CN113201520A - Beta-mannase heat-resistant mutant M15, recombinant bacterium and application thereof - Google Patents
Beta-mannase heat-resistant mutant M15, recombinant bacterium and application thereof Download PDFInfo
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- CN113201520A CN113201520A CN202110697983.9A CN202110697983A CN113201520A CN 113201520 A CN113201520 A CN 113201520A CN 202110697983 A CN202110697983 A CN 202110697983A CN 113201520 A CN113201520 A CN 113201520A
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- gly
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- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
- C12N9/2477—Hemicellulases not provided in a preceding group
- C12N9/2488—Mannanases
- C12N9/2494—Mannan endo-1,4-beta-mannosidase (3.2.1.78), i.e. endo-beta-mannanase
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- 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)
- C12Y302/01078—Mannan endo-1,4-beta-mannosidase (3.2.1.78), i.e. endo-beta-mannanase
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Abstract
本发明涉及β‑甘露聚糖酶耐热突变体M15、重组菌及其应用，属于基因工程和酶工程领域，本发明采用蛋白质表面电荷优化策略，理性设计并筛选突变位点，将白曲霉来源的β‑甘露聚糖酶ManAK进行改良，再通过高通量筛选方法挑选正向突变，最终获得热稳定性提高的23株β‑甘露聚糖酶突变体。本发明获得的β‑甘露聚糖酶突变体耐热性是原始基因的1.2～3.5倍，耐热性均有显著提高，有利于实现其在商业中的应用，从而降低生产成本。
The invention relates to β-mannanase heat-resistant mutant M15, recombinant bacteria and applications thereof, and belongs to the fields of genetic engineering and enzyme engineering. The invention adopts a protein surface charge optimization strategy, rationally designs and screens mutation sites, and uses Aspergillus albicans source The β-mannanase ManAK was improved, and then forward mutations were selected by high-throughput screening method, and finally 23 β-mannanase mutants with improved thermostability were obtained. The heat resistance of the β-mannanase mutant obtained by the invention is 1.2 to 3.5 times that of the original gene, and the heat resistance is significantly improved, which is conducive to realizing its commercial application, thereby reducing the production cost.
Description
Technical Field
The invention belongs to the field of genetic engineering and enzyme engineering, and particularly relates to a beta-mannase heat-resistant mutant, a recombinant strain and application thereof.
Background
Mannan is the second major component of hemicellulose, and is formed by the linkage of mannopyranose through beta-1, 4 glycosidic bonds. The complete degradation of mannan requires the synergistic action of mannanase, mannosidase, glucosidase, galactosidase and deacetylation esterases. Among them, mannanase is a key enzyme in the degradation process.
Beta-mannanases (EC 3.2.1.78) are able to hydrolyze mannans to mannose and mannooligosaccharides by catalyzing beta-1, 4 mannosidic bonds. The enzyme is used as an important industrial enzyme preparation and is widely applied to the fields of animal feed, food processing, medicines, textiles, biological bleaching, petroleum and other renewable energy sources. In some application fields, particularly processes such as feed granulation and renewable energy production, high-temperature treatment is required, so that the developed beta-mannase is required to have good heat resistance. The optimal action temperature of the heat-resistant enzyme is above 60 ℃, the reaction temperature is higher, the pollution of mixed bacteria is reduced, the interference of metabolites of the mixed bacteria is reduced, the catalytic capability and the catalytic efficiency of the enzyme can be improved, and the production cost is greatly reduced. At present, the heat resistance of the beta-mannase on the market often cannot meet the industrial requirement, so that the biological activity of the beta-mannase is unstable in the production and transportation processes, and the application of the beta-mannase is severely limited. Therefore, the development of the beta-mannase with excellent heat resistance has important industrial significance and economic value.
Disclosure of Invention
The invention aims to provide a beta-mannase mutant with improved heat resistance. The invention improves beta-mannase ManAK (amino acid sequence SEQIDNO: 1 and nucleotide sequence SEQIDNO: 2) from aspergillus kawachii by rational design, adopting a protein surface charge optimization strategy and screening design sites to obtain the beta-mannase single-site mutant with improved thermal stability. Another object of the present invention is to provide a recombinant vector comprising the above-mentioned mutated β -mannanase gene. The beta-mannase gene obtained by mutation is inserted into a proper site of an expression vector, so that the nucleotide sequence of the beta-mannase gene can be operably connected with an expression regulatory sequence to obtain a recombinant yeast expression plasmid. The invention also provides a recombinant strain containing the beta-mannase gene, wherein the strain is saccharomycete.
In order to achieve the above object, the present invention provides the following technical solutions:
a single-site beta-mannase mutant with improved heat resistance is M7(D97N) and is prepared from a polypeptide with an amino acid sequence of SEQ ID NO: 1 from the beta-mannanase from the amino acid aspartic acid at position 97 (D) to asparagine (N);
or the beta-mannase mutant is M8(D107N) and is formed by amino acid sequence SEQ ID NO: 1 from the beta-mannanase from the amino acid aspartic acid (D) at position 107 to asparagine (N);
or the beta-mannase mutant is M9(D113N) and is formed by amino acid sequence SEQ ID NO: 1 from the beta-mannanase from amino acid 113, aspartic acid (D), to asparagine (N);
or the beta-mannase mutant is M10(D123N) and is formed by amino acid sequence SEQ ID NO: 1 from the beta-mannanase from amino acid 123 (aspartic acid) (D) to asparagine (N);
or the beta-mannase mutant is M11(D136N) and is formed by amino acid sequence SEQ ID NO: 1 from the beta-mannanase from the amino acid aspartic acid at position 136 (D) to asparagine (N);
or the beta-mannase mutant is M12(D151N) and is formed by amino acid sequence SEQ ID NO: 1 from the amino acid aspartic acid (D) at position 151 of the β -mannanase to asparagine (N);
or the beta-mannase mutant is M13(D154N), and the mutant is formed by amino acid sequence SEQ ID NO: 1 from the amino acid aspartic acid (D) at position 154 of the β -mannanase to asparagine (N);
or the beta-mannase mutant M14(D159N) is a mutant which has the amino acid sequence of SEQ ID NO: 1 from the beta-mannanase from the 159 th amino acid aspartic acid (D) to asparagine (N);
or the beta-mannase mutant is M15(D197N), and the amino acid sequence is SEQ ID NO: 1 from the beta-mannanase from which the amino acid aspartic acid (D) at position 197 is changed to asparagine (N), the amino acid sequence being as shown in SEQ ID NO:3 is shown in the specification;
or the beta-mannase mutant is M16(D203N) and is formed by amino acid sequence SEQ ID NO: 1 from the amino acid aspartic acid (D) at position 203 of the β -mannanase to asparagine (N);
or the beta-mannase mutant is M17(D216N) and is formed by amino acid sequence SEQ ID NO: 1 from the beta-mannanase from which the amino acid aspartic acid (D) at position 216 is changed to asparagine (N);
or the beta-mannase mutant is M18(D218N) and is formed by amino acid sequence SEQ ID NO: 1 from the beta-mannanase from the amino acid aspartic acid at position 218 (D) to asparagine (N);
or the beta-mannase mutant is M19(D225N) and is formed by amino acid sequence SEQ ID NO: 1 from the beta-mannanase from which the amino acid aspartic acid at position 225 (D) is changed to asparagine (N);
or the beta-mannase mutant is M20(D233N) and is formed by amino acid sequence SEQ ID NO: 1 from the amino acid aspartic acid at position 233 of the β -mannanase to asparagine (N);
or the beta-mannase mutant is M21(D235N) and is formed by amino acid sequence SEQ ID NO: 1 from the beta-mannanase from the 235 th amino acid aspartic acid (D) to asparagine (N);
or the beta-mannase mutant is M22(D255N), and the mutant M22 consists of amino acid sequence SEQ ID NO: 1 from the amino acid aspartic acid at position 255 of the beta-mannanase to asparagine (N);
or the beta-mannase mutant is M23(D258N), and the mutant is formed by amino acid sequence SEQ ID NO: 1 from the beta-mannanase from the amino acid aspartic acid at position 258 (D) to asparagine (N);
or the beta-mannase mutant is M24(D267N) and is formed by amino acid sequence SEQ ID NO: 1 from the beta-mannanase from the amino acid aspartic acid at position 267 (D) to asparagine (N);
or the beta-mannase mutant is M25(D273N), and the beta-mannase mutant is formed by amino acid sequences of SEQ ID NO: 1 from the amino acid aspartic acid at position 273 (D) of the β -mannanase to asparagine (N);
or the beta-mannase mutant is M26(D274N), and the mutant is formed by amino acid sequence SEQ ID NO: 1 from the amino acid aspartic acid (D) at position 274 of the β -mannanase to asparagine (N);
or the beta-mannase mutant is M30(D341N) and is formed by amino acid sequence SEQ ID NO: 1 from the amino acid aspartic acid (D) at position 341 of the β -mannanase to asparagine (N);
or the beta-mannase mutant is M33(D359N), and the mutant is formed by amino acid sequence SEQ ID NO: 1 from the amino acid aspartic acid (D) at position 359 of the β -mannanase to asparagine (N);
or the beta-mannase mutant is M41(E244Q) and is formed by amino acid sequence SEQ ID NO: 1, the amino acid glutamic acid (E) at position 244 of the β -mannanase is changed to glutamine (Q).
The invention provides application of the beta-mannase mutant
The invention provides a recombinant strain containing the beta-mannase mutant coding gene, wherein the recombinant strain is pichia pastoris X33.
Compared with the prior art, the invention has the beneficial effects that:
the invention provides amino acid sequences for respectively producing the beta-mannanase mutants. Based on SEQ ID NO: 1, designing a screening site through rational design, wherein the beta-mannase ManAK is shown in SEQ ID NO: 1, replacing aspartic acid (D) with asparagine (N) and glutamic acid (E) with glutamine (Q) at different sites, reasonably optimizing the surface charge distribution of protein, and obtaining 23 beta-mannase mutants with remarkably improved heat resistance.
According to the substituted position and amino acid, compared with the original beta-mannase ManAK, the modified mutant is treated at 75 ℃, and the enzyme activity is lost for half the required time (t)1/2) Is that1.2-3.5 times of the original enzyme ManAK, which shows that the beta-mannase carbohydrase mutant obtained by the invention has obviously improved heat resistance compared with the wild type, and is beneficial to realizing the commercial application of the mutant, thereby reducing the production cost.
Drawings
FIG. 1 shows the comparison of the heat resistance of the beta-mannanase mutant and the original gene ManAK.
Detailed Description
The invention discloses a mannase mutant, a preparation method and application thereof, and a DNA molecule, a vector and a host cell for coding the mannase mutant. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. The technical solutions of the present invention are further described in detail with reference to the drawings and the specific embodiments, but the embodiments are only for explaining the present invention and do not limit the scope of the present invention.
1. Strains and vectors:
coli DH5 a, Pichia pastoris X33, vector pPICZ α A, Amp, Zeocin were purchased from Invitrogen.
2. Enzyme and kit:
PCR enzymes and ligase were purchased from Takara, and plasmid extraction kit and gel recovery kit were purchased from Omega.
3. The formula of the culture medium is as follows:
coli medium (LB medium): 1% tryptone, 0.5% yeast extract, 1% NaCl;
LB-AMP medium: adding 100 mu g/mLAMP into LB culture medium;
YPD medium: 1% yeast extract, 2% tryptone, 2% glucose;
YPDZ culture medium, namely YPD culture medium added with 100 mu g/mLzeocin;
MD culture medium: 1.34% YNB, 0.4mg/L biotin, 2% glucose;
BMGY medium: 1% yeast extract, 2% peptone, 100mM potassium phosphate buffer (pH6.0), 1% glycerol;
when the culture medium is solid, 2% agar powder is added.
4. The experimental method comprises the following steps:
the strain culture conditions are as follows: escherichia coli was cultured at 37 ℃ and yeast was cultured at 30 ℃. The rotation speed of the shaker during liquid culture is 200 rpm.
The transformation method of Pichia pastoris X33 comprises the following steps:
inoculating activated Pichia pastoris X33 into a 50mLYPD liquid culture medium, carrying out shake flask culture at 30 ℃ until the 0D600 is 1.2-1.5, then carrying out low-temperature centrifugation to collect thalli, sequentially washing the thalli with 40mL of ice-cold sterile water and 10mL of ice-cold sorbitol solution with the concentration of 1mol/L, and finally carrying out heavy suspension on the thalli with 5mL of sorbitol solution to prepare yeast competent suspension. 100uL of yeast competence and 10uL of linearized vector are uniformly mixed and transferred to a precooled electric rotating cup for electric shock conversion, and the condition of electric conversion is 1.5kV and 5 msec. After the shock, 1mL of sorbitol solution was added and transferred to a 1.5mL centrifuge tube and incubated at 30 ℃ for 1 h. Centrifuging at 5000rpm for 5min, collecting thallus, coating onto MD screening plate, and culturing upside down until positive monoclonal antibody grows out.
The detection of the activity of the beta-mannase is carried out according to the national standard GB T36861-2018 of the people's republic of China; the mannanase activity is defined as the amount of enzyme required for the degradation of a sample at pH 5.5 and 37 ℃ to release 1. mu. mol reducing sugars from a mannan (Sigma G0753) solution at a concentration of 3mg/mL per minute, expressed as U.
5. Example 1: rational design of beta-mannase thermal stability site and primer design
Based on the amino acid sequence of ManAK (as shown in SEQ ID NO: 1), Asp in ManAK is respectively added97Mutation to Asn97、Asp107Mutation to Asn107、Asp113Mutation to Asn113、Asp123Mutation to Asn123、Asp136Mutation to Asn136、Asp151Mutation to Asn151、Asp154Mutation to Asn154、Asp159Mutation to Asn159、Asp197Mutation to Asn197、Asp203Mutation to Asn203、Asp216Mutation to Asn216、Asp218Mutation to Asn218、Asp225Mutation to Asn225、Asp233Mutation to Asn233、Asp235Mutation to Asn235、Asp255Mutation to Asn255、Asp258Mutation to Asn258、Asp267Mutation to Asn267、Asp273Mutation to Asn273、Asp274Mutation to Asn274、Asp341Mutation to Asn341、Asp359Mutation to Asn359、Glu244Mutation to Gln244Mutants M7, M8, M9, M10, M11, M12, M13, M14, M15, M16, M17, M18, M19, M20, M21, M22, M23, M24, M25, M26, M30, M33 and M41 containing corresponding mutation sites are sequentially obtained, and 23 mutants are calculated. PCR primers were designed based on the ManAK nucleotide sequence (shown in SEQ ID NO: 2) and synthesized by Celastri Borcico, Inc.
6. Example 2: construction of beta-mannase site-directed mutant recombinant vector
A recombinant vector pPICZ alpha A-ManAK containing a ManAK gene is taken as a template, a primer of a mutation site is added, PCR amplification is carried out by using high fidelity enzyme, a product is subjected to Dpn I enzyme digestion treatment, DH5 alpha escherichia coli competent cells are transformed by heat shock, positive clones are screened on an LB plate containing Amp, and sequencing work is completed by Qingdao Ruo Boxing Ke Co.
7. Example 3: construction of beta-mannase site-directed mutant expression vector
Extracting plasmids from the positive clones with correct sequencing after mutation in example 2, purifying, linearizing the corresponding plasmids by Sac I single enzyme digestion, electrically transferring into pichia pastoris X33, screening by an MD plate to obtain transformants of each mutant expression strain, and transferring into a YPD plate for activation.
8. Example 4: high-throughput expression validation of beta-mannase mutant in pichia pastoris
The transformant activated in example 3 was selected and inoculated into a medium containing 1mLBMGY, shake-cultured in 48-well plates at 30 ℃ under 200rmp, and after 24 hours, methanol of 1% of the culture volume was added for the first time to induce the expression of the enzyme, and then the induction was performed every 24 hours during the post-culture. After the induction expression is carried out for 96 hours, the culture solution is centrifuged to obtain a supernatant, and the activity and the thermal stability of the beta-mannase in the supernatant of the fermentation liquor are measured.
9. Example 5: amplification culture expression verification of beta-mannase mutant in pichia pastoris
The beta-mannase mutant with improved thermal stability verified in example 4 was activated, inoculated into 20mLBMGY medium, shake-cultured at 30 ℃ in a shake flask at 200rmp, and after 24h, 1% methanol of the culture volume was added for the first time to induce the expression of the enzyme, and then the induction was performed every 24h during the culture. After the induction expression is carried out for 96 hours, the culture solution is centrifuged to obtain a supernatant, and the activity and the thermal stability of the beta-mannase in the supernatant of the fermentation liquor are measured. As shown in FIG. 1, the results show that after mutation, the obtained beta-mannase M7, M8, M9, M10, M11, M12, M13, M14, M15, M16, M17, M18, M19, M20, M21, M22, M23, M24, M25, M26, M30, M33 and M41 are treated at 75 ℃, the time required for half of the enzyme activity loss is 1.2-3.5 times of that of the original enzyme Man, and the mutant AK bacteria have improved heat resistance.
The above results show that Asp of ManAK97Mutation to Asn97、Asp107Mutation to Asn107、Asp113Mutation to Asn113、Asp123Mutation to Asn123、Asp136Mutation to Asn136、Asp151Mutation to Asn151、Asp154Mutation to Asn154、Asp159Mutation to Asn159、Asp197Mutation to Asn197、Asp203Mutation to Asn203、Asp216Mutation to Asn216、Asp218Mutation to Asn218、Asp225Mutation to Asn225、Asp233Mutation to Asn233、Asp235Mutation to Asn235、Asp255Mutation to Asn255、Asp258Mutation to Asn258、Asp267Mutation to Asn267、Asp273Mutation to Asn273、Asp274Mutation to Asn274、Asp341Mutation to Asn341、Asp359Mutation to Asn359、Glu244Mutation to Gln244The resulting mutant has further improved heat resistance.
Sequence listing
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Gly Val Ser Ala Asp Leu Phe Trp Gln Tyr Gly Asp Asp Leu Ser Thr
325 330 335
Gly Lys Ser Pro Asp Asp Gly Asn Thr Ile Tyr Tyr Gly Thr Ser Asp
340 345 350
Tyr Glu Cys Leu Val Thr Asp His Val Ala Ala Ile Gly Ser Ala
355 360 365
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| 赵梅: "耐热β-甘露聚糖酶基因的克隆与表达及酶学性质", 《食品与生物技术学报》 * |
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