CN112481237A - Alkali-resistant lipase - Google Patents

Alkali-resistant lipase Download PDF

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CN112481237A
CN112481237A CN202011427117.XA CN202011427117A CN112481237A CN 112481237 A CN112481237 A CN 112481237A CN 202011427117 A CN202011427117 A CN 202011427117A CN 112481237 A CN112481237 A CN 112481237A
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王兴吉
钱娟娟
曹世源
郭庆文
王金余
王克芬
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Shandong Lonct Enzymes Co ltd
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    • C12Y301/01003—Triacylglycerol lipase (3.1.1.3)

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Abstract

The invention belongs to the technical field of biology, and particularly relates to alkali-resistant lipase. The invention obtains the alkali-resistant lipase mutant gene B-2 from rhizopus mutant bacteria by PCR, constructs the B-2 on pPIC9 plasmid to obtain recombinant plasmid pPIC9-B-2, converts the recombinant plasmid pPIC9-B-2 into pichia pastoris GS200, and screens to obtain the recombinant bacteria C-2 of high-expression alkaline lipase, wherein the alkaline lipase fermentation enzyme activity can reach 85000U/ml. When the alkaline lipase expressed by C-2 is treated for 9 hours under the condition of pH 11.5, the relative activity of the alkaline lipase still remains about 80 percent, and the alkaline resistance is better.

Description

Alkali-resistant lipase
The technical field is as follows:
the invention relates to the technical field of biology, in particular to a pichia pastoris engineering bacterium for high yield of alkali-resistant lipase and lipase produced by the same.
Background art:
lipases are biological enzymes capable of hydrolyzing long chain fatty glycerides to glycerol and long chain fatty acids (or the reverse reaction). The lipase can perform chemical reactions such as ester exchange, hydrolysis, esterification and the like in a water-oil interface or a non-aqueous solvent system, and has the characteristics of substrate specificity, site selectivity and the like. The lipase can be applied to the industries of grease processing, food processing, leather processing, feed, biological energy and the like, and has wider application value.
Alkaline lipase is an enzyme which hydrolyzes ester bonds in fat under alkaline conditions, is a novel enzyme for detergents and is mainly used as a novel enzyme variety of detergents. It can decompose the greasy dirt of clothes into diglyceride, monoglyceride and fatty acid, etc. which are easily dissolved in water, so that it can obviously raise the washing effect of washing powder, specially can remove yellow spot. We commonly use various enzyme-containing washing powders, in which the enzyme preparations mainly include alkaline protease and alkaline lipase. Alkaline protease is used for decomposing protein dirt such as sweat stain, blood stain, etc. Alkaline lipase acts mainly on fatty acid and ester dirt, which we usually refer to as oil stains.
The lipase is mainly from plants, animals and microorganisms, the microbial lipase is widely existed in bacteria, yeast and mould, has the characteristics of multiple types, short period, quick propagation and easy genetic variation, has wider action temperature, action pH and substrate specificity than animal and plant lipases, can catalyze the hydrolysis, alcoholysis, acidolysis, ester exchange, synthesis and the like of ester compounds under the condition of no coenzyme, has the characteristics of mild catalysis condition, low energy consumption, less byproducts, high efficiency, high selectivity, environmental friendliness and the like, and changes the relatively harsh conditions of high temperature, strong acid, strong base and the like required by the traditional esterification or transesterification reaction. Although various lipases are available in nature, these natural enzymes often fail to meet biocatalytic requirements, including the range of substrate selection, pH, temperature profile and stability (stability of long-term reaction, organic solvent resistance), etc.
With the continuous development of genetic engineering technology, the pichia pastoris engineering bacteria are constructed by combining the genetic engineering means with the required lipase genes, so that the pichia pastoris engineering bacteria can be better expressed, and the pichia pastoris engineering bacteria have great significance for meeting the requirements of industrial production and reducing the production cost.
The invention content is as follows:
the invention aims to provide a lipase mutant with improved alkali resistance and a pichia pastoris engineering strain thereof. The lipase mutant is specifically a lipase mutant B-2, is derived from rhizopus ZF056 obtained by ultraviolet mutagenesis, and is expressed in Pichia pastoris GS200 after a lipase mutant coding gene is cloned from the ZF056 and a recombinant plasmid is constructed, so that a Pichia pastoris engineering strain is obtained.
The enzymatic properties of the lipase mutant B-2 are as follows:
(1) pH 10.0-11.5, the enzyme activity is stable, and the optimum action pH is 10.5.
(2) Temperature: the enzyme activity is stable at 45-60 ℃, and the optimal action temperature is 50 ℃.
(3) Alkali resistance: the enzyme activity of the enzyme is still more than 80% after 9 hours under the condition of pH 11.5.
The following definitions are used in the present invention:
1. nomenclature for amino acid and DNA nucleic acid sequences
The accepted IUPAC nomenclature for amino acid residues is used, in the form of a three letter code. DNA nucleic acid sequences employ the accepted IUPAC nomenclature.
2. Identification of lipase mutants
In the present invention, B-1 represents a wild-type lipase, B-2 represents a lipase mutant, and the information is shown in the following table.
The amino acid sequence of the wild lipase B-1 is shown in a sequence table SEQ ID No. 2;
the nucleotide sequence of the wild lipase B-1 encoding gene is shown in SEQ ID No. 1;
the amino acid sequence of the lipase mutant B-2 is shown in a sequence table SEQ ID No. 4;
the nucleotide sequence of the lipase mutation B-2 coding gene is shown in SEQ ID No. 3.
The invention also provides an expression vector or a recombinant strain containing the mutant gene;
preferably, the expression vector for expressing the mutant is pPIC9, and the host cell is Pichia pastoris GS 200.
Preferably, the recombinant strain is obtained by connecting a lipase mutation coding gene B-2 with an expression vector pPIC9 and expressing the gene in Pichia pastoris GS 200.
The construction method of the recombinant strain comprises the following steps:
1. carrying out enzyme digestion on the encoding gene B-2 of the lipase mutant, and connecting the encoding gene B-2 to an expression vector pPIC9 to obtain a recombinant vector;
2. the recombinant vector is transformed into Pichia pastoris GS200 to obtain a novel lipase production strain GS200/pPIC 9-B-2.
The invention also provides application of the recombinant strain in fermentation production of lipase strains, in particular application of GS200/pPIC9-B-2 recombinant strain serving as a production strain in fermentation production of lipase.
Specifically, the method for producing lipase by using the recombinant bacterium through fermentation (50L fermentation tank) comprises the following steps:
seed tank culture: the culture temperature is 30-31 ℃, the rotation speed is 200-3/h-6m3Ventilating, stirring and culturing, wherein the pH is 5.0-5.5, the dissolved oxygen is 20-30%, and the wet weight is increased to 60-80g/L for seed transferring;
culturing in a fermentation tank: the inoculation amount is 10-15%, the culture temperature is 30-31 ℃, the rotation speed is 200-3/h-6m3Ventilating and stirring for culturing, wherein the pH is 5.0-5.5, the period is 0-20h, 40-50% of glucose is fed-batch carbon source, the feeding-batch speed is 800g/h, the dissolved oxygen is 20-30%, and the culturing is carried out for 20h until the wet weight of the thalli is 180 g/L; stopping supplying the carbon source, rebounding the dissolved oxygen to more than 80%, and keeping for 1 hour; adding methanol at a flow rate of 50-200g/h, controlling dissolved oxygen at 20-30%, and culturing for about 130h until fermentation is finished.
The culture medium formula of the seeding tank comprises: 4-5% of glycerol, 1-2% of ammonium dihydrogen phosphate, 0.5-1% of potassium dihydrogen phosphate, 0.5-1% of magnesium sulfate, 0.5-0.8% of potassium sulfate, 0.05-0.1% of calcium sulfate, 0.2-0.5% of potassium hydroxide and the balance of water, wherein the pH value is 5.0-5.5;
the fermentation tank culture medium formula is as follows: 4-5% of glycerol, 1-2% of ammonium dihydrogen phosphate, 0.5-1% of potassium dihydrogen phosphate, 0.5-1% of magnesium sulfate, 0.5-0.8% of potassium sulfate, 0.05-0.1% of calcium sulfate, 0.2-0.5% of potassium hydroxide and the balance of water, and the pH value is 5.0-5.5.
Has the advantages that:
1. the invention discloses a brand-new lipase mutant which has the characteristics of high enzyme activity and high alkali resistance. The enzyme activity of the mutant fermentation liquor can reach more than 85000U/mL;
2. the lipase obtained by the invention still has the enzyme activity of more than 80 percent after 9 hours under the condition of pH 11.5, and compared with wild lipase B-1, the alkali resistance is obviously improved.
Description of the drawings:
FIG. 1 shows a PCR identification electrophoretogram of a colony;
FIG. 2 is a pH optimum curve of alkaline lipase;
FIG. 3 is a temperature optimum curve for alkaline lipase;
FIG. 4 shows the alkali resistance curve of alkaline lipase.
The specific implementation mode is as follows:
the present invention is illustrated in greater detail by the specific examples, which are given by way of illustration only and are not intended to limit the scope of the invention. Modifications may be made by those skilled in the art, which would be within the principles of the invention, and such modifications are to be considered within the scope of the invention. The molecular biological experiment method not specifically described in this example can be referred to "molecular cloning Experimental Manual".
The present invention is further explained below by means of specific embodiments.
EXAMPLE 1 acquisition of Gene encoding lipase mutant B-2
A strain ZF056 with alkali-resistant lipase activity is obtained by ultraviolet mutagenesis and screening of a rhizopus LD037 for producing alkali lipase, a PCR primer is designed according to a nucleotide sequence of a wild-type lipase B-1 encoding gene from the rhizopus LD037, a restriction enzyme site Xho I is added to the 5 'end, a restriction enzyme site Not I is added to the 3' end, a mutant gene B-2 is obtained by PCR, and the nucleotide sequence is SEQ ID No.3 is obtained by sequencing.
In the present invention, the mutation site information is obtained by comparing the nucleotide sequences of the wild type and the mutant as shown in the following table.
Figure BDA0002825404210000041
EXAMPLE 2 construction of recombinant vector pPIC9-B-2
Respectively carrying out Xho I and Not I enzyme digestion on a B-2 encoding gene and a plasmid pPIC9, recovering a product, mixing the recovered B-2 encoding gene and pPIC9 in proportion, connecting the mixture at 16 ℃ overnight by using T4 ligase, transforming Escherichia coli DH5 alpha competent cells by the connecting product, coating the transformed product on an LB (ampicillin-containing) solid plate, carrying out inverted culture at 37 ℃ overnight, picking a single colony to an LB liquid culture medium, carrying out culture at 37 ℃, carrying out colony PCR on a bacterial liquid, and carrying out electrophoresis identification on the result as shown in figure 1, wherein the sequencing result shows that the sequence is correct and the sequence size is 1.6 kb. The recombinant plasmid pPIC9-B-2 was extracted for use.
EXAMPLE 3 transformation of Pichia pastoris with recombinant plasmids
1. Preparation of Pichia pastoris GS200 competent cell
1) Selecting a single colony of a pichia pastoris plate, inoculating the single colony in 5mL of YPD culture medium, and oscillating overnight at 30 ℃ at 220 r/min;
2) inoculating 0.5mL of overnight-cultured bacterial liquid into 50mL of freshly prepared YPD medium, and performing shaking culture at 30 ℃ and 220r/min to enable the OD600 value to reach 1.3-1.5;
3) centrifuging the culture solution at 4 deg.C and 3000r/min for 5 min;
4) discarding the supernatant, adding 50mL of ice-precooled sterile water, and oscillating to resuspend the thalli;
5) centrifuging at 4 ℃ and 3000r/min for 5min, removing supernatant, sucking residual liquid on the tube wall, adding 25mL of ice-precooled sterile water, and oscillating to resuspend thalli;
6) centrifuging at 4 ℃ and 3000r/min for 5min, removing supernatant, sucking residual liquid on the tube wall, adding 10mL of 1mol/L sterile sorbitol solution precooled on ice, and resuspending thalli;
7) centrifuging at 4 deg.C and 3000r/min for 5min, discarding supernatant, sucking off residual liquid on tube wall, adding 1mL of 1mol/L sterile sorbitol solution precooled on ice (adding glycerol to final concentration of 15%), shaking and mixing.
8) 100 μ L/tube into sterile EP jar, and freezing at-70 deg.C (fresh competent cells are more effective).
2. Transformation of linearized plasmids
The recombinant plasmid pPIC9-B-2 obtained by extraction is subjected to single enzyme digestion by Sal I to obtain a linearized plasmid. Freshly prepared (or frozen at-70 ℃) competent cells were placed in an ice bath and allowed to thaw completely.
1) Removing 100 μ L of competent cells into a new sterile EP tube, adding 10 μ L of linearized plasmid, mixing by gentle blowing, sucking out and transferring into a 0.2 cm-type electroporation transfer cup;
2) the transformation cup was placed in an ice bath for 5-10 minutes, maintaining the low temperature.
3) Electroporation transformation shock conditions: 1500V, 200 omega, 25 muF, discharge time of about 5ms, one electric shock.
4) After electric shock, immediately adding 1mL of 1mol/L sorbitol solution precooled at 4 ℃ into an electric shock conversion cup, uniformly blowing by using a liquid transfer gun, and placing in an ice bath;
5) MD medium (1.34% YNB; 4X 10-5% biotin; 2% glucose plate), 100-;
6) two recombinant bacteria are obtained by screening on an MD plate, a target sequence is obtained by colony PCR, and the sequence comparison shows that the target sequence is the nucleotide sequence of a target gene B-2, namely the obtained strains are recombinant bacteria containing pPIC9-B-2 and are respectively named as C-1 and C-2.
