CN104651334B - The mould lipase Variant of branch spore that anionic surfactant tolerance improves - Google Patents

Abstract

The present invention provides a kind of lipase, which has good anionic surfactant tolerance, and the enzyme preparation that can be used as detergent uses, and has good prospects for commercial application.

Description

The mould lipase Variant of branch spore that anionic surfactant tolerance improves

Technical field

The invention belongs to field of biotechnology, in particular relate to the mould rouge of branch spore of anionic surfactant tolerance raising Fat enzyme variants.

Background technique

Lipase (lipase, EC3.1.1.3) is a kind of special ester linkage hydrolyzing enzyme, is widely present in animals and plants and micro- life In object.Lipase can be catalyzed the reaction such as hydrolysis, alcoholysis, esterification, transesterification and synthesis of compound of ester type compound. It can be applied to multiple industry such as food, medicine, detergent, weaving, biodiesel, papermaking, leather, cosmetics and environmental protection Field (Hasan F, Ali SA, Hameed A.Industrial applications of microbial Lipases.Enzyme Microb.Technol.2006,39:235-251.).Detergent is mainly used in tableware and clothing Cleaning, bleaching, dry-cleaning, leather cleaning, contact lenses cleaning, the cleaning of the industrial wastes such as cosmetics and food processing, exhaust pipe With the degradation of water closet debirs etc. (Enzymes used in detergents:Lipases).In low temperature, alkalescent Under wash environment, oily dirt is difficult to remove, and the lipase that cut grease is incorporated in laundry detergents and dish washing detergent, can obtain (application of the lipase in detergent, Ban Kou is rich to be repaired relatively good clean effect, daily chemical industry collected translation, nineteen ninety the 2nd Phase, 11-14).

The lipase being commercialized at present has: the lipase Lipolase of Novozymes Company, the Lumafast of Genencor Company, And Ji Site-Brocades Co., Ltd Lipomax.Although three kinds of commercial lipases are applied relatively extensively on detergent industry, But the tolerance in anion and nonionic surfactant is poor.And surfactant be in detergent it is indispensable at Point, the removal of dirt in washing process is played a crucial role, wherein anionic surfactant is in detergent Main component, therefore, application of these three the existing commercial lipases on detergent industry is by larger limitation.

Therefore, industry is badly in need of a kind of lipase that anionic surfactant tolerance is good, to overcome drawbacks described above, improves Application of the lipase on detergent industry.

Summary of the invention

The present inventor obtains a kind of lipase by mutation, which has good anionic surface Activating agent tolerance, the enzyme preparation that can be used as detergent use, and have good prospects for commercial application.

Therefore, the first aspect of the present invention is, provides a kind of polypeptide with lipase active.

Polypeptide provided by the invention with lipase active includes amino acid sequence shown in the SEQ ID No:42 of mutation Column or its active fragment, wherein the mutation includes by the 377th bad ammonia of amino acid sequence shown in SEQ ID No:42 Acid mutation is acidic amino acid or its amide.

In one embodiment of the invention, by the 377th of amino acid sequence shown in SEQ ID No:42 rely Histidine mutations are glutamic acid (E), aspartic acid (D), glutamine (Q) or asparagine (N), preferably sport glutamic acid (E) Or aspartic acid (D).

The second aspect of the invention is, provides the nucleic acid molecules of the polypeptide of coding first aspect.

The third aspect of the invention is, provides the carrier of the nucleic acid molecules comprising second aspect.

In one embodiment of the invention, the carrier is expression vector.

In one embodiment of the invention, the carrier is designed to express in eukaryocyte or prokaryotic cell. In one embodiment of the invention, the carrier be further designed for bacterial cell, fungal cell, yeast cells, It is expressed in mammalian cell, insect cell or plant cell.

The fourth aspect of the invention is, provides the load of the nucleic acid molecules comprising second aspect of the present invention or the third aspect The cell of body.

In one embodiment of the invention, the cell is eukaryocyte or prokaryotic cell.At of the invention one In embodiment, the cell is that bacterial cell, fungal cell, yeast cells, mammalian cell, insect cell or plant are thin Born of the same parents.

The fifth aspect of the invention is, provides the lipase that the cell for the use of the present invention is the 4th generates.

The sixth aspect of the invention is, provides the lipase enzyme that the cell for the use of the present invention is the 4th generates Liquid.

In one embodiment of the invention, the lipase enzyme solution is the fermentation supernatant of the cell.

