CN104651334A

CN104651334A - Cladosporium lipase variant being improved in resistance of anionic surfactant

Info

Publication number: CN104651334A
Application number: CN201310586664.6A
Authority: CN
Inventors: 陈苗苗; 苏春阳; 许骏
Original assignee: Wilmar Shanghai Biotechnology Research and Development Center Co Ltd
Current assignee: Wilmar Shanghai Biotechnology Research and Development Center Co Ltd
Priority date: 2013-11-20
Filing date: 2013-11-20
Publication date: 2015-05-27
Anticipated expiration: 2033-11-20
Also published as: CN104651334B

Abstract

The invention provides a lipase, which is excellent in resistance of anionic surfactant, can be employed as an enzyme preparation in a detergent and is excellent in industrial application prospect.

Description

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

Technical field

The invention belongs to biological technical field, relate to the mould lipase Variant of branch spore that anion surfactant tolerance improves specifically.

Background technology

Lipase (lipase, EC3.1.1.3) is the special ester linkage hydrolyzing enzyme of a class, is extensively present in animals and plants and microbe.Lipase can the hydrolysis of catalysis ester compound, alcoholysis, esterification, transesterify and compound the reaction such as synthesis.It can be applicable to multiple industrial circle (Hasan F such as food, medicine, washing composition, weaving, biofuel, papermaking, leather, makeup and environment protection; Ali SA; Hameed A.Industrial applications ofmicrobial lipases.Enzyme Microb.Technol.2006,39:235-251.).Washing composition is mainly used in the cleaning of tableware and clothing, bleaching, dry-cleaning, leather cleaning, contact lenses is cleaned, the cleaning of the industrial waste such as makeup and food-processing, the degraded of vapor pipe and water closet organic waste etc. (Enzymes used in detergents:Lipases).Under low temperature, weakly alkaline wash environment, oily dirt is difficult to remove, and allocates the lipase that cut grease in scouring agent and dish washing detergent into, can obtain reasonable clean effect (application of lipase in washing composition, Ban Kou is rich to be repaiied, daily chemical industry collected translation, phase nineteen ninety the 2nd, 11-14).

Current commercial lipase has: the lipase Lipolase of Novozymes Company, the Lumafast of Genencor Company, and the Lipomax of Ji Site-Brocades Co., Ltd.Although three kinds of commercial lipases are applied comparatively wide on detergent industry, the tolerance in negatively charged ion and nonionogenic tenside is poor.And tensio-active agent is indispensable composition in washing composition, vital effect is played to the removal of dirt in washing process, wherein anion surfactant is the main component in washing composition, therefore, these three kinds of commercial lipases application on detergent industry existing are subject to larger restriction.

Therefore, industry is badly in need of a kind of lipase of anion surfactant better tolerance, to overcome above-mentioned defect, improves the application of lipase on detergent industry.

Summary of the invention

The present inventor is by sudden change, and obtain a kind of lipase, this lipase has good anion surfactant tolerance, and the zymin that can be used as washing composition uses, and has good prospects for commercial application.

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

The polypeptide with lipase activity provided by the invention comprises the aminoacid sequence shown in SEQ ID No:42 or its active fragments of sudden change, and it is acidic amino acid or its acid amides that wherein said sudden change comprises the lysine mutation of the 377th of the aminoacid sequence shown in SEQ ID No:42.

In one embodiment of the invention, be L-glutamic acid (E), aspartic acid (D), glutamine (Q) or l-asparagine (N) by the lysine mutation of the 377th of the aminoacid sequence shown in SEQ ID No:42, preferably sport L-glutamic acid (E) or aspartic acid (D).

Second aspect of the present invention is, provides the nucleic acid molecule of the polypeptide of coding first aspect.

3rd aspect of the present invention is, providing package is containing the carrier of the nucleic acid molecule of second aspect.

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

In one embodiment of the invention, described carrier is designed to express in eukaryotic cell or prokaryotic cell prokaryocyte.In one embodiment of the invention, described carrier is designed to express in bacterial cell, fungal cell, yeast cell, mammalian cell, insect cell or vegetable cell further.