Example 4 inducible expression of Yeast containing recombinant plasmid pPIC9-B-2
BMGY medium formulation: 1% yeast extract, 2% peptone, 0.1mol/L pH6.0 phosphate buffer, 1.34% YNB, 4X 10-5% of a mixture of biotin and at least one other substance,1% of glycerol.
BMMY culture medium formula: 1% yeast extract, 2% peptone, 0.1mol/L pH6.0 phosphate buffer, 1.34% YNB, 4X 10-5% biotin, 0.5% methanol.
Respectively inoculating recombinant bacteria C-1 and C-2 and recombinant strain YB-1 constructed by using wild type lipase B-1 encoding gene by the same method into a triangular flask filled with 30mL of BMGY culture medium, culturing at 30 ℃ and 220r/min until OD600 is about 10, centrifugally collecting thalli, resuspending the thalli by using 35mL of BMMY induction culture medium, continuously culturing for 48 hours at 30 ℃ and 220r/min, and measuring the lipase activity in supernatant after the fermentation broth is centrifuged, wherein the results are shown in the table below.
Bacterial strains Lipase activity/U.mL-1
YB-1 5412
C-1 12215
C-2 12209
Example 5 fermentation Performance verification of recombinant bacterium C-2
Seed tank culture: the culture temperature is 30 ℃, the rotating speed is 300rpm, and the air volume is 2.5m3Ventilating, stirring and culturing, wherein the pH is 5.1, the dissolved oxygen content is 25 percent, and the wet weight is increased to 75g/L for seed transferring;
culturing in a fermentation tank: the inoculation amount is 12 percent, the culture temperature is 30 ℃, the rotating speed is 300rpm, and the air volume is 2.5m3Per hour, aeration-agitation culture, pH5.1, weekThe period is 0-20h, the carbon source of 45 percent of glucose is fed-batch at the feeding speed of 700g/h and the dissolved oxygen of 25 percent, and the culture is carried out for 20h until the wet weight of the thalli is 170 g/L; stopping supplying the carbon source, rebounding the dissolved oxygen to more than 80%, and keeping for 1 hour; adding methanol at a flow rate of 150g/h, controlling dissolved oxygen at 25%, and culturing for about 130h until fermentation is finished.
The culture medium formula of the seeding tank comprises: 4.5% glycerol, 1.5% ammonium dihydrogen phosphate, 0.7% potassium dihydrogen phosphate, 0.7% magnesium sulfate, 0.6% potassium sulfate, 0.07% calcium sulfate, 0.3% potassium hydroxide, pH 5.1;
the fermentation tank culture medium formula is as follows: 4.5% glycerol, 1.5% ammonium dihydrogen phosphate, 0.6% potassium dihydrogen phosphate, 0.6% magnesium sulfate, 0.6% potassium sulfate, 0.07% calcium sulfate, 0.3% potassium hydroxide, pH 5.1;
A50L amplification fermentation tank verification experiment is carried out by adopting the fermentation method, the total fermentation period is 150h, the average enzyme production level of 3 batches of fermentation enzyme production conditions is 85470U/mL, and the following table shows that the strain not only produces high-yield lipase, but also has certain stability in fermentation performance and enzyme activity of the produced lipase.
Fermentation enzyme production of 3-batch high-yield alkaline lipase strain
Batches of Fermentation period (h) Fermentation vigor (U/mL)
1 150 85825
2 150 85560
3 150 85025
Example 6 method for measuring enzyme Activity
Referring to GB/T23535-2009, olive oil is mixed with 4% (w/v) polyvinyl alcohol (PVA) in a ratio of 1:3(v/v) and treated with a high speed homogenizer to give a milky white emulsion. Using the emulsified olive oil as a lipase hydrolysis substrate. Each reaction system included 4mL of olive oil emulsion, 5mL of 50mmol/L buffer, and 1mL of diluted enzyme solution. After 15min at 40 ℃, the reaction was stopped by 15mL of 95% ethanol solution. Meanwhile, a blank group is set, and the enzyme solution inactivated in advance is added into the reaction system as a blank. Respectively adding 2 drops of phenolphthalein indicator into the experimental group and the blank group, titrating by using 0.1mol/L NaOH standard solution until the liquid is reddish and does not fade within 30s, and recording the volume of the consumed 0.1mol/L NaOH standard solution. The average of three experiments was taken.
Definition of unit lipase activity: an amount of enzyme that produces 1. mu. mol of fatty acid per minute under certain reaction conditions.