The seventh aspect of the invention is, provides the polypeptide of the first aspect of the invention or the core of the second aspect Acid molecule coding polypeptide or third in terms of vector encoded polypeptide or the 4th in terms of cell give expression to polypeptide, Or the lipase enzyme solution of the lipase or the 6th aspect in terms of the 5th is for the purposes in washing process.

The eighth aspect of the invention is, provides the polypeptide of the first aspect of the invention or the core of the second aspect The cell of carrier or the 4th aspect in terms of acid molecule or third is preparing the purposes in lipase.

The ninth aspect of the invention is, provides a kind of cleaning product, and the cleaning product includes of the invention first The cell of carrier in terms of the polypeptide of a aspect or the nucleic acid molecules of the second aspect or third or the 4th aspect or The lipase enzyme solution of lipase or the 6th aspect in terms of 5th.

Detailed description of the invention

Fig. 1 shows the SDS tolerance comparison result of the lipase of 377 different mutation.

Fig. 2 shows that the cation and nonionic surfactant tolerance of lipase mutant 496 and lipase wt compare As a result.

Fig. 3 shows the LAS tolerance comparison result of lipase mutant 496 and lipase wt.

Fig. 4 shows the AOS tolerance comparison result of lipase mutant 496 and lipase wt.

Fig. 5 shows the AES tolerance comparison result of lipase mutant 496 and lipase wt.

Fig. 6 shows the SDS tolerance comparison result of lipase mutant 496 and lipase wt.

Mould (Cladosporium sp.) WBRD3.10062425 of branch spore of the invention is in preservation on the 17th in 06 month in 2011 In (the BeiChen West Road, Chaoyang District, BeiJing City 1 " China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC) " No. 3 Institute of Microorganism, Academia Sinica, institute, postcode 100101), deposit number is CGMCC No.4962, classification naming are as follows: branch spore Mould Cladosporium sp..

Specific embodiment

Below in conjunction with specific embodiment, the invention will be further described.It should be understood that following embodiment is merely to illustrate this Invention is not for limiting the scope of the invention.

In the present invention, if without particularly illustrating, percentage (%) or part all refer to the weight relative to composition Percentage or parts by weight.

In the present invention, if without particularly illustrating, related each component or its preferred ingredient be can be combined with each other Form new technical solution.

In the present invention, if without particularly illustrating, all embodiments mentioned in this article and preferred implementation side Formula can be combined with each other to form new technical solution.

In the present invention, if without particularly illustrating, all technical characteristics and preferred feature mentioned in this article can New technical solution is formed to be combined with each other.

In the present invention, if not opposite explanation, the sum of content of each component is 100% in composition.

In the present invention, if not opposite explanation, the sum of number of each component can be 100 weight in composition Part.

In the present invention, unless otherwise indicated, numberical range " a-b " indicates the contracting of any real combinings between a to b Sketch form shows that wherein a and b is real number.Such as numberical range " 0-5 " expression has all listed between " 0-5 " herein Whole real numbers, " 0-5 " are that the breviary of these combinations of values indicates.

In the present invention, unless otherwise indicated, integer numberical range " a-b " indicates the arbitrary integer combination between a to b Breviary indicate, wherein a and b is integer.Such as integer numberical range " 1-N " indicates 1,2 ... N, wherein N is integer.

In the present invention, unless otherwise indicated, " a combination thereof " indicates the multicomponent mixture of each element, such as two Kind, three kinds, four kinds and until maximum possible multicomponent mixture.

If be not specifically stated, term "an" used in this specification refers to "at least one".

If be not specifically stated, the benchmark of percentage (including weight percent) of the present invention is all the combination The total weight of object.

" range " disclosed herein is in the form of lower and upper limit.It can be respectively one or more lower limits and one Or multiple upper limits.Given range is defined by a selected lower limit and a upper limit.Selected lower and upper limit limit The boundary of special range is determined.All ranges that can be defined in this way comprising and can combine, i.e., any lower limit It can combine to form a range with any upper limit.For example, the range of 60-120 and 80-110 are listed for special parameter, The range for being interpreted as 60-110 and 80-120 is also to expect.In addition, if the minimum zone value 1 and 2 listed, and if List maximum magnitude value 3,4 and 5, then below range can all expect: 1-3,1-4,1-5,2-3,2-4 and 2-5.

Herein, unless otherwise indicated, the ratio or weight of each component all refer to dry weight.

Herein, unless otherwise indicated, each reaction all carries out at normal temperatures and pressures.

Herein, unless otherwise indicated, each reaction step can be carried out sequentially, can also be carried out out of order.Example Such as, other steps be may include between each reaction step, and can also be with reversed order between reaction step.Preferably, originally Reaction method in text is that sequence carries out.

In following embodiments of the invention, Assay of lipase activity using p-nitrophenyl palmitate (p-NPP) method come Measurement, this method generate color products p-nitrophenyl using p-nitrophenyl palmitate as reaction substrate, by the catalysis of lipase Phenol (p-NP), the product have strong absorb under 405-410nm wavelength.Enzyme activity unit is defined as: 1 unit, which refers to, to be marked Enzyme amount needed for the p-NP of 1 μm of ol of catalysis release per minute under the conditions of quasi-experiment.The specific method is as follows:

It is pre-configured with substrate and buffer, substrate: the dissolution of 6mg/ml p-NPP(isopropanol), buffer: 0.05mol/ LTris(pH8.0,0.1% Arabic gum).By substrate and buffer with 1:9(v/v) it is made into reaction mixture.Take two 2ml from Heart pipe, respectively control tube and sample cell.Addition 400ul reaction mixture is to two centrifuge tubes respectively, in 35 DEG C of pre- warm bath 5min. A certain amount of dilution enzyme solution is added into sample cell, after mixing, continues warm bath 15min.1.5ml ethyl alcohol is added to above-mentioned two Centrifuge tube terminates reaction, and adds the dilution enzyme solution of same amount to control tube.12000rpm is centrifuged 2min, takes supernatant, surveys 405nm The light absorption value at place.

According to enzyme activity calculation formula obtained by standard curve are as follows: A=- ([A1-A0] × 0.7885-0.0118) × V1 × n/(V2 × t).

Wherein, A: sample enzyme activity (U/ml) A1: the OD405 of sample enzyme solution, A0: compares the OD405 of enzyme solution, V1: overall reaction The volume (ml) of liquid, n: the extension rate of enzyme solution, V2: the volume (ml) of enzyme solution, t: reaction time (min).

In following embodiments of the invention, the culture medium that uses are as follows:

LB liquid medium: 1% peptone, 0.5% yeast extract, 1% sodium chloride adjust pH to 7.0.

PDA liquid medium: 20% potato (is cut into small pieces, adds boiling rotten, with eight layers of filtered through gauze, collect liquid), and 2% Glucose, natural pH.

YPD fluid nutrient medium: 1% yeast extract, 2% peptone, 1% glucose, natural pH.

Growth medium BMGY:1% yeast extract, 2% peptone, 100mM potassium phosphate (pH6.0), 1.34% yeast basic Nitrogen source, 4 × 10^-5% biotin, 1% glycerol.

Induced medium BMMY:1% yeast extract, 2% peptone, 100mM potassium phosphate (pH6.0), 1.34% yeast basic Nitrogen source, 4 × 10^-5% biotin, 0.5% methanol.

Lipase identifies culture medium: 1% yeast extract, 2% peptone, 100mM potassium phosphate (pH6.0), 1.34% yeast base Plinth nitrogen source, 4 × 10^-5% biotin, 0.5% methanol, 0.001% rhodamine B, 4% polyvinyl alcohol, 2% olive oil, 2% agar.

In following embodiments of the invention, WBRD3.10062425 is for the branch spore used mould (Cladosporium sp.) (the north " China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC) " was deposited on 06 17th, 2011 No. 3 Institute of Microorganism, Academia Sinica, institute of the Chaoyang District Jing Shi North Star West Road 1, postcode 100101), deposit number is CGMCC No.4962, classification naming are as follows: the mould Cladosporium sp. of branch spore.

1,377 saturation mutation of embodiment

1.1, the mould genome of branch spore extracts

The mould WBRD3.10062425 of branch spore is inoculated with PDA liquid medium, 28 DEG C, cultivate 2 days under the conditions of 200rpm, is passed through Buchner funnel suction method collects cladosporium sp body, quick-frozen in liquid nitrogen.

Thallus after taking freezing uses precious company MiniBEST Universal according to the method that manufacturer provides Genomic DNA Extraction Kit kit extracts genome, obtains the mould genome of branch spore.

The mould genome of branch spore obtained will be extracted and carry out agarose electrophoresis, by (white good purchased from Shanghai with DNA standard sample Development in science and technology Co., Ltd) banding pattern and brightness compare, verify genome quality and concentration.

