Mutant enzyme of type I pullulanase and preparation method and application thereof

文档序号：1016147 发布日期：2020-10-27 浏览：42次中文

阅读说明：本技术 I型普鲁兰酶的突变体酶及其制备方法与应用 (Mutant enzyme of type I pullulanase and preparation method and application thereof ) 是由逄晓阳 *** 杨洋芦晶张书文李伟勋朱青徐琛于 2020-07-23 设计创作，主要内容包括：本发明公开了I型普鲁兰酶的突变体酶,突变体酶的氨基酸序列如SEQ ID No.1所示。本发明还公开了编码所述突变体酶的基因的核苷酸序列如SEQ ID No.2所示。本发明还公开了突变体酶的制备方法,包括根据I型普鲁兰酶的氨基酸序列进行同源建模,基于序列比对分析和蛋白结构分析,选取突变位点并设计对突变引物对；以携带I型普鲁兰酶基因的载体为模板,分别进行定点突变反应,得到突变质粒；于突变质粒转化工程菌中复制表达对应的突变体酶。本发明的突变体酶具有更优的耐热性能和比酶活。(The invention discloses a mutant enzyme of pullulanase I, wherein the amino acid sequence of the mutant enzyme is shown in SEQ ID No. 1. The invention also discloses a nucleotide sequence of the gene for coding the mutant enzyme, which is shown as SEQ ID No. 2. The invention also discloses a preparation method of the mutant enzyme, which comprises the steps of carrying out homologous modeling according to the amino acid sequence of the pullulanase I, selecting mutation sites and designing mutation primer pairs based on sequence comparison analysis and protein structure analysis; respectively carrying out site-directed mutagenesis reaction by taking a vector carrying the type I pullulanase gene as a template to obtain mutant plasmids; and replicating and expressing the corresponding mutant enzyme in the mutant plasmid transformation engineering bacteria. The mutant enzyme has better heat resistance and specific enzyme activity.)

A mutant enzyme of pullulanase type I, characterized in that the amino acid sequence of the mutant enzyme is shown in SEQ ID No. 1.

2. The gene encoding the mutant enzyme according to claim 1, wherein the nucleotide sequence of the gene encoding the mutant enzyme is represented by SEQ ID No. 2.

3. An engineered bacterium comprising a gene encoding the mutant enzyme of claim 1.

4. A vector comprising a gene encoding the mutant enzyme of claim 1.

5. A method of preparing the mutant enzyme of claim 1, comprising the steps of:

firstly, performing homologous modeling according to an amino acid sequence of the pullulanase type I disclosed in an NCBI database, selecting amino acid residues within 10 angstroms away from a catalytic site of the pullulanase type I as an object based on sequence alignment analysis and protein structure analysis, excluding very conservative amino acid residues through sequence alignment, selecting 1 non-conservative amino acid mutation site, and designing a corresponding mutation primer pair for the mutation site, wherein the mutation primer pair is 5'-CGTATCTGAATGAAAGCGGCTGCGGCAATGTTATG-3' and 5'-GCAGCCGCTTTCATTCAGATACGCGCCGGTTTTATC-3';

secondly, taking a vector carrying the type I pullulanase gene as a template, and carrying out point mutation reaction by using a designed mutation primer pair and a PCR mutation method to obtain a mutation plasmid with a mutant enzyme gene;

and step three, converting the mutant plasmid obtained in the step two into engineering bacteria capable of expressing target genes, and expressing corresponding mutant enzymes along with the replication of the engineering bacteria.

6. The method for preparing the mutant enzyme according to claim 5, wherein the point mutation reaction conditions in the second step are: pre-denaturation at 94 ℃ for 5 min; melting at 94 ℃ for 20s, annealing at 65 ℃ for 20s, and extending at 72 ℃ for 4min for 25 cycles; extending for 10min at 72 ℃; keeping the temperature at 4 ℃.

7. The method of claim 5, wherein the engineered bacterium in step two is E.coli.

8. The method for preparing the mutant enzyme according to claim 5, wherein the vector in the second step is pET-22b (+).

9. The method of making the mutant enzyme according to claim 5, further comprising using Ni²⁺And (3) separating and purifying the mutant enzyme by an affinity chromatography mode.

10. Use of a mutant enzyme according to claim 1 for the preparation of a product for enzymatic hydrolysis of polysaccharides containing a-1, 6 glycosidic linkages.

Technical Field

The present invention relates to the technical field of genetic engineering and enzyme engineering. More specifically, the invention relates to a mutant enzyme of pullulanase I and a preparation method and application thereof.

Background

Many pullulanases have been discovered at present, and even pullulanases have been studied at the genetic level. However, there are few pullulanases that can be used in industrial production, and pullulanase derived from Bacillus acidopululyticus is currently most commercialized. The main application mode of the pullulanase is used in the saccharification process of starch, the industrial application condition is that the pH is 4.5-5.5, the temperature is 55-65 ℃ or even higher, and the sustained action time is up to 48-60 h (NISHA and SATYANARAYANA, 2016). Most of the reported pullulanase type I cannot adapt to the industrial condition, some enzymes have poor heat resistance, and some enzymes have low or even inactivated enzyme activity under the condition of pH5.5 or lower. Even commercial b.acetidopululilyticus pullulanase presents a significantly shorter plate with a half-life of only 34.9min at 60 ℃, without sufficiently strong thermostability.

In view of the above situation, the present invention is to provide pullulanase having high heat resistance, taking the heat resistance of pullulanase as a primary consideration. The heat-resistant pullulanase is mostly derived from thermophilic or extreme thermophilic microorganisms, and the growth conditions and the culture conditions are severe, so that the sampling and screening are not facilitated. Therefore, it is worth trying to obtain pullulanase with good heat resistance by adopting a genetic engineering technical mode.

Disclosure of Invention

An object of the present invention is to solve at least the above problems and to provide at least the advantages described later.

Still another object of the present invention is to provide a heat-resistant pullulanase type I mutant enzyme having excellent heat-resistant property and excellent specific enzyme activity, and a method for preparing the same.

To achieve these objects and other advantages in accordance with the present invention, there is provided a mutant enzyme of pullulanase type I having an amino acid sequence shown in SEQ ID No. 1.

Provides a gene for coding the mutant enzyme, and the nucleotide sequence of the gene for coding the mutant enzyme is shown as SEQ ID No. 2.

An engineered bacterium comprising a gene encoding the mutant enzyme is provided.

There is provided a vector comprising a gene encoding the above mutant enzyme.

There is provided a method of preparing the mutant enzyme, comprising the steps of:

step one, performing homologous modeling according to an amino acid sequence of the pullulanase type I disclosed in an NCBI database, selecting amino acid residues within 10 angstroms from a catalytic site of the pullulanase type I as an object based on sequence alignment analysis and protein structure analysis, excluding very conservative amino acid residues through sequence alignment, selecting 1 non-conservative amino acid mutation site, designing a corresponding mutation primer pair for the mutation site, wherein the mutation primer pair is a mutation primer pair

5'-CGTATCTGAATGAAAGCGGCTGCGGCAATGTTATG-3' and

5′-GCAGCCGCTTTCATTCAGATACGCGCCGGTTTTATC-3′；

Preferably, the point mutation reaction conditions in step two are: pre-denaturation at 94 ℃ for 5 min; melting at 94 ℃ for 20s, annealing at 65 ℃ for 20s, and extending at 72 ℃ for 4min for 25 cycles; extending for 10min at 72 ℃; keeping the temperature at 4 ℃.

Preferably, the engineering bacterium in the second step is Escherichia coli.

Preferably, the vector in step two is pET-22b (+).

Preferably, Ni is used²⁺And (3) separating and purifying the mutant enzyme by an affinity chromatography mode.

Provides an application of the mutant enzyme in preparing products for enzymolysis of polysaccharide containing a-1, 6 glycosidic bonds.

The invention at least comprises the following beneficial effects:

firstly, a mutant enzyme with heat resistance and specific enzyme activity superior to those of the existing pullulanase I is obtained.

Secondly, compared with the existing pullulanase PulF, the mutant enzyme has higher enzyme activity in a wider pH range, and particularly, the relative enzyme activity of the mutant enzyme is more than 85% in the pH range of 5.0-5.5. Within the pH range of 6.0-7.0, the relative enzyme activity of the mutant enzyme is obviously higher than that of Pullulan enzyme PulF.

Third, the relative enzyme activities of the pullulanase PulF and the mutant enzyme V481C have similar trend, and the enzyme activities of the two enzymes are rapidly reduced when the pH value is far away from the optimum value. And different buffer systems have great influence on the two enzymes, and the relative enzyme activity in the phosphate buffer solution with the pH value of 6.0 is nearly 3 times that in the acetic acid buffer solution with the pH value of 6.0. Therefore, in combination with the analysis of the industrial application conditions, the mutant enzyme has the greatest advantages compared with the pullulanase PulF: the enzyme activity is higher within the range of pH5.0-5.5, and the requirement on the pH environment of reaction liquid in the saccharification process is not harsh.

Fourthly, when pullulanase PulF is mutated into V481C, the optimal temperature is not changed and is still 80 ℃. In addition, by comparing the relative enzyme activity change trends of the pullulanase PulF and the mutant enzyme with the change of temperature, it can be seen that the relative enzyme activity change trend of the mutant enzyme V481C is almost consistent with that of the pullulanase PulF.

The pullulanase in the sugar industry has the advantage that the pullulanase has higher relative enzyme activity in the temperature range of 60-75 ℃ by combining the analysis of the industrial application condition because the reaction temperature is 55-65 ℃ or even higher, wherein the most common reaction temperature is 60 ℃.

Fifth, the thermostability of the mutant enzyme was enhanced at both 75 ℃ and 80 ℃. The half-life of V481C is longest under the condition of 75 ℃, and is improved by nearly 13 times compared with the half-life of pullulanase PulF under the same condition. At 80 ℃ the half-lives of all enzymes are very short.

Sixth, K of mutant enzyme compared to Pullulan enzyme_mThe value is reduced, which indicates that the combination ability of the mutant enzyme and the pullulan is enhanced; catalytic constant K of mutant enzyme V481C_cat/K_mThe increase indicates that the catalytic efficiency of the mutant enzyme on the pullulan is improved.

Additional advantages, objects, and features of the invention will be set forth in part in the description which follows and in part will become apparent to those having ordinary skill in the art upon examination of the following or may be learned from practice of the invention.

Drawings

FIG. 1 is a nucleic acid electrophoresis of the point mutation PCR product of the present invention;

FIG. 2 is a standard curve of the protein of the present invention;

FIG. 3 is a diagram of denaturing polyacrylamide gel electrophoresis of the purified mutant enzyme of the present invention;

FIG. 4 is a graph showing half-lives at 75 ℃ of the mutant enzyme of the present invention and Pullulan enzyme PulF;

FIG. 5 is a graph showing half-lives of the mutant enzymes of the present invention at 80 ℃.

Detailed Description

The present invention is further described in detail below with reference to the attached drawings so that those skilled in the art can implement the invention by referring to the description text.

It is to be noted that the experimental methods described in the following embodiments are all conventional methods unless otherwise specified, and the reagents and materials are commercially available unless otherwise specified.

1. Raw material preparation

Biological material: as shown in Table 1

TABLE 1 biomaterials

The method for constructing pET-22b (+) -pulF comprises the following steps:

the accession number and the microbial source of WP-011994577.1 (Fervidobacterium nodosum Rt17-B1) are selected as original enzymes, and the pullulanase is named as PulF for convenience of expression.

After a signal peptide coding sequence is removed from a gene sequence of the pullulanase PulF, the total length of a gene fragment derived from Fervidobacteriumdosum is 2460bp, and the gene of the 2460 total length fragment is synthesized from the Huada gene after codon optimization. The synthesized gene sequences were ligated into expression vector pET-22b (+) via NdeI/XhoI double cleavage sites and transformed into competent cells BL21(DE 3). Positive clones were picked from LB plates supplemented with ampicillin (final concentration 100. mu.g/mL) and the successfully sequenced recombinant bacteria were stored in 15% glycerol tubes. The successfully constructed recombinant plasmid was named pET-22b (+) -pulF. The nucleotide sequence for coding the pullulanase PulF is shown in SEQ ID No.3, the nucleotide sequence for coding the pullulanase PulF after codon optimization is shown in SEQ ID No.4, and the amino acid sequences of the pullulanase PulF and the pullulanase PulF after codon optimization are the same and are shown in SEQ ID No. 5.

Culture medium:

LB liquid Medium (g/L): tryptone 10, yeast extract powder 5, sodium chloride 10, pH7.0, 121 ℃, sterilized for 15 min.

LB solid Medium (g/L): tryptone 10, yeast extract powder 5, sodium chloride 10, agar powder 20, pH7.0, 121 ℃, sterilized for 15 min.

The main reagents are as follows:

analytically pure reagents such as sodium dihydrogen phosphate, disodium hydrogen phosphate, sodium chloride, imidazole, Ethylene Diamine Tetraacetic Acid (EDTA), glacial acetic acid, sodium acetate, anhydrous glucose, phenol, sodium hydroxide, potassium sodium tartrate, sodium bisulfite, 3, 5-dinitrosalicylic acid, n-butyl alcohol, anhydrous ethanol, concentrated sulfuric acid, chloride salts of various metal ions and the like are purchased from Beijing GmbH (national chemical group of chemical reagents); precast gel (SurePAGE)^TMBis-Tris, 8%, 10 well) and SDS-PAGE running buffer purchased from kasei biotechnology limited; SDS-PAGE protein loading buffer and protein marker, purchased from Dalibao bioengineering company; affinity chromatography column packing (Ni)

High Performance), available from general electric medical Group (GE) of america; BCA protein quantification kit, plasmid miniprep kit, BL21(DE3) competence and DH5 alpha competence, all purchased from Tiangen Biochemical technology (Beijing) Co., Ltd; fast Mutagenesis System kit, available from TransGenBiotech, Inc., of Beijing, all-purpose gold Biotechnology.

Cell disruption buffer (lysine buffer): 50mM phosphate buffer, 100mM sodium chloride, pH 7.0;

loading buffer (Binding buffer): 50mM phosphate buffer, 500mM sodium chloride, pH 7.0;

washing buffer (Washing buffer): 50mM phosphate buffer, 500mM sodium chloride, 100mM imidazole, pH 7.0;

elution buffer (elusion buffer): 50mM phosphate buffer, 500mM sodium chloride, 500mM imidazole, pH 7.0;

dialysate a (dialysate a): 50mM phosphate buffer, 10mM EDTA, pH 7.0;

dialysate b (dialysate b): 50mM phosphate buffer, pH 7.0.

The main apparatus is as follows:

avanti JXN-26 intelligent high efficiency centrifuge, beckmann coulter ltd, usa; ultrasonic cell disruption instrument JY92-IIN, Ningbo Xinzhi Biotech GmbH;a nucleic acid protein detector HD-2 and a constant flow pump HL-2, Shanghai Kaishi Instrument Analyzer; PowerPac Basic and Mini-PROTECTAN Tetra Cell electrophoresis System, Bio-Rad, USA; constant temperature shaking incubator, shanghai-chang scientific instruments ltd; gel imaging system, shanghai volitang scientific instruments ltd; model TP600 PCR Instrument, TaKaRa, Japan;20M multifunctional microplate reader, Tekken company, Switzerland.

2. Experimental methods

2.1 site-directed mutagenesis:

a mutation primer is designed by using pET-22b (+) -PulF as a template, and a gene encoding PulF is subjected to point mutation by using a Fast Mutagenesis System kit. The mutant primer sequences are shown in Table 2, the point mutation reaction system is shown in Table 3, and the reaction conditions of the point mutation PCR are as follows: pre-denaturation at 94 ℃ for 5 min; melting at 94 ℃ for 20s, annealing at 65 ℃ for 20s, and extending at 72 ℃ for 4min for 25 cycles; extending for 10min at 72 ℃; keeping the temperature at 4 ℃.

TABLE 2 mutant primer sequences

TABLE 3 Point mutation reaction System

After the point mutation PCR reaction is finished, 5 mu L of PCR product is taken and subjected to 1% agarose gel electrophoresis detection, if bright and clear target bands appear and the size is consistent with the expected size, the successful amplification is indicated. To the PCR product successfully amplified, 0.5. mu.L of DMT enzyme was added and incubated at 37 ℃ for 1 hour to remove the plasmid template.

2.2 transformation and preservation of PCR products:

after the PCR product is digested by DMT enzyme, DMT competent cells are transformed for replication and verification, and the transformation steps are as follows:

(1) taking out DMT competent cells, slightly thawing, adding 5 μ L of PCR product digested with DMT enzyme under aseptic condition, gently mixing, and ice-cooling for 30 min;

(2) performing heat shock in 42 ℃ water bath for 45s, then rapidly placing competent cells on ice, and standing for 3min, wherein the action needs to be gentle without shaking a centrifuge tube;

(3) adding 500 μ L of SOC culture medium (without antibiotic) at room temperature into each tube of competent cells under sterile condition, and resuscitating at 37 deg.C and 200rpm for 1 h;

(4) under the aseptic condition, the recovered bacterial liquid is blown and beaten uniformly, 200 mu L of the liquid is taken and coated on an LB solid culture medium containing ampicillin, and the liquid is cultured for 12h at 37 ℃;

(5) the colonies were picked and the plasmids were extracted for sequencing.

The plasmid with error-free sequence alignment is the plasmid with successful site-directed mutagenesis, and then E.coli BL21(DE3) competent cells are transformed for subsequent expression. The successfully transformed recombinant bacterium E.coli BL21(DE3) is frozen and stored in a glycerol tube for later use.

2.3 purification of mutant enzymes:

induced expression of mutant enzymes:

and taking out the successfully constructed mutant recombinant bacteria from a glycerol cryopreservation tube, streaking the recombinant bacteria on an LB solid culture medium containing ampicillin, and culturing the recombinant bacteria at 37 ℃ for 12 h. Single colonies were picked and inoculated into 20mL of LB liquid medium (Amp)⁺100. mu.g/mL) was cultured at 37 ℃ for 10 h at 200rpm to form a seed solution. The seed liquid was inoculated in LB liquid medium (Amp) at an inoculum size of 2%⁺100 μ g/mL, liquid loading 400mL/2L), culturing at 37 ℃ and 200rpm until OD600nm reaches 0.6-0.8. IPTG was added to the medium at a final concentration of 1mM, and the culture was continued at 28 ℃ and 200 rpm. After 6h, the fermentation broth is centrifuged for 15min at 6000rpm and 4 ℃, and thalli are collected.

Separation and purification of mutant enzyme protein:

the cells were resuspended in cell disruption buffer (Lysis buffer) and disrupted by sonication at 150W for 15min under ice bath conditions. Centrifuging at 8000rpm for 20min at 4 deg.C after crushing, filtering the supernatant with 0.22 μm water system filter membrane, and collecting filtrate;

sample the filtrate to Ni²⁺In the affinity column, the column is previously equilibrated with a loading buffer (Binding buffer). After the filtrate is completely loaded, continuously balancing the column by using a loading buffer solution (Binding buffer) until no protein is detected in the effluent liquid;

washing the column with Washing buffer (Washing buffer) until no protein is detected in the effluent;

eluting the target protein by using an Elution buffer (Elution buffer), and collecting the eluent until the protein cannot be detected in the eluent;

dialyzing the eluent in the dialysate A to remove salt ions and metal ions in the eluent, and dialyzing for 2 times, each time for 2-3 h;

and (4) continuously dialyzing the eluent in the dialysate B to remove the EDTA in the eluent, and dialyzing for 2 times, 2-3 h each time. Collecting the eluent to obtain the purified mutant enzyme solution. For convenience of description, the resulting mutant enzyme was named V481C. Wherein the amino acid sequence of the mutant enzyme V481C is shown as SEQ ID No.1, and the nucleotide sequence of the gene coding the mutant enzyme V481C is shown as SEQ ID No.2

The above processes are all carried out at 4 ℃.

Protein content determination of mutant enzymes:

determining the protein concentration of the purified mutant enzyme solution using a BCA protein quantification kit,

denaturing polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the mutant enzymes:

mu.L of 5 Xloading buffer and 20. mu.L of mutant enzyme solution were mixed and boiled for 10 min. Centrifuging to obtain 10 μ L supernatant, loading to 8% prefabricated gel sample well, and running gel at 150V voltage. After running the glue, dyeing with Coomassie brilliant blue R250 for not less than 3 h. Then decolorized until the background is clear and viewed by a gel imaging system.

3. Determination of enzymatic Properties of mutant enzymes

The enzyme activity determination method comprises the following steps:

pullulan polysaccharide with the final concentration of 0.5% (w/v) is used as a substrate, and the mutant enzyme to be detected catalyzes and reacts for 10min under the optimal condition. After the reaction is finished, the content of reducing sugar released during the enzyme reaction is measured by a 3, 5-dinitrosalicylic acid method (DNS method) to indirectly calculate the enzyme activity. The specific experimental steps are as follows: 20 mu L of 1% (w/v) pullulan polysaccharide dissolved in buffer solution with the optimal pH value is preheated for 5min under the optimal temperature condition; adding 20 μ L of diluted enzyme solution into preheated substrate solution rapidly, and reacting at optimum temperature for 10 min; after 10min, 3, 5-dinitrosalicylic acid reagent (DNS reagent) is rapidly added to stop the reaction, the temperature of the mixed solution is raised to 99.9 ℃, and the color development is carried out for 10 min. After the color development is finished, 100 mu L of deionized water is added and mixed fully, 100 mu L of the mixture is added into a 96-hole enzyme label plate, and the absorbance is measured at the wavelength of 540 nm. Definition of enzyme activity (U): under certain reaction conditions, the pullulanase required for releasing 1 mu mol of reducing sugar from the pullulan in unit time (1min) is obtained.

Wherein, the optimal enzyme reaction conditions are as follows: V481C, 80 ℃ and pH 5.5.

Influence of pH on enzyme Activity:

under the optimal temperature, 1% (w/v) pullulan dissolved in buffer solutions with different pH values is used as a substrate to determine the enzyme activity. The pH range is 3.5-7.5, and two buffer solution systems are adopted: 0.2M acetate buffer solution with the pH value of 3.5-6.0; 0.2M phosphate buffer solution with pH of 6.0-7.5. The enzyme activities measured under different pH conditions are expressed by relative enzyme activities, and the measured highest enzyme activity is 100%.

Wherein, the optimum reaction temperature of the enzyme is as follows: V481C, 80 ℃.

Influence of temperature on enzyme activity:

taking 1% (w/v) pullulan dissolved in buffer solution with the optimal pH as a substrate, and determining the enzyme activity of the mutant enzyme at different temperatures (4-90 ℃). The enzyme activities measured at different temperatures are expressed by relative enzyme activities, and the measured highest enzyme activity is 100%.

Wherein, the optimum reaction pH of the enzyme is as follows: V481C is pH 5.5.

Effect of temperature on enzyme stability:

adding pullulanase solution into the buffer solution with the optimal pH value according to the volume ratio of 1:1, incubating at different temperatures, and sampling at intervals to measure the enzyme activity. The residual enzyme activity was calculated with the initial enzyme activity of the enzyme solution without incubation as 100%.

Determination of enzyme kinetic parameters:

preparing a pullulan series solution with the concentration range of 0.5-1.0 mg/mL and dissolved in a buffer solution with the optimal pH value, and using the solution to determine the enzyme kinetic parameters of pullulanase. Except for different pullulan concentrations, the enzyme activity of the enzyme under the series of substrate concentrations is determined under the standard condition, and K is calculated by a Mie equation (Linweaver-Burk equation)_mAnd V_maxValue, and then K is calculated_catAnd K_cat/K_mThe value is obtained.

4. Results of the experiment

As shown in FIG. 1, PCR was carried out using pET-22b (+) -pulF as a template and the pair of mutant primers shown in Table 1, and the PCR product was observed by electrophoresis on 1% agarose gel, and the band marked 1 in FIG. 1 was the electrophoretic band of mutant enzyme V481C, and it can be seen from FIG. 1 that the mutant primers amplified a band having the same size as expected, and although a plurality of amplified bands appeared, it did not affect the subsequent experiments.

The protein concentration of the purified mutant enzyme was measured by BCA protein quantification kit method, the standard curve was shown in FIG. 2, and the protein concentration of the mutant enzyme was calculated according to the linear equation of the standard curve, as shown in Table 4.

TABLE 4 protein concentration of mutant enzymes

The results of denaturing polyacrylamide gel electrophoresis (SDS-PAGE) of the purified mutant enzyme are shown in FIG. 3. As can be seen from the figure, Ni²⁺The affinity chromatographic column plays a good role in separating and purifying the mutant enzyme, a band marked as 1 in figure 3 is an electrophoresis band of the mutant enzyme V481C, the mutant enzyme is presented as a single band in gel, and the size of the mutant enzyme estimated according to the molecular weight of a protein Marker is consistent with the expected size.

Effect of pH on mutant enzyme Activity

The relative enzyme activities of the mutant enzymes under different pH conditions were determined separately, and the experimental results are listed in Table 5.

TABLE 5 relative enzyme Activity of mutant enzymes at different pH conditions

Note: WT means PulF which is a pullulanase

As can be seen from Table 5, when Pullulan enzyme PulF was mutated to V481C, the optimum pH was changed from 5.0 to 5.5. However, compared with pullulanase PulF, the mutant enzyme has higher enzyme activity in a wider pH range, and particularly, the relative enzyme activity of the mutant enzyme is more than 85% in the pH range of 5.0-5.5. Within the pH range of 6.0-7.0, the relative enzyme activity of the mutant enzyme is obviously higher than that of Pullulan enzyme PulF.

In addition, except for the pH range of 5.0-5.5, the relative enzyme activities of the pullulanase PulF and the mutant enzyme V481C have similar trend, and the enzyme activities of the pullulanase PulF and the mutant enzyme V481C are rapidly reduced when the pH value is far away from the optimal value. And different buffer systems have great influence on the two enzymes, and the relative enzyme activity in the phosphate buffer solution with the pH value of 6.0 is nearly 3 times that in the acetic acid buffer solution with the pH value of 6.0.

Because the pH range of the pullulanase reaction in the sugar industry is 4.5-5.5, the mutant enzyme has the greatest advantages compared with pullulanase PulF by combining the analysis of the industrial application condition: the enzyme activity is higher within the range of pH5.0-5.5, and the requirement on the pH environment of reaction liquid in the saccharification process is not harsh.

Influence of temperature on the enzymatic activity of the mutants:

the relative enzyme activities of the mutant enzymes at different temperatures were determined separately, and the experimental results are listed in table 6.

TABLE 6 relative enzyme Activity of mutant enzymes at different temperatures

Note: WT means PulF which is a pullulanase

As can be seen from Table 6, when Pullulan enzyme PulF is mutated to V481C, the optimum temperature is not changed and is still 80 ℃; the relative enzyme activity of the mutant enzyme is greater than that of Pullulan enzyme PulF at the temperature, except that of V481C at 60 ℃. Under the condition of 60 ℃, the relative enzyme activities of V481C and pullulanase PulF are respectively 18.19% and 18.68%, and the difference is not large. In addition, by comparing the relative enzyme activity change trends of the pullulanase PulF and the mutant enzyme with the change of temperature, it can be seen that the relative enzyme activity change trend of the mutant enzyme V481C is almost consistent with that of the pullulanase PulF.

To further explore how the thermostability of the mutant enzymes varied, the half-lives of the mutant enzymes were determined at 75 ℃ and 80 ℃.

Effect of temperature on mutant enzyme stability:

the residual enzyme activity of the mutant enzyme V481C after incubation at 75 ℃ and 80 ℃ for different periods of time was determined, and the natural logarithm of the residual enzyme activity was taken as the curve of the change of the residual enzyme activity with the incubation time, as shown in FIG. 4 and FIG. 5. And half-lives at two temperatures were calculated for each mutant enzyme, and the results are listed in table 7.

As is apparent from FIGS. 4 and 5, the thermostability of the mutant enzyme was enhanced at 75 ℃ and 80 ℃.

TABLE 7 half-lives of Pullulan enzyme PulF and mutant enzymes at different temperatures

Note: WT means PulF which is a pullulanase

The half-life of V481C is longest under the condition of 75 ℃, and is improved by nearly 13 times compared with the half-life of pullulanase PulF under the same condition. At 80 ℃ the half-lives of all enzymes are very short.

Enzyme kinetic parameters of the mutant enzymes:

the enzyme kinetic parameters of the mutant enzyme V481C and the pullulanase PulF are listed in table 8.

TABLE 8 comparison of enzyme kinetic parameters

Note: WT means PulF which is a pullulanase

As can be seen from Table 8, K of the mutant enzyme V481C compared to PulF, a pullulanase_mThe value is reduced, which indicates that the combination ability of the mutant enzyme V481C and pullulan is enhanced; catalytic constant K of mutant enzyme V481C_cat/K_mAll are increased, which shows that the mutant enzyme V481C has improved catalytic efficiency on pullulan.

By comprehensively comparing the reaction kinetic parameters, heat resistance and specific enzyme activity, the mutant enzyme V481C has the best enzymological properties. Compared with pullulanase PulF, the mutant enzyme V481C has obviously improved thermal stability, obviously improved combination capability and catalytic efficiency on pullulanase and greatly increased specific enzyme activity which is nearly 1.9 times that of the pullulanase PulF.

While embodiments of the invention have been described above, it is not limited to the applications set forth in the description and the embodiments, which are fully applicable in various fields of endeavor to which the invention pertains, and further modifications may readily be made by those skilled in the art, it being understood that the invention is not limited to the details shown and described herein without departing from the general concept defined by the appended claims and their equivalents.

SEQUENCE LISTING

<110> institute for agricultural product processing of Chinese academy of agricultural sciences

Mutant enzyme of <120> type I pullulanase and preparation method and application thereof

<130>1

<160>1

<170>PatentIn version 3.5

<210>1

<211>820

<212>PRT

<213> Artificial sequencing

<400>1

Met Ala Thr Glu Leu Val Ile His Tyr His Arg Trp Asp Gly Asn Tyr

1 5 10 15

Asp Gly Trp Asn Leu Trp Ile Trp Trp Val Glu Pro Ile Ser Lys Asp

20 25 30

Gly Ala Ala Tyr Gln Phe Thr Glu Lys Asp Asp Phe Gly Val Val Ala

35 40 45

Arg Val Lys Phe Asp Glu Thr Leu Thr Lys Val Gly Ile Ile Val Arg

50 55 60

Leu Asn Glu Trp Lys Glu Lys Asp Val Ala Met Asp Arg Phe Ile Ser

65 70 75 80

Ile Lys Asp Gly Lys Ala Glu Val Trp Leu Leu Gln Gly Ile Glu Gln

85 90 95

Ile Tyr Thr Thr Lys Pro Asp Thr Ser Pro Arg Val Leu Phe Ala Gln

100 105 110

Ala Arg Ala Gln Asp Val Ile Glu Ala Tyr Leu Thr Gly Gln Val Asp

115 120 125

Thr Thr Lys Val Ser Ala Lys Val Thr Val Asp Gly Val Glu Arg Lys

130 135 140

Val Ala Lys Val Glu Lys Ala Asn Pro Thr Asp Ile Ser Lys Thr Asn

145 150 155 160

His Val Lys Ile Thr Leu Ala Glu Pro Ile Lys Leu Asp Glu Val Asn

165 170 175

Lys Asp Val Gln Val Glu Ile Glu Gly Tyr Lys Pro Ala Arg Val Ile

180 185 190

Met Met Glu Ile Leu Asp Lys Ile Tyr Tyr Asp Gly Pro Leu Gly Phe

195 200 205

Glu Tyr Ser Pro Thr Lys Thr Thr Ile Arg Val Trp Ser Pro Val Ser

210 215 220

Lys Thr Val Asp Leu Leu Leu Tyr Lys Asn Trp Asp Asp Lys Glu Pro

225 230 235 240

Thr Lys Val Val Pro Met Lys Tyr Ile Gly Asn Gly Ala Trp Glu Ala

245 250 255

Val Leu Asp Gly Asp Trp Glu Gly Trp Phe Tyr Arg Ile Arg Tyr Phe

260 265 270

Ser Tyr Gly Glu Tyr Arg Glu Gly Val Asp Tyr Phe Ser Lys Ala Val

275 280 285

Thr Lys Asn Ser Ala Lys Ser Ala Ile Ile Asp Leu Lys Lys Thr Asn

290 295 300

Pro Ser Asp Trp Asp Lys Asp Val Arg Pro Thr Met Lys Ala Leu Glu

305 310 315 320

Asp Ala Ile Ile Tyr Glu Ile His Ile Ala Asp Met Thr Gly Leu Asp

325 330 335

Asn Ser Asn Val Lys Asn Lys Ala Thr Tyr Leu Gly Leu Thr Glu Lys

340 345 350

Gly Thr Arg Gly Pro Asn Gly Val Thr Thr Gly Leu Asp His Leu Val

355 360 365

Glu Leu Gly Val Thr His Val His Ile Leu Pro Met Phe Asp Phe Tyr

370 375 380

Thr Gly Asp Glu Ser Glu Arg Asp Phe Glu Lys Ser Tyr Asn Trp Gly

385 390 395 400

Tyr Asp Pro Tyr Leu Phe Thr Val Pro Glu Gly Arg Tyr Ser Thr Asn

405 410 415

Pro Ile Asp Pro His Val Arg Ile Asn Glu Val Lys Gln Met Val Lys

420 425 430

Ala Leu His Glu Asn Gly Ile Arg Val Ile Leu Asp Met Val Phe Pro

435 440 445

His Thr Phe Gly Ile Gly Val Leu Ser Pro Phe Asp Thr Ala Val Pro

450 455 460

Tyr Tyr Phe Tyr Arg Ile Asp Lys Thr Gly Ala Tyr Leu Asn Glu Ser

465 470 475 480

Gly Cys Gly Asn Val Met Ala Thr Glu Arg Pro Met Leu Arg Lys Tyr

485 490 495

Val Ile Asp Thr Leu Lys Trp Trp Val Leu Glu Tyr His Val Asp Gly

500 505 510

Phe Arg Phe Asp Gln Met Gly Leu Met Asp Lys Lys Thr Met Leu Asp

515 520 525

Leu Glu Lys Glu Leu His Ala Ile Asp Pro Thr Ile Leu Leu Tyr Gly

530 535 540

Glu Pro Trp Gly Gly Trp Gly Ala Pro Ile Arg Phe Gly Lys Ser Asp

545 550 555 560

Val Gly Gly Thr His Ile Ala Ala Phe Asn Asp Glu Phe Arg Asp Ala

565 570 575

Met Arg Gly Ser Val Phe Asn Ala Thr Val Lys Gly Phe Leu Met Gly

580 585 590

Ala Leu Ala Lys Glu Thr Ala Ile Lys Arg Gly Val Val Gly Ser Ile

595 600 605

Glu Tyr Asp Asp Val Ile Arg Ser Phe Ala Lys Asp Pro Glu Glu Thr

610 615 620

Ile Asn Tyr Val Ala Cys His Asp Asn His Thr Leu Trp Asp Lys Asn

625 630 635 640

Tyr Leu Ala Ala Gln Ala Asp Thr Asn Ile Lys Trp Thr Glu Glu Met

645 650 655

Leu Lys Asn Ala Gln Lys Leu Ala Gly Ala Ile Leu Leu Thr Ser Gln

660 665 670

Gly Ile Pro Phe Leu His Ala Gly Gln Asp Phe Ala Arg Thr Lys Lys

675 680 685

Gly Asp Glu Asn Ser Tyr Asn Ser Pro Ile Ser Ile Asn Gly Leu Asp

690 695 700

Tyr Ala Arg Lys Ala Glu Phe Ile Asp Val Phe Asn Tyr Tyr Lys Gly

705 710 715 720

Leu Ile Glu Ile Arg Lys Ala His Pro Ala Phe Arg Gln Arg Thr Ala

725 730 735

Asp Asp Ile Lys Lys Lys Ile Thr Phe Leu Pro Thr Thr Arg Lys Met

740 745 750

Val Ala Phe Thr Ile Lys Asp Glu Asn Asp Ser Trp Lys Glu Ile Leu

755 760 765

Val Ile Tyr Asn Gly Asp Thr Lys Asp Gln Asp Phe Thr Leu Pro Glu

770 775 780

Gly Thr Trp Asn Val Val Val Asp Gln Gln Asn Ala Gly Thr Lys Val

785 790 795 800

Leu Tyr Gln Val Ser Gly Lys Ile Thr Val Lys Ser Ile Ser Ala Met

805 810 815

Val Met Tyr Lys

820

<210>2

<211>2460

<212>DNA

<213> Artificial sequencing

<400>2

atggcgaccg aactggtgat tcattatcat cgctgggatg gcaattatga tggctggaat 60

ctgtggattt ggtgggtgga accgattagc aaagatggcg cggcgtatca gtttaccgag 120

aaggatgatt ttggcgtggt ggcgcgcgtg aaatttgatg aaaccctgac caaagtgggc 180

attattgtgc gcctgaacga atggaaagaa aaggatgtgg cgatggatcg ctttattagc 240

atcaaagacg gcaaagcgga agtttggctg ctgcagggca ttgaacagat ttataccacc 300

aaaccggata ccagtccgcg tgtgctgttt gcacaagcgc gtgcgcaaga tgtgattgaa 360

gcgtatctga ccggccaagt ggataccacc aaagtgagcg cgaaagtgac cgtggatggc 420

gtggaacgca aagtggcgaa agtggaaaaa gcgaatccga ccgatattag caaaaccaac 480

catgtgaaaa ttaccctggc ggaaccgatt aaactggatg aggtgaacaa agatgtgcag 540

gtggaaattg aaggctataa accggcgcgc gtgattatga tggaaatcct ggacaagatt 600

tactatgatg gcccgctggg ctttgaatat agcccgacca aaaccaccat tcgcgtgtgg 660

agcccggtta gcaaaaccgt ggatctgctg ctgtataaga attgggatga taaggaaccg 720

accaaagtgg tgccgatgaa gtatattggc aatggcgcgt gggaagcagt tctggatggc 780

gattgggaag gctggtttta tcgcattcgc tactttagct atggcgaata tcgcgaaggc 840

gtggattatt ttagcaaagc ggtgaccaaa aatagcgcga aaagcgcgat tattgacctg 900

aaaaagacca atccgagcga ttgggataaa gatgtgcgcc cgaccatgaa agcgctggaa 960

gatgcgatta tctacgaaat ccacatcgcg gatatgaccg gcctggataa tagcaatgtg 1020

aaaaacaagg cgacctatct gggcctgacc gaaaaaggta cccgcggtcc gaatggtgtt 1080

accaccggcc tggatcatct ggttgaactg ggcgtgaccc atgtgcatat tctgccgatg 1140

ttcgattttt ataccggcga tgaaagcgaa cgcgattttg agaagagcta caattggggc 1200

tatgacccgt atctgtttac cgttccggaa ggccgctata gcaccaatcc gattgatccg 1260

catgtgcgca ttaatgaagt gaagcagatg gtgaaagcgc tgcatgaaaa tggcattcgc 1320

gtgattctgg atatggtgtt tccgcatacc tttggcattg gcgtgctgag cccgtttgat 1380

accgcggtgc cgtattattt ttaccgcatc gataaaaccg gcgcgtatct gaatgaaagc 1440

ggctgcggca atgttatggc gaccgaacgt ccgatgctgc gcaaatatgt gattgatacc 1500

ctgaaatggt gggtgctgga atatcatgtg gatggctttc gctttgatca gatgggcctg 1560

atggataaaa aaaccatgct ggacctggaa aaagaactgc atgcgattga tccgaccatt 1620

ctgctgtatg gcgaaccttg gggtggttgg ggcgcgccaa ttcgttttgg caaaagcgat 1680

gtgggcggca cccatattgc ggcgttcaat gatgaatttc gcgatgcgat gcgcggcagc 1740

gtttttaatg cgaccgtgaa aggctttctg atgggcgcgc tggcaaaaga aaccgcgatt 1800

aaacgcggtg tggtgggcag cattgaatat gatgatgtga ttcgcagctt tgcgaaagat 1860

ccggaagaga ctattaatta tgtggcgtgc catgataatc ataccctgtg ggacaaaaat 1920

tatctggcgg cgcaggcgga taccaatatt aaatggaccg aggagatgct gaaaaatgcg 1980

cagaaactgg cgggtgcgat tttgttaacc agccagggca ttccgttttt gcatgcgggc 2040

caggattttg cgcgcaccaa aaaaggcgat gagaacagct ataatagccc gattagcatt 2100

aatggcctgg attatgcgcg caaagcggaa tttatcgacg tgttcaacta ttataagggc 2160

ctgatcgaaa ttcgcaaagc gcatccggcg tttcgtcaac gtaccgcgga tgatattaag 2220

aagaagatca ccttcctgcc gaccacccgt aaaatggtgg cgttcaccat taaagatgag 2280

aacgacagct ggaaagagat cctggtgatt tataacggcg ataccaagga tcaggatttt 2340

accctgccgg aaggcacctg gaatgtggtg gtggatcagc aaaatgcggg caccaaagtg 2400

ctgtatcagg tgagcggcaa aattaccgtg aaaagcatta gcgcgatggt gatgtataaa 2460

<210>3

<211>2460

<212>DNA

<213> Artificial sequencing

<400>3

atggcaacag aactggtcat ccattatcat cgctgggatg gtaattacga tggttggaac 60

ctatggattt ggtgggtaga gcctatctct aaagacggcg cggcatatca atttactgaa 120

aaagatgatt tcggtgtcgt agcaagagtt aaattcgacg aaactttgac aaaagttgga 180

atcattgtta gacttaatga atggaaggaa aaagatgttg cgatggatag atttatatcg 240

ataaaagatg gaaaagcgga ggtgtggttg ttgcaaggta tcgaacagat atacacaaca 300

aagccagata caagcccaag agtacttttc gctcaagcta gagcacagga tgttatcgaa 360

gcttacttaa caggacaagt tgatactaca aaagttagcg caaaagttac agtcgacggt 420

gttgaaagaa aagttgcaaa ggttgaaaaa gcaaatccaa cggatatatc aaaaacaaac 480

cacgtaaaaa tcactcttgc tgaaccaata aagcttgacg aagtcaacaa agatgtccaa 540

gttgaaatcg aaggctacaa accagcaaga gtaatcatga tggaaatctt agacaaaata 600

tactacgatg gtccacttgg ttttgagtat tctccaacta aaactacaat cagagtatgg 660

tctcctgtct caaagacagt agatttacta ctttacaaaa attgggatga caaagaacct 720

acaaaagtcg tacctatgaa atacatcgga aatggcgcgt gggaagccgt tctcgatggt 780

gattgggaag gttggttcta tagaataaga tatttcagtt acggagagta tagggaagga 840

gtagattact tctccaaagc agtcacgaaa aactctgcaa agagcgcaat catagatctg 900

aaaaagacaa acccatctga ctgggataaa gatgtaagac caaccatgaa agctcttgaa 960

gacgctataa tctacgaaat tcatattgca gatatgacag gacttgacaa ctccaatgtc 1020

aaaaataaag ctacatacct tggccttacc gaaaagggta caagaggacc aaatggcgtt 1080

acaactggtt tagaccacct tgttgaatta ggcgttacgc atgtgcatat ccttccaatg 1140

tttgatttct acacaggtga tgaatcagaa agagattttg aaaagagcta caattgggga 1200

tacgatcctt acttattcac agtaccagaa ggtagatact caacaaaccc aattgaccca 1260

cacgttagga taaacgaagt caagcaaatg gtcaaagcat tacacgaaaa tggaataaga 1320

gtaatactcg atatggtatt cccacataca tttggtatcg gcgttctttc accatttgac 1380

acagcggtcc cttactattt ctacagaatc gataagacag gcgcttattt gaacgaaagc 1440

ggcgttggaa atgtcatggc aacagaaaga ccaatgctta gaaaatacgt tattgataca 1500

ttgaagtggt gggtattaga ataccatgtt gatggattca gattcgacca aatgggtctc 1560

atggataaga aaacaatgct agatttggaa aaagaattgc acgctataga tccaacgata 1620

ttgctctacg gtgaaccatg gggtggttgg ggtgctccaa taagattcgg aaaatcagat 1680

gttggtggaa cacacatcgc tgcatttaac gatgaattca gagacgctat gaggggttcc 1740

gtgttcaacg caacagtcaa aggtttctta atgggcgcgc ttgcaaaaga aacagcgatc 1800

aaacgtggcg ttgtcggaag cattgagtac gacgatgtaa taagaagctt tgcaaaagac 1860

ccagaagaaa caattaatta cgttgcatgc catgataacc acacactttg ggataaaaat 1920

tacttagcag ctcaagcaga tacaaacata aaatggacag aagaaatgct caaaaacgct 1980

caaaaattag ctggagccat actattaact tctcaaggta taccattctt acacgctggc 2040

caagattttg caagaaccaa aaaaggtgat gaaaattcat ataactcacc aatttcaata 2100

aacggtcttg attatgcaag aaaagcagaa ttcatagatg tcttcaatta ctacaaagga 2160

ctcatagaaa tcagaaaagc tcatccagca ttcagacaaa gaacagcaga tgatataaaa 2220

aagaaaatca catttttgcc aaccacaagg aaaatggttg cctttacaat caaagatgaa 2280

aacgatagct ggaaagagat actcgtcata tacaacggtg atacaaaaga ccaagatttt 2340

acattaccag aaggcacatg gaacgtagta gtcgaccaac aaaacgcagg tacaaaagta 2400

ttgtatcaag ttagtggaaa aataactgta aaatccatat ccgcgatggt aatgtacaag 2460

<210>4

<211>2460

<212>DNA

<213> Artificial sequencing

<400>4