Calculating enzyme activity X ═ (B-A) × C × 100 ×/0.1/15
In the formula, X: enzyme activity of the sample, U/mL; b: titrating the volume of NaOH consumed by the sample, mL; a: titrating the volume of NaOH consumed by the blank; c: concentration of NaOH standard solution, mol/L; 100: consumption of 1mL of 0.1mol/L NaOH standard solution corresponds to the production of 100. mu. mol of fatty acid; n, sample dilution factor; 0.1: a conversion coefficient of the concentration of the NaOH standard solution; 15: the reaction time was 15 min.
Example 7 enzymatic Properties of Lipase
(1) Optimum pH for action
And taking the supernatant of the C-2 fermentation liquor obtained in the example 5 as a sample, and determining the enzyme activity under different pH conditions at the temperature of 50 ℃ on the basis of the determined highest enzyme activity of the lipase. As can be seen from FIG. 2, the lipase has stable enzyme activity in the pH range of 10.0-11.5, and the optimum action pH is 10.5.
(2) Optimum temperature of action
Taking the supernatant of the C-2 fermentation broth obtained in example 5 as a sample, measuring the enzyme activity under different temperature conditions under the condition of pH 10.5 by taking the measured highest enzyme activity of the lipase as a reference, and calculating the relative enzyme activity, wherein the result is shown in figure 3, the enzyme activity of the lipase produced by C-2 is stable at 45-60 ℃, and the optimal action temperature is 50 ℃.
(3) Alkali resistance
Taking supernatant of YB-1 and C-2 fermentation liquids obtained in example 5 as samples respectively, taking the enzyme activity of lipase without treatment as 100% standard, carrying out heat preservation treatment on the samples at 50 ℃ under the condition of pH 11.5 respectively, measuring the enzyme activity every 1h, and calculating the residual enzyme activity, wherein as can be seen from figure 4, after 9h, the relative activity of the enzyme produced by C-2 still remains more than 80%, and compared with the lipase produced by recombinant strain YB-1 constructed by the original lipase B-1 encoding gene, the alkali resistance is greatly improved, which indicates that the lipase produced by the recombinant strain C-2 has good alkali resistance.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the patent. It should be noted that, for those skilled in the art, various changes, combinations and improvements can be made in the above embodiments without departing from the patent concept, and all of them belong to the protection scope of the patent. Therefore, the protection scope of this patent should be subject to the claims.
SEQUENCE LISTING
<110> Shandonglongket enzyme preparations Co., Ltd
<120> an alkali-resistant lipase
<130> 1
<160> 4
<170> PatentIn version 3.5
<210> 1
<211> 1578
<212> DNA
<213> Rhizopus LD037
<400> 1
atgtcccggt ggtccgcggc gactgccact gccaatggcg gggcacctgg tagcagagcc 60
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gtgacgccgg tatctcaggc cacgccgggt tccggtgcgg gggatccgac ccgtgtgtac 180
tgggaggtgt ccacagacgc cagctttgac gtagccaccg cagctgggga aatgaccacc 240
gaagcggacc gcgaccacac cgtgaaaatc aatgtcacgg gacttgcccc gtccaccacc 300
tactattacc ggttcacggt ggtggacggt ccgtccgcag gtgaggtatc ccgcacgggt 360
cgaacgcgga cgacgccggc ggacgacgcc gcaccggacc acctccgctt cggggtgtgt 420
tcgtgttcga actacgaggc aggacacttc cgtgcctacc gtgagcttgc tgaccgcgac 480
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ggcgcggcct acggcacaac agtacggacc gtggaaccgc gcgaacgtac ccgcacactc 600
gtcggttacc ggatccgaca ggggcactac caccgagacc ctgacctcgc agacctgcat 660
gccgctaagc cgatgatctg catctgggat gaccacgagt ttttcgacaa tgcgtggcgt 720
gacggtgcca ccggggattc cgactacggc ggggcgcagg agtacgcggc ggtgaggcag 780
gctgcgaccg aggcctacta cgaatggatg ccggttcgtg cgggagaggg gttcggcacg 840
gacggtggac gtcatctcta ccggcaccta cgctacggta cgctcgcaga gctcatcctc 900
cccgaccttc gcacttaccg cgacatgcag agctccccgg ccaccgctgc cgcggacggc 960
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accacctggc aactggtcgg gaactcggtg atgttcgctc cgatgacttt gcctcactca 1080
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atcatgaaca gggggtccgg gcctcacggt cgtggaatga acgtggtctt cctcaccggt 1260
gacatccact cctcgtgggc tgcagatatc cccgcgcagg ccggagagta tcggctgggc 1320
cgggacacga ccgtggcggc cgcggagttc atcgtcccgt cggtgaccgc tgcgagcgcc 1380
ttcgattcga ttgttccgac cagtgccgcg gcacctgcgg tcagggaggc tctgcggctg 1440
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<210> 2
<211> 530
<212> PRT
<213> Rhizopus LD037
<400> 2
Met Ser Arg Trp Ser Ala Ala Thr Ala Thr Ala Asn Gly Gly Ala Pro
1 5 10 15
Gly Ser Arg Ala Phe Leu His Ser Val Ala Ser Gly Asp Pro Trp Pro
20 25 30
Asp Ser Val Val Ile Trp Thr Arg Val Thr Pro Val Ser Gln Ala Thr
35 40 45
Pro Gly Ser Gly Ala Gly Asp Pro Thr Arg Val Tyr Trp Glu Val Ser
50 55 60
Thr Asp Ala Ser Phe Asp Val Ala Thr Ala Ala Gly Glu Met Thr Thr
65 70 75 80
Glu Ala Asp Arg Asp His Thr Val Lys Ile Asn Val Thr Gly Leu Ala
85 90 95
Pro Ser Thr Thr Tyr Tyr Tyr Arg Phe Thr Val Val Asp Gly Pro Ser
100 105 110
Ala Gly Glu Val Ser Arg Thr Gly Arg Thr Arg Thr Thr Pro Ala Asp
115 120 125
Asp Ala Ala Pro Asp His Leu Arg Phe Gly Val Cys Ser Cys Ser Asn
130 135 140
Tyr Glu Ala Gly His Phe Arg Ala Tyr Arg Glu Leu Ala Asp Arg Asp
145 150 155 160
Asp Val Glu Phe Val Leu His Leu Gly Asp Tyr Thr Tyr Glu Tyr Glu
165 170 175
Ser Gly Glu Tyr Gly Ala Ala Tyr Gly Thr Thr Val Arg Thr Val Glu
180 185 190
Pro Arg Glu Arg Thr Arg Thr Leu Val Gly Tyr Arg Ile Arg Gln Gly
195 200 205
His Tyr His Arg Asp Pro Asp Leu Ala Asp Leu His Ala Ala Lys Pro
210 215 220
Met Ile Cys Ile Trp Asp Asp His Glu Phe Phe Asp Asn Ala Trp Arg
225 230 235 240
Asp Gly Ala Thr Gly Asp Ser Asp Tyr Gly Gly Ala Gln Glu Tyr Ala
245 250 255
Ala Val Arg Gln Ala Ala Thr Glu Ala Tyr Tyr Glu Trp Met Pro Val
260 265 270
Arg Ala Gly Glu Gly Phe Gly Thr Asp Gly Gly Arg His Leu Tyr Arg
275 280 285
His Leu Arg Tyr Gly Thr Leu Ala Glu Leu Ile Leu Pro Asp Leu Arg
290 295 300
Thr Tyr Arg Asp Met Gln Ser Ser Pro Ala Thr Ala Ala Ala Asp Gly
305 310 315 320
Arg Thr Met Met Gly Gln Asn Gln Phe Asp Trp Phe Ala Gly Val Leu
325 330 335
Thr Thr Ser Thr Thr Thr Trp Gln Leu Val Gly Asn Ser Val Met Phe
340 345 350
Ala Pro Met Thr Leu Pro His Ser Leu Asp Pro Arg Leu His Asp Trp
355 360 365
Leu Val Asp Lys Ile Gly Leu Pro Pro Asp Gly Ile Ala Leu Asn Thr
370 375 380
Asp Gln Trp Asp Gly Tyr Met Val Glu Arg Gln Arg Ile Ile Asp Val
385 390 395 400
Ile Met Asn Arg Gly Ser Gly Pro His Gly Arg Gly Met Asn Val Val
405 410 415
Phe Leu Thr Gly Asp Ile His Ser Ser Trp Ala Ala Asp Ile Pro Ala
420 425 430
Gln Ala Gly Glu Tyr Arg Leu Gly Arg Asp Thr Thr Val Ala Ala Ala
435 440 445
Glu Phe Ile Val Pro Ser Val Thr Ala Ala Ser Ala Phe Asp Ser Ile
450 455 460
Val Pro Thr Ser Ala Ala Ala Pro Ala Val Arg Glu Ala Leu Arg Leu
465 470 475 480
Gly Glu Gly Leu Leu Met Asp Val Asp His Trp Tyr Lys Tyr Val Asp
485 490 495
Leu Ser Arg His Gly Met Met Val Val Asp Val Asp Pro Ser Pro Gly
500 505 510
Val Arg Pro Ala Gly Gln Glu Leu Asp Arg Ser Arg Thr Val Tyr Thr
515 520 525
Pro Ala
530
<210> 3
<211> 1578
<212> DNA
<213> Rhizopus ZF056
<400> 3
atgtcccggt ggtccgcggc gactgccact gccgatggcg gggcacctgg tagcagagcc 60
ttcctgcatt ccgtcgcttc cggagatccc tggcccgaca gcgtcgtcat ctggacccag 120
gtgacgccgg tatctcaggc cacgccgggt tccggtgcgg gggatccgac ccgtgtgtac 180
tgggaggtgt ccacagacgc cagctttgac gtagccaccg cagctgggga aatgaccacc 240
gaagcggacc gcgaccacac cgtgaaaatc aatgtcacgg gacttgcccc gtccaccacc 300
tactattacc ggttcacggt ggtggacggt ccgtccgcag gtgaggtatc ccgcacgggt 360
cgaacgcgga cgacgccggc ggacgacgcc gcaccggacc acctccgctt cggggtgtgt 420
tcgtgttcga actacgaggc aggacacttc cgtgcctacc gtgagcttgc tgaccgcgac 480
gacgttgagt tcgtcctgca tctcggcgac tacacctatg aatacgagtc cggggaatac 540
ggcgcggcct acggcacaac agtacggacc gtggaaccgc gcgaacgtac ccgcacactc 600
gtcggttacc ggatccgaca ggggcactac caccgagacc ctgacctcgc agacctgcat 660
gccgctaagc cgatgatctg catctgggat gaccacgagt ttttcgacaa tgcgtggcgt 720
gacggtgcca ccggggattc cgactacggc ggggcgcagg agtacgcggc ggtgaggcag 780
gctgcgaccg aggcctacta cgaatggatg ccggttcgtg cgggagaggg gttcggcacg 840
gacggtggac gtcatctcta ccggcaccta cgctacggta cgctcgcaga gctcatcctc 900
cccgaccttc gcacttaccg cgacatgcag agctccccgg ccaccgctgc cgcggacggc 960
cgcacgatga tgggacagaa ccagttcgac tggttcgcgg gcgtcctgac gacctcgacg 1020
accacctggc aactggtcgg gaactcggtg atgttcgctc cgatgacttt gcctcactca 1080
ctggatccac ggttgcatga ctggttggtc gacaagatcg gcctgccgcc ggacggaatt 1140
gcactgaata cggaccagtg ggacgggtac atggtggaac gccagcgcat catcgacgtc 1200
atcatgaaca gggggtccgg gcctcacggt cgtggaatga acgtggtctt cctcaccggt 1260
gacatccact cctcgtgggc tgcagatatc cccgcgcagg ccggagagta tcggctgggc 1320
cgggacacga ccgtggcggc cgcggagttc atcgtcccgt cggtgaccgc tgcgagcgcc 1380
ttcgattcga ttgttccgac cagtgccgcg gcacctgcgg tcagggaggc tctgcggctg 1440
ggggaagggt tgctcatgga tgtggaccat tggtacaagt acgtcgacct gagccgacac 1500
ggaatgatgg tggtccccgg ggttcgtcct gccggacaag aactcgaccg gtcccggacg 1560
gtctacaccc cggcgtga 1578
<210> 4
<211> 530
<212> PRT
<213> Rhizopus ZF056
<400> 4
Met Ser Arg Trp Ser Ala Ala Thr Ala Thr Ala Leu Gly Gly Ala Pro
1 5 10 15
Gly Ser Arg Ala Phe Leu His Ser Val Ala Ser Gly Asp Pro Trp Pro
20 25 30
Asp Ser Val Val Ile Trp Thr Gln Val Thr Pro Val Ser Gln Ala Thr
35 40 45
Pro Gly Ser Gly Ala Gly Asp Pro Thr Arg Val Tyr Trp Glu Val Ser
50 55 60
Thr Asp Ala Ser Phe Asp Val Ala Thr Ala Ala Gly Glu Met Thr Thr
65 70 75 80
Glu Ala Asp Arg Asp His Thr Val Lys Ile Asn Val Thr Gly Leu Ala
85 90 95
Pro Ser Thr Thr Tyr Tyr Tyr Arg Phe Thr Val Val Asp Gly Pro Ser
100 105 110
Ala Gly Glu Val Ser Arg Thr Gly Arg Thr Arg Thr Thr Pro Ala Asp
115 120 125
Asp Ala Ala Pro Asp His Leu Arg Phe Gly Val Cys Ser Cys Ser Asn
130 135 140
Tyr Glu Ala Gly His Phe Arg Ala Tyr Arg Glu Leu Ala Asp Arg Asp
145 150 155 160
Asp Val Glu Phe Val Leu His Leu Gly Asp Tyr Thr Tyr Glu Tyr Glu
165 170 175
Ser Gly Glu Tyr Gly Ala Ala Tyr Gly Thr Thr Val Arg Thr Val Glu
180 185 190
Pro Arg Glu Arg Thr Arg Thr Leu Val Gly Tyr Arg Ile Arg Gln Gly
195 200 205
His Tyr His Arg Asp Pro Asp Leu Ala Asp Leu His Ala Ala Lys Pro
210 215 220
Met Ile Cys Ile Trp Asp Asp His Glu Phe Phe Asp Asn Ala Trp Arg
225 230 235 240
Asp Gly Ala Thr Gly Asp Ser Asp Tyr Gly Gly Ala Gln Glu Tyr Ala
245 250 255
Ala Val Arg Gln Ala Ala Thr Glu Ala Tyr Tyr Glu Trp Met Pro Val
260 265 270
Arg Ala Gly Glu Gly Phe Gly Thr Asp Gly Gly Arg His Leu Tyr Arg
275 280 285
His Leu Arg Tyr Gly Thr Leu Ala Glu Leu Ile Leu Pro Asp Leu Arg
290 295 300
Thr Tyr Arg Asp Met Gln Ser Ser Pro Ala Thr Ala Ala Ala Asp Gly
305 310 315 320
Arg Thr Met Met Gly Gln Asn Gln Phe Asp Trp Phe Ala Gly Val Leu
325 330 335
Thr Thr Ser Thr Thr Thr Trp Gln Leu Val Gly Asn Ser Val Met Phe
340 345 350
Ala Pro Met Thr Leu Pro His Ser Leu Asp Pro Arg Leu His Asp Trp
355 360 365
Leu Val Asp Lys Ile Gly Leu Pro Pro Asp Gly Ile Ala Leu Asn Thr
370 375 380
Asp Gln Trp Asp Gly Tyr Met Val Glu Arg Gln Arg Ile Ile Asp Val
385 390 395 400
Ile Met Asn Arg Gly Ser Gly Pro His Gly Arg Gly Met Asn Val Val
405 410 415
Phe Leu Thr Gly Asp Ile His Ser Ser Trp Ala Ala Asp Ile Pro Ala
420 425 430
Gln Ala Gly Glu Tyr Arg Leu Gly Arg Asp Thr Thr Val Ala Ala Ala
435 440 445
Glu Phe Ile Val Pro Ser Val Thr Ala Ala Ser Ala Phe Asp Ser Ile
450 455 460
Val Pro Thr Ser Ala Ala Ala Pro Ala Val Arg Glu Ala Leu Arg Leu
465 470 475 480
Gly Glu Gly Leu Leu Met Asp Val Asp His Trp Tyr Lys Tyr Val Asp
485 490 495
Leu Ser Arg His Gly Met Met Val Val Asp Val Asp Pro Ser Pro Gly
500 505 510
Val Arg Pro Ala Gly Gln Glu Leu Asp Arg Ser Arg Thr Val Tyr Thr
515 520 525
Pro Ala
530

Claims (4)

1. An alkali-resistant lipase is characterized in that the amino acid sequence of the lipase is shown as a sequence table SEQ ID NO. 4.
2. The lipase-encoding gene of claim 1.
3. The encoding gene of claim 2, which is represented by SEQ ID No.3 of the sequence Listing.
4. Use of the alkali-resistant lipase of claim 1.
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114958807A (en) * 2022-06-17 2022-08-30 山东隆科特酶制剂有限公司 Alkali-resistant lipase mutant and application thereof

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6020180A (en) * 1992-07-06 2000-02-01 Novo Nordisk A/S C. antarctica lipase and lipase variants
CN103667091A (en) * 2013-12-05 2014-03-26 青岛蔚蓝生物集团有限公司 Alkaline lipase production bacterial strain and application
CN109576244A (en) * 2018-12-06 2019-04-05 天津科技大学 A kind of novel lipase and its preparation and application

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6020180A (en) * 1992-07-06 2000-02-01 Novo Nordisk A/S C. antarctica lipase and lipase variants
CN103667091A (en) * 2013-12-05 2014-03-26 青岛蔚蓝生物集团有限公司 Alkaline lipase production bacterial strain and application
CN109576244A (en) * 2018-12-06 2019-04-05 天津科技大学 A kind of novel lipase and its preparation and application

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114958807A (en) * 2022-06-17 2022-08-30 山东隆科特酶制剂有限公司 Alkali-resistant lipase mutant and application thereof
CN114958807B (en) * 2022-06-17 2023-06-09 山东隆科特酶制剂有限公司 Alkali-resistant lipase mutant and application thereof

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