CN102876643A

CN102876643A - New application of GDSL protein to preparation of lipase

Info

Publication number: CN102876643A
Application number: CN201210356229XA
Authority: CN
Inventors: 周志刚; 何夙旭; 徐俐; 杨雅麟; 张美超; 李青
Original assignee: Feed Research Institute of Chinese Academy of Agricultural Sciences
Current assignee: Feed Research Institute of Chinese Academy of Agricultural Sciences
Priority date: 2012-09-21
Filing date: 2012-09-21
Publication date: 2013-01-16

Abstract

The invention discloses new application of GDSL protein to preparation of lipase. The invention provides application of the GDSL protein to (1) preparation of lipases or (2) degradation of a substrate of the lipase. The GDSL protein is the following protein in (a) or (b): (a) the protein comprises the amino acid sequence shown as the sequence 2; and (b) the protein is formed in the way that the amino acid sequence shown as the sequence 2 is subjected to substitution and/or deletion and/or addition of one or more amino acid residues, has lipase activity and is derived from the sequence 2. The GDSL protein has lipase activity. The application of the GDSL protein as the lipase has the advantages of higher reaction temperature, high heat stability, action under the alkaline condition, high pH stability, effects on the substrates of C2-C12 and high enzyme activity. The invention opens up a new way for the process flow of the lipase and has a great economic value.

Description

The new purposes of GDSL albumen in preparation lipase

Technical field

The present invention relates to the new purposes of GDSL albumen in preparation lipase.

Background technology

Lipase (1ipase; triacylglycerolacylhydrolases; EC3.1.1.3) be Lipase; it is one of most important enzyme in the industrial enzyme preparation; can be at water-oil interface catalysis natural substrate fat hydrolysis; generate lipid acid, glycerine and monoglyceride or diester, simultaneously can also catalytic transesterification, the number of chemical reactions such as synthetic, the alcoholysis of transesterification, ester, acidolysis, ammonia solution, have usually that catalytic efficiency is high, the simple advantage of catalytic condition.

The lipase of present industrial application mostly is greatly the extracellular enzyme of microorganisms, warm enzyme from temperature of reaction, mostly being (optimal reactive temperature 37-40 ℃), and at high temperature reaction is restricted.Zimadzhunt L 340 refers to best catalytic temperature 60-85 ℃ enzyme, have advantages of that chemical catalyst is incomparable,, operative temperature high such as catalytic efficiency extensively (stability at high temperature is also better), action pH is extensive etc., thereby can overcome the unsettled phenomenon of biological property that middle temperature enzyme (20-55 ℃) and cold-adapted enzyme (2-20 ℃) occur in application process, can replace traditional middle temperature enzyme catalysis and chemical catalysis, for optimization technological process has been opened up a new way.

Utilize the lipase of Heat stability is good to have following advantage as biological catalyst: (1) because this fat Enzymic stability is high, at room temperature separating-purifying and packed and transported, and can keep for a long time active, thereby the cost of preparation lipase; (2) to the reactor cooling system require standard low, thereby reduced energy expenditure, and because its high-temperature stability, do not need aborning complicated refrigerating unit, not only saved spending but also reduced the process of cooling pollution on the environment; (3) with the raising of temperature of reaction, enzyme molecular motion speed is accelerated, and catalytic capability is strengthened, and accelerated kinetic reaction, thereby service efficiency is improved; (4) owing to adopting the pyroreaction condition, reduced the danger of mesophilic microorganism pollution reaction system, thereby improved product purity, the pyroreaction condition has also improved bioavailability and the solubility of organic compound simultaneously, thereby has realized effective biological restoration.

The thermal stability problems of lipase is the bottleneck problem of lipase research and production and application always, and the present Research of just present lipase is only improved the lipase characteristic by protein engineering, far can not solve every field to the requirement of lipase.Therefore, the lipase that seek to find to have new features, heat-resisting, high vigor, meets the modern biological project requirement is a very significant job.

Summary of the invention

The purpose of this invention is to provide the new purposes of GDSL albumen in preparation lipase.

The invention provides the application of GDSL albumen, be following (1) or (2):

(1) preparation lipase; (2) substrate of degraded lipase;

Described GDSL albumen is following (a) or (b):

(a) protein that is formed by the aminoacid sequence shown in the sequence in the sequence table 2;

(b) with aminoacid sequence shown in the sequence 2 of sequence table through replacement and/or disappearance and/or the interpolation of one or several amino-acid residue and have lipase activity by the derivative protein of sequence 2.

When using the substrate of described GDSL proteolytic degradation lipase, the reaction conditions of described degraded is 30-80 ℃, pH6.5 – 8.0, and the reaction conditions of described degraded is preferably 65 ℃, pH7.5.

The substrate of described lipase is at least a in p-nitrophenyl yl acetate, butyric acid p-nitrophenyl ester, 4-nitrophenyl octanoate, 4-nitrophenyl certain herbaceous plants with big flowers acid esters, 4-nitrophenyl laurate and the 4-nitrophenyl cetylate.

