WO2021248363A1 - 一种Thermobifida fusca角质酶突变体及其可溶性表达的方法 - Google Patents

一种Thermobifida fusca角质酶突变体及其可溶性表达的方法 Download PDF

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WO2021248363A1
WO2021248363A1 PCT/CN2020/095350 CN2020095350W WO2021248363A1 WO 2021248363 A1 WO2021248363 A1 WO 2021248363A1 CN 2020095350 W CN2020095350 W CN 2020095350W WO 2021248363 A1 WO2021248363 A1 WO 2021248363A1
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cutinase
mutant
seq
coli
cutinase mutant
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吴敬
王蕾
朱方剑
陈晟
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江南大学
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Priority to CN202080101956.4A priority patent/CN116171323A/zh
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Priority to US18/063,732 priority patent/US20230287367A1/en

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    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
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    • C12Y301/00—Hydrolases acting on ester bonds (3.1)
    • C12Y301/01—Carboxylic ester hydrolases (3.1.1)
    • C12Y301/01074—Cutinase (3.1.1.74)
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    • C12Y503/00—Intramolecular oxidoreductases (5.3)
    • C12Y503/04—Intramolecular oxidoreductases (5.3) transposing S-S bonds (5.3.4)
    • C12Y503/04001—Protein disulfide-isomerase (5.3.4.1), i.e. disufide bond-forming enzyme
    • D—TEXTILES; PAPER
    • D06—TREATMENT OF TEXTILES OR THE LIKE; LAUNDERING; FLEXIBLE MATERIALS NOT OTHERWISE PROVIDED FOR
    • D06M—TREATMENT, NOT PROVIDED FOR ELSEWHERE IN CLASS D06, OF FIBRES, THREADS, YARNS, FABRICS, FEATHERS OR FIBROUS GOODS MADE FROM SUCH MATERIALS
    • D06M16/00—Biochemical treatment of fibres, threads, yarns, fabrics, or fibrous goods made from such materials, e.g. enzymatic
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    • C12N2800/00—Nucleic acids vectors
    • C12N2800/10—Plasmid DNA
    • C12N2800/101—Plasmid DNA for bacteria

Definitions

  • the invention relates to a Thermobifida fusca cutinase mutant and a method for soluble expression thereof, and belongs to the technical field of enzyme engineering.
  • Thermobifida fusca cutinase belongs to the ⁇ / ⁇ hydrolase family.
  • the ⁇ sheet is located in the center, surrounded by ⁇ helices and loop regions, forming a sandwich structure.
  • the active center is composed of the Ser-His-Asp catalytic triangle. It is a multifunctional enzyme that can Hydrolysis of macromolecular polyesters, insoluble triglycerides and small molecular soluble esters.
  • T. fusca cutinase can also catalyze the esterification of various acids and alcohols, as well as the transesterification of esters and alcohols.
  • cutinase and lipase The characteristic difference between cutinase and lipase is that the active center of cutinase is not covered by a "lid" structure, which catalyzes the exposure of the key amino acid Ser to the solvent. Therefore, some cutinases can also hydrolyze high molecular weight polyester PET (Polyethylene terephthalate), but PET is highly crystallized, which reduces the accessibility of cutinase to the polyester chain in PET, and seriously affects the catalytic efficiency of cutinase. After reaching the glass transition temperature (69-80°C), the movement of PET's molecular chain intensifies and forms Many gaps increase the accessibility between the active center of the enzyme molecule and the catalytic effect becomes more obvious.
  • polyester PET Polyethylene terephthalate
  • T. fusca cutinase in the prior art cannot maintain activity under the glass transition temperature of PET. Therefore, improving the thermal stability of T. fusca cutinase is an urgent problem to be solved in realizing industrial production.
  • An object of the present invention is to provide a cutinase mutant with strong thermal stability.
  • the amino acids at at least one of the following positions are mutated: 89th, 204th, and 253rd.
  • the cutinase mutant is (a) or (b):
  • the second object of the present invention is to provide a gene encoding the cutinase mutant.
  • the gene encoding the mutant D204C/E253C contains the nucleotide sequence shown in SEQ ID NO.4.
  • the third object of the present invention is to provide an expression vector carrying the gene.
  • the expression vector is a pET series plasmid.
  • the expression vector is pSCDsbC, and its nucleotide sequence is shown in SEQ ID NO.5.
  • the fourth object of the present invention is to provide microbial cells expressing the cutinase mutant.
  • the microbial cell is Escherichia coli.
  • the E. coli is E. coli BL21, E. coli BL21(DE3), E. coli JM109, E. coli DH5 ⁇ , or E. coli TOP10.
  • the fifth object of the present invention is to provide a method for soluble expression of the cutinase mutant, which is to co-express the cutinase mutant and the periplasmic protein disulfide bond oxidoreductase DsbC.
  • amino acid sequence of the periplasmic protein disulfide bond oxidoreductase DsbC is shown in SEQ ID NO.6.
  • the method is to link the gene encoding the cutinase mutant and the gene encoding the periplasmic protein disulfide bond oxidoreductase to a vector, respectively, and transform them into microbial cells for expression.
  • the method also adds the RBS sequence of the ribosome binding site upstream of the gene.
  • the method uses the plasmid pSC as the expression vector and E. coli Origami B (DE3) as the host to co-express the cutinase mutant and the periplasmic protein disulfide bond oxidoreductase.
  • nucleotide sequence of the plasmid pSC is shown in SEQ ID NO.7.
  • the sixth objective of the present invention is to provide the application of the cutinase mutant in the chemical or textile fields.
  • the application is to modify PET fiber or its fabric.
  • the modification is to subject the cutinase mutant to an enzymatic hydrolysis reaction on PET fiber at a dose of 10 U/g substrate, with a bath ratio of 1:40; the enzymatic hydrolysis reaction is performed at 80°C. Proceed under.
  • Triton X-100 with a final concentration of 0.5-2% is also added to the enzymatic hydrolysis reaction.
  • the present invention selects the mutation of glutamic acid at position 204 and aspartic acid at position 253 near the catalytic triangle to cysteine to introduce a pair of disulfide bonds to stabilize the catalytic triangle; It is mutated to cysteine to form a pair of disulfide bonds to stabilize the N-terminal domain of cutinase, which makes the cutinase mutants have higher thermal stability.
  • the mutant can catalyze the hydrolysis of the ester bond of PET fiber under the reaction conditions of the glass transition temperature (Tg) of PET to achieve the purpose of hydrophilization modification.
  • the present invention aims at the defect that the mutant D204C/E253C is easily misfolded during the expression process to form a large number of inclusion bodies due to the introduction of disulfide bonds, and the soluble expression ratio is extremely low, and provides a soluble expression mutant D204C/E253C Methods.
  • the mutant D204C/E253C and the periplasmic protein disulfide bond oxidoreductase DsbC were co-expressed in E. coli Origami B (DE3) to isomerize abnormal disulfide bonds and achieve high-efficiency and soluble expression.
  • Figure 1 is the SDS-PAGE of different proteins; among them, M is the protein molecular weight standard; lanes 1 to 3 in (A) are the cells of recombinant E. coli BL21(DE3)/pET-20b(+)-cut Outer supernatant, broken wall supernatant, broken wall precipitate, lanes 4 to 6 are the extracellular supernatant, broken wall supernatant of recombinant E.coli BL21(DE3)/pET-20b(+)-T61C/T89C, respectively Wall-broken precipitation; Lanes 1 to 3 in (B) are the extracellular supernatant, wall-broken supernatant, and wall-broken sediment of recombinant E.coli BL21(DE3)/pET-20b(+)-D204C/E253C, respectively.
  • Figure 2 is the SDS-PAGE image of purified wild-type cutinase and mutant; among them, M is the protein molecular weight standard; lane 2 is wild-type T.fusca cutinase, lanes 3 and 4 are mutants T61C/T89C, Lanes 5 and 6 are mutants D204C/E253C.
  • FIG 3 shows the enzyme activities of cutinase and mutants at different temperatures; among them, WT is the wild enzyme.
  • Figure 4 shows the thermal stability of mutant D204C/E253C at 80°C (A) and 90°C (B), respectively.
  • Figure 5 shows the absorbance at 240 nm of the wild-type cutinase and mutant D204C/E253C treatment residue.
  • Figure 6 shows the difference in wettability of PET fibers treated with different cutinase.
  • Figure 7 is the electrophotograph of PET fiber under different treatment conditions; among them, (A) and (B) are the electron micrographs of PET fiber treated with buffer at 50°C; (C) and (D) are wild-type cutinase at 50°C Electron micrographs of treated PET fibers; Figures (E) and (F) are the electron micrographs of PET fibers treated with 80°C buffer solution; (H) and (G) are the PET fibers treated with mutant D204C/E253C at 80°C Electron micrograph.
  • Figure 8 shows the construction process of the recombinant plasmid pSCDsbC-D204C/E253C.
  • Figure 9 shows the double restriction digestion verification of the recombinant plasmid pSCDsbC-D204C/E253C; M: 5000bp DNA marker; 1: Sample.
  • Figure 10 shows the expression of recombinant bacteria E.coli BL21(DE3)/pET20b-D204C/E253C and E.coli Origami B(DE3)/pSCDsbC-D204C/E253C;
  • A pET20b-D204C/E253C in the host E.coli BL21 (DE3) expression;
  • B pSCDsbC-D204C/E253C expression in host E.coli Origami B (DE3);
  • M protein Marker molecular weight standard (10-200kDa); lanes 1, 2 and 3 represent cells, respectively Outer supernatant, broken wall supernatant, broken wall sedimentation.
  • Enzyme activity determination method use continuous spectrophotometry to determine cutinase activity.
  • the reaction system is 1.5mL, including 30 ⁇ L of appropriately diluted enzyme solution and 1470 ⁇ L of 10mmol ⁇ L -1 Tris-HCl buffer (pH8.0) containing 50mmol ⁇ L -1 sodium sulfodeoxycholate and 50mmol ⁇ L -1 pNPB, The production rate of p-nitrophenol was recorded at 405nm.
  • Enzyme activity definition At 37°C, the amount of enzyme required to hydrolyze p-nitrophenyl butyrate to produce 1 ⁇ mol p-nitrophenol per minute is one unit of enzyme activity.
  • Thermal stability analysis method properly dilute the enzyme solution with a buffer of 10mmol ⁇ L -1 Tris-HCl (pH 8.0), and then incubate it at 90°C for 10 minutes to accurately determine the enzymes in the enzyme solution before and after the incubation.
  • the cutinase activity before the incubation is expressed as 100%, and the residual enzyme activity of the cutinase after the incubation is calculated using this as a standard to determine the thermal stability of the cutinase at 90°C.
  • Site-directed mutagenesis Using rapid PCR technology, based on the cutinase gene sequence of Thermobifida fusca (NCBI database accession number: AAZ54921.1), and the plasmid pET20b(+) carrying the cutinase gene -cut and pET24a(+)-cut are templates (plasmid is disclosed in the paper "Gene Identification, High-efficiency Expression and Molecular Modification of Thermomonas Cutinase”), respectively, design primers and synthesize site-directed mutation of cutinase gene ( The mutated bases are underlined).
  • the site-directed mutagenesis primers for introducing the sequence T61C mutation are:
  • Reverse primer CCAGGCGATGGAAGCCTCAGTGCCACAGTAGCCGGGGGA
  • the site-directed mutagenesis primers for introducing the sequence T89C mutation are:
  • Reverse primer TGCCCGGCTGTCCGGCTGGTCGAGACAGGTGATGGTGTC
  • the site-directed mutagenesis primers for introducing the sequence D204C mutation are:
  • Reverse primer GTTCGGGGCGAAGTGGGTTGCGCC ACA CAGCTCCAGGTA
  • the primers for site-directed mutagenesis to introduce mutations in sequence E253C are:
  • Reverse primer GAACGGGCAGGTGGAGCGGTACTC ACA GACCTCGCCGAA
  • the site-directed mutagenesis primers for introducing the sequence D204C mutation are:
  • Reverse primer CGGGGCAAAATGGGTGGCACCACACAGTTCCAGATAGGC
  • the primers for site-directed mutagenesis to introduce mutations in sequence E253C are:
  • Reverse primer GCTGCGGTATTCACACACTTCGCCGAACAG
  • PCR reaction systems are: 5 ⁇ PS buffer 10 ⁇ L, dNTPs Mix (2.5mM) 4 ⁇ L, forward primer (10 ⁇ M) 1 ⁇ L, reverse primer (10 ⁇ M) 1 ⁇ L, template DNA 1 ⁇ L, Prime STAR HS DNA polymerase (5U/ ⁇ L) 0.5 ⁇ L, add double distilled water to 50 ⁇ L.
  • PCR amplification conditions were: 94°C pre-denaturation for 4 minutes; followed by 30 cycles (98°C for 10s, 58°C for 5s, 72°C for 6min); 72°C for 10 minutes of extension.
  • the PCR product was digested by Dpn I (Fermentas) and transformed into E. coli JM109 competent cells.
  • DEAE-Sepharose FF anion exchange column was pre-equilibrated with buffer A and loaded, and then washed with buffer A to remove unbound components, and then mixed with buffer A and buffer B (buffer A containing 1M NaCl) Linear gradient elution, the flow rate of the whole process is 1mL/min, the detection wavelength is 280nm, the eluate with absorption peak is collected, and the cutinase activity and protein electrophoresis are detected.
  • the enzyme activity components are separated and purified by monoQ pre-packed anion exchange column under similar conditions as above, and finally purified products are obtained through cutinase activity and protein electrophoresis detection (as shown in Figure 2).
  • the cutinase enzyme activity was measured every 10°C within the range of 20 ⁇ 90°C to determine the optimum temperature of the enzyme, and the highest enzyme activity was counted as 100%. Calculate the relative enzyme activity at each temperature. The results were shown at 70 °C, wild and mutant enzymes were the highest in 3 enzymatic activity, can be up to 56U ⁇ mL -1 and 9U ⁇ mL -1, respectively; at 90 °C, the mutant enzyme relative activity It was 92.2%, and the wild enzyme was 8.7%.
  • the mutant D204C/E253C was incubated at 80°C and 90°C, and samples were taken regularly to determine the residual enzyme activity to determine the thermal stability of the enzyme.
  • the results showed that the wild-type cutinase was inactivated after being incubated at 80°C and 90°C for 10 minutes, and the residual enzyme activity was 10.7% after being incubated at 70°C for 10 minutes.
  • mutant D204C/E253C can reach 16h at 80°C, and it has 55.6% activity even if it is incubated at 90°C for 10min ( Figure 4). This indicates that the thermal stability of mutant D204C/E253C at 80°C and 90°C is good.
  • Enzyme modification 1g of polyester fabric (PET fiber) was shaken and washed in a 60°C water bath for 30 minutes, and then placed in a phosphate buffer (pH 7.0) at a bath ratio of 1:40. Take the cutinase or cutinase mutant fermentation culture supernatant prepared in Example 1 with an enzyme content of 400 U into the above-mentioned treatment solution, so that the final enzyme concentration of cutinase is 10 U/mL.
  • a fiber penetrant Triton X-100 with a final concentration of 1% is also added to the enzymatic hydrolysis system. The entire system was sealed and placed in a water bath constant temperature shaker for 24h. After the treatment, the fabric was taken out and washed thoroughly with distilled water at 60°C.
  • UV absorbance test polyester and PET film hydrolyzate Dilute the wild-type cutinase and mutant treatment residue 10 times. Use an ultraviolet spectrophotometer to test the ultraviolet absorbance value of the cutinase treatment residue at 240 nm under different treatment times. The blank sample is the cutinase treatment solution without adding polyester fiber under the same conditions.
  • FIG. 7 (A) and (B) are electron micrographs of PET fiber treated with buffer at 50°C. It can be seen from the picture that the surface of the PET fiber is clear and smooth.
  • Figure 7 (C) and (D) are electron micrographs of wild-type cutinase treated at 50°C. It can be seen that there are obvious etching marks on the surface of the fiber.
  • Figure 7 (E) and (F) are electron micrographs of PET fiber treated with 80°C buffer solution.
  • Figure 7 (H) and (G) are electron micrographs of mutant D204C/E253C treated at 80°C. It can be seen that there are obvious etching marks on the surface of the fiber, and the PET fiber surface etching marks treated by mutant D204C/E253C More than wild-type cutinase, more obvious. This indicates that the modification effect of mutant D204C/E253C on PET fiber is more effective than wild-type cutinase.
  • Example 4 Construction and highly soluble expression of recombinant strain E. coli Origami B(DE3)/pSCDsbC-D204C/E253C.
  • the plasmid pET-20b(+)-D204C/E253C was digested with double enzymes, and the gel was recovered.
  • the recovered mutant D204C/E253C genes were respectively ligated to the cloning vector pSCDsbC at 16°C, and then transferred to E.coli JM109, and plated After overnight culture, a single colony was picked and cultured for about 10 hours, and the two recombinant plasmids were extracted separately for restriction enzyme digestion verification.
  • the detailed construction process is shown in Figure 8.
  • the plasmid pSCDsbC-D204C/E253C was extracted, and the plasmid pSCDsbC-D204C/E253C was transformed into E. coli Origami B (DE3) competent. Then the competent cells were coated on an ampicillin-resistant LB plate, and the monoclonal colonies were picked to 10mL LB liquid medium, 8-10h after culturing, 5% inoculum transferred to 50mL of ampicillin-resistant TB medium, 37 [deg.] C to 1.5 OD 600 culture, the final concentration of 0.4mmol ⁇ L - 1 IPTG was cooled to 25°C at the same time to induce expression for 24h. The result of SDS-PAGE identification is shown in Figure 10.
  • the mutant pET20b-D204C/E253C is in host E.coli BL21(DE3).
  • a large number of inclusion bodies ( Figure 1(B)) will be formed when expressed in D204C/E253C. This is due to the abnormal folding of the mutant D204C/E253C, which leads to the formation of intermolecular disulfide bonds.
  • the mutants are large in the form of dimers. Aggregate to form inclusion bodies.
  • the expression of mutant pET20b-D204C/E253C in the host E is due to the abnormal folding of the mutant D204C/E253C.
  • Lane 1 has two obvious bands between 25-35kD, each with a molecular weight of 28.2. KD cutinase mutant D204C/E253C and DsbC with a molecular weight of 25kD, the presence of DsbC can isomerize the intermolecular disulfide bonds generated by incorrect oxidation. The results proved that the co-expression of mutant D204C/E253C and molecular chaperonin DsbC in E. coli Origami B (DE3) resulted in the correct folding and expression of mutant D204C/E253C.
  • the recombinant strain E.coli Origami B(DE3)/pSCDsbC-D204C/E253C was fermented in TB medium at 25°C for 24 hours, the cutinase mutants in the fermentation broth were collected, and the expression was obtained according to the method in Example 3.
  • the thermostability of the extracellular enzymes was determined, and the results are shown in Table 1.
  • E.coli BL21(DE3) By combining the activity of wild-type T.fusca cutinase in E.coli BL21(DE3) and the mutant D204C/E253C in two expression systems (E.coli BL21(DE3), E.coli Origami B(DE3) )
  • E.coli Origami B(DE3) the enzyme activity of E. coli Origami B(DE3)/pSCDsbC-D204C/E253C is 6.8 times that of E. coli BL21(DE3)/pET20b-D204C/E253C, while at 90
  • the thermal stability at °C is basically the same as E.coli BL21(DE3)/pET20b-D204C/E253C.
  • the enzyme activity was measured after the cells were disrupted, and the residual enzyme activity after incubation at 70°C for 10 minutes was measured. The results are shown in Table 3.

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Abstract

提供了一种热稳定性强的角质酶突变体，其是在SEQ ID NO.1所示的角质酶的基础上将第61位和第89位突变成半胱氨酸获得氨基酸序列如SEQ ID NO.2所示的角质酶突变体，或者是在SEQ ID NO.1所示的角质酶的基础上，将第204位谷氨酸和第253位天冬氨酸突变成半胱氨酸获得的氨基酸序列如SEQ ID NO.3所示的角质酶突变体。还提供了编码该角质酶突变体的基因，携带该基因的表达载体，表达该角质酶突变体的微生物细胞，可溶性表达该角质酶突变体的方法，以及该角质酶突变体在化工或纺织领域的应用和利用该角质酶突变体对PET纤维或其织物的改性方法。

Description

一种Thermobifida fusca角质酶突变体及其可溶性表达的方法 技术领域
本发明涉及一种Thermobifida fusca角质酶突变体及其可溶性表达的方法，属于酶工程技术领域。
背景技术
Thermobifida fusca角质酶属于α/β水解酶家族，β折叠位于中心，四周被α螺旋及loop区域包围，形成三明治结构，活性中心由Ser-His-Asp催化三角组成，是一种多功能酶，能够水解大分子聚酯、不溶性的甘油三酯和小分子可溶性酯类物质。此外，T.fusca角质酶还可以催化多种酸和醇的酯化反应，以及酯和醇的酯交换反应。角质酶与脂肪酶特征性的区别在于角质酶的活性中心无“盖子”结构覆盖，催化关键氨基酸Ser暴露于溶剂，因此部分角质酶也可以水解高分子量的聚酯PET(Poly ethylene terephthalate)，但PET高度结晶，降低了角质酶对PET中聚酯链的可及性，严重影响角质酶的催化效率，在达到玻璃态转变温度(69-80℃)后，PET的分子链运动加剧，会形成许多空隙，增大和酶分子活性中心之间的可及性，催化效果愈加明显。
现有技术中的T.fusca角质酶无法在PET的玻璃态转变温度条件下保持活性。因此，提高T.fusca角质酶热稳定性是实现工业化生产亟待解决的问题。
发明内容
本发明的一个目的是提供一种热稳定性强的角质酶突变体，在SEQ ID NO.1所示的角质酶的基础上，对以下至少一个位点的氨基酸进行突变：第61位、第89位、第204位、第253位。
在一种实施方式中，所述角质酶突变体为(a)或(b)：
(a)在SEQ ID NO.1所示的角质酶的基础上，将第61位和第89位突变成半胱氨酸，获得的突变体的氨基酸序列如SEQ ID NO.2所示；
(b)在SEQ ID NO.1所示的角质酶的基础上，将第204位谷氨酸和第253位天冬氨酸突变成半胱氨酸，获得的突变体的氨基酸序列如SEQ ID NO.3所示。
本发明的第二个目的是提供编码所述角质酶突变体的基因。
在一种实施方式中，编码突变体D204C/E253C的基因含有SEQ ID NO.4所示的核苷酸序列。
本发明的第三个目的是提供携带所述基因的表达载体。
在一种实施方式中，所述表达载体为pET系列的质粒。
在一种实施方式中，所述表达载体为pSCDsbC，其核苷酸序列如SEQ ID NO.5所示。
本发明的第四个目的是提供表达所述角质酶突变体的微生物细胞。
在一种实施方式中，所述微生物细胞为大肠杆菌。
在一种实施方式中，所述大肠杆菌为E.coli BL21、E.coli BL21(DE3)、E.coli JM109、E.coli DH5α或E.coli TOP10。
本发明的第五个目的是提供一种可溶性表达所述角质酶突变体的方法，是将所述角质酶突变体与周质蛋白二硫键氧化还原酶DsbC共表达。
在一种实施方式中，所述周质蛋白二硫键氧化还原酶DsbC的氨基酸序列如SEQ ID NO.6所示。
在一种实施方式中，所述方法是将编码角质酶突变体的基因与编码所述周质蛋白二硫键氧化还原酶的基因分别与载体连接，转化至微生物细胞中表达。
在一种实施方式中，所述方法还在基因的上游添加核糖体结合位点的RBS序列。
在一种实施方式中，所述方法以质粒pSC为表达载体，以E.coli Origami B(DE3)为宿主，共表达角质酶突变体与周质蛋白二硫键氧化还原酶。
在一种实施方式中，所述质粒pSC的核苷酸序列如SEQ ID NO.7所示。
本发明的第六个目的是提供所述角质酶突变体在化工或纺织领域的应用。
在一种实施方式中，所述应用是对PET纤维或其织物进行改性。
在一种实施方式中，所述改性是将所述角质酶突变体以10U/g底物的剂量对PET纤维进行酶解反应，浴比为1:40；所述酶解反应在80℃下进行。
在一种实施方式中，所述酶解反应中还添加终浓度为0.5～2％的Triton X-100。
有益效果：
1、本发明选择催化三角附近的第204位谷氨酸和第253位天冬氨酸突变成半胱氨酸，以引入一对二硫键，稳定催化三角；将第61位和第89位突变成半胱氨酸，形成一对二硫键稳定角质酶的N端结构域，使得角质酶突变体具有较高的热稳定性。突变体可在PET的玻璃化转变温度(Tg)的反应条件下催化PET纤维酯键水解，以达到亲水化改性的目的。
2、本发明针对因二硫键的引入，导致突变体D204C/E253C在表达过程中易错误折叠形成大量包涵体，可溶表达比率极低的缺陷，提供了一种可溶性表达突变体D204C/E253C的方法。通过将突变体D204C/E253C和周质蛋白二硫键氧化还原酶DsbC在大肠杆菌突变株E.coli Origami B(DE3)中共表达，来异构化异常的二硫键，实现了高效可溶性表达。
附图说明
图1为不同蛋白的SDS-PAGE图；其中，M，蛋白质分子量标准；(A)中的泳道1～3分别为重组菌E.coli BL21(DE3)/pET-20b(+)-cut的胞外上清、破壁上清、破壁沉淀，泳道4～6分别为重组菌E.coli BL21(DE3)/pET-20b(+)-T61C/T89C的胞外上清、破壁上清、破壁沉淀；(B)中的泳道1～3分别为重组菌E.coli BL21(DE3)/pET-20b(+)-D204C/E253C的胞外上清，破壁上清，破壁沉淀。
图2为纯化后的野生型角质酶与突变体的SDS-PAGE图；其中，M，蛋白质分子量标准；泳道2为野生型T.fusca角质酶，泳道3、4均为突变体T61C/T89C，泳道5、6均为突变体D204C/E253C。
图3为不同温度下角质酶及突变体的酶活；其中，WT为野生酶。
图4为突变体D204C/E253C分别在80℃(A)和90℃(B)下的热稳定性。
图5为野生型角质酶和突变体D204C/E253C处理残液在240nm处的吸光值。
图6为用不同角质酶处理后的PET纤维润湿性性差异。
图7为不同处理条件下PET纤维的电照片；其中，(A)、(B)是50℃缓冲液处理的PET纤维的电镜照片；(C)、(D)是50℃下野生型角质酶处理的PET纤维的电镜照片；图(E)、(F)是80℃缓冲液处理的PET纤维的电镜照片；(H)、(G)是80℃下突变体D204C/E253C处理的PET纤维的电镜照片。
图8为重组质粒pSCDsbC-D204C/E253C的构建过程。
图9为重组质粒pSCDsbC-D204C/E253C的双酶切验证；M：5000bp DNA marker；1：样品。
图10为重组菌E.coli BL21(DE3)/pET20b-D204C/E253C及E.coli Origami B(DE3)/pSCDsbC-D204C/E253C的表达；(A)pET20b-D204C/E253C在宿主E.coli BL21(DE3)中的表达；(B)pSCDsbC-D204C/E253C在宿主E.coli Origami B(DE3)中的表达；M:蛋白Marker分子量标准(10-200kDa)；泳道1,2,3分别代表胞外上清,破壁上清,破壁沉淀。
具体实施方式
酶活测定方法：采用连续分光光度法测定角质酶活力。反应体系为1.5mL，包括30μL适当稀释的酶液和1470μL含50mmol·L -1硫磺脱氧胆酸钠和50mmol·L -1pNPB的10mmol·L -1Tris-HCl缓冲液(pH8.0)，在405nm处记录对硝基酚的生成速率。
酶活定义：在37℃，每分钟水解对硝基苯丁酸酯生成1μmol对硝基酚所需要的酶量为一个酶活力单位。
热稳定性分析方法：将酶液用10mmol·L -1 Tris-HCl(pH8.0)的缓冲液进行适当的稀释，然后在90℃的条件下保温10min，分别精确测定保温前后酶液的酶活，在分析过程中，将保温前的角质酶酶活表示为100％，以此为标准来计算角质酶在保温后的残留酶活，以确定角质酶在90℃条件下的热稳定性。
实施例1突变体酶及野生酶的制备。
(1)定点突变：利用快速PCR技术，根据嗜热单孢菌(Thermobifida fusca)的角质酶的基因序列(NCBI数据库登录号：AAZ54921.1)，以携带有角质酶基因的质粒pET20b(+)-cut和pET24a(+)-cut为模板(质粒公开于论文《嗜热单孢菌角质酶的基因鉴定、高效表达及分子改造》中)，分别设计引物并合成对角质酶基因进行定点突变(下划线为突变的碱基)。
(a)以携带有角质酶基因的质粒pET20b/cut为模板：
引入序列T61C突变的定点突变引物为：
正向引物：GCGGTGGCGATCTCCCCCGGCTACTGTGGCACTGAGGCT
反向引物：CCAGGCGATGGAAGCCTCAGTGCCACAGTAGCCGGGGGA
引入序列T89C突变的定点突变引物为：
正向引物：GTCATCACCATCGACACCATCACCTGTCTCGACCAGCCG
反向引物：TGCCCGGCTGTCCGGCTGGTCGAGACAGGTGATGGTGTC
引入序列D204C突变的定点突变引物为：
正向引物：ATCAGCAAGGCCTACCTGGAGCTG TGTGGCGCAACCCAC
反向引物：GTTCGGGGCGAAGTGGGTTGCGCC ACACAGCTCCAGGTA
引入序列E253C突变的定点突变引物为：
正向引物：CGCGACGGACTCTTCGGCGAGGTC TGTGAGTACCGCTCC
反向引物：GAACGGGCAGGTGGAGCGGTACTC ACAGACCTCGCCGAA
(b)以携带有角质酶基因的质粒pET24a/cut为模板：
引入序列D204C突变的定点突变引物为：
正向引物：GCCTATCTGGAACTGTGTGGTGCCACCCATTTTGCCCCG
反向引物：CGGGGCAAAATGGGTGGCACCACACAGTTCCAGATAGGC
引入序列E253C突变的定点突变引物为：
正向引物：CTGTTCGGCGAAGTGTGTGAATACCGCAGC
反向引物：GCTGCGGTATTCACACACTTCGCCGAACAG
PCR反应体系均为：5×PS buffer 10μL，dNTPs Mix(2.5mM)4μL，正向引物(10μM)1μL，反向引物(10μM)1μL，模板DNA 1μL，Prime STAR HS DNA polymerase(5U/μL)0.5 μL，加入双蒸水至50μL。PCR扩增条件均为：94℃预变性4min；随后进行30个循环(98℃10s，58℃5s，72℃6min)；72℃继续延伸10min。PCR产物经Dpn I(Fermentas公司)消化，转化大肠杆菌JM109感受态细胞，感受态细胞在LB固体培养基(含100μg/mL氨苄/卡娜青霉素)培养过夜后，挑单克隆于LB液体培养基(含100μg/mL氨苄/卡娜青霉素)中培养，后提取质粒，测序。
(2)突变体酶与野生酶的表达与纯化：将测序为正确突变的质粒(对于野生酶，直接用模版质粒)转化表达宿主大肠杆菌BL21(DE3)感受态细胞，挑取转入表达宿主大肠杆菌BL21(DE3)的单克隆于LB液体培养基(含100μg/mL氨苄青霉素)生长8～10h，按4％接种量将种子发酵液接到TB液体培养基(含100μg/mL氨苄青霉素)；大肠杆菌在37℃摇床培养至OD 600＝0.6～0.8，加入0.01mM终浓度的IPTG诱导胞外表达，并在25℃摇床继续培养发酵48小时后，将发酵液于4℃、10000rpm离心15min除菌体，收集上清液。
将按照上述方法获得的上清液中加入70％(w/v)的固体硫酸铵，于4℃条件下盐析过夜后，10,000rpm离心20min，取沉淀物加入适量的缓冲液A(20mmol/L Tris-HCl，pH8.0)溶解，并在缓冲液A中透析过夜。将透析后的样品于12,000rpm离心20min，再经过0.45μm膜过滤后制成上样样品。DEAE-Sepharose FF阴离子交换柱用缓冲液A预平衡后上样，然后用缓冲液A洗去未结合组分后，用缓冲液A和缓冲液B(含1MNaCl的缓冲液A)的混合液进行线性梯度洗脱，全程流速为1mL/min，检测波长为280nm，收集有吸收峰的洗脱液，进行角质酶活性和蛋白电泳检测。将酶活力组分再经monoQ预装阴离子交换柱以上述类似的条件分离纯化，经角质酶活性和蛋白电泳检测最终获得纯化制品(如图2所示)。
实施例2野生型角质酶及其突变体的热稳定性分析
采用10mmol/L Tris-HCl(pH8.0)缓冲液，在20～90℃范围内每间隔10℃测定角质酶酶活，以确定酶的最适温度，将酶活最高的计为100％，计算各温度下的相对酶活。结果如图3所示，在70℃时，野生酶和突变酶的酶活均最高，可分别达56U·mL -1和9U·mL -1；温度为90℃时，突变酶的相对酶活为92.2％，野生酶为8.7％。
在10mmol/L Tris-HCl(pH8.0)缓冲液中，将突变体D204C/E253C分别置于80℃和90℃下保温，定期取样测定残留酶活，以确定酶的热稳定性。结果显示，野生型角质酶在80℃以及90℃条件下保温10min后均已失活，在70℃保温10min后残留酶活为10.7％。
突变体D204C/E253C在80℃条件下的半衰期可达16h,即使在90℃保温10min仍具有55.6％的活性(图4)。这表明突变体D204C/E253C在80℃和90℃的热稳定性良好。
实施例3角质酶用于PET纤维的改性处理
(1)酶改性：将1g涤纶织物(PET纤维)在60℃水浴振荡清洗30min后，按浴比1:40放入磷酸盐缓冲液(pH7.0)中。取酶含量为400U的实施例1制备的角质酶或角质酶突变体的发酵培养液上清至上述处理液中，使角质酶最终酶浓度为10U/mL。可选地，还向酶解体系中加入终浓度为1％的纤维渗透剂Triton X-100。将整个体系密封并置于水浴恒温振荡器中处理24h。处理完成后将织物取出用60℃蒸馏水充分洗涤。
(2)紫外吸光度测试涤纶和PET薄膜的水解产物：将野生型角质酶和突变体处理残液稀释10倍。用紫外分光光度计测试角质酶处理残液在不同处理时间下240nm处的紫外吸光度值。空白样为相同条件下不加入涤纶纤维的角质酶处理液。
在80℃条件下，突变体D204C-E253C处理残液在240nm的吸光值是突变体D204C-E253C在50℃条件下的3.13倍，野生型在50℃条件下的3.24倍(图5)。
(3)润湿性：将经过角质酶处理后的PET纤维清洗干净后放入烘箱进行烘干，烘干后的PET纤维平铺开放置，然后将PET纤维在温度25℃、相对湿度为65％的恒温恒湿条件下平衡24h。然后用移液枪吸取20μL的去离子水滴于PET纤维上，在同一块PET纤维上需要连续滴9个不同的区域，在此过程中分别记录下从水滴刚接触到PET纤维开始到水滴完全浸入PET纤维为止所用的时间，然后将这些数据取平均值。结果如图6所示，在Triton X-100的存在下，突变体D204C/E253C处理的PET纤维润湿性相比于野生型T.fusca角质酶提高了1.62倍。
(4)SEM分析酶处理引起的PET纤维表面微观形态变化：酶处理引起PET纤维表面微观形态发生改变。经过酶处理的PET纤维用SEM拍照，纤维表面形态如图7所示。图7(A)、(B)是50℃缓冲液处理的PET纤维电镜照片，由图可以看到PET纤维表面清晰光滑。图7(C)、(D)是野生型角质酶在50℃条件下处理后的电镜照片，可以看出纤维表面有明显的蚀刻痕迹。图7(E)、(F)是80℃缓冲液处理的PET纤维电镜照片，由图可以看到PET纤维表面清晰光滑。图7(H)、(G)是突变体D204C/E253C在80℃条件下处理后的电镜照片，可以看出纤维表面有明显的蚀刻痕迹,并且突变体D204C/E253C处理的PET纤维表面蚀刻痕迹比野生型角质酶更多，更加明显。这表明突变体D204C/E253C对PET纤维的改性效果比野生型角质酶更加有效。
实施例4重组菌E.coli Origami B(DE3)/pSCDsbC-D204C/E253C的构建及高可溶性表达。
(1)重组菌E.coli Origami B(DE3)/pSCDsbC-D204C/E253C的构建
将质粒pET-20b(+)-D204C/E253C双酶切，并进行胶回收，回收得到的突变体D204C/E253C基因在16℃分别连接至克隆载体pSCDsbC，然后转入E.coli JM109，涂板过夜培养后挑取单 菌落培养10h左右，分别提取这两种重组质粒进行酶切验证，详细构建过程如图8。
经酶切验证，电泳出现两条带，一条在783bp左右，为突变体D204C/E253C基因，另一条在6001bp左右，为pSCDsbC载体(图9)。将酶切验证正确的质粒送去测序，测序结果正确，因此，构建成功。
(2)重组菌E.coli Origami B(DE3)/pSCDsbC-D204C/E253C的表达
提取质粒pSCDsbC-D204C/E253C，将质粒pSCDsbC-D204C/E253C转化至E.coli Origami B(DE3)感受态中，然后将上述感受态细胞涂布氨苄抗性的LB平板，挑取单克隆菌落到10mL LB液体培养基中，培养8-10h后，以5％的接种量转接到50mL氨苄抗性的TB培养基中，37℃培养OD 600至1.5时，加入终浓度为0.4mmol·L -1的IPTG同时降温至25℃诱导表达24h。经SDS-PAGE鉴定，结果如图10所示。
经电泳分析，与野生型角质酶pET20b-cut在宿主E.coli BL21(DE3)中的表达相比(图1(A))，突变体pET20b-D204C/E253C在宿主E.coli BL21(DE3)中的表达时会形成大量的包涵体(图1(B))，这是由于突变体D204C/E253C的非正常折叠，导致了分子间二硫键的形成，突变体以二聚体的形式大量聚集形成包涵体。另外，突变体pET20b-D204C/E253C在宿主E.coli Origami B(DE3)中的表达情况与在宿主E.coli BL21(DE3)中一致。突变体pSCDsbC-D204C/E253C在宿主E.coli Origami B(DE3)中可溶性表达大幅度提高(图10)，其中1泳道在25-35kD之间有两条明显的条带，分别是分子量为28.2kD的角质酶突变体D204C/E253C和分子量为25kD的DsbC，DsbC的存在可使错误氧化生成的分子间二硫键异构化。结果证明了将突变体D204C/E253C与分子伴侣蛋白DsbC在大肠杆菌突变株E.coli Origami B(DE3)中的共表达，使得突变体D204C/E253C得以正确的折叠表达。
将重组菌E.coli Origami B(DE3)/pSCDsbC-D204C/E253C在TB培养基中，于25℃条件下发酵24h，收集发酵液中的角质酶突变体，按照实施例3的方法对表达获得的胞外酶的热稳定性进行测定，结果如表1所示。
表1重组菌E.coli Origami B(DE3)/pSCDsbC-D204C/E253C在25℃条件下摇瓶发酵24h后的胞外酶的酶活及90℃条件下的热稳定性
Figure PCTCN2020095350-appb-000001
通过将野生型T.fusca角质酶在E.coli BL21(DE3)中的酶活和突变体D204C/E253C分别在两种表达系统(E.coli BL21(DE3)，E.coli Origami B(DE3))中的酶活进行比较(表1)，E.coli Origami B(DE3)/pSCDsbC-D204C/E253C的酶活是E.coli BL21(DE3)/pET20b-D204C/E253C的6.8倍，而在90℃条件下的热稳定性与E.coli BL21(DE3)/pET20b-D204C/E253C基本持平。
对比例 不同位点的角质酶突变体的构建
按照实施例1相同的策略，分别对不同位点进行突变，并将突变后的角质酶按照实施例1的方法表达。引物如下：
表2 不同突变位点及引物
Figure PCTCN2020095350-appb-000002
Figure PCTCN2020095350-appb-000003
破碎细胞后测定酶活，并在70℃下测定保温10min后的残留酶活，结果如表3所示。
表3 不同突变酶的酶活及热稳定性
Figure PCTCN2020095350-appb-000004
注:a,未检测出突变体对pNPB的活性；b,无残留酶活。
虽然本发明已以较佳实施例公开如上，但其并非用以限定本发明，任何熟悉此技术的人，在不脱离本发明的精神和范围内，都可做各种的改动与修饰，因此本发明的保护范围应该以权利要求书所界定的为准

Claims (19)

  1. 一种热稳定性强的角质酶突变体，其特征在于，氨基酸序列如SEQ ID NO.3所示。
  2. 一种热稳定性强的角质酶突变体，在SEQ ID NO.1所示的角质酶的基础上，对以下至少一个位点的氨基酸进行突变：第61位、第89位、第204位、第253位。
  3. 根据权利要求2所述的角质酶突变体，其特征在于，所述角质酶突变体为(a)或(b)：
    (a)在SEQ ID NO.1所示的角质酶的基础上，将第61位和第89位突变成半胱氨酸，获得的突变体的氨基酸序列如SEQ ID NO.2所示；
    (b)在SEQ ID NO.1所示的角质酶的基础上，将第204位谷氨酸和第253位天冬氨酸突变成半胱氨酸，获得的突变体的氨基酸序列如SEQ ID NO.3所示。
  4. 编码权利要求1～3任一所述角质酶突变体的基因。
  5. 根据权利要求4所述的基因，其特征在于，编码突变体D204C/E253C的基因含有SEQ ID NO.4所示的核苷酸序列。
  6. 携带权利要求4或5所述基因的表达载体。
  7. 根据权利要求6所述的表达载体，其特征在于，所述表达载体为pET系列的质粒。
  8. 根据权利要求6所述的表达载体，其特征在于，所述表达载体为pSCDsbC，其核苷酸序列如SEQ ID NO.5所示。
  9. 表达所述角质酶突变体的微生物细胞。
  10. 根据权利要求9所述的微生物细胞，其特征在于，所述微生物细胞为大肠杆菌。
  11. 根据权利要求10所述的微生物细胞，茄子在于，所述大肠杆菌为E.coli BL21、E.coli BL21(DE3)、E.coli JM109、E.coli DH5α或E.coli TOP10。
  12. 一种可溶性表达所述角质酶突变体的方法，其特征在于，将权利要求1～3任一所述的所述角质酶突变体与周质蛋白二硫键氧化还原酶DsbC共表达。
  13. 根据权利要求12所述的方法，其特征在于，所述周质蛋白二硫键氧化还原酶DsbC的氨基酸序列如SEQ ID NO.6所示。
  14. 根据权利要求12所述的方法，其特征在于，所述方法是将编码角质酶突变体的基因与编码所述周质蛋白二硫键氧化还原酶的基因分别与载体连接，转化至微生物细胞中表达。
  15. 根据权利要求14所述的方法，其特征在于，所述方法还在基因的上游添加核糖体结合位点的RBS序列。
  16. 根据权利要求12所述的方法，其特征在于，以质粒pSC为表达载体，以E.coli Origami B(DE3)为宿主，共表达角质酶突变体与周质蛋白二硫键氧化还原酶。
  17. 根据权利要求16所述的方法，其特征在于，所述质粒pSC的核苷酸序列如SEQ ID NO.7所示。
  18. 权利要求1～3任一所述的角质酶突变体在化工或纺织领域的应用。
  19. 一种对PET纤维或其织物的改性方法，其特征在于，将所述角质酶突变体以≥10U/g底物的剂量对PET纤维在50～80℃进行酶解。
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