The results show that extracting, the genome integrity degree obtained is good, and concentration reaches 50ng/ μ l.

The building of 1.2 wild type lipase recombinant yeast pichia pastoris

The mould genome of branch spore prepared using embodiment 1.1 is template, using lipcs-U and lipcs-D as primer pair, carries out PCR amplification, to clone lipase gene, wherein lipcs-U and lipcs-D sequence is respectively such as SEQ ID NO.1 and SEQ ID Shown in NO.2.

PCR reaction system are as follows: rTaq(is purchased from Takara company) 0.25 μ l, 10 × PCR buffer, 5 μ l, dNTP mixture (being purchased from Takara company) 4 μ l, lipcs-U1 μ l, lipcs-D1 μ l, 0.1 μ l of genome add water polishing to 50 μ l.

PCR program are as follows: 95 DEG C of 30s, (95 DEG C of 30s, 51 DEG C of 30s, 72 DEG C of 1min30s) 28 circulations, 72 DEG C of 7min.

The PCR product that Omega plastic recovery kit will be used to recycle uses Takara company restriction enzyme EcoR I With I digestion of Not, and with through with the connection of the pPIC9k plasmid of identical enzymatic treatment, the connection product of acquisition is converted DH5 α competence Cell is incubated overnight to transformant in 37 DEG C and grows.Picking transformant is inoculated into LB liquid medium, in 37 DEG C, 160rpm Lower culture 14-16h collects thallus.According to the method that manufacturer provides, (Shanghai is purchased from Axygen small amount plasmid extraction kit Hundred match companies) extracting plasmid, double digestion experiment is carried out with restriction enzyme EcoR I and Not I, digestion verification is correctly positive Property clone send to Sangon Biotech (Shanghai) Co., Ltd. be sequenced, the sequence as shown in SEQ ID NO.41, compile The amino acid sequence of code is as shown in SEQ ID NO.42.With restriction enzyme Bgl II by plasmid linearization, linearizes product and use The purifying of Axygen cleaning agents box, and be 300ng/ μ l by the concentration that electrophoresis verifies linearization plasmid.

With reference to Shixuan W., Geoffrey J.L..High efficiency transformation by electroporation of Pichia pastoris pretreated with lithium acetate and Dithiothreitol.Biotechniques, 2004,36:152-154 method finish red ferment by the preparation of sorbierite ablution Female GS115 competent cell.

1 μ g linearization plasmid is taken, 100 μ l GS115 competent cells are added, is transferred to electric shock cup, electric shock cup is stood on ice After five minutes, it is placed on electroporation and shocks by electricity, condition: voltage 2000v, time 5s.It is rapidly added 1ml1mol/l sorbitol solution, is inhaled Beat uniformly after, by bacterium solution be coated with RBD plate (1.34% yeast basic nitrogen source, 4 × 10^-5% biotin, 2% glucose, the mountain 1mol/l Pears alcohol, 1.5% agar).In 28 DEG C of culture a couple of days, until transformant is grown.

Transformant on picking RBD plate is seeded on lipase identification plate, in 28 DEG C of culture a couple of days, until occurring Color ring obtains the transformant with lipase activity, which is named as " wt ", lipase expressed by the transformant strain It is named as " Lipwt ".Picking monoclonal, is seeded to YPD fluid nutrient medium, 28 DEG C, 200rpm culture for 24 hours, by 600 μ l bacterium solutions and 400 μ l50% glycerol (w/v%) are mixed and made into glycerol tube, are stored in -80 DEG C.

1.3,377 lysine mutations are phenylalanine

Using the mould genome of branch spore as template, with Lipcs-U, F-D(sequence as shown in SEQ ID NO.3), F-U(sequence such as Shown in SEQ ID NO.4) and Lipcs-D be primer, through PCR amplification, the lipase that preparation 377 sports phenylalanine is prominent Variant, detailed process is as follows:

First round PCR, using the mould genome of branch spore as template, respectively using Lipcs-U and F-D or F-U and Lipcs-D as primer It is right, carry out PCR amplification, obtain PCR product respectively, wherein PCR system are as follows: Primstar(is purchased from Takara company) 0.25 μ l, 5 × buffer, 10 μ l, dNTP mixture, 4 μ l, lipcs-U1 μ l, F-D1 μ l(either F-U1 μ l, Lipcs-D1 μ l), genome 0.1 μ l adds water polishing to 50 μ l.PCR program are as follows: 95 DEG C of 30s, (95 DEG C of 30s, 51 DEG C of 30s, 72 DEG C of 1min) 28 circulations, 72 ℃7min。

First round PCR product is diluted 10 times by the second wheel PCR, and isometric mixing is used as template, with Lipcs-U/Lipcs- D carries out PCR amplification, wherein PCR system as primer pair are as follows: Primstar(is purchased from Takara company) 0.25 μ l, 5 × PCR be slow 10 μ l, dNTP mixture of fliud flushing 4 μ l, lipcs-U1 μ l, lipcs-D1 μ l, 0.5 μ l of template add water polishing to 50 μ l.PCR program Are as follows: 95 DEG C of 30s, (95 DEG C of 30s, 51 DEG C of 30s, 72 DEG C of 1min30s) 28 circulations, 72 DEG C of 7min.

PCR product is recycled using Omega plastic recovery kit, with Takara company restriction enzyme EcoR I and Not Connection product is converted DH5 α competent cell, 37 DEG C overnight, turns with the pPIC9k plasmid connection with identical enzymatic treatment by I digestion Beggar grows.Picking transformant is inoculated into LB liquid medium, cultivates 14-16h under 37 DEG C, 160rpm, until transformant is long Out.Fetch transformant, 10000rpm, 4 DEG C, thalline were collected by centrifugation by 5min, plasmid is extracted according to 1.2 method of embodiment, with limit Property restriction endonuclease EcoR I and Not I processed carry out double digestion experiment, and the correct positive colony of digestion verification is sent to raw work bioengineering The sequencing of (Shanghai) limited liability company.The results show that obtaining the clone that 377 lysine mutations are phenylalanine.

Using the method for embodiment 1.2, cultivates institute's clone, extraction plasmid, progress plasmid linearization, electricity and turn to finish red ferment Mother obtains recombinant yeast pichia pastoris, and makes its glycerol tube bacterium solution.

1.4,377 mutant lipase preparations

By the method for embodiment 1.3, the fatty enzyme mutant that 377 lysine mutations are other amino acid is prepared respectively The clone of body, and make the glycerol tube bacterium solution accordingly cloned, wherein lipase mutant and its primer pair are as shown in table 1.

Mould 377 site mutant of lipase of table 1, branch spore and its mutant primer

Mutating acid	Upstream primer	Sequence number	Downstream primer	Sequence number
					L	L-U	SEQ ID NO.5	L-D	SEQ ID NO.6
S	S-U	SEQ ID NO.7	S-D	SEQ ID NO.8
					Y	Y-U	SEQ ID NO.9	Y-D	SEQ ID NO.10

C	C-U	SEQ ID NO.11	C-D	SEQ ID NO.12
					W	W-U	SEQ ID NO.13	W-D	SEQ ID NO.14
P	P-U	SEQ ID NO.15	P-D	SEQ ID NO.16
					H	H-U	SEQ ID NO.17	H-D	SEQ ID NO.18
Q	Q-U	SEQ ID NO.19	Q-D	SEQ ID NO.20
					R	R-U	SEQ ID NO.21	R-D	SEQ ID NO.22
I	I-U	SEQ ID NO.23	I-D	SEQ ID NO.24
					M	M-U	SEQ ID NO.25	M-D	SEQ ID NO.26
T	T-U	SEQ ID NO.27	T-D	SEQ ID NO.28
					N	N-U	SEQ ID NO.29	N-D	SEQ ID NO.30
V	V-U	SEQ ID NO.31	V-D	SEQ ID NO.32
					A	A-U	SEQ ID NO.33	A-D	SEQ ID NO.34
D	D-U	SEQ ID NO.35	D-D	SEQ ID NO.36
					G	G-U	SEQ ID NO.37	G-D	SEQ ID NO.38
E	E-U	SEQ ID NO.39	E-D	SEQ ID NO.40

1.5, the expression of wt and each saturation mutation

The glycerol tube bacterium solution of 100 μ l wt and each saturation mutation is taken to be seeded to YPD fluid nutrient medium, 28 DEG C of cultures respectively After for 24 hours, growth medium BMGY is inoculated in 0.1% inoculum concentration, in 28 DEG C of cultures to thalli growth to OD₆₀₀For 2-6,1500g, 4 DEG C, centrifugation fermentation liquid 5min, remove supernatant, and thallus is resuspended with the induced medium BMMY of former culture volume, adds daily 0.5% methanol, induction 3 days after, 15000rpm, 4 DEG C be centrifuged 15 minutes, collect fermentation supernatant.

1.6, the tolerance detection of surfactant

The fermented supernatant fluid prepared in embodiment 1.5 is subjected to protein electrophoresis, and passes through Millipore centrifugal filter device (being purchased from Sinopharm Chemical Reagent Co., Ltd.) carries out protein concentration, and destination protein concentration is adjusted to 0.05 μ g/ μ l.

Final concentration of 0.5% SDS is added into the measurement system of the lipase activity of detection fermentation supernatant, and adds and waits bodies The reaction tube of ponding tests the SDS tolerance of lipase as control.Detailed process is as follows:

By 50mM Tris-Cl(pH8.0) buffer and 6mg/ml pNPP substrate with 9:1 proportional arrangement at mixed liquor.It takes 400 μ l mixed liquors are managed to 2ml Eppendorf, and SDS to final concentration of 0.5%, oscillation mixing is added.It is incubated for 5 minutes at 45 DEG C, 1 μ l fermentation supernatant is added to be reacted.After reaction 15 minutes, it is 2ml that ethyl alcohol to final volume, which is added, and 12000rpm is centrifuged 2 minutes, Test 405nm absorbance value.Two controls are set in this experiment, wherein first control is to change SDS into isometric water, His test condition is constant.Second control after reacting and terminating ethyl alcohol addition, then adds equivalent enzyme solution to start that enzyme solution is not added, Other test conditions are constant.

Circular is: according to above-mentioned pNPP method, calculating the vigor of the enzyme under the reaction condition for being added to 0.5%SDS With the vigor that SDS is replaced as to enzyme under the reaction condition of isometric water, the ratio of the two is multiplied by the residual activity that 100 be enzyme Percentage.To measure Lipwt(K) the residual activity percentage of lipase is 100%, calculate the residual activity hundred of other lipase Divide ratio, as Fig. 1.

According to Fig. 1's as a result, when 377 amino acids are basic amino acid, such as arginine (R), lysine (K), histidine (H) tolerance declines when, when 377 amino acids are acidic amino acid and its amide, such as glutamic acid (E), aspartic acid (D), paddy Glutamine (Q) and tolerance rises when asparagine (N), especially when 377 amino acids are glutamic acid (E), aspartic acid (D) When, tolerance reaches highest.When 377 amino acids are neutral fat race amino acid, such as glycine (G), alanine (A) and bright ammonia Tolerance declines whens sour (L) etc..When 377 amino acids be hydroxyl or sulphur amino acid, such as when serine (S), threonine (T) it is resistance to It is slightly improved by property.When 377 amino acids be aromatic amino acid, when such as tyrosine (Y), tryptophan (W) tolerance slightly on Tolerance declines when rising, but sporting phenylalanine (F).Particularly, when 377 amino acids sport proline, by albumen Concentration and Lipwt adjust consistent (0.05 μ g/ μ l), and the 1/50 of Lipwt is down in specific enzyme activity.Illustrate that proline affects lipase Activated centre.

In the present invention, compared with Lipwt, when 377 amino acids are glutamic acid (E), aspartic acid (D), anion table Face activating agent tolerance increase rate is maximum, and Glutamic Acid is optimal.According to above-mentioned saturation mutation as a result, analyzing reason such as Under: 377 amino acids are located at Lipcs protein three-dimensional structure surface, encounter the anionic surfactant in environment, basic amine group Acid performance affinity interaction, causes albumen to be damaged, and acidic amino acid cashes repulsive interaction, and albumen is caused to obtain to a certain extent To protection.

The Pichia pastoris recombinant bacterial strain that 377 amino acid are glutamic acid is named as " 496 ", the mutant lipase life of expression Entitled " Lip496 ".

Embodiment 2, the detection of the tolerance of surfactant

Prepare mass concentration be 10% cationic surfactant (CTAB), nonionic surfactant (AEO-9, Triton X-100 and Tween-80) and anionic surfactant (LAS, AOS, AES and SDS) storage liquid.

According to the method for embodiment 1.6, the Lip496 from embodiment 1.5 is tested to various surfactant tolerances, As a result as shown in figures 2-6.Wherein specific calculation method is: according to above-mentioned pNPP method, calculating is being added to surfactant Under reaction condition the vigor of enzyme and by surfactant replacement at the vigor of enzyme under the reaction condition of isometric water, the ratio of the two Multiplied by 100 be enzyme residual activity percentage.

According to fig. 2 as a result, lipase mutant 496 is resistance to cationic surfactant compared with wild type lipase It is reduced by property, the tolerance of nonionic surfactant Triton X-100, Tween-80 and AEO-9 is changed less substantially, Wherein Triton X-100 is declined slightly.

According to Fig. 3's -6 as a result, lipase mutant 496 is better than wild type rouge to the tolerance of LAS, AOS, AES and SDS Fat enzyme, wherein being twice or more of wild type lipase to the tolerance of LAS, AOS and SDS, the tolerance to AES is wild type 1.5 times or more of lipase.Anionic surfactant tolerance is greatly improved.

Since anionic surfactant accounts for major part in current detergent component, and 496 pairs of yin of lipase mutant from Sub- tolerance greatly enhances, and therefore, the enzyme preparation that can be used as detergent uses, and has good prospects for commercial application.Lip496 The advantages of using as detergent enzyme preparation shows both ways: (1) lipase of the present invention can be improved the enzyme of enzyme-containing detergent Preparation vigor improves washing effect, and the reduction of enzyme activity loss, causes the cost of expensive lipase is opposite to drop It is low；(2) with scientific and technological progress, the market scope of liquid detergent will constantly expand, the rouge that Surfactant tolerance improves The demand of fat enzyme will be more urgent, and Lip496 has complied with the needs of market development, solve the technical problem that lipase is easy inactivation, It paves the way for the further genralrlization application of liquid detergent.

Claims (17)

1. the polypeptide with lipase active, polypeptide sequence amino acid sequence as shown in the SEQ ID No:42 being mutated It is shown, wherein described sport the 377th lysine mutation of amino acid sequence shown in SEQ ID No:42 is acidity Amino acid or its amide.

2. polypeptide as described in claim 1, wherein the 377th of amino acid sequence shown in SEQ ID No:42 is relied Histidine mutations are glutamic acid (E), aspartic acid (D), glutamine (Q) or asparagine (N).

3. polypeptide as claimed in claim 2, wherein the 377th of amino acid sequence shown in SEQ ID No:42 is relied Histidine mutations are glutamic acid (E) or aspartic acid (D).

4. encoding the nucleic acid molecules of polypeptide described in any one of claims 1 to 3.

5. including the carrier of nucleic acid molecules as claimed in claim 4.

6. carrier as claimed in claim 5, which is characterized in that the carrier is expression vector.

7. carrier as claimed in claim 6, which is characterized in that the carrier is designed in eukaryocyte or prokaryotic cell Expression.

8. carrier as claimed in claim 7, which is characterized in that the carrier is designed to bacterial cell, fungal cell, the food in one's mouth It is expressed in newborn zooblast, insect cell or plant cell.

9. the cell comprising carrier described in any one of nucleic acid molecules as claimed in claim 4 or claim 5-8.

10. cell as claimed in claim 9, which is characterized in that the cell is eukaryocyte or prokaryotic cell.

11. cell as claimed in claim 10, which is characterized in that the cell is bacterial cell, fungal cell, mammal Cell, insect cell or plant cell.

12. the lipase generated using cell described in any one of claim 9-11.

13. the lipase enzyme solution generated using cell described in any one of claim 9-11.

14. lipase enzyme solution as claimed in claim 13, which is characterized in that the lipase enzyme solution is the fermentation of the cell Supernatant.

15. the polypeptide or power of polypeptide of any of claims 1-3 or nucleic acid molecule encoding as claimed in claim 4 Benefit requires cell described in any one of polypeptide or claim 9-11 of vector encoded described in any one of 5-8 to give expression to Polypeptide or claim 12 described in lipase enzyme solution described in lipase or claim 13 or 14 be used for washing process In purposes.

16. polypeptide of any of claims 1-3 or nucleic acid molecules as claimed in claim 4 or claim 5-8 Any one of described in carrier or any one of claim 9-11 described in cell preparing the purposes in lipase.

17. a kind of cleaning product, the cleaning product includes polypeptide of any of claims 1-3 or claim 4 It is thin described in any one of carrier or claim 9-11 described in any one of described nucleic acid molecules or claim 5-8 Lipase enzyme solution described in lipase described in born of the same parents or claim 12 or claim 13 or 14.