The present invention also protects recombinant plasmid is imported the recombinant bacterium that intestinal bacteria obtain; The recombinant plasmid that described recombinant plasmid obtains for the multiple clone site with the encoding gene insertion vector pET-28a (+) of described GDSL albumen.

The encoding gene of described GDSL albumen specifically can be following 1) to 4) in arbitrary described dna molecular:

1) sequence 3 of sequence table is from the dna molecular shown in 5 ' terminal the 1st to 792 Nucleotide;

2) dna molecular shown in the sequence 3 of sequence table;

3) under stringent condition with 1) or 2) dna molecular with albumen of lipase activity of the dna sequence dna hybridization that limits and coding;

4) with 1) or 2) dna sequence dna that limits has the dna molecular that 90% above homology and coding have the albumen of lipase activity.

Described intestinal bacteria are preferably e. coli bl21 (DE3).

The present invention also protects a kind of method of the GDSL of preparation albumen, is to cultivate above-described recombinant bacterium, obtains described GDSL albumen.In the described method, the concrete steps of described cultivation are: recombinant bacterium is seeded to LB liquid nutrient medium (containing 100 μ g/mL kantlex), and shaking culture is to OD ₆₀₀Reach 0.8, then add the IPTG(inductor) to concentration be 1mmo/L, 20 ℃, 200rpm shaking culture 12 hours.In the described method, after cultivating recombinant bacterium, also comprise ultrasonication and the supernatant of ultrasonication is carried out the step of affinity chromatography.The design parameter of described ultrasonication is: power 300W, net cycle time 90min, every work 5s is 8s intermittently), the centrifugal 10min of 12000rpm collects supernatant liquor.Described affinity chromatography specifically can be affinity chromatography.

The present invention also protects a kind of method of substrate of the lipase of degrading, and is the substrate that adopts described GDSL proteolytic degradation lipase.The reaction conditions of described degraded is 30-80 ℃, pH6.5-8.0.The reaction conditions of described degraded is preferably 65 ℃, pH7.5.The substrate of described lipase specifically can be at least a in p-nitrophenyl yl acetate, butyric acid p-nitrophenyl ester, 4-nitrophenyl octanoate, 4-nitrophenyl certain herbaceous plants with big flowers acid esters, 4-nitrophenyl laurate and the 4-nitrophenyl cetylate.

The present invention finds that described GDSL albumen has lipase activity, and described GDSL albumen as lipase, is had following advantage: can adopt higher temperature of reaction; Has good thermostability; Can under alkaline condition, act on; Has good pH stability; Substrate to C2-C12 all can play a role; Enzyme activity is higher.The present invention relates to the technical process of lipase to have opened up a new way, has great economic worth.

Description of drawings

Fig. 1 sieves the acquisition lipase strains under 60 ℃ of culture condition among the embodiment 1 again.

Fig. 2 is the enzyme of each bacterial strain among the embodiment 1 preliminary survey result alive.

Fig. 3 is the three-dimensional structure prognostic chart of GDSL albumen.

Fig. 4 is the structural representation of recombinant plasmid pET-28a – B2.

Fig. 5 is the SDS-PAGE of abduction delivering GDSL albumen in intestinal bacteria; 1, molecular weight marker, 2, the supernatant liquor, 3 that obtains of contrast bacterium, the supernatant liquor that recombinant bacterium obtains.

Fig. 6 is p-NP-absorbancy typical curve.

Fig. 7 is the electrophorogram of albumen behind the purifying.

Fig. 8 is the Mass Spectrometric Identification result of target protein.

Fig. 9 is optimal pH and the pH Stability Determination result among the embodiment 3.

Figure 10 is optimum temperuture and the thermal stability determination result among the embodiment 3.

Figure 11 is the impact that each reagent is lived on enzyme among the embodiment 3.

Figure 12 is the impact that each organic solvent is lived on enzyme among the embodiment 3.

Figure 13 is the impact that stain remover is lived on enzyme among the embodiment 3.

Figure 14 is the impact that proteolytic enzyme is lived on enzyme among the embodiment 3.

Figure 15 is to the different carbon chain lengths substrate specificity among the embodiment 4.

Embodiment

Following embodiment is convenient to understand better the present invention, but does not limit the present invention.Experimental technique among the following embodiment if no special instructions, is ordinary method.Quantitative test in following examples all arranges repeated experiments three times, results averaged.Used test materials among the following embodiment if no special instructions, is and purchases available from routine biochemistry reagent shop.Carrier pET-28a (+): Novagen, GR201023.E. coli bl21 (DE3): Beijing Quanshijin Biotechnology Co., Ltd, CD601-01.P-NP cetylate (p-NPP): Sigma, 1492-30-4.P-NP (PNP): Sigma, 4043-96-3.The English full name of CHAPS is 3-[(3-cholamidopropyl) dimethylammonio] propanesulfonate).

The acquisition of embodiment 1, wild mushroom and the discovery of lipase

One, yielding lipase bacterial strain screening

Primary dcreening operation substratum (g/L): (NH ₄) ₂SO ₄1g, NH ₄NO ₃1g, NaCl 1g, MgSO ₄7H ₂O 0.1g, K ₂HPO ₃1g, FeSO ₄7H ₂O 0.01g, Na ₂CO ₃Transfer PH to 8.0, add again 1.0% sweet oil, sterilization.

Liquid fermentation medium (g/L): Zulkovsky starch 10g, (NH ₄) ₂SO ₄5g, K ₂HPO ₃1g, corn starch 20g, soybean cake powder 20g, triolein 10g, pH8.0.

Primary dcreening operation: the sediment of pond sample is taken from the mixed piscina of the joyful feed corporation,Ltd of Zhejiang Jiaxing, and be in July, 2009 sample time, and 4 ℃ of conditions are preserved sample.The sediment of pond sample is carried out ten times of gradient dilutions with aseptic PBS damping fluid, get 100 microlitres and coat the primary dcreening operation substratum, be put under 60 ℃ of temperature and cultivate, the yielding lipase bacterial strain sweet oil of can degrading produces transparent circle, and its lipase activity of the large examination of transparent circle diameter and colony diameter ratio is strong and weak according to producing.

Multiple sieve: the inoculation with lipase activity of separation and purification is entered to sieve again solid (repetition measurement produces true fat enzyme bacterial strain) or liquid fermentation medium (inducing the product enzyme to survey active), solid plate is put under 30 or 60 ℃ of temperature and is cultivated, and observes the transparent bacterium of degraded.

Produce bacterium by high temperature screening strategy and the preliminary high temperature resistant fatty enzyme of 26 strains that obtains of rhodamine sweet oil screening method, sieve again strategy acquisition 12 strain bacterium by triolein again and demonstrate larger transparent circle.Multiple sieve acquisition lipase strains is seen Fig. 1 under 60 ℃ of culture condition.Wherein B2, B6, B9, B12 are the lipase bacterium that the high-temperature cultivation conditional filtering arrives.

Adopt liquid fermentation medium at 200r/min, 60 ℃ of shaking tables are cultivated 48h.With thalline 12, the centrifugal 2min of 000rpm gets supernatant, and thalline is resuspended in 50mmol/L Tris-HCl (pH 8.0) damping fluid, with ultrasonic disruption instrument smudge cells (4s/ time, interval 10s, broken 30 times), 13,000rpm, 4 ℃ of centrifugal 10min namely get thalline intracellular enzyme supernatant.Supernatant is carried out enzyme preliminary survey alive.Live and all be higher than intracellular enzyme and live by born of the same parents being reached enzyme that bacterial isolates born of the same parents that intracellular enzyme live to measure finds that screening obtains detect outward outward.Enzyme preliminary survey alive the results are shown in Figure 2.Get enzyme the highest B2 bacterial strain alive and carry out follow-up test.

Two, the evaluation of bacterial strain

Extract the B2 strain gene group DNA, and adopt 16S rDNA universal primer 27F and 1492R to carry out the pcr amplification of 16S rDNA.27F：5’-AGAGTTTGATCCTGGCTCAG-3’1492R：5’-TACCTTGTTACGACTT-3’。

Reaction system: 10 * PCR buffer, 5 μ L, dNTP mix (2.5mmol/L) 4 μ L, Taq (5U/ μ L) 0.5 μ L, 27F (25 μ mol/L) 1 μ L, 1492R (25 μ mol/L) 1 μ L, template DNA 1 μ L, ddH ₂O 8 μ L, cumulative volume 50 μ L.

PCR reaction conditions: 95 ℃ of 5min; 94 ℃ of 30s, 55 ℃ of 30s, 72 ℃ of 1min30s are after 32 circulations; 72 ℃ are extended 10min.

The about 1500bp of order-checking gained 16S rDNA gene order length is shown in the sequence 1 of sequence table.The 16S rDNA gene order of bacterial strain is compared and Geobacillus toebii(EU391540 at the GenBank nucleic acid database) have higher similarity, judge that tentatively it belongs to Geobacillus sp..

Three, the discovery of lipase

From the B2 bacterial strain, find an albumen, have the function of lipase, with its called after GDSL albumen, shown in the sequence 2 of sequence table.Be the GDSL gene with the unnamed gene of coding GDSL albumen, its open reading frame is shown in the sequence 3 of sequence table.The three-dimensional structure prognostic chart of GDSL albumen is seen Fig. 3.

The preparation of embodiment 2, GDSL albumen

One, the structure of recombinant expression vector

1, the double chain DNA molecule shown in the sequence 3 of composition sequence table.

2, take the synthetic double chain DNA molecule of step 1 as template, the primer pair that forms with B2F and B2R carries out PCR and increases, and obtains pcr amplification product.

B2F:5 '-GAC GAATTCThe restriction endonuclease recognition sequence of ATGAGACGCGGTATTGTAAGCAC-3'(glissade mark restriction enzyme EcoRI);

B2R:5 '-GAC GCGGCCGCThe restriction endonuclease recognition sequence of TTGTTTGTCCTCCTCCGTCCAC-3'(underscore mark restriction enzyme NotI).

PCR reaction conditions: 95 ℃ of denaturation 5min; 94 ℃ of 30sec, 60 ℃ of 30sec, 72 ℃ of 1min, 30 circulations; Last 72 ℃ are extended 10min.

3, with the pcr amplification product of restriction enzyme EcoRI and NotI double digestion step 2, reclaim enzyme and cut product.

4, with restriction enzyme EcoRI and NotI double digestion carrier pET-28a (+), reclaim carrier framework (approximately 5300bp).

5, the enzyme of step 3 is cut the carrier framework connection that product is connected with step, obtained recombinant plasmid pET-28a – B2.According to sequencing result, recombinant plasmid pET-28a – B2 is carried out structrual description as follows: in the sequence 3 of having inserted sequence table between the EcoRI of carrier pET-28a (+) and the NotI restriction enzyme site from the double chain DNA molecule shown in 5 ' terminal the 1st to 792 Nucleotide (the His label coding gene fusion on 3 ' terminal and the carrier of the double chain DNA molecule of insertion is so that carry out follow-up protein purification).The structural representation of recombinant plasmid pET-28a – B2 is seen Fig. 4.

Two, the structure of recombinant bacterium

Recombinant plasmid pET-28a – B2 is imported e. coli bl21 (DE3), obtain recombinant bacterium.

Carrier pET-28a (+) is imported e. coli bl21 (DE3), obtain contrasting bacterium.

Three, GDSL protein expression and evaluation

1, recombinant bacterium or contrast bacterium are seeded to 5mL LB liquid nutrient medium (containing 100 μ g/mL kantlex), 37 ℃, 200rpm shaking culture spend the night.

2, the bacterium liquid of step 1 is seeded to 20mL LB liquid nutrient medium (containing 100 μ g/mL kantlex) with 1/100 volume ratio, 37 ℃, 200rpm shaking culture are to OD ₆₀₀Reach 0.6; Then add the IPTG(inductor) to concentration be 1mmo/L, 20 ℃, 200rpm shaking culture 12 hours.

3, get bacterium liquid that step 2 obtains centrifugal and collect thalline, then thalline is carried out ultrasonication (power 300W, net cycle time 90min, every work 5s is 8s intermittently), the centrifugal 10min of 12000rpm collects supernatant liquor.

4, the supernatant liquor that the supernatant liquor that recombinant bacterium step 3 is obtained and contrast bacterium step 3 obtain carries out SDS-PAGE, the results are shown in Figure 5.Have molecular weight in the supernatant liquor that recombinant bacterium obtains and be about the expection protein band of 29.5kD, and the supernatant liquor that the contrast bacterium obtains does not show this protein band.Target protein is scaled off from SDS-PAGE, send Tianjin biochip skill to have company limited to carry out the one-level Mass Spectrometric Identification.The result shows, this albumen is the GDSL albumen shown in the sequence 2 of sequence table really.

5, the supernatant liquor that the supernatant liquor that recombinant bacterium step 3 is obtained and contrast bacterium step 3 obtain carries out lipase activity as solution to be measured respectively and detects.

Adopt p-NP production standard curve, see Fig. 6.The typical curve equation is y=57.548x-0.9233, R ²=0.9993, y represents enzyme and lives (unit is μ mol/min), the 405nm absorbance value that the x representative adopts spectrophotometric determination to obtain.

By lipase p-NP cetylate (p-NPP) is degraded to yellow p-NP (PNP) principle, adopts the lipase activity of spectrophotometry solution to be measured (or its diluent).Specific operation process is as follows: will be sequentially added into l.8mL solution B and 0.1mL solution A, 37 ℃ of water bath heat preservation 5min in the test tube; Test tube is divided into control tube and sample hose, the solution to be measured (or its diluent) 0.1mL and the mixing that add high-temperature boiling deactivation in the control tube, add solution to be measured (or its diluent) 0.lmL and mixing in the sample hose, immediately timing, each test tube adds 1mL 95% aqueous ethanolic solution with termination reaction behind 37 ℃ of reaction 10min; Adopt spectrophotometric determination 405nm absorbance value.Solution A: take by weighing 90mg p-NP cetylate and be dissolved in the 30mL Virahol.Solution B: 50mmol/L Tris-HCl damping fluid (pH8.0).

The live definition of unit lipase of l enzyme: under pH8.0,37 ℃ of conditions, per minute discharges the needed enzyme amount of 1 μ mol p-nitrophenol.

Formula is calculated in enzyme work: A=([A _t-A ₀] * K+C ₀) * Vl * n/ (V2 * t)

The enzyme of A one solution to be measured is lived (U/mL), A _tThe absorbance value of sample hose after one reaction, A ₀The absorbance value of control tube after one reaction, K=57.4, Co=0.9233, n one extension rate, the volume (2mL) of Vl one reaction solution, V2 one adds the volume (0.1mL) of the solution to be measured (or its diluent) of test tube, t one reaction times (10min).

The enzyme of the supernatant liquor that recombinant bacterium step 3 obtains is lived and is 4.48U/mL, and contrast bacterium step 3 obtains the enzyme 0U/mL of being alive of supernatant liquor.

Four, GDSL Protein expression and purification

1, recombinant bacterium is seeded to 50mL LB liquid nutrient medium (containing 100 μ g/mL kantlex), 37 ℃, 200rpm shaking culture spend the night.

2, the bacterium liquid with 4mL step 1 is seeded to 200mL LB liquid nutrient medium (containing 100 μ g/mL kantlex), and 37 ℃, 200rpm shaking culture are to OD ₆₀₀Reach 0.8; Then add the IPTG(inductor) to concentration be 1mmo/L, 20 ℃, 200rpm shaking culture 12 hours.

4, the supernatant liquor that step 3 is obtained carries out affinity chromatography (nickel post).

Adopt the NTA post of 1mL.

Damping fluid in the elution process (pH is 7.6) is as follows: NTA-0: contain 20mmol/L Tris-HCl, 0.5mol/LNaCl and 10g/100mL glycerine; NTA-60: contain 20mmol/L Tris-HCl, 60mmol/L imidazoles, 0.5mol/LNaCl and 10g/100mL glycerine; NTA-200: contain 20mmol/L Tris-HCl, 200mmol/L imidazoles, 0.5mol/LNaCl and 10g/100mL glycerine.

Elution process: (1) pillar 10-20mL water balance, add 5mL at every turn and after stream is clean, add again, all same later on; (2) with NTA-0 balance 15-20mL, the supernatant liquor that obtains of loading 5mL step 3 then; (3) carry out wash-out with 5mL NTA-60, to remove foreign protein; (4) with 5mL NTA-200 wash-out target protein, collected the solution behind the post, be the GDSL protein liquid.

5, the GDSL protein liquid that step 4 is obtained carries out SDS-PAGE, the results are shown in Figure 7.The result shows, has obtained electrophoretically pure GDSL albumen after the affinity chromatography.

6, downcut the purpose band in the step 5 and carry out the mass spectrum determined amino acid, the results are shown in Figure 8, the result shows that this target protein is the GDSL albumen shown in the sequence 2 of sequence table really.

Embodiment 3, GDSL albumen are as the Property Identification of lipase

Adopt the 4 GDSL protein liquids that prepare of the step 4 of embodiment 2 to carry out each experiment of embodiment 3.

One, optimal pH and pH Stability Determination

1, optimal pH

Detect the optimal pH of GDSL protein liquid, method is referring to 5 of the step 3 of embodiment 2, difference only is to adopt different damping fluids as solution B, adopts respectively the 50mmol/L Gly-HCl damping fluid of following damping fluid: pH2.0-3.6, the 50mmol/L HAc-NaAc damping fluid of pH3.6-5.0,50mmol/L citric acid-Na of pH5.0-8.0 ₂HPO ₄The 50mmol/L Gly-NaOH damping fluid of the 50mmol/L Tris-HCl damping fluid of damping fluid, pH8.0-9.0 and pH9.0-12.0.

Adopt citric acid-Na of pH7.5 ₂HPO ₄Damping fluid during as solution B enzyme live the highlyest, this highest enzyme work as 100%, is calculated and is adopted other damping fluid to live as the relative enzyme under the condition of solution B, partial results is seen Fig. 9 (a).Among Fig. 9 (a), selecting of pH8.0 is the Tris-HCl damping fluid, and selecting of pH9.0 is the Gly-NaOH damping fluid.

The result shows, GDSL albumen is 7.5 as the optimal pH of lipase, and enzymic activity can maintain more than 60% in pH6.5 – 8.0 scopes, and pH is below 5 or 9 do not have the enzyme biopsy to go out when above.

2, pH stability

Pre-treatment: 1 parts by volume GDSL protein liquid damping fluids different from 10 parts by volume are mixed, process 60min for 37 ℃.Adopt respectively the 50mmol/L HAc-NaAc damping fluid of following damping fluid: pH3.0-5.0,50mmol/L citric acid-Na of pH5.0-8.0 ₂HPO ₄The 50mmol/L Gly-NaOH damping fluid of the 50mmol/L Tris-HCl damping fluid of damping fluid, pH8.0-9.0 and pH9.0-12.0.

Pretreated protein liquid is carried out enzyme activity determination, and method is referring to 5 of the step 3 of embodiment 2, and difference only is to adopt 50mmol/L citric acid-Na of pH7.5 ₂HPO ₄Damping fluid.

Enzyme was alive the highest when the Gly-NaOH damping fluid of employing pH12.0 carried out pre-treatment, and this highest enzyme work as 100%, is calculated the relative enzyme that adopts under other pretreatment condition and lived, and partial results is seen Fig. 9 (b).Among Fig. 9 (a), the point of pH5.0 is citric acid-Na ₂HPO ₄Damping fluid, selecting of pH8.0 is the Tris-HCl damping fluid, selecting of pH9.0 is Gly-NaOH 0 damping fluid.

The result shows, GDSL albumen is more stable in pH 4.0 – 12.0 scopes, and the residual enzyme activity can keep more than 70%, and to increase this enzyme more stable along with the pH value, illustrates that this enzyme has preferably pH stability under neutrality and alkaline condition.

Two, optimum temperuture and thermal stability determination

1, optimum temperuture

The GDSL protein liquid is carried out enzyme activity determination, and method is referring to 5 of the step 3 of embodiment 2, and difference only is to adopt 50mmol/L citric acid-Na of pH7.5 ₂HPO ₄Damping fluid and adopt different temperature of reaction (20-80 ℃).

Enzyme is alive the highest when adopting 65 ℃ to react, and this highest enzyme work as 100%, is calculated the relative enzyme that adopts under other temperature of reaction and lived, and the results are shown in Figure 10(a).The result shows, GDSL albumen is 65 ℃ as the optimal reactive temperature of lipase, and has higher activity between 30-80 ℃, keeps the activity more than 50.0%; GDSL albumen also increases as lipase increase enzymic activity along with temperature of reaction 30-65 ℃ the time thereupon, and enzyme was lived on a declining curvely after temperature of reaction surpassed 65 ℃, and when 80 ℃ of temperature of reaction, residual enzyme work is 62.0%.

2, thermostability

Pre-treatment is processed the GDSL protein liquid 3 hours under differing temps, and respectively at sampling in 0.5,1,2,3 hour, puts on frozen water immediately.

To not carry out pretreated enzyme work as 100%, as 100%, the relative enzyme that calculates under each pretreatment temperature condition of employing is lived, and the results are shown in Figure 10(b with this highest enzyme work).The result shows, GDSL albumen 60-80 ℃ process 3h after residual enzyme work reach more than 60%, enzyme under 60 ℃ of conditions live hardly influenced up to 92%, 90 ℃ of processing after enzyme work can reach 47%, show that GDSL albumen is high temperature lipase.

Three, different metal ion and relevant chemical reagent are on enzyme impact alive

Add different reagent (metal ion or chemical reagent) in reaction system, come detection reagent on the impact of enzymic activity, method is referring to 5 of the step 3 of embodiment 2, and difference only is to adopt 50mmol/L citric acid-Na of pH7.5 ₂HPO ₄Damping fluid.Reagent final concentration in reaction system is 1mmol/L or 10mmol/L.With the reaction system that do not add reagent in contrast (CK).

, calculate the relative enzyme of each treatment group and live as 100% with the enzyme work of CK treatment group, the results are shown in Figure 11.When lower concentration (1mM), Zn ²⁺With the β mercaptoethanol lipase activity of GDSL albumen is had activation, and other cation recognition is not obvious.Under the 10mM concentration, Zn ²⁺, K ⁺, Li ⁺, Na ⁺With the β mercaptoethanol lipase activity of GDSL albumen is had activation, the lipase activity of other ion pair GDSL albumen all can have restraining effect in various degree, and Ca ²⁺, Co ²⁺, Ni ²⁺, Cu ²⁺, Mn ²⁺And Pb ²⁺Inhibition the most obvious, residual enzyme is lived and all to be lower than 30%.

Four, organic solvent is on enzyme impact alive

Pre-treatment: in the GDSL protein liquid, add different organic reagents (volumn concentration is 10% or 30%), 40 ℃ of reaction 10min.With the reaction system that adds equal-volume water in contrast (CK).

, calculate the relative enzyme of each treatment group and live as 100% with the enzyme work of CK treatment group, the results are shown in Figure 12.When concentration 10%, methyl alcohol, Virahol and n-Octanol have faintly activation to the lipase activity of GDSL albumen, and isopropylcarbinol is inhibited, and other organic solvent is lived almost without impact on enzyme.When concentration 30%, methyl alcohol, Virahol and n-Octanol are to the lipase activity of GDSL albumen tool activation faintly still, and work has stronger restraining effect to enzyme for isopropylcarbinol and ethanol, other organic broad dose enzyme are lived still without affecting.

Five, different stain removers are on enzyme impact alive

Pre-treatment: in the GDSL protein liquid, add different stain removers (the quality percentage composition that makes stain remover is 0.01% or 0.10%), 40 ℃ of reaction 10min.With the reaction system that adds equal-volume water in contrast (CK).

, calculate the relative enzyme of each treatment group and live as 100% with the enzyme work of CK treatment group, the results are shown in Figure 13.CTAB when lower concentration or high density all the lipase activity to GDSL albumen have activation, can improve enzyme and live approximately 20%.0.1% Txrion 100 and PTT0 have faintly restraining effect to the lipase activity of GDSL albumen, still reach more than 80% but residual enzyme is alive.Other tensio-active agent is lived almost without impact on enzyme.

Six, the protease inhibitor ability is measured

Pre-treatment: in the GDSL protein liquid, add different proteolytic enzyme, processed 2 hours.When proteolytic enzyme was trypsinase, adding dosage was 150U, and treatment condition are pH 7.0,25 ℃.When proteolytic enzyme was Proteinase K, adding dosage was 10U, and treatment condition are pH7.5,37 ℃.When proteolytic enzyme was subtilopeptidase A, adding dosage was 5U, and treatment condition are 7.5,37 ℃ of pH.When proteolytic enzyme was stomach en-, adding dosage was 18U, and reaction conditions is 2.0,37 ℃ of pH.When proteolytic enzyme was Chymetin, adding dosage was 10U, and reaction conditions is 7.5,25 ℃ of pH.

Pretreated protein liquid is carried out enzyme activity determination, and method is referring to 5 of the step 3 of embodiment 2, and difference only is to adopt 50mmol/L citric acid-Na of pH7.5 ₂HPO ₄Damping fluid.Arrange and do not carry out pretreated contrast (CK).

, calculate the relative enzyme of each treatment group and live as 100% with the enzyme work of CK treatment group, the results are shown in Figure 14.GDSL albumen still has the lipase activity more than 60% after Proteinase K, stomach en-, Chymotrypsin, subtilisin and trypsinase and processing, illustrate that this enzyme has certain protease resistant.

Embodiment 4, GDSL albumen are as the mensuration of lipase to the different chain length substrate specificity

The GDSL protein liquids of 4 preparations of the step 4 of embodiment 2 are carried out enzyme activity determination as solution to be measured, method is referring to 5 of the step 3 of embodiment 2, difference is as follows: during (1) preparation solution A, replacing the p-NP cetylate and make concentration of substrate with other substrate is 1mM; (2) 50mmol/L citric acid-Na of employing pH7.5 ₂HPO ₄Damping fluid.

Each substrate is as follows: 4-Nitrophenyl acetate(C2, the p-nitrophenyl yl acetate, Sigma N8130), 4-Nitrophenyl butyrate(C4, the butyric acid p-nitrophenyl ester, Sigma N9876), 4-Nitrophenyl caproate (C6, the caproic acid p-nitrophenyl ester, Sigma 956-75-2) 4-Nitrophenyl caprylate(C8,4-nitrophenyl octanoate, Sigma 21742), 4-Nitro phenyl decanoate(C10,4-nitrophenyl certain herbaceous plants with big flowers acid esters, Sigma N0252), 4-Nitrophenyl dodecanoate (C12,4-nitrophenyl laurate, Sigma61716).

When substrate was 4-Nitrophenyl acetate, the enzyme of GDSL protein liquid was lived and is 166U/mL.

When substrate was 4-Nitrophenyl butyrate, the enzyme of GDSL protein liquid was lived and is 190U/mL.

When substrate was 4-Nitrophenyl caproate, the enzyme of GDSL protein liquid was lived and is 246U/mL.

When substrate was 4-Nitrophenyl caprylate, the enzyme of GDSL protein liquid was lived and is 297U/mL.

When substrate was 4-Nitrophenyl decanoate, the enzyme of GDSL protein liquid was lived and is 231U/mL.

When substrate was 4-Nitrophenyl dodecanoate, the enzyme of GDSL protein liquid was lived and is 109U/mL.

The enzyme slip-knot fruit that obtains as substrate with 4-Nitrophenyl caprylate is calculated and adopts the relative enzyme of other substrate to live as 100%.The results are shown in Figure 15.The maximum degradable chain length of GDSL albumen is C12 carbochain, and degradation capability is along with the increase activity of chain length also correspondingly increases when substrate is C2-C8, and relative reactivity reached 56% when substrate was C2.The chain length of substrate greater than C8 after degrading activity descend thereupon, be 37% when being 78%, C12 during C10.

Embodiment 5, recombinase kinetic constant are measured

The GDSL protein liquids of 4 preparations of the step 4 of embodiment 2 are carried out enzyme activity determination as solution to be measured, method is referring to 5 of the step 3 of embodiment 2, difference is as follows: (1) preparation is during solution A, with 4-Nitrophenyl caprylate(or 4-Nitrophenyl decanoate) replacement p-NP cetylate and to make its concentration be 1mM; (2) 50mmol/L citric acid-Na of employing pH7.5 ₂HPO ₄Damping fluid; (3) successively 2,3,5,7,10,15,20, stop the enzyme reaction of living during 30min, and measure absorbance value.

By calculating enzymic activity and the ratio in reaction times size, when ratio was constant within certain time period such as this enzyme, then the enzymatic reaction within this time period was first order reaction, determined in this time for surveying K _mAnd V _MaxThe reaction Best Times.

According to the first order reaction time of determining, 4-Nitrophenyl caprylate measures K _mValue and V _MaxReaction times be 10min, 4-Nitrophenyl decanoate measures K _mValue and V _MaxReaction times be 10min.

The GDSL protein liquids of 4 preparations of the step 4 of embodiment 2 are carried out enzyme activity determination as solution to be measured, method is referring to 5 of the step 3 of embodiment 2, difference is as follows: during (1) preparation solution A, 4-Nitrophenyl caprylate(or 4-Nitrophenyl decanoate with the difference amount) replace the p-NP cetylate, making its concentration is 1,0.8,0.4,0.2,0.15 or 0.1mM; (2) 50mmol/L citric acid-Na of employing pH7.5 ₂HPO ₄Damping fluid; The reaction times of (3) adopting leading portion to determine.

Calculate speed of response by formula, adopt the two methods reciprocal of Michaelis-Menton equation to try to achieve K _mValue and V _MaxTransform its structural formula by double-reciprocal plot method (Lineweaver-Burk method) again:

\frac{1}{v} = \frac{K_{m}}{V_{\max}} \times \frac{1}{[S]} + \frac{1}{V_{\max}}

GDSL albumen is to the K of C8 substrate _m=0.26mmol/L, V _Max=149.25 μ mol/min/mg.GDSL albumen is to the K of C10 substrate _m=0.41mmol/L, V _Max=71.1 μ mol/min/mg.

The mensuration of embodiment 6, recombinase B2 specific activity

Be defined as than unit of activity: every milligram of enzyme activity unit that zymoprotein is contained.

The GDSL protein liquids of 4 preparations of the step 4 of embodiment 2 are carried out enzyme activity determination as solution to be measured, method is referring to 5 of the step 3 of embodiment 2, difference is as follows: during (1) preparation solution A, replace the p-NP cetylate with 4-Nitrophenyl caprylate, making its concentration is 1mM; (2) 50mmol/L citric acid-Na of employing pH7.5 ₂HPO ₄Damping fluid.By the protein concentration in the protein quantification kit measurement GDSL protein liquid of Bole company.

Enzyme work and the ratio of protein concentration are the ratio vigor of GDSL albumen.The ratio vigor that calculates GDSL albumen take C8 as substrate is 498U/mg.

Claims

1.GDSL the application of albumen is following (1) or (2):

(1) preparation lipase; (2) substrate of degraded lipase;

Described GDSL albumen is following (a) or (b): the protein that (a) is comprised of the aminoacid sequence shown in the sequence in the sequence table 2; (b) with aminoacid sequence shown in the sequence 2 of sequence table through replacement and/or disappearance and/or the interpolation of one or several amino-acid residue and have lipase activity by the derivative protein of sequence 2.

2. application as claimed in claim 1 is characterized in that: in described (2), the reaction conditions of described degraded is 30-80 ℃, pH6.5-8.0.

3. application as claimed in claim 1 or 2, it is characterized in that: in described (2), the substrate of described lipase is at least a in p-nitrophenyl yl acetate, butyric acid p-nitrophenyl ester, 4-nitrophenyl octanoate, 4-nitrophenyl certain herbaceous plants with big flowers acid esters, 4-nitrophenyl laurate and the 4-nitrophenyl cetylate.

4. such as arbitrary described application in the claims 1 to 3, it is characterized in that: described GDSL albumen is the protein that the described method of claim 7 prepares.

5. recombinant plasmid is imported the recombinant bacterium that intestinal bacteria obtain; The recombinant plasmid that described recombinant plasmid obtains for the multiple clone site with the encoding gene insertion vector pET-28a (+) of GDSL albumen;

6. recombinant bacterium as claimed in claim 5, it is characterized in that: the encoding gene of described GDSL albumen is following 1) to 4) in arbitrary described dna molecular: 1) sequence 3 of sequence table is from the dna molecular shown in 5 ' terminal the 1st to 792 Nucleotide; 2) dna molecular shown in the sequence 3 of sequence table; 3) under stringent condition with 1) or 2) dna molecular with albumen of lipase activity of the dna sequence dna hybridization that limits and coding; 4) with 1) or 2) dna sequence dna that limits has the dna molecular that 90% above homology and coding have the albumen of lipase activity.

7. a method for preparing GDSL albumen is to cultivate claim 5 or 6 described recombinant bacteriums, obtains GDSL albumen; Described GDSL albumen is following (a) or (b): the protein that (a) is comprised of the aminoacid sequence shown in the sequence in the sequence table 2; (b) with aminoacid sequence shown in the sequence 2 of sequence table through replacement and/or disappearance and/or the interpolation of one or several amino-acid residue and have lipase activity by the derivative protein of sequence 2.

8. the method for the substrate of the lipase of degrading is the substrate that adopts GDSL proteolytic degradation lipase;

9. method as claimed in claim 8, it is characterized in that: the reaction conditions of described degraded is 30-80 ℃, pH6.5-8.0; The substrate of described lipase is at least a in p-nitrophenyl yl acetate, butyric acid p-nitrophenyl ester, 4-nitrophenyl octanoate, 4-nitrophenyl certain herbaceous plants with big flowers acid esters, 4-nitrophenyl laurate and the 4-nitrophenyl cetylate.

10. method as claimed in claim 8 or 9, it is characterized in that: described GDSL albumen is the protein that the described method of claim 7 prepares.