CN104130988A - 1,3-1,4-Beta-glucanase mutant - Google Patents
1,3-1,4-Beta-glucanase mutant Download PDFInfo
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Abstract
The invention discloses a 1,3-1,4-beta-glucanase mutant, and belongs to the fields of gene engineering and enzyme engineering. Lysine in the 20th position, 117th position and 165th position of 1,3-1,4-beta-glucanase from Bacillus terquilensis mutates through an overlapping extension PCR method to form serine in order to obtain single mutants K20S, K117S and K165S respectively. The above three mutation sites are integrally mutated to obtain a K20S/K117S/K165S triple mutant enzyme. Four mutant enzymes have higher catalysis activity and better thermal stability. Compared with wild enzymes, the above mutant enzymes are in favor of realizing the industrial application.
Description
Technical field
The present invention relates to a kind of 1,3-1,4-beta-glucan enzyme mutant, especially a kind of have 1 of more a high catalytic activity and thermostability, 3-1,4-beta-glucan enzyme mutant, belongs to genetically engineered and enzyme engineering field.
Background technology
Beta-glucan is a kind of non-starchiness polysaccharide that is present in grass cell walls, and in the economic class cereal such as barley, wheat, rice, content is very high.It is by up to thousands of β-D-Glucose residues by β-1,3 or β-Isosorbide-5-Nitrae glycosidic link the linear alignment form, there is very high molecular weight.It can be dissolved in the water, and the solution viscosity of formation is very high, and it is many unfavorable that this has brought to beer industry and feedstuff industry.In the main raw material Fructus Hordei Germinatus of brewing industry, contain a large amount of beta-glucans, undegradable beta-glucan is present in wheat juice and can causes wheat juice viscosity excessive, causes filtration difficulty, extends wheat wine with dregs filtration time, reduce extract content, for finished beer, can affect its non-biostability.And when producing draft beer, too much beta-glucanase can cause the Pore Blocking of filter membrane, filtration capacity declines.In feedstuff industry, beta-glucan in wheat class feed all can not directly be digested and assimilated in people's enteron aisle or the enteron aisle of animal, must after enzymatic degradation, just can be utilized, and it has stoped the absorption of effective constituent in animal intestinal, reduced effective constituent transformation efficiency in feed, it is a kind of antinutritional factor, and for microorganism particularly pathogenic bacterium live away from home breeding provide abundant nutrition to cause a large amount of harmful microorganisms to breed in animal intestinal, cause livestock and poultry diarrhea, can competitiveness consume large quantity of material and reduce efficiency of feed utilization simultaneously.
Derive from 1 of Te Jila genus bacillus (Bacillus terquilensis) CGX5-1,3-1,4-beta-glucanase, is called for short beta-glucanase.1,3-1,4-beta-glucanase is that a class can be trisaccharide and tetrose at 3-O-Glucopyranose site-specific nature cutting beta-glucan, this is the basis of its industrial application.In beer industry wheat juice saccharifying, temperature is to be increased to 78 ℃ from 48 ℃, and the temperature of curing in feedstuff industry is also more than 65 ℃.And the major part that at present screening obtains is wild 1,3-1, the optimum temperuture of 4-beta-glucanase mainly concentrates on 45 ℃ and 55 ℃, can not meet industrial demand.And catalytic activity not high be also its major reason that can not promote the use of.And Ji Jia zymin company is as expensive in the beta-glucan zymin of Novi's letter, DMS research and development production in the market, and the current domestic part zymin company production beta-glucan zymin that also has, but its level is far away from external zymin company, and the production bacterial classification of beta-glucan zymin and technique are all trade secrets, domestic research and development speed is slow, so domestic market dependence on import still to a great extent.Therefore, if can improve wild-type 1,3-1, the catalytic activity of 4-beta-glucanase and thermostability, the beta-glucanase of acquisition high reactivity high heat stability, so just can reduce costs, and promotes it in industrial application.
In order to improve 1,3-1, the thermostability of 4-beta-glucanase, has carried out some researchs both at home and abroad.Research is at present found, 1,3-1, and in 4-beta-glucanase, a pair of disulfide linkage of unique existence is little for the impact of protein thermostability.And the sudden change of Gln1, Thr2, Ser5 and Phe7 has improved the thermostability of beta-glucanase significantly.The results of hybridization of beta-glucanase is shown, the hybrid enzyme H (A16-M) that front 16 amino acid that are bacillus amyloliquefaciens (B.amyloliquefaciens) by front 16 amino acid substitutions that derive from Bacillus macerans (B.macerans) obtain and between the two front 12 amino acid are replaced, or hybrid enzyme H (A12-M)-△ 13 that deletion Tyr13 forms at high temperature has good stability.
Used in the present inventionly derive from the wild 1 of Te Jila genus bacillus (B.terquilensis) CGX5-1,3-1, the optimum temperuture of 4-beta-glucanase is 45 ℃, than vigor, is 2490.1U/mg, can not adapt to the requirement of industrial application.Therefore, further improve catalysis activity and the thermostability of this enzyme significant in industrial application to it.
Summary of the invention
The object of this invention is to provide a kind of 1,3-1,4-beta-glucan enzyme mutant, especially a kind of have 1 of more high catalytic activity and thermostability, 3-1, a 4-beta-glucan enzyme mutant.
The aminoacid sequence of described mutant is as shown in following (1) or (2) or (3) or (4) or (5):
(1) aminoacid sequence shown in SEQ ID NO.1,
(2) aminoacid sequence shown in SEQ ID NO.2,
(3) aminoacid sequence shown in SEQ ID NO.3,
(4) aminoacid sequence shown in SEQ ID NO.4,
(5) process one or several aminoacid replacement, disappearance or interpolation on the aminoacid sequence basis of (1) or (2) or (3) or (4), and there is 1,3-1, the aminoacid sequence of 4-1,4 beta-glucanase activity.
The nucleotide sequence of coding said mutation body is the nucleotide sequence as shown in SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7 or SEQ ID NO.8 preferably.
Aminoacid sequence as the mutant of SEQ ID NO.1 be wild 1 as shown in SEQ ID NO.9 by nucleotide sequence, 3-1, the Methionin Lys of the 20th of 4-beta-glucanase is mutated into Serine Ser.Gained mutant called after K20S.
Aminoacid sequence as the mutant of SEQ ID NO.2 be wild 1 as shown in SEQ ID NO.9 by nucleotide sequence, 3-1, the Methionin Lys of the 117th of 4-beta-glucanase is mutated into Serine Ser.Gained mutant called after K117S.
Aminoacid sequence as the mutant of SEQ ID NO.3 be wild 1 as shown in SEQ ID NO.9 by nucleotide sequence, 3-1, the Methionin Lys of the 165th of 4-beta-glucanase is mutated into Serine Ser.Gained mutant called after K165S.
Aminoacid sequence as the mutant of SEQ ID NO.4 be wild 1 as shown in SEQ ID NO.9 by nucleotide sequence, 3-1, the Methionin Lys of the 20th, 117,165 of 4-beta-glucanase is all mutated into Serine Ser.Gained mutant called after K20S/K117S/K165S.
Another object of the present invention is to provide a kind of structure described 1,3-1, the method of 4-beta-glucan enzyme mutant, is to be template by the wild enzyme gene that derives from B.terquilensis CGX5-1 (SEQ ID NO.9), adopts the method for overlapping extension PCR to carry out rite-directed mutagenesis acquisition; Obtain encoding the nucleotide sequence of gene of beta-glucan enzyme mutant respectively as shown in SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7 or SEQ ID NO.8.Through all mutational sites of order-checking evaluation are all successful, according to goal-selling, suddenlyd change, subsequently the gene fragment of encoding mutant enzyme is carried out to recombinant expressed acquisition mutant.
Describedly recombinant expressedly preferably take pET28a (+) as expression vector, take intestinal bacteria as expressive host.
For the construction process of three strain beta-glucanase single mutation enzyme K20S, K117S and K165S, the wild enzyme gene of preferably take is template, and by 20,117 and 165 s' Methionin Lys, the method by overlapping extension PCR sports Serine Ser respectively.
For the construction process of three mutant enzyme K20S/K117S/K165S, the K20S single mutation enzyme of preferably take is template, and the method by overlapping extension PCR sports Serine Ser by 117 and 165 Methionin Lys respectively through two steps.
The 3rd technical problem that the present invention will solve is to provide described in fermentative production 1,3-1, and the method for 4-beta-glucan enzyme mutant, preferably take pET28a (+) as expression vector, take intestinal bacteria as expressive host.Take TB liquid nutrient medium as fermention medium, and recombinant bacterium is cultured to OD at 37 ℃ of 200rpm
600be about 1.0, add 0.06mM final concentration IPTG and and 8mM final concentration alpha-lactose abduction delivering, and cultivate 6h under 24 ℃ of 150rpm.After gained bacterium liquid is centrifugal, abandon microorganism collection supernatant.Supernatant is sloughed imidazoles through Ni-NTA affinity chromatography column purification and GEPD-10 post, obtains 1,3-1,4-beta-glucan enzyme mutant.
Preferably on flat board, picking list bacterium colony is in receiving the LB liquid nutrient medium of mycin containing 100 μ g/mL sulfuric acid cards for the activation method of described recombinant bacterium, and 37 ℃ of 200rpm cultivate 10-12h.
Beneficial effect of the present invention: four strain beta-glucan enzyme mutants provided by the invention are compared with wild enzyme, have improved 98.2% than vigor; Optimum temperuture is brought up to 55-60 ℃ by 45 ℃; T
50value is increased to 76 ℃ of left and right by 62 ℃; Transformation period at 50 ℃, by the highest 214min that extends to of 95min, higher than wild enzyme, has improved 125.3% far away; Transformation period at 60 ℃, extends at most 59min by 32.5min, has improved 81.5%.The catalytic activity of visible mutation enzyme and thermostability all to some extent significantly improve, and are more conducive in industrial application.
Accompanying drawing explanation
The optimum temperuture comparison of the wild enzyme of Fig. 1 and four strain beta-glucan enzyme mutants; : wild enzyme, zero: K20S, △: K117S, ▽: K165S, ◇: K20S/K117S/K165S.
The T of the wild enzyme of Fig. 2 and four strain beta-glucan enzyme mutants
50value relatively; : wild enzyme, zero: K20S, △: K117S, ▽: K165S, ◇: K20S/K117S/K165S.
The wild enzyme of Fig. 3 and four strain beta-glucan enzyme mutants the transformation period comparison of 50 ℃; : wild enzyme, zero: K20S, △: K117S, ▽: K165S, ◇: K20S/K117S/K165S.
Fig. 4 wild fish four strain beta-glucan enzyme mutants the transformation period comparison of 60 ℃; : wild enzyme, zero: K20S, △: K117S, ▽: K165S, ◇: K20S/K117S/K165S.
Embodiment
The selection in embodiment 1 mutational site
The putative amino acid sequence of beta-glucanase is committed to I-TASSER line server and carries out homology modeling, and calculate the position of Methionin in structure in the PDB file that modeling is obtained input Voronoia software.As can be seen from Table 1, in beta-glucanase three-dimensional structure, the average packing value of all 12 Methionins, between 0.47 to 0.62, all in protein surface, and has the highest solution accessibility.This means that the Methionin that is positioned at protein surface may have impact for beta-glucan enzyme heat stability.
The average packing density value of 12 Methionins in table 1 beta-glucanase
The Te Jila genus bacillus CGX5-1 genome of take is template, obtain the encoding bglt gene of beta-glucanase of the method amplification by PCR, and its nucleotide sequence is as shown in SEQ ID NO.9.Utilize overlap extension pcr, take expression vector pET28a (+)-bglt as template, 12 sites in table 1 are sported respectively to Serine.
The putative amino acid sequence of 12 plant mutant strains is committed to I-TASSER line server and carries out homology modeling.And its total energy of the PDB file that modeling is obtained input Gromacs computed in software.In order to measure its catalytic property, recombinant expressed 12 plant mutant enzymes in intestinal bacteria.As can be seen from Table 2, the ratio vigor of K20S, K117S and K165S is compared with wild enzyme, have increased significantly, and all decreases to some degree of ratio vigor of other nine plant mutant enzymes.For total energy, except K58S, K80S and K214S mutant enzyme, the total energy of all the other mutant strains is all lower than wild enzyme.And total energy is lower, represent that enzyme heat stability is better.In conjunction with above-mentioned 2 points, select K20S, K117S and K165S tri-plant mutant strains for the mensuration of follow-up character.And the ratio vigor of K20S/K117S/K165S tri-mutant enzymes that obtain after three site simultaneous mutations is compared with wild enzyme, have increased significantly, and total energy is also lower than wild enzyme, this is indicating that it may also have better thermostability.
Ratio vigor and the total energy comparison of the wild enzyme of table 2 and mutant enzyme
The preparation of embodiment 2 beta-glucan enzyme mutant K20S, K117S, K165S and K20S/K117S/K165S
(1) rite-directed mutagenesis
1) structure of carrier pET28a (+)-bglt
The Te Jila genus bacillus CGX5-1 genome of take is template, and the method amplification by PCR obtains bglt gene.Primer is as follows:
Forward primer: 5 '-C
gGATCCaTGAAACGAGTGTTGCTAATT-3 ', underscore is BamHI restriction enzyme site,
Reverse primer: 5 '-T
cTCGAGgTATTTTTTTGTATAGCGCAC-3 ', underscore is XhoI restriction enzyme site, lowercase is mutational site;
PCR reaction system is: 5U/ μ L rTaq 1 μ L, and 10 * rTaq Buffer, 5 μ L, 2.5mM dNTPs 4 μ L, 100 μ M forward primer 1 μ L, 100 μ M reverse primer 1 μ L, second step PCR product 20 μ L, add distilled water polishing to 50 μ L;
PCR reacts amplification condition: 94 ℃ of denaturation 5min; Carry out subsequently 94 ℃ of 1min, 56 ℃ of 50s, 72 ℃ 50s35 circulation;
Pcr amplification product is used restriction enzyme BanHI and XhoI to carry out after double digestion through cutting after glue reclaims, be connected with pET28a (+) plasmid through same digestion with restriction enzyme, and be converted in e. coli bl21 (DE3) competent cell.The template of gained recombinant plasmid pET28a (+)-bglt rite-directed mutagenesis research.The nucleotide sequence of bglt gene is (containing signal peptide sequence) as shown in SEQ ID NO.9.
2) rite-directed mutagenesis of single mutation enzyme K20S, K117S and K165S; Utilize overlap extension pcr, take expression vector pET28a (+)-bglt as template,
The rite-directed mutagenesis primer of introducing K20S codon is:
Forward primer: 5 '-GGTTTTTGGCAA
aGTgCAGATGGTTATT-3 ', underscore is mutating alkali yl,
Reverse primer: 5 '-AATAACCATCTGC
aCTtTGCCAAAAACC-3 ', underscore is mutating alkali yl; The rite-directed mutagenesis primer of introducing K117S codon is:
Forward primer: 5 '-AAAAGACACAACG
aGTgTTCAATTTAAC-3 ', underscore is mutating alkali yl,
Reverse primer: 5 '-AGTTAAATTGAAC
aCTcGTTGTGTCTTTT-3 ', underscore is mutating alkali yl; The rite-directed mutagenesis primer of introducing K165S codon is:
Forward primer: 5 '-ACGGGCAATTA
aGTcATACTGCAACA-3 ', underscore is mutating alkali yl,
Reverse primer: 5 '-TGTTGCAGTATG
aCTtAATTGCCCGTCGA-3 ', underscore is mutating alkali yl;
3) rite-directed mutagenesis of three mutant enzyme K20S/K117S/K165S: utilize overlapping extension PCR method, the single mutation enzyme K20S gene of take is template, the rite-directed mutagenesis primer of introducing K117S codon is:
Forward primer: 5 '-AAAAGACACAACG
aGTgTTCAATTTAAC-3 ', underscore is mutating alkali yl,
Reverse primer: 5 '-AGTTAAATTGAAC
aCTcGTTGTGTCTTTT-3 ', underscore is mutating alkali yl; The two mutant enzyme genes of the K20S/K117S of take are afterwards template, and the rite-directed mutagenesis primer of introducing K165S is:
Forward primer: 5 '-ACGGGCAATTA
aGTcATACTGCAACA-3 ', underscore is mutating alkali yl,
Reverse primer: 5 '-TGTTGCAGTATG
aCTtAATTGCCCGTCGA-3 ', underscore is mutating alkali yl;
Overlapping extension PCR is divided into three steps, and the concrete implementation condition of three steps is as follows:
The first step PCR reaction system is: 2 * PrimeSTAR max premix25 μ L, 100 μ M forward primer 1 μ L, 100 μ M reverse primer 1 μ L, template DNA 1 μ L, distilled water polishing to 50 μ L;
The first step PCR reacts amplification condition: 94 ℃ of denaturation 5min; Carry out subsequently 94 ℃ of 1min, 56 ℃ of 50s, 72 ℃ 50s30 circulation; Finally be kept at 4 ℃.
Second step PCR reaction system is: 2 * PrimeSTAR max premix, 10 μ L, the first step PCR product 14 μ L, the first step PCR product 24 μ L, distilled water polishing to 20 μ L;
Second step PCR reacts amplification condition: 94 ℃ of denaturation 5min; Carry out subsequently 94 ℃ of 1min, 56 ℃ of 50s, 72 ℃ 50s15 circulation; Finally be kept at 4 ℃.
The 3rd step PCR reaction system is: 5U/ μ L rTaq 1 μ L, and 10 * rTaq Buffer, 5 μ L, 2.5mM dNTPs 4 μ L, 100 μ M forward primer 1 μ L, 100 μ M reverse primer 1 μ L, second step PCR product 20 μ L, add distilled water polishing to 50 μ L;
The 3rd step PCR reaction amplification condition: 94 ℃ of denaturation 5min; Carry out subsequently 94 ℃ of 1min, 56 ℃ of 50s, 72 ℃ 50s35 circulation; Finally be kept at 4 ℃.
Use restriction enzyme BanHI and XhoI to carry out after double digestion the above-mentioned fragment obtaining by pcr amplification, be connected with pET28a (+) plasmid through same digestion with restriction enzyme, and be converted in e. coli bl21 (DE3) competent cell.
(2) expression and purification of mutant
On flat board, picking is containing the single bacterium colony of the intestinal bacteria of above-mentioned recombinant plasmid in receiving the LB liquid nutrient medium of mycin containing 100 μ g/mL sulfuric acid cards, and 37 ℃ of 200rpm cultivate 10-12h, by 4% inoculum size, is forwarded to containing 100 μ g/mL sulfuric acid cards and receives the TB liquid nutrient medium of mycin.Recombinant bacterium is cultured to OD at 37 ℃ of 200rpm
600be about 1.0, add 0.06mM final concentration IPTG and and 8mM final concentration alpha-lactose abduction delivering, and cultivate 6h under 24 ℃ of 150rpm.Bacterium liquid after expressing, at 4 ℃, the centrifugal 20min of 9000rpm, is abandoned to microorganism collection supernatant.The supernatant liquor of acquisition is added to Ni-NTA affinity column, after loading, use 1 * Binding Buffer wash-out until light absorption value is steady, add respectively the imidazoles eluant solution target protein of 50mM, 100mM and 250mM final concentration.
By enzyme activity determination and SDS-PAGE, analyze, find that mutant enzyme mainly appears in 100mM imidazoles elutriant, and band is single.By GEPD-10 desalting column, use 20mM phosphoric acid buffer (pH6.5) to wash lower target protein the elutriant that contains target protein.Use afterwards albumen ultra-filtration centrifuge tube concentrated, obtain respectively single mutation enzyme K20S, K117S, K165 and three mutant enzyme K20S/K117S/K165S goods.
Embodiment 3 enzymes are lived and protein concentration analysis
(1) enzyme activity determination method:
3,5-dinitrosalicylic acid (DNS) method and improvement AZO measuring method combine and measure the method for activity of beta-glucanase:
Enzyme is lived and is defined: 1mL enzyme liquid is under 6.5 conditions at 40 ℃ with pH value, and the Reduction of Glucose amount of substance that the generation of per minute hydrolysis beta-glucan is equivalent to 1 μ mol is 1 enzyme activity unit, with U/mL, represents.
Fermentation clear liquid enzyme activity determination: fermented liquid, after centrifugal, dilutes suitable multiple by supernatant liquor, measures its enzyme activity.
The drafting of glucose typical curve: draw respectively 1% glucose standardized solution 2.0,3.0,4.0,5.0,6.0mL in 50mL volumetric flask, with distilled water, be settled to scale, make every milliliter of rare reference liquid that contains respectively glucose 200,400,600,800,1000,1200 μ g.Respectively get rare reference liquid 0.5mL of different concns in test tube, add pH6.5 Sodium phosphate dibasic-phosphate sodium dihydrogen buffer solution 1.5mL, then add DNS reagent 3.0mL, in boiling water bath, boil 7min, after being cooled to rapidly room temperature after taking-up, add distilled water 10mL, shake up.With distilled water 0.5mL, replace grape malt sugar reference liquid in contrast, use 10mm cuvette, at wavelength 550nm place, with spectrophotometer, measure respectively its absorbancy.Take absorbancy as ordinate zou, and corresponding glucose concn is X-coordinate, drawing standard curve.
Sample enzyme activity determination: accurately draw dilution enzyme liquid 0.5mL to be measured (3 parallel test tubes of each sample), and pH6.5 SODIUM PHOSPHATE, MONOBASIC-Sodium phosphate dibasic damping fluid 1.0mL, be placed in 40 ℃ of water-bath preheating 5min, add the 1.0% beta-glucan solution 0.5mL through preheating, start immediately timing, in 40 ℃ of water-baths, accurately react 10min, add immediately 3.0mlDNS liquid termination reaction, then be placed in boiling water bath 7min, take out after cooling rapidly and add 10mL deionized water, after shaking up, measure the light absorption value of the reaction solution under 550nm.Carry out blank mensuration simultaneously, its step is for drawing dilution enzyme liquid 0.5mL to be measured, add 1.0mL pH5.0 Sodium phosphate dibasic-citrate buffer solution, then first add 3.0mL DNS liquid to make enzyme deactivation, 40 ℃ of water-bath preheatings, add again the same 1.0% beta-glucan solution 0.5mL through preheating, 40 ℃ of water-bath 10min, are then placed in boiling water bath 7min, and later step is same as sample determination, during by sample determination, obtain light absorption value, according to typical curve, can obtain corresponding enzyme activity unit.
(2) determination of protein concentration:
Bradford method is measured the method for protein concentration in solution:
Get 200 μ L testing samples and add 2mLBradford reagent, mix and under 595nm, measure light absorption value rapidly afterwards, blank is pH6.5 phosphate buffered saline buffer.Every group of three, sample is parallel, and gained light absorption value reference standard curvilinear equation y=0.0042x+0.0082 can obtain the protein concentration in solution.
(3) than vigour: experimental result is listed in table 3.Wild enzyme preparation is compared with mutant enzyme goods, can be found, the ratio vigor of four plant mutant enzymes is compared with wild enzyme all to have significantly and is promoted.Wherein, K20S, K117S and K165S single mutation enzyme than enzyme work, reach 4675.1U/mg, compare and improved respectively 88.3%, 59.6% and 84.1% with wild enzyme.And three mutant enzyme K20S/K117S/K165S than enzyme work, reach 4936.4U/mg, compare and improved 98.2% with wild enzyme.Obviously, the ratio enzyme of three strain single mutation enzymes and strain three mutant enzymes is lived in comparing with wild enzyme all to have had significantly and is promoted.
The wild enzyme of table 3 is lived relatively with the ratio enzyme of four strain beta-glucan enzyme mutants
The thermostability of the wild enzyme of embodiment 4 and mutant enzyme
(1) the optimum temperuture measuring method of wild enzyme and mutant enzyme:
Get the enzyme preparation 100 μ L of acquisition, respectively at differing temps (35,40,45,50,55,, 60,65,70 ℃), measure enzyme activity.The enzyme of usining maximum value alive is lived as 100% relative enzyme, and the relative enzyme that the enzyme value alive at other temperature is at this temperature divided by maximum value gained percentage ratio is lived.The optimum temperuture of wild enzyme and mutant enzyme is shown in Fig. 1.As can be seen from Figure 1, the optimum temperuture of wild enzyme is 45 ℃, and the optimum temperuture of K165S reaches 55 ℃, and the optimum temperuture of K20S, K117S and K20S/K117S/K165S reaches 60 ℃.
(2) T of wild enzyme and mutant enzyme
50values determination method:
Get the enzyme preparation 2mL of acquisition, respectively at differing temps (40,45,50,55,60,65,70,75,80 ℃), process 15min, take out and be placed in cooled on ice 10min immediately, get 100 μ L and measure beta glucan enzyme activity.The enzyme of usining maximum value alive is lived as 100% relative enzyme, and the relative enzyme that the enzyme value alive at other temperature is at this temperature divided by maximum value gained percentage ratio is lived.T
50value is defined as the temperature that is reduced to an initial enzyme half alive through above-mentioned treat enzyme work.As can be seen from Figure 2, the inactivation curve of mutant enzyme all relaxes than wild enzyme.The T of wild enzyme
50value is 62 ℃, the T of K20S, K117S and K165S single mutation enzyme
50value is respectively 76 ℃, 76 ℃ and 75 ℃, and the T of three mutant enzyme K20S/K117S/K165S
50value is 76 ℃.The T of mutant enzyme
50value is all higher than wild enzyme.
(3) wild enzyme and mutant enzyme are at the transformation period measuring method of 50 ℃:
Get the enzyme preparation 2mL of acquisition, respectively at 50 ℃, process different times (20,40,60,80,100,120,140,160,180,200,220,240min), take out immediately and be placed in cooled on ice 10min, treatment solution is diluted and gets 100 μ L after reasonable multiple and measure activity of beta-glucanase.The processing primary fermentation liquid enzyme of usining is lived in being worth as 100% relative enzyme and is lived, and the relative enzyme that the enzyme of different treatment under time value alive is under this condition divided by maximum value gained percentage ratio is alive.Transformation period is defined as enzyme and lives the needed time of half gone through enzyme after above-mentioned processing.The inactivation curve of mutant enzyme at 50 ℃ compared relatively mild with wild enzyme as can be seen from Figure 3.The transformation period of wild enzyme at 50 ℃ is 95min, and three strain single mutation enzyme K20S, the transformation period of K117S and K165S reaches respectively 149min, 210min and 102min, and the transformation period of three mutant enzyme K20S/K117S/K165S reaches 214min especially, higher than wild enzyme, improved 125.3% far away.
(4) wild enzyme and mutant enzyme are at the transformation period measuring method of 60 ℃:
Get the enzyme preparation 2mL of acquisition, process different times (10,20,30,40,50,60,70min) respectively at 60 ℃, take out and be placed in cooled on ice 10min immediately, treatment solution is diluted and gets 100 μ L after reasonable multiple and measure activity of beta-glucanase.The processing primary fermentation liquid enzyme of usining is lived in being worth as 100% relative enzyme and is lived, and the relative enzyme that the enzyme of different treatment under time value alive is under this condition divided by maximum value gained percentage ratio is alive.At 60 ℃, the inactivation curve of mutant enzyme is compared milder with wild enzyme as can be seen from Figure 4.The transformation period of wild enzyme at 60 ℃ is determined as 32.5min, and three strain single mutation enzyme K20S, the transformation period of K117S and K165S reaches respectively 46min, 58min and 37min.The transformation period of three mutant enzyme K20S/K117S/K165S reaches 59min especially, compares and has improved 81.5% with wild enzyme.
Although the present invention with preferred embodiment openly as above, but it is not in order to limit the present invention, any person skilled in the art, without departing from the spirit and scope of the present invention, can be through one or several aminoacid replacement, disappearance or interpolation on aminoacid sequence of the present invention basis, obtain thering is 1,3-1, the aminoacid sequence of 4-1,4 beta-glucanase activity.Therefore protection scope of the present invention should be with being as the criterion that claims were defined.
Claims (10)
1. one kind 1,3-1,4-beta-glucan enzyme mutant, is characterized in that, the aminoacid sequence of described mutant is as shown in following (1) or (2) or (3) or (4) or (5):
(1) aminoacid sequence shown in SEQ ID NO.1,
(2) aminoacid sequence shown in SEQ ID NO.2,
(3) aminoacid sequence shown in SEQ ID NO.3,
(4) aminoacid sequence shown in SEQ ID NO.4,
(5) process one or several aminoacid replacement, disappearance or interpolation on the aminoacid sequence basis of (1) or (2) or (3) or (4), and there is 1,3-1, the aminoacid sequence of 4-1,4 beta-glucanase activity.
2. the gene of mutant described in the claim 1 of encoding.
3. carry carrier or the reconstitution cell of gene described in claim 2.
4. one kind obtains described in claim 11,3-1, the method of 4-beta-glucan enzyme mutant, it is characterized in that, with wild 1,3-1, the gene of 4-beta-glucanase is template, adopt the method for overlapping extension PCR to carry out the gene that rite-directed mutagenesis obtains encoding mutant body, subsequently the gene fragment of encoding mutant body is carried out to recombinant expressed acquisition mutant.
5. method according to claim 4, it is characterized in that, for aminoacid sequence beta-glucanase single mutant K20S, the K117S as shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 and the structure of K165S respectively, the wild enzyme gene of take is template, respectively the 20th, 117 and 165 s' Methionin is sported to Serine by overlapping extension PCR.
6. method according to claim 4, it is characterized in that, the structure of Trimutant K20S/K117S/K165S for aminoacid sequence as shown in SEQ ID NO.4, successively sports Serine by the 20th, 117 and 165 s' Methionin by overlapping extension PCR.
7. according to the arbitrary described method of claim 4-6, it is characterized in that, take pET28a (+) as expression vector, take intestinal bacteria as expressive host.
8. method according to claim 8, is characterized in that, take TB liquid nutrient medium as fermention medium, and recombinant bacterium is cultured to OD at 37 ℃
600=0.8-1.0, adds IPTG and alpha-lactose abduction delivering, abandons thalline after gained bacterium liquid is centrifugal, collects and contains 1,3-1, the supernatant of 4-beta-glucan enzyme mutant.
9. method according to claim 8, is characterized in that, supernatant is sloughed imidazoles through Ni-NTA affinity chromatography column purification and GE PD-10 post, obtains 1,3-1,4-beta-glucan enzyme mutant.
10. the application of mutant in beer production described in claim 1.
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| CN104862290A (en) * | 2015-06-12 | 2015-08-26 | 江南大学 | 1,3-1,4-beta-glucanase mutant |
| CN104862290B (en) * | 2015-06-12 | 2017-11-17 | 江南大学 | A kind of 1,3 1,4 beta glucan enzyme mutants |
| CN105907749A (en) * | 2016-05-24 | 2016-08-31 | 东北林业大学 | Gene mutation, deletion, insertion and recombinant fragment obtaining method based on overlapping extension PCR process |
| CN111269905A (en) * | 2020-03-26 | 2020-06-12 | 汪利平 | Heat-stable β -1,3-1,4-glucanase for reducing non-biological turbidity of beer |
| CN111269905B (en) * | 2020-03-26 | 2023-09-01 | 广东燕京啤酒有限公司 | Thermostable beta-1,3-1, 4-glucanases for reducing the abiotic haze of beer |
| CN112481240A (en) * | 2020-12-10 | 2021-03-12 | 江苏科技大学 | GH16 family heat-resistant glucanase mutant and construction method and application thereof |
| CN112481240B (en) * | 2020-12-10 | 2021-11-09 | 江苏科技大学 | GH16 family heat-resistant glucanase mutant and construction method and application thereof |
| CN113234705A (en) * | 2021-04-21 | 2021-08-10 | 江南大学 | Acid-tolerant 1,3-1, 4-beta-glucanase mutants |
| CN113234705B (en) * | 2021-04-21 | 2022-09-27 | 江南大学 | Acid-resistant 1,3-1, 4-beta-glucanase mutant |
| CN113699136A (en) * | 2021-06-04 | 2021-11-26 | 江苏科技大学 | Beta-1, 3-1, 4-glucanase mutant with high catalytic activity at animal body temperature and application thereof |
| CN113699136B (en) * | 2021-06-04 | 2023-02-03 | 江苏科技大学 | Animal body beta-1, 3-1, 4-glucanase mutant with high catalytic activity at room temperature and application thereof |
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