4th aspect of the present invention is, providing package contains the cell of the nucleic acid molecule of second aspect present invention or the carrier of the third aspect.

In one embodiment of the invention, described cell is eukaryotic cell or prokaryotic cell prokaryocyte.In one embodiment of the invention, described cell is bacterial cell, fungal cell, yeast cell, mammalian cell, insect cell or vegetable cell.

5th aspect of the present invention is, provides the lipase that the cell using the present invention the 4th aspect produces.

6th aspect of the present invention is, provides the lipase enzyme liquid that the cell using the present invention the 4th aspect produces.

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

7th aspect of the present invention is, the lipase enzyme liquid of the polypeptide providing the cell expressing of the polypeptide of the vector encoded of the polypeptide of the polypeptide of first aspect of the present invention or the nucleic acid molecule encoding of second aspect or the 3rd aspect or the 4th aspect to go out or the lipase of the 5th aspect or the 6th aspect is for the purposes in washing process.

8th aspect of the present invention is, provides the cell of the carrier of the polypeptide of first aspect of the present invention or the nucleic acid molecule of second aspect or the 3rd aspect or the 4th aspect preparing the purposes in lipase.

9th aspect of the present invention is, there is provided a kind of cleaning product, described cleaning product comprises the lipase of the carrier of the polypeptide of first aspect of the present invention or the nucleic acid molecule of second aspect or the 3rd aspect or the cell of the 4th aspect or the 5th aspect or the lipase enzyme liquid of the 6th aspect.

Accompanying drawing explanation

Fig. 1 shows the SDS tolerance comparative result of the lipase of the difference sudden change of 377.

Fig. 2 shows positively charged ion and the nonionogenic tenside tolerance comparative result of lipase mutant 496 and lipase wt.

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

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

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

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

Branch spore of the present invention mould (Cladosporium sp.) WBRD3.10062425 was deposited in " China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC) " (No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City Institute of Microorganism, Academia Sinica on 06 17th, 2011, postcode 100101), preserving number is CGMCC No.4962, and Classification And Nomenclature is: the mould Cladosporium sp. of branch spore.

Embodiment

Below in conjunction with specific embodiment, the invention will be further described.Should be understood that following examples only for illustration of the present invention but not for limiting scope of the present invention.

In the present invention, if do not illustrated especially, percentage ratio (%) or part all refer to weight percentage relative to composition or weight part.

In the present invention, if do not illustrated especially, involved each component or its preferred ingredient can be combined to form new technical scheme mutually.

In the present invention, if do not illustrated especially, all embodiments mentioned in this article and preferred implementation can be combined to form new technical scheme mutually.

In the present invention, if do not illustrated especially, all technical characteristics mentioned in this article and preferred feature can be combined to form new technical scheme mutually.

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

In the present invention, if do not have contrary explanation, in composition, the number sum of each component can be 100 weight parts.

In the present invention, unless otherwise indicated, the breviary of any real combinings that numerical range " a-b " represents between a to b represents, wherein a and b is real number.Such as numerical range " 0-5 " represents the whole real numbers all listed between " 0-5 " herein, and the breviary of " 0-5 " just these combinations of values represents.

In the present invention, unless otherwise indicated, the breviary of the arbitrary integer combination that integer numerical range " a-b " represents between a to b represents, wherein a and b is integer.Such as integer numerical range " 1-N " represents 1,2 ... N, wherein N is integer.

In the present invention, unless otherwise indicated, " its combination " represents the multicomponent mixture of described each element, such as two kinds, three kinds, four kinds and until the multicomponent mixture of maximum possible.

If do not particularly not pointed out, this specification sheets term " one " used refers to " at least one ".

If do not particularly not pointed out, the benchmark of percentage ratio of the present invention (comprising weight percentage) is all the gross weight of described composition.

" scope " disclosed herein is with the form of lower limit and the upper limit.One or more lower limit can be respectively, and one or more upper limit.Given range is limited by a selected lower limit and a upper limit.Selected lower limit and the upper limit define the border of special scope.All scopes that can carry out by this way limiting comprise and may be combined with, and namely any lower limit can be combined to form a scope with any upper limit.Such as, list the scope of 60-120 and 80-110 for special parameter, be interpreted as that the scope of 60-110 and 80-120 also expects.In addition, if the minimum extent value listed 1 and 2, and if list maximum range value 3,4 and 5, then the scope below can all expect: 1-3,1-4,1-5,2-3,2-4 and 2-5.

In this article, except as otherwise noted, the ratio of each component or weight all refer to dry weight.

In this article, except as otherwise noted, each reaction is carried out all at normal temperatures and pressures.

In this article, except as otherwise noted, each reactions steps can sequentially be carried out, and also can not carry out in order.Such as, between each reactions steps, other steps can be comprised, and also can reversed order between reactions steps.Preferably, reaction method is herein that order is carried out.

In following embodiment of the present invention, Assay of lipase activity adopts p-nitrophenyl cetylate (p-NPP) method to measure, the method with p-nitrophenyl cetylate for reaction substrate, produce color products p-NP (p-NP) by the catalysis of lipase, this product has strong absorption under 405-410nm wavelength.Enzyme activity unit is defined as: namely 1 unit refers to that per minute catalysis discharges the enzyme amount needed for the p-NP of 1 μm of ol under standard laboratory conditions.Concrete grammar is as follows:

Pre-configured substrate and damping fluid, substrate: 6mg/ml p-NPP(Virahol dissolves), damping fluid: 0.05mol/lTris(pH8.0,0.1% gum arabic).By substrate and damping fluid with 1:9(v/v) be made into reaction mixture.Get two 2ml centrifuge tubes, be respectively control tube and sample hose.Add 400ul reaction mixture to two centrifuge tube respectively, at 35 DEG C of pre-temperature bath 5min.In sample hose, add a certain amount of dilution enzyme liquid, after mixing, continue temperature bath 15min.Add 1.5ml ethanol to above-mentioned two centrifuge tube termination reactions, and the dilution enzyme liquid adding same amount is to control tube.The centrifugal 2min of 12000rpm, gets supernatant, surveys the light absorption value at 405nm place.

According to typical curve gained enzyme calculation formula alive be: A=-([A1-A0] × 0.7885-0.0118) × V1 × n/(V2 × t).

Wherein, A: sample enzyme is lived (U/ml), A1: the OD405 of sample enzyme liquid, A0: the OD405 of contrast enzyme liquid, V1: the volume (ml) of total reaction liquid, n: the extension rate of enzyme liquid, V2: the volume (ml) of enzyme liquid, t: reaction times (min).

In following embodiment of the present invention, the substratum of use is:

LB liquid nutrient medium: 1% peptone, 0.5% yeast extract, 1% sodium-chlor, adjustment pH to 7.0.

PDA liquid nutrient medium: 20% potato (be cut into small pieces, add water well-done, by eight layers of filtered through gauze, collect liquid), 2% glucose, natural pH.

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

Growth medium BMGY:1% yeast extract, 2% peptone, 100mM potassiumphosphate (pH6.0), 1.34% yeast basic nitrogen source, 4 × 10 ^-5% vitamin H, 1% glycerine.

Inducing culture BMMY:1% yeast extract, 2% peptone, 100mM potassiumphosphate (pH6.0), 1.34% yeast basic nitrogen source, 4 × 10 ^-5% vitamin H, 0.5% methyl alcohol.

Lipase qualification substratum: 1% yeast extract, 2% peptone, 100mM potassiumphosphate (pH6.0), 1.34% yeast basic nitrogen source, 4 × 10 ^-5% vitamin H, 0.5% methyl alcohol, 0.001% rhodamine B, 4% polyvinyl alcohol, 2% sweet oil, 2% agar.

In following embodiment of the present invention, branch spore mould (Cladosporium sp.) WBRD3.10062425 used was deposited in " China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC) " (No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City Institute of Microorganism, Academia Sinica on 06 17th, 2011, postcode 100101), preserving number is CGMCC No.4962, and Classification And Nomenclature is: the mould Cladosporium sp. of branch spore.

Embodiment 1,377 saturation mutations

1.1, the mould genome of branch spore extracts

Mould for branch spore WBRD3.10062425 is inoculated PDA liquid nutrient medium, 28 DEG C, cultivate 2 days under 200rpm condition, collect cladosporium sp body by Büchner funnel suction method, quick-frozen in liquid nitrogen.

Get freezing after thalline, according to the method that manufacturer provides, use precious company MiniBEST UniversalGenomic DNA Extraction Kit test kit extracting genome, obtain branch spore mould genome.

Agarose electrophoresis is carried out by extracting the mould genome of branch spore obtained, by the banding pattern and luminance factor with DNA standard model (purchased from Shanghai Hao Jia development in science and technology company limited) comparatively, checking genome quality and concentration.

Result shows, and extract the genome integrity degree obtained good, concentration reaches 50ng/ μ l.

The structure of 1.2 wild type lipase recombinant yeast pichia pastoris

The mould genome of branch spore prepared with embodiment 1.1 is for template, and with lipcs-U and lipcs-D for primer pair, carry out pcr amplification, to clone lipase gene, wherein lipcs-U and lipcs-D sequence is respectively as shown in SEQ ID NO.1 and SEQ ID NO.2.

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

PCR program is: 95 DEG C of 30s, (95 DEG C of 30s, 51 DEG C of 30s, 72 DEG C of 1min30s) 28 circulation, 72 DEG C of 7min.

By the PCR primer using Omega glue to reclaim test kit recovery, Takara company restriction enzyme EcoR I and Not I enzyme is used to cut, and with through being connected with the pPIC9k plasmid of same enzyme process, by the connection product conversion DH5 α competent cell obtained, grow in 37 DEG C of incubated overnight to transformant.Picking transformant is inoculated in LB liquid nutrient medium, in 37 DEG C, cultivate 14-16h under 160rpm, collect thalline.According to the method that manufacturer provides, with Axygen plasmid Mini Kit (matching company purchased from Shanghai hundred) extracting plasmid, double digestion experiment is carried out with restriction enzyme EcoR I and Not I, positive colony correct for digestion verification is delivered to Sangon Biotech's order-checking, described sequence is as shown in SEQ ID NO.41, and the aminoacid sequence of its coding is as shown in SEQ ID NO.42.With restriction enzyme Bgl II by plasmid linearization, linearizing product Axygen cleaning agents box is purified, and is 300ng/ μ l by the concentration of electrophoresis checking linearization plasmid.

With reference to Shixuan W., Geoffrey J.L..High efficiency transformation by electroporationof Pichia pastoris pretreated with lithium acetate and dithiothreitol.Biotechniques, 2004, the method of 36:152-154, prepares Pichia pastoris GS115 competent cell by sorbyl alcohol washing out method.

Get 1 μ g linearization plasmid, add 100 μ l GS115 competent cells, proceed to electric shock cup, electric shock cup, after leaving standstill 5 minutes on ice, is placed on electroporation and shocks by electricity, condition: voltage 2000v, time 5s.Add rapidly 1ml1mol/l Sorbitol Solution USP, inhale and beat evenly, by the coating of bacterium liquid RBD flat board (1.34% yeast basic nitrogen source, 4 × 10 ^-5% vitamin H, 2% glucose, 1mol/l sorbyl alcohol, 1.5% agar).A couple of days is cultivated, until transformant grows in 28 DEG C.

Transformant on picking RBD flat board, is seeded on lipase qualification flat board, cultivates a couple of days in 28 DEG C, until there is color ring, obtain the transformant with lipase activity, by this transformant called after " wt ", lipase called after " Lipwt " expressed by this transformant strain.Picking mono-clonal, is seeded to YPD liquid nutrient medium, 28 DEG C, 200rpm cultivates 24h, 600 μ l bacterium liquid and 400 μ l50% glycerine (w/v%) is mixed and made into glycerine pipe, is stored in-80 DEG C.

1.3,377 lysine mutations are phenylalanine

With the mould genome of branch spore for template, with Lipcs-U, F-D(sequence as shown in SEQ ID NO.3), F-U(sequence is as shown in SEQ ID NO.4) and Lipcs-D for primer, through pcr amplification, prepare the lipase mutant that 377 sport phenylalanine, detailed process is as follows:

First round PCR, with the mould genome of branch spore for template, respectively with Lipcs-U and F-D or F-U and Lipcs-D for primer pair, carry out pcr amplification, obtain PCR primer respectively, wherein, PCR system is: Primstar(is purchased from Takara company) 0.25 μ l, 5 × damping fluid 10 μ l, dNTP mixture 4 μ l, lipcs-U1 μ l, F-D1 μ l(or F-U1 μ l, Lipcs-D1 μ l), genome 0.1 μ l, add water polishing to 50 μ l.PCR program is: 95 DEG C of 30s, (95 DEG C of 30s, 51 DEG C of 30s, 72 DEG C of 1min) 28 circulation, 72 DEG C of 7min.

Second takes turns PCR, first round PCR primer is diluted 10 times, and equal-volume mixes as template, using Lipcs-U/Lipcs-D as primer pair, carry out pcr amplification, wherein PCR system is: Primstar(is purchased from Takara company) 0.25 μ l, 5 × PCR damping fluid 10 μ l, dNTP mixture 4 μ l, lipcs-U1 μ l, lipcs-D1 μ l, template 0.5 μ l, add water polishing to 50 μ l.PCR program is: 95 DEG C of 30s, (95 DEG C of 30s, 51 DEG C of 30s, 72 DEG C of 1min30s) 28 circulation, 72 DEG C of 7min.

PCR primer used Omega glue to reclaim test kit to reclaim, cut with Takara company restriction enzyme EcoR I and Not I enzyme, be connected with the pPIC9k plasmid of same enzyme process, will connect product conversion DH5 α competent cell, 37 DEG C are spent the night, and transformant grows.Picking transformant is inoculated in LB liquid nutrient medium, in 37 DEG C, cultivate 14-16h under 160rpm, grow to transformant.A peek transformant, 10000rpm, 4 DEG C, 5min collected by centrifugation thalline, plasmid is extracted according to embodiment 1.2 method, carry out double digestion experiment with restriction enzyme EcoR I and Not I, positive colony correct for digestion verification is delivered to Sangon Biotech's order-checking.Result shows, and obtaining 377 lysine mutations is the clone of phenylalanine.

Adopt the method for embodiment 1.2, cultivate institute's DCRP, extract plasmid, carry out plasmid linearization, electricity turns pichia spp, obtain recombinant yeast pichia pastoris, and make its glycerine pipe bacterium liquid.

1.4,377 mutant lipase preparations

By the method for embodiment 1.3, prepare the clone that 377 lysine mutations are other amino acid whose lipase mutants respectively, and make the glycerine pipe bacterium liquid of corresponding clone, wherein, lipase mutant and primer pair as shown in table 1.

Table 1, branch spore mould lipase 377 site mutant and mutant primer thereof

Mutating acid	Upstream primer	Sequence numbering	Downstream primer	Sequence numbering
					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 glycerine pipe bacterium liquid getting 100 μ l wt and each saturation mutation is respectively seeded to YPD liquid nutrient medium, after 28 DEG C of cultivation 24h, is inoculated in growth medium BMGY, is cultured to thalli growth to OD in 28 DEG C with 0.1% inoculum size ₆₀₀for 2-6,1500g, 4 DEG C, centrifugal fermented liquid 5min, remove supernatant, with the resuspended thalline of inducing culture BMMY of former culture volume, add 0.5% methyl alcohol every day, induce after 3 days, 15000rpm, 4 DEG C centrifugal 15 minutes, collection fermentation supernatant.

1.6, the tolerance of tensio-active agent detects

The fermented supernatant fluid of preparation in embodiment 1.5 is carried out protein electrophoresis, and carries out protein concentration by Millipore centrifugal filter device (purchased from Chemical Reagent Co., Ltd., Sinopharm Group), target protein concentration is adjusted to 0.05 μ g/ μ l.

To detect fermentation supernatant lipase activity mensuration system in add the SDS that final concentration is 0.5%, and the reaction tubes adding equal-volume water is in contrast, the SDS tolerance of test lipase.Detailed process is as follows:

By 50mM Tris-Cl(pH8.0) damping fluid becomes mixed solution with 6mg/ml pNPP substrate with 9:1 proportional arrangement.Get 400 μ l mixed solutions to manage to 2ml Eppendorf, adding SDS to final concentration is 0.5%, vibration mixing.Hatch 5 minutes at 45 DEG C, add 1 μ l fermentation supernatant and react.React after 15 minutes, adding ethanol to final volume is centrifugal 2 minutes of 2ml, 12000rpm, test 405nm absorbance value.Arrange two contrasts in this experiment, wherein first contrast is for change SDS into equal-volume water, and other test conditions are constant.Second contrast is for starting not enzyme-added liquid, and question response terminates after ethanol adds, then adds equivalent amounts of enzyme liquid, and other test conditions are constant.

Circular is: according to above-mentioned pNPP method, and the vigor of enzyme under the vigor calculating enzyme under the reaction conditions that with the addition of 0.5%SDS and reaction conditions SDS being replaced as equal-volume water, the ratio of the two is multiplied by the residual activity per-cent that 100 are enzyme.To measure Lipwt(K) the residual activity per-cent of lipase for 100%, calculate the residual activity per-cent of other lipase, be Fig. 1.

According to the result of Fig. 1, when 377 amino acids are basic aminoacids, as tolerance when arginine (R), Methionin (K), Histidine (H) declines, when 377 amino acids are acidic amino acid and acid amides thereof, as L-glutamic acid (E), aspartic acid (D), glutamine (Q) and l-asparagine (N) time tolerance rise, particularly when 377 amino acids are L-glutamic acid (E), aspartic acid (D), tolerance reaches the highest.When 377 amino acids are neutral fat race amino acid, as tolerance when glycine (G), L-Ala (A) and leucine (L) etc. declines.When 377 amino acids are hydroxyl or sulphur amino acid, as tolerance when Serine (S), Threonine (T) etc. improves slightly.When 377 amino acids are die aromatischen Aminosaeuren, as tolerance when tyrosine (Y), tryptophane (W) rises slightly, but when sporting phenylalanine (F), tolerance declines.Especially, when 377 amino acids sport proline(Pro), protein concentration is adjusted consistent (0.05 μ g/ μ l) with Lipwt, live than enzyme and be down to 1/50 of Lipwt.Illustrate that proline(Pro) have impact on the active centre of lipase.

In the present invention, compared with Lipwt, when 377 amino acids are L-glutamic acid (E), aspartic acid (D), anion surfactant tolerance increase rate is maximum, and its Glutamic Acid is optimum.According to the result of above-mentioned saturation mutation; analysis reason is as follows: 377 amino acids are positioned at Lipcs protein three-dimensional structure surface; run into the anion surfactant in environment; basic aminoacids performance affinity interaction; albumen is caused to be damaged; repulsive interaction cashed by acidic amino acid, causes albumen to be protected to a certain extent.

Be the pichia spp recombinant bacterial strain called after " 496 " of L-glutamic acid by 377 amino acid, its mutant lipase called after " Lip496 " of expressing.

The tolerance of embodiment 2, tensio-active agent detects

Preparation quality concentration is cats product (CTAB), nonionogenic tenside (AEO-9, Triton X-100 and Tween-80) and anion surfactant (LAS, AOS, AES and the SDS) stock solution of 10%.

According to the method for embodiment 1.6, test Lip496 from embodiment 1.5 to various tensio-active agent tolerance, result as shown in figures 2-6.Wherein concrete method of calculation are: according to above-mentioned pNPP method, the vigor of enzyme under the reaction conditions that with the addition of tensio-active agent that calculates becomes the vigor of enzyme under the reaction conditions of equal-volume water with by surfactant replacement, the ratio of the two is multiplied by the residual activity per-cent that 100 are enzyme.

According to the result of Fig. 2, compared with wild type lipase, lipase mutant 496 pairs of cats product tolerances reduce, and substantially change not quite the tolerance of nonionogenic tenside Triton X-100, Tween-80 and AEO-9, wherein Triton X-100 slightly declines.

According to the result of Fig. 3-6, the tolerance of lipase mutant 496 couples of LAS, AOS, AES and SDS is better than wild type lipase, being wherein more than the twice of wild type lipase to the tolerance of LAS, AOS and SDS, is more than 1.5 times of wild type lipase to the tolerance of AES.Anion surfactant tolerance is improved greatly.

Because in current detergent component, anion surfactant accounts for major part, and lipase mutant 496 pairs of negatively charged ion tolerances strengthen greatly, and therefore, the zymin that can be used as washing composition uses, and has good prospects for commercial application.The advantage that Lip496 uses as detergent enzyme preparation shows both ways: (1) lipase of the present invention can improve the zymin vigor of enzyme-containing detergent, improve washing effect, and the reduction of enzyme activity loss, the cost of the lipase causing price high reduces relatively; (2) along with scientific-technical progress, the market scope of liquid washing agent will constantly expand, the demand of the lipase that Surfactant tolerance improves will be more urgent, Lip496 has complied with the needs of market development, solve the technical barrier of the easy inactivation of lipase, for the further genralrlization application of liquid washing agent is paved the way.

Claims

1. there is the polypeptide of lipase activity, described polypeptide comprises the aminoacid sequence shown in SEQ ID No:42 or its active fragments of sudden change, and it is acidic amino acid or its acid amides that wherein said sudden change comprises the lysine mutation of the 377th of the aminoacid sequence shown in SEQ ID No:42.

2. polypeptide as claimed in claim 1, be wherein L-glutamic acid (E), aspartic acid (D), glutamine (Q) or l-asparagine (N) by the lysine mutation of the 377th of the aminoacid sequence shown in SEQ ID No:42, preferably sport L-glutamic acid (E) or aspartic acid (D).

3. the nucleic acid molecule of the polypeptide of coding described in claim 1 or 2.

4. comprise the carrier of nucleic acid molecule according to claim 3, preferably, described carrier is expression vector, more preferably, described carrier is designed to express in eukaryotic cell or prokaryotic cell prokaryocyte, further preferably, for expressing in bacterial cell, fungal cell, yeast cell, mammalian cell, insect cell or vegetable cell.

5. comprise the cell of nucleic acid molecule according to claim 3 or carrier according to claim 4, be preferably eukaryotic cell or prokaryotic cell prokaryocyte, be more preferably bacterial cell, fungal cell, yeast cell, mammalian cell, insect cell or vegetable cell.

6. use the lipase that the cell described in claim 5 produces.

7. use the lipase enzyme liquid that the cell described in claim 5 produces, preferably, described lipase enzyme liquid is the fermentation supernatant of described cell.

8. the polypeptide that the polypeptide of the polypeptide described in claim 1 or 2 or nucleic acid molecule encoding according to claim 3 or the polypeptide of vector encoded according to claim 4 or cell expressing according to claim 5 go out or lipase according to claim 6 or lipase enzyme liquid according to claim 7 are for the purposes in washing process.

9. the polypeptide described in claim 1 or 2 or nucleic acid molecule according to claim 3 or carrier according to claim 4 or cell according to claim 5 are preparing the purposes in lipase.

10. a cleaning product, described cleaning product comprises polypeptide described in claim 1 or 2 or nucleic acid molecule according to claim 3 or carrier according to claim 4 or cell according to claim 5, or lipase according to claim 6 or lipase enzyme liquid according to claim 7.