show obvious yellow color.

Based on the screening of the mutant library constructed by error-prone PCR, 6 strains with significantly improved enzyme activity were obtained, the nucleotide sequence of lipase was determined, the amino acid sequence of lipase was deduced by using triplet codons, and the amino acid substitution of lipase mutants And K _cat value increase times are shown in Table 1. The K _cat value of the lipase mutant was determined according to the method in Example 3.

Table 1 Sequencing results of mutants

lipase mutant amino acid substitution position K _cat (/min) The multiple of K _cat improvement Starting strain lipase - 1138 1.0 Mutant 2-1 Ala129Ser/Lys161Arg 2278 2 Mutant 2-4 Lys161Arg/Thr195Tyr 2845 2.5 Mutant 2-7 Ala230Thr /Ser373Asn 1707 1.5 Mutant 2-8 Val261Gly/ Ser373Asn 3726 3.3 Mutant 3-2 Ala129Ser/Ala230Thr/Lys322Arg 2278 2 Mutant 3-4 Lys161Arg/Ala230Thr/Ser373Asn 2278 2

The K _cat value determination of embodiment 3 lipase mutants

In order to determine the K _cat value of lipase, it is necessary to separate and purify the enzyme.

Shake flask fermentation: Inoculate the starting strain and each lipase mutant into 25 mL of BMGY medium, shake culture at 30°C for 16-20 h until the OD ₆₀₀ is 2-6, collect the bacteria by centrifugation, and dilute with BMMY medium to The OD ₆₀₀ was 1, and the expression was induced by adding 0.5% methanol every 24 h. After culturing for 3-4 days, the fermentation supernatant was collected.

Separation and purification: the fermentation supernatant of the mutant strain was concentrated through a 10 KD ultrafiltration membrane, followed by SP-Sepharose FF strong cation exchange chromatography and Phenyl-Sepharose 6 FF hydrophobic chromatography column chromatography to obtain the purified active component of mutant lipase. Specific operation reference Yu Xiao-Wei et al. J Mol Catal B: Enzym , 2009, 57:304-311.

test methods:

The assay method of lipase activity is pNPP method (Pencreach G et al. Enzyme and Microbial Technol. 1996, 18: 417-422.). Enzyme activity is defined as the amount of enzyme that produces 1 μmol p-nitrophenol per minute under certain reaction conditions, which is one international unit of lipase hydrolysis activity. When the substrate p-nitrophenol palmitate concentration ranged from 0 to 25 mmol/L, the enzyme activity was measured, and the kinetic parameter K _cat of lipase was calculated.

Example 4 Gene mutation site of site-directed mutagenesis combination lipase mutant

After multiple rounds of error-prone PCR for mutation library construction, seven mutants containing amino acid mutation sites Ala129Ser, Lys161Arg, Thr195Tyr, Ala230Thr, Val261Gly, Lys322Arg, and Ser373Asn were obtained. In order to investigate the effect of one mutation and the combination of mutations on the viability of lipase mutant strains, multiple lipase mutants were obtained by performing site-directed mutation combinations on the discovered mutation sites. (Site-directed mutagenesis can be performed using a commercially available kit.)

The gene containing the above combination of mutation sites was connected to the vector pPIC9K, and electrotransformed into Pichia pastoris GS115 to obtain high-efficiency secretion and expression of lipase. The K _cat value of the lipase mutant was determined in the same manner as in Example 3. Table 2 shows the combination site of each mutant enzyme and the fold of K _cat improvement.

Table 2 The sequencing results of the mutants and their K _cat increased folds

lipase mutant amino acid substitution position K _cat (/min) The multiple of K _cat improvement Starting strain lipase - 1138 1.0 Mutant 1-1 Ala129Ser 1252 1.1 Mutant 1-2 Lys161Arg 1479 1.3 Mutant 1-3 Thr195Tyr 1479 1.3 Mutant 1-4 Ala230Thr 1821 1.6 Mutant 1-5 Val261Gly 1935 1.7 Mutant 1-6 Lys322Arg 1366 1.2 Mutant 1-7 Ser373Asn 1707 1.5 Mutant 2-2 Ala129Ser / Ala230Thr 1365 1.2 Mutant 2-3 Ala129Ser/Val261Gly 1593 1.4 Mutant 2-5 Lys161Arg / Lys322Arg 1707 1.5 Mutant 2-6 Lys161Arg/Ser373Asn 2048 1.8 Mutant 3-1 Ala129Ser/Ala230Thr/Val261Gly 3186 2.8 Mutant 3-3 Lys161Arg/Thr195Tyr/Ser373Asn 2617 2.3 Mutant 3-5 Lys161Arg/Val261Gly/Lys322Arg 2731 2.4 Mutant 3-6 Ala230Thr/Lys322Arg/Ser373Asn 2845 2.5 Mutant 4-1 Ala129Ser/Lys161Arg/Ala230Thr/Lys322Arg 2073 1.8

<210> SEQ ID NO: 1

<211> 389

<212> PRT

<213> Rhizopuschinensis CCTCC M 201021 lipase amino acid

the

<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

the

<210> SEQ ID NO: 2

<211> 389

<212> PRT

<213> Rhizopuschinensis CCTCC M 201021 lipase amino acid mutant

the

<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

the

<210> SEQ ID NO: 3

the

<400> 3

F: 5'-TCAAGATCCCTAGGGTTCCTGTTGGTCATAAAGGTTC-3';

R: 5'-AATTCCAGTGCGGCCGCTTACAAACAGCTTCCTTCG-3'.

the

Claims (1)

1. high catalysis activity lipase mutant, it is characterized in that using the fallibility PCR method by the lipase gene that the Genbank accession number that zhizopchin (Rhizopus chinensis) CCTCC M201021 obtains is EF405962, by many wheel restructuring and rite-directed mutagenesis, the lipase mutant that obtains through orthogenesis, in the aminoacid sequence of mutant, comprise amino acid mutation Lys322Arg; With turnover number K _catExpression, the enzyme work of lipase mutant is improved than starting strain lipase; The sequencing result of mutant and K thereof _catThe multiple that improves is: