CN114752583A - Heat-resistant beta-1, 3-1, 4-glucanase mutant and preparation method and application thereof - Google Patents
Heat-resistant beta-1, 3-1, 4-glucanase mutant and preparation method and application thereof Download PDFInfo
- Publication number
- CN114752583A CN114752583A CN202210333541.0A CN202210333541A CN114752583A CN 114752583 A CN114752583 A CN 114752583A CN 202210333541 A CN202210333541 A CN 202210333541A CN 114752583 A CN114752583 A CN 114752583A
- Authority
- CN
- China
- Prior art keywords
- mutant
- glucanase
- enzyme
- beta
- nucleotide sequence
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
- C12N9/2405—Glucanases
- C12N9/2434—Glucanases acting on beta-1,4-glucosidic bonds
- C12N9/244—Endo-1,3(4)-beta-glucanase (3.2.1.6)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/70—Vectors or expression systems specially adapted for E. coli
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
- C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
- C12Y302/01006—Endo-1,3(4)-beta-glucanase (3.2.1.6)
Abstract
The invention relates to a heat-resistant beta-1, 3-1, 4-glucanase mutant and a preparation method and application thereof, belonging to the technical field of enzyme engineering. The amino acid sequence of the heat-resistant beta-1, 3-1, 4-glucanase mutant is shown as SEQ ID NO. 3. The beta-1, 3-1, 4-glucanase mutant is obtained by mutating threonine (Thr) at the 9 th position and glycine (Gly) at the 43 th position of the amino acid sequence of the mutant into cysteine (Cys) on the basis of original enzyme. Compared with the original enzyme, the optimum reaction temperature of the enzyme is obviously improved from 60 ℃ to 75 ℃, the gold metal ion tolerance of the mutant enzyme EccslG69 is increased to a certain extent, and the application range and the application potential of the heat-resistant beta-1, 3-1, 4-glucanase mutant are widened.
Description
Technical Field
The invention belongs to the technical field of enzyme engineering, and particularly relates to a heat-resistant beta-1, 3-1, 4-glucanase mutant and a preparation method and application thereof.
Background
The beta-1, 3-1, 4-glucanase (PlicA) is obtained from a strain of isolated soil bacteria, is preliminarily identified as bacillus pseudochinensis (Paenibacillus), has alkali-resistant and salt-resistant characteristics, and can hydrolyze barley beta-glucan and laminarin. The characteristics of alkali resistance and salt resistance enable the enzyme to become a good candidate for further research and industrial application, can be applied to the fields of industrial papermaking, wine brewing and the like, and has wide application prospect. However, the glucanase can be rapidly inactivated at high temperature, and the loss is possibly large in practical application, so that the improvement of the heat stability of the glucanase has very important significance for widening the practical application of the glucanase.
The disulfide bond is related to the biological activity of a higher-order structure of the protein, is the only covalent bond in the protein, needs energy of 209.3-418.6kJ/mo1 to break the bond, and has a key effect on maintaining the structure and the function of the protein. The formed disulfide bonds become hydrophobic cores of folded proteins, and local hydrophobic residues are condensed at the periphery of the disulfide bonds and are linked together through hydrophobic effect. The construction of disulfide bonds in proteins is one of the main strategies to improve their stability and has been successfully applied in various enzymatic studies. Chinese patent publication No. CN107177581 (application No. 201710456875.6) discloses a modified nitrile hydratase, and a method for modifying nitrile hydratase, wherein Pro at position 133 of the α subunit and Asp at position 215 of the β subunit in the amino acid sequence of nitrile hydratase are each replaced by Cys, and a disulfide bond is formed between the two subunits. The stress resistance, heat resistance and product tolerance of the modified nitrile hydratase are all obviously improved, and the activity of the nitrile hydratase is not reduced. Chinese patent document CN104531729A (application No. 201410758084.5) discloses a mutant feruloyl esterase a, which is obtained by designing mutations a126C and N152C according to the spatial structure of ferulic acid esterase a from a. usamii E001, and obtaining the mutant feruloyl esterase a: AuFaeAA126C-N152C, C126 and C152 may form a disulfide bond. The experimental result shows that the thermal stability of the enzyme is obviously improved after mutation. Chinese patent document CN103642776A (application No. 201310666411.X) discloses a mutant xylanase, wherein mutations S108C and N152C are designed according to the spatial structure derived from a.oryzae cic 40186 xylanase Aoxyn11A, and the mutant xylanase is obtained: the mature peptides 108C and 152C of Aoxyn11AELJ can form a disulfide bond across secondary structures. The experimental result shows that the thermal stability of the enzyme is obviously improved after mutation.
The correct formation of disulfide bonds is one of the rate-limiting steps in protein folding, and it is essential to control the position of the introduced disulfide bonds. Therefore, when a disulfide bond is introduced, the thermal stability of the enzyme molecule can be maximized by adding it properly. The invention aims to overcome the defect that the prior beta-1, 3-1, 4-glucanase (PlicA) loses activity under the condition of high temperature, and the addition of disulfide bonds is designed in a targeted manner, so that the heat resistance of the enzyme is improved.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a heat-resistant beta-1, 3-1, 4-glucanase mutant and a preparation method and application thereof.
The technical scheme of the invention is as follows:
a heat-resistant beta-1, 3-1, 4-glucanase mutant has an amino acid sequence shown in SEQ ID NO. 3.
The primase of the beta-1, 3-1, 4-glucanase mutant is beta-1, 3-1, 4-glucanase (PlicA), and the amino acid sequence of the mutant is GenBank ACCESSION NO: AGK07610.1(SEQ ID NO:1), the nucleotide sequence of which GenBank ACCESSION NO: KC513762.1(SEQ ID NO: 2).
The beta-1, 3-1, 4-glucanase mutant of the present invention is obtained by mutating threonine (Thr) at the 9 th position and glycine (Gly) at the 43 th position of the amino acid sequence to cysteine (Cys) on the basis of the original enzyme.
The nucleotide sequence of the beta-1, 3-1, 4-glucanase mutant is shown as SEQ ID NO. 4.
A recombinant plasmid containing the nucleotide sequence of the beta-1, 3-1, 4-glucanase mutant.
Preferably, the vector of the recombinant plasmid is pET28a (+).
A gene engineering bacterium containing the nucleotide sequence of the beta-1, 3-1, 4-glucanase mutant or the recombinant plasmid.
According to the invention, the expression host of the genetic engineering bacteria is preferably Escherichia coli.
More preferably, the expression host bacterium is escherichia coli BL 21.
The preparation method of the heat-resistant beta-1, 3-1, 4-glucanase mutant comprises the following steps:
(1) artificially synthesizing plasmid pET28a (+) EccslG containing nucleotide sequence of primary enzyme beta-1, 3-1, 4-glucanase (PlicA), designing primers G35-69U and G35-69D and G35-69WU and G35-69WD, taking plasmid pET28a (+) EccslG as a template and G35-69U and G35-69D as primers, and carrying out PCR synthesis on a target gene EccslG69TThe target gene EccslG69 is synthesized by inverse PCR with plasmid pET28a (+) EccslG as a template and G35-69WU and G35-69WD as primersG；
(2) Synthesizing the target gene EccslG69 synthesized in the step (1)GAnd EccslG69TAnd (3) connecting and cyclizing to construct a mutant plasmid, transferring the mutant plasmid into escherichia coli BL21 to construct recombinant escherichia coli, and performing induced expression on the heat-resistant beta-1, 3-1, 4-glucanase mutant.
According to a preferred embodiment of the present invention, the nucleotide sequence of G35-69U in step (1) is: 5'-CCGTTAtgcTACCACAACTCCTCCACCT-3' (SEQ ID NO:9),
the nucleotide sequence of G35-69D is: 5'-AGCTTgcaATCGTTGGTAAAAGTTGCATT-3' (SEQ ID NO:10),
the nucleotide sequence of the G35-69WU is as follows: 5'-TTTTACCAACGATtgcAAGCTGCGT-3' (SEQ ID NO:7),
the nucleotide sequence of the G35-69WD is as follows: 5'-GAGTTGTGGTAgcaTAACGGTTCCCA-3' (SEQ ID NO: 8).
The heat-resistant beta-1, 3-1, 4-glucanase mutant is applied to the fields of food, feed and paper making.
The heat-resistant beta-1, 3-1, 4-glucanase mutant can act on 1,3 and 1,4 glycosidic bonds of beta-glucan under the high-temperature condition to generate oligosaccharide and glucose, and can be applied to the fields of food, feed, paper making and the like.
The experimental procedures not described in detail in the present invention were carried out according to the methods described in the prior art.
The invention has the beneficial effects that:
the invention provides a heat-resistant beta-1, 3-1, 4-glucanase mutant, which is obtained by mutating threonine (Thr) at the 9 th site and glycine (Gly) at the 43 th site of an amino acid sequence of an original enzyme (SEQ ID NO:1) into cysteine (Cys), obviously improves the optimal reaction temperature of the enzyme compared with the original enzyme, increases the temperature from 60 ℃ to 75 ℃, also increases the metal ion tolerance of a mutant enzyme EccslG69 to a certain extent, and widens the application range and the application potential of the heat-resistant beta-1, 3-1, 4-glucanase mutant.
Drawings
FIG. 1 is a graph showing the activity change of wild enzyme and mutant enzyme at different temperatures.
FIG. 2 is a bar graph of the relative activities of wild-type enzyme and mutant enzyme in the presence of different metal ions.
Detailed Description
The method of operation of the present invention is further illustrated below with reference to specific examples. These examples are only for illustrating the present invention in detail and are not intended to limit the scope of the present invention. The experimental procedures referred to in the examples are those conventional in the art unless otherwise specified.
Material source:
materials and reagents: plasmid pET28a (+) EccslG, which contains the nucleotide sequence of the original enzyme β -1,3-1, 4-glucanase (PlicA), was synthesized from the entire gene of King-Shirui, Japan. The plasmid extraction kit is purchased from Novovovoxel trade company, and BL21 escherichia coli competent cells are purchased from Novoxel biology company; the primer is synthesized by Shanghai Bioengineering company; dpn I restriction enzymes were purchased from Shanghai bioengineering; the PCR product purification and recovery kit is purchased from Tiangen biology company; the LB culture medium comprises 1% of peptone, 0.5% of yeast extract powder, 1% of sodium chloride and pH 7.0-7.4; the nickel column purification kit is purchased from Qihai biology company; the other reagents are purchased at home and abroad and are of analytical pure grade.
Example 1
Taking beta-1, 3-1, 4-glucanase (PlicA) as a template, and the amino acid sequence of the protein GenBank ACCESSION NO: AGK07610.1(SEQ ID NO:1), nucleotide sequence GenBank ACCESSION NO: KC513762.1(SEQ ID NO:2), modeling the secondary structure of beta-1, 3-1, 4-glucanase (PlicA) By utilizing Swiss Model software, predicting the position of Disulfide bond addition By utilizing Disulfide By Design, avoiding substrate binding part according to specific sites in the tertiary structure, and finally selecting two different combinations to construct Disulfide bond in Thr9And Gly43And Ser76And Asp205Disulfide bonds are constructed between them.
Example 2 preparation of beta-1, 3-1, 4-glucanase mutants
At Thr9And Gly43Constructing disulfide bond between the two, specifically the encoding Thr9Codon ACC of (1) and coding for Gly43The codon GGC is mutated into a codon TGC for coding Cys, and the mutation is carried out to obtain a mutant enzyme EccslG69, wherein the amino acid sequence of the mutant enzyme EccslG69 is shown as SEQ ID NO. 3, and the nucleotide sequence is shown as SEQ ID NO. 4;
at Ser76And Asp205Constructing disulfide bond between the two, specifically encoding Ser76Codon AGC of (1) and coding for Asp205The codon GAT of (A) is mutated into a codon TGC for coding Cys, and the mutation is carried out to obtain a mutant enzyme EccslG102, wherein the amino acid sequence of the mutant enzyme EccslG102 is shown as SEQ ID NO. 5, and the nucleotide sequence is shown as SEQ ID NO. 6;
The specific preparation method of the mutant enzyme comprises the following steps:
(1) primers were designed from an artificially synthesized plasmid pET28a (+) EccslG containing the nucleotide sequence of the original enzyme β -1,3-1, 4-glucanase (PlicA), and the nucleotide sequence of β -1,3-1, 4-glucanase in the plasmid pET28a (+) EccslG was codon-optimized and suitable for expression in E.coli, wherein the primers for the mutant enzyme EccslG69 were G35-69U and G35-69D and G35-69WU and G35-69WD, the primers for the mutant enzyme EccslG102 were G102-231U and G102-231D and G102-231WD, and the nucleotide sequence information of the primers are as follows:
TABLE 1 nucleotide sequence information of primers
Note: the underlined bold italics in the table indicate the introduction of the cysteine mutation.
PCR amplification is carried out by taking plasmid pET28a (+) EccslG as a template and G35-69U and G35-69D as primers to obtain target gene EccslG69T；
The plasmid pET28a (+) EccslG is taken as a template, G35-69WU and G35-69WD are taken as primers, reverse PCR amplification is carried out, and the target gene EccslG69 is obtainedG；
Using plasmid pET28a (+) EccslG as template and G102-231U and G102-231D as primer to make PCR amplification to obtain target gene EccslG102S；
Using plasmid pET28a (+) EccslG as template and G102-231WU and G102-231WD as primer to make inverse PCR amplification so as to obtain the target gene EccslG102 D；
The reaction systems for the PCR amplification and the reverse PCR amplification are shown in Table 2:
TABLE 2 reaction systems for PCR amplification and reverse PCR amplification
| Composition (I) | Volume of |
| Form panel | 2μL |
| Upstream primer | 2μL |
| Downstream primer | 2μL |
| Phanta enzyme | 19μL |
| Double distilled water | 25μL |
| General System | 50μL |
The reaction conditions of the PCR amplification and the reverse PCR amplification are as follows: 3min at 95 ℃; at 95 ℃ for 15s, at 70 ℃ for 15s, at 72 ℃ for 3min, for 30 cycles; keeping the temperature at 72 ℃ for 5min and keeping the temperature at 4 ℃.
(2) The product EccslG69 obtained by the reverse PCR amplification in the step (1)GAnd EccslG102DRespectively digesting the templates by Dpn I enzyme, recovering target bands, and respectively reacting with a corresponding PCR amplification product EccslG69 by using a C112 kitTAnd EccslG102SPerforming connection and cyclization to construct a mutant plasmid, then transferring the mutant plasmid into BL21 escherichia coli competent cells by using a chemo-transfer method, coating the competent cells on an LB (LB) plate (kanamycin concentration is 30 mu g/mL) containing kanamycin resistance, performing overnight culture at 37 ℃, and obtaining a positive transformant, namely recombinant escherichia coli, after the sequencing is correct;
(3) and (3) inoculating the recombinant escherichia coli constructed in the step (2) into 50mL LB liquid culture medium containing kanamycin (the concentration of kanamycin is 30 mug/mL), culturing at 37 ℃ and 250r/min for 4 hours until the OD value is 0.8-1.0, adding an inducer IPTG25 muL, carrying out induced expression for 8 hours, centrifuging, retaining precipitates, and purifying target proteins by adopting a nickel column purification kit to obtain the mutant enzyme EccslG69 and the mutant enzyme EccslG 102.
Example 3 analysis of the Properties of the mutant enzymes
(1) Enzyme activity assay
The enzyme activity determination method is a DNS method, namely a 3, 5-dinitrosalicylic acid method. Adding 2.0mL of diluted enzyme solution into 2.0mL of dextran solution (pH6.0), reacting at 37 deg.C for 30min, adding 5.0mL of LDNS reagent, developing in boiling water bath for 5min, and measuring OD with spectrophotometer540。
Definition of enzyme activity: 1 enzyme activity unit (U) is defined as the amount of enzyme required to release 1. mu. mol reducing sugar per minute.
The protein concentration was determined by the Bradford method using bovine serum albumin as a standard.
(2) Determination of optimum temperature
The enzyme activities of the original enzyme, the mutant enzyme EccslG102 and EccslG69 are measured at different temperatures (55-90 ℃). The optimum temperature is defined as the temperature corresponding to the highest enzyme activity (based on 100% relative activity). As shown in FIG. 1, the optimum temperature of the original enzyme EccslG was 60 ℃ and the optimum temperature of the mutant enzyme EccslG102 was 50 ℃ which was somewhat lower than that of the original enzyme, and the optimum temperature of the mutant enzyme EccslG69 was 75 ℃ which was significantly higher than that of the original enzyme.
(3) Enzyme stability assay
Keeping the temperature of the enzyme solution at 55 ℃, 65 ℃ and 75 ℃ for 2h, and measuring the residual enzyme activity by taking the initial enzyme activity as 100%. The results show that the original enzyme EccslG, the mutant enzymes EccslG102 and EccslG69 have stable enzyme activity at 55 ℃, 65 ℃ and 75 ℃, and can keep more than 90% of activity in 1 hour.
(4) Determination of optimum pH
The optimum pH values of the original enzyme, the mutant enzyme EccslG102 and EccslG69 were measured by formulating 0.5% (w/v, g/mL) dextran solutions with 50mmol/L acetic acid-sodium acetate buffers at different pH values (4.0-11.0). The results show that the three enzymes have the highest activity at pH9-10, and the pH characteristics before and after mutation of the original enzyme are consistent.
(5) Determination of Metal ion tolerance
Zn with 0.1M concentration is prepared2+、Ni+、Mn2+、Co2+、Fe3+、Fe2+、Na+、Ca2+、K+、Mg2+The metal ion solution of (1) is added into a reaction system by 40 mu L, and the enzyme activities of the original enzyme, the mutant enzyme EccslG69 and EccslG102 are respectively measured under different metal ion conditions, wherein the reaction system of the original enzyme without any metal ions is taken as a blank control group, the relative activity of the original enzyme of the blank control group is calculated by 100%, and the relative activities of the enzymes of other experimental groups are calculated by taking the blank control as a reference, and the result is shown in figure 2.
The results show that the mutant enzyme EccslG69 is specific to Ni compared to the original enzyme+、Mn2+、Fe3+、Fe2+、Na+、Ca2+、K+、Mg2+With respect to Mn only, while the mutant enzyme had improved tolerance to Mn to various degrees2+、Fe3+、Na+、Ca2+、K+The tolerance of (2) is improved to various degrees. Thus, the disulfide bonds are increased, and the mutant enzyme EccslG69 metal ion tolerance is also increased to some extent.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention and are equivalent to the replacement of the above embodiments are included in the scope of the present invention.
SEQUENCE LISTING
<110> university of Qilu industry
<120> heat-resistant beta-1, 3-1, 4-glucanase mutant and preparation method and application thereof
<160> 14
<170> PatentIn version 3.5
<210> 1
<211> 238
<212> PRT
<213> Paenibacillus sp.
<400> 1
Met Lys Gln Lys Lys Ile Val Thr Ile Gly Leu Val Leu Val Leu Ser
1 5 10 15
Thr Met Leu Trp Ala Ser Ser Ala Leu Gly Ala Thr Val Phe Trp Glu
20 25 30
Pro Leu Thr Tyr His Asn Ser Ser Thr Trp Gln Lys Ala Asp Gly Tyr
35 40 45
Ser Asn Gly Gly Met Phe Asn Cys Thr Trp Arg Ala Asn Asn Ala Thr
50 55 60
Phe Thr Asn Asp Gly Lys Leu Arg Leu Ala Leu Thr Ser Ser Ser Tyr
65 70 75 80
Asn Lys Phe Asp Gly Ala Glu Tyr Arg Thr Thr Phe Lys Thr Gly Tyr
85 90 95
Gly Lys Cys Glu Val Ser Met Lys Pro Ala Lys Asn Pro Gly Ile Val
100 105 110
Ser Ser Phe Phe Ile Tyr Thr Gly Pro Ser Asp Gly Thr Pro Trp Asp
115 120 125
Glu Ile Asp Ile Gly Phe Leu Gly Lys Asp Thr Thr Lys Val Gln Phe
130 135 140
Asn Tyr Phe Thr Asn Gly Val Gly Gly His Glu Lys Val Ile Asp Leu
145 150 155 160
Gly Phe Asp Ala Ser Gln Gly Tyr His Thr Tyr Ala Phe Asp Trp Gln
165 170 175
Pro Gly Ser Ile Thr Trp Tyr Val Asp Gly Val Gln Lys His Lys Ala
180 185 190
Thr Thr Asn Ile Pro Thr His Pro Gly Lys Ile Met Met Asn Leu Trp
195 200 205
Asn Gly Ile Gly Val Asp Ser Trp Leu Gly Ala Tyr Asn Gly Ala Asn
210 215 220
Pro Leu Tyr Ala Tyr Tyr Asp Trp Val Lys Tyr Thr Ser Asn
225 230 235
<210> 2
<211> 717
<212> DNA
<213> Paenibacillus sp.
<400> 2
atgaagcaaa aaaagatcgt gaccatcggc ttagtgttgg tattgtcgac gatgttgtgg 60
gcatcgagcg cgctgggggc tacggtgttc tgggagccgc tgacgtatca taacagcagc 120
acctggcaaa aggcggacgg gtattccaat ggcgggatgt ttaattgcac ctggcgggcc 180
aacaacgcaa ccttcaccaa cgacggcaag ctgcgtctcg ccttgaccag ctcctcgtac 240
aacaagttcg acggcgccga gtatcggacc acattcaaga cgggctacgg caagtgtgag 300
gttagcatga agccggccaa aaacccgggc attgtctcct cgttcttcat ctataccggg 360
ccgtcggacg ggacgccttg ggatgagatc gacattggat ttttgggcaa ggacacgacc 420
aaggtgcaat tcaattattt tacgaacggc gtcggcggtc atgagaaagt gatcgacctg 480
ggcttcgatg cctcccaagg atatcacacg tatgccttcg attggcagcc gggctcgatc 540
acctggtacg tggatggcgt gcagaagcac aaggccacga ccaacattcc gacccatccg 600
ggcaagatca tgatgaatct gtggaatggc atcggcgtgg acagctggct cggagcttat 660
aatggtgcca acccgctcta tgcctattac gattgggtca agtatacgag caactaa 717
<210> 3
<211> 213
<212> PRT
<213> Artificial sequence
<400> 3
Ala Thr Val Phe Trp Glu Pro Leu Cys Tyr His Asn Ser Ser Thr Trp
1 5 10 15
Gln Lys Ala Asp Gly Tyr Ser Asn Gly Gly Met Phe Asn Cys Thr Trp
20 25 30
Arg Ala Asn Asn Ala Thr Phe Thr Asn Asp Cys Lys Leu Arg Leu Ala
35 40 45
Leu Thr Ser Ser Ser Tyr Asn Lys Phe Asp Gly Ala Glu Tyr Arg Thr
50 55 60
Thr Phe Lys Thr Gly Tyr Gly Lys Cys Glu Val Ser Met Lys Pro Ala
65 70 75 80
Lys Asn Pro Gly Ile Val Ser Ser Phe Phe Ile Tyr Thr Gly Pro Ser
85 90 95
Asp Gly Thr Pro Trp Asp Glu Ile Asp Ile Gly Phe Leu Gly Lys Asp
100 105 110
Thr Thr Lys Val Gln Phe Asn Tyr Phe Thr Asn Gly Val Gly Gly His
115 120 125
Glu Lys Val Ile Asp Leu Gly Phe Asp Ala Ser Gln Gly Tyr His Thr
130 135 140
Tyr Ala Phe Asp Trp Gln Pro Gly Ser Ile Thr Trp Tyr Val Asp Gly
145 150 155 160
Val Gln Lys His Lys Ala Thr Thr Asn Ile Pro Thr His Pro Gly Lys
165 170 175
Ile Met Met Asn Leu Trp Asn Gly Ile Gly Val Asp Ser Trp Leu Gly
180 185 190
Ala Tyr Asn Gly Ala Asn Pro Leu Tyr Ala Tyr Tyr Asp Trp Val Lys
195 200 205
Tyr Thr Ser Asn Leu
210
<210> 4
<211> 642
<212> DNA
<213> Artificial sequence
<400> 4
gcgaccgtgt tttgggaacc gttatgctac cacaactcct ccacctggca aaaggcggac 60
ggttacagca acggcggtat gttcaactgc acctggcgtg ctaataatgc aacttttacc 120
aacgattgca agctgcgttt ggcgctgacg tcttcaagct ataacaaatt cgacggcgct 180
gagtatcgta ccacctttaa aaccggttac ggcaagtgcg aagtgagcat gaaaccggcg 240
aaaaacccgg gcatcgtgag ctcttttttc atctatactg gtccaagtga tggcaccccg 300
tgggatgaaa tcgacatcgg tttcctgggt aaggatacca ccaaagttca atttaactac 360
ttcaccaacg gtgtgggtgg tcatgagaaa gtgatcgacc tgggcttcga cgcttcgcaa 420
ggttaccaca cctacgcctt cgactggcag ccgggcagta ttacctggta tgttgatggg 480
gtgcagaagc acaaagctac gactaacatt ccgacccatc cgggcaagat catgatgaat 540
ctgtggaatg gcattggtgt tgatagctgg ctgggtgcgt acaatggtgc caacccgctg 600
tatgcgtact atgattgggt taagtacacc tcaaacctcg ag 642
<210> 5
<211> 214
<212> PRT
<213> Artificial sequence
<400> 5
Ala Thr Val Phe Trp Glu Pro Leu Thr Tyr His Asn Ser Ser Thr Trp
1 5 10 15
Gln Lys Ala Asp Gly Tyr Ser Asn Gly Gly Met Phe Asn Cys Thr Trp
20 25 30
Arg Ala Asn Asn Ala Thr Phe Thr Asn Asp Gly Lys Leu Arg Leu Ala
35 40 45
Leu Thr Ser Ser Ser Tyr Asn Lys Phe Asp Gly Ala Glu Tyr Arg Thr
50 55 60
Thr Phe Lys Thr Gly Tyr Gly Lys Cys Glu Val Cys Met Lys Pro Ala
65 70 75 80
Lys Asn Pro Gly Ile Val Ser Ser Phe Phe Ile Tyr Thr Gly Pro Ser
85 90 95
Asp Gly Thr Pro Trp Asp Glu Ile Asp Ile Gly Phe Leu Gly Lys Asp
100 105 110
Thr Thr Lys Val Gln Phe Asn Tyr Phe Thr Asn Gly Val Gly Gly His
115 120 125
Glu Lys Val Ile Asp Leu Gly Phe Asp Ala Ser Gln Gly Tyr His Thr
130 135 140
Tyr Ala Phe Asp Trp Gln Pro Gly Ser Ile Thr Trp Tyr Val Asp Gly
145 150 155 160
Val Gln Lys His Lys Ala Thr Thr Asn Ile Pro Thr His Pro Gly Lys
165 170 175
Ile Met Met Asn Leu Trp Asn Gly Ile Gly Val Asp Ser Trp Leu Gly
180 185 190
Ala Tyr Asn Gly Ala Asn Pro Leu Tyr Ala Tyr Tyr Cys Trp Val Lys
195 200 205
Tyr Thr Ser Asn Leu Glu
210
<210> 6
<211> 642
<212> DNA
<213> Artificial sequence
<400> 6
gcgaccgtgt tttgggaacc gttaacctac cacaactcct ccacctggca aaaggcggac 60
ggttacagca acggcggtat gttcaactgc acctggcgtg ctaataatgc aacttttacc 120
aacgatggca agctgcgttt ggcgctgacg tcttcaagct ataacaaatt cgacggcgct 180
gagtatcgta ccacctttaa aaccggttac ggcaagtgcg aagtgtgcat gaaaccggcg 240
aaaaacccgg gcatcgtgag ctcttttttc atctatactg gtccaagtga tggcaccccg 300
tgggatgaaa tcgacatcgg tttcctgggt aaggatacca ccaaagttca atttaactac 360
ttcaccaacg gtgtgggtgg tcatgagaaa gtgatcgacc tgggcttcga cgcttcgcaa 420
ggttaccaca cctacgcctt cgactggcag ccgggcagta ttacctggta tgttgatggg 480
gtgcagaagc acaaagctac gactaacatt ccgacccatc cgggcaagat catgatgaat 540
ctgtggaatg gcattggtgt tgatagctgg ctgggtgcgt acaatggtgc caacccgctg 600
tatgcgtact attgctgggt taagtacacc tcaaacctcg ag 642
<210> 7
<211> 25
<212> DNA
<213> Artificial sequence
<400> 7
ttttaccaac gattgcaagc tgcgt 25
<210> 8
<211> 26
<212> DNA
<213> Artificial sequence
<400> 8
gagttgtggt agcataacgg ttccca 26
<210> 9
<211> 28
<212> DNA
<213> Artificial sequence
<400> 9
ccgttatgct accacaactc ctccacct 28
<210> 10
<211> 29
<212> DNA
<213> Artificial sequence
<400> 10
agcttgcaat cgttggtaaa agttgcatt 29
<210> 11
<211> 31
<212> DNA
<213> Artificial sequence
<400> 11
gtactattgc tgggttaagt acacctcaaa c 31
<210> 12
<211> 30
<212> DNA
<213> Artificial sequence
<400> 12
ggtttcatgc acacttcgca cttgccgtaa 30
<210> 13
<211> 23
<212> DNA
<213> Artificial sequence
<400> 13
tgcgaagtgt gcatgaaacc ggc 23
<210> 14
<211> 27
<212> DNA
<213> Artificial sequence
<400> 14
cttaacccag caatagtacg catacag 27
Claims (10)
1. A heat-resistant beta-1, 3-1, 4-glucanase mutant has an amino acid sequence shown in SEQ ID NO. 3.
2. The nucleotide sequence of the beta-1, 3-1, 4-glucanase mutant of the claim 1 is shown as SEQ ID NO. 4.
3. A recombinant plasmid comprising the nucleotide sequence of the β -1,3-1, 4-glucanase mutant of claim 2.
4. The recombinant plasmid of claim 3, wherein the vector of the recombinant plasmid is pET28a (+).
5. A genetically engineered bacterium comprising a nucleotide sequence of the mutant β -1,3-1, 4-glucanase of claim 2 or the recombinant plasmid of claim 3.
6. The genetically engineered bacterium of claim 5, wherein the expression host of the genetically engineered bacterium is Escherichia coli;
more preferably, the expression host bacterium is escherichia coli BL 21.
7. The method for preparing the thermostable beta-1, 3-1, 4-glucanase mutant according to claim 1, comprising the following steps:
(1) artificially synthesizing plasmid pET28a (+) EccslG containing nucleotide sequence of primase beta-1, 3-1, 4-glucanase (PlicA), designing primers G35-69U and G35-69D and G35-69WUAnd G35-69WD, using plasmid pET28a (+) EccslG as template, using G35-69U and G35-69D as primer, synthesizing objective gene EccslG69 by PCRTThe target gene EccslG69 is synthesized by reverse PCR by taking the plasmid pET28a (+) EccslG as a template and G35-69WU and G35-69WD as primersG；
(2) The target gene EccslG69 synthesized in the step (1)GAnd EccslG69TConnecting and cyclizing to construct a mutant plasmid, transferring the mutant plasmid into escherichia coli BL21 to construct recombinant escherichia coli, and performing induced expression on the heat-resistant beta-1, 3-1, 4-glucanase mutant.
8. The method according to claim 7, wherein the nucleotide sequence of the primase β -1,3-1, 4-glucanase (PlicA) in step (1) is shown in SEQ ID NO 2.
9. The method according to claim 7, wherein the nucleotide sequence of G35-69U in step (1) is: 5'-CCGTTAtgcTACCACAACTCCTCCACCT-3' (SEQ ID NO:9),
the nucleotide sequence of G35-69D is as follows: 5'-AGCTTgcaATCGTTGGTAAAAGTTGCATT-3' (SEQ ID NO:10),
The nucleotide sequence of the G35-69WU is as follows: 5'-TTTTACCAACGATtgcAAGCTGCGT-3' (SEQ ID NO:7),
the nucleotide sequence of the G35-69WD is as follows: 5'-GAGTTGTGGTAgcaTAACGGTTCCCA-3' (SEQ ID NO: 8).
10. The thermostable beta-1, 3-1, 4-glucanase mutant of claim 1 for use in the fields of food, feed, and paper.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210333541.0A CN114752583B (en) | 2022-03-30 | 2022-03-30 | Heat-resistant beta-1, 3-1, 4-glucanase mutant and preparation method and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210333541.0A CN114752583B (en) | 2022-03-30 | 2022-03-30 | Heat-resistant beta-1, 3-1, 4-glucanase mutant and preparation method and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN114752583A true CN114752583A (en) | 2022-07-15 |
| CN114752583B CN114752583B (en) | 2023-07-21 |
Family
ID=82329516
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202210333541.0A Active CN114752583B (en) | 2022-03-30 | 2022-03-30 | Heat-resistant beta-1, 3-1, 4-glucanase mutant and preparation method and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN114752583B (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2023225459A2 (en) | 2022-05-14 | 2023-11-23 | Novozymes A/S | Compositions and methods for preventing, treating, supressing and/or eliminating phytopathogenic infestations and infections |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CA2811206A1 (en) * | 2010-09-15 | 2012-03-22 | The Regents Of The University Of California | Thermophilic mutants of trichoderma reesei endoglucanase i |
| US8192974B1 (en) * | 2011-03-09 | 2012-06-05 | Genozyme Biotech, Inc. | Glucanase having increased enzyme activity and thermostability |
| CN104862290A (en) * | 2015-06-12 | 2015-08-26 | 江南大学 | 1,3-1,4-beta-glucanase mutant |
| CN108048430A (en) * | 2018-01-08 | 2018-05-18 | 中国农业科学院饲料研究所 | Endoglucanase NfEG12A mutant and its encoding gene and application |
| 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 |
-
2022
- 2022-03-30 CN CN202210333541.0A patent/CN114752583B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CA2811206A1 (en) * | 2010-09-15 | 2012-03-22 | The Regents Of The University Of California | Thermophilic mutants of trichoderma reesei endoglucanase i |
| US8192974B1 (en) * | 2011-03-09 | 2012-06-05 | Genozyme Biotech, Inc. | Glucanase having increased enzyme activity and thermostability |
| CN104862290A (en) * | 2015-06-12 | 2015-08-26 | 江南大学 | 1,3-1,4-beta-glucanase mutant |
| CN108048430A (en) * | 2018-01-08 | 2018-05-18 | 中国农业科学院饲料研究所 | Endoglucanase NfEG12A mutant and its encoding gene and application |
| 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 |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2023225459A2 (en) | 2022-05-14 | 2023-11-23 | Novozymes A/S | Compositions and methods for preventing, treating, supressing and/or eliminating phytopathogenic infestations and infections |
Also Published As
| Publication number | Publication date |
|---|---|
| CN114752583B (en) | 2023-07-21 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN111748537B (en) | Uridine phosphatase mutant and application thereof | |
| CN110791494A (en) | Aspartic enzyme mutant, recombinant expression vector and recombinant bacterium containing aspartic enzyme mutant and application | |
| CN112391365B (en) | Starch branching enzyme mutant with improved catalytic activity and application thereof | |
| CN112175916B (en) | L-amino acid ligase mutant, recombinant vector, recombinant bacterium and application thereof | |
| CN111172142B (en) | Cephalosporin C acylase mutant with high thermal stability | |
| CN114752583B (en) | Heat-resistant beta-1, 3-1, 4-glucanase mutant and preparation method and application thereof | |
| CN114525268A (en) | Glutamic acid decarboxylase mutant with improved pH tolerance and application thereof in synthesis of gamma-aminobutyric acid | |
| CN112301012B (en) | Cyclodextrin glucosyltransferase mutant and construction method thereof | |
| CN110129305B (en) | Cephalosporin C acylase mutant for preparing 7-ACA | |
| CN115058408B (en) | Metagenome-derived high-specific-activity acid-resistant D-psicose 3-epimerase and encoding gene and application thereof | |
| CN111117987A (en) | High-specific-activity acidic mannase mutant | |
| CN114703165A (en) | Beta-glucosidase mutant and application thereof | |
| CN110628745B (en) | Mutant enzyme Xynh31-K210R and application thereof | |
| CN113322247A (en) | Beta-mannase heat-resistant mutant M19, recombinant bacterium and application thereof | |
| CN113201521A (en) | Beta-mannase heat-resistant mutant M14, recombinant bacterium and application thereof | |
| CN108165538B (en) | Kidney bean epoxide hydrolase mutant with improved catalytic activity | |
| CN114395543B (en) | Trehalose synthase mutant and application thereof | |
| CN112481245B (en) | Recombinant D-tagatose 3 epimerase DTE-CM and construction method and application thereof | |
| CN112210544B (en) | Cyclodextrin glucosyltransferase mutant and application thereof | |
| CN114752582B (en) | Beta-glucosidase mutant with improved glucose tolerance and application thereof | |
| CN112680425B (en) | Alcohol dehydrogenase mutant and application thereof | |
| CN112111506B (en) | Method for improving expression quantity of gamma-glutamine transpeptidase by RBS optimization | |
| CN109280651B (en) | Lactate dehydrogenase mutant gene LbLDH1 and fermentation method for efficient expression of lactate dehydrogenase mutant gene LbLDH1 in escherichia coli | |
| CN114438057A (en) | Heat-resistant alkali-resistant xylanase and application thereof | |
| CN114621944A (en) | Arginine deiminase mutant with improved enzyme activity |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |