CN102260655B - Cutinase mutants and preparation method thereof - Google Patents

Abstract

The present invention provides a mutant of cutinase from Thermobifida fusca and a preparation method thereof. The mutant includes one or two cutinase active center sites corresponding to Thermobifida fusca The substitution of nearby amino acid residues, the catalytic efficiency of I218A, Q132A/T101A and W195A/F249A on cotton fiber was improved, and the catalytic efficiency of mutant I218A on cutin was 50% higher than that of the original cutinase; Q132A/T101A on synthetic The catalytic efficiency of fiber ethylene terephthalate (PET) is 3 times higher than that of pro-cutinase. These three mutants have broad application prospects in the pretreatment of textile fibers.

Description

A mutant of cutinase and its preparation method

technical field

The present invention relates to an enzyme mutant and a preparation method thereof, in particular to a cutinase mutant and a preparation method thereof. A technique for the substrate specificity of thermocutinases. the

Background technique

Cutinases are hydrolytic enzymes capable of hydrolyzing macromolecules of cutin. As a multifunctional enzyme, it has been widely used in many fields such as textile industry, food industry and chemical industry. Especially in the textile industry, cutinase can be used in cotton fiber bioscouring to remove wax and cutin on the surface of cotton fiber, and help to further remove impurities such as pectin and protein, so as to achieve the purpose of scouring. Studies in recent years have shown that cutinase can also be used to modify synthetic fiber polyethylene terephthalate (PET), increase fabric water absorption, improve hand feel, and improve fabric quality. Both of the above two processes currently adopt the traditional alkali treatment process, which has disadvantages such as high water consumption and energy consumption, strong alkalinity of discharged wastewater, deep color, high COD value, and great damage to fibers. Enzyme scouring process, as an energy-saving and environmentally friendly textile clean production technology, has become a new trend in the development of dyeing and finishing industries at home and abroad. the

A large number of studies on cutinase have been carried out abroad, mainly focusing on cutinase derived from fungi such as Fusarium solani. However, fungal cutinase has poor thermal stability and is not suitable for application in the textile industry. And the heat-resistant cutinase produced by Thermobifida fusca developed by our laboratory can effectively hydrolyze polyesters such as cutin and PET, but the hydrolysis efficiency is low (Chen Jian, Wu Jing, Chen Sheng. A kind of heat-resistant cutin Enzyme and its coding gene and expression. Application No. 200710026074.2). Therefore, it is extremely important to determine the mutation site by structural simulation and improve the catalytic efficiency of thermostable cutinase on cutin and PET by site-directed mutagenesis technology, which has strong industrial application value. the

Contents of the invention

A technical problem to be solved by the present invention is to provide a cutinase mutant, comprising one or two substitutions corresponding to the amino acid residues near the cutinase active center site of thermophilic actinomycete (Thermobifida fusca), and Compared with the parent cutinase, it has a stronger ability to hydrolyze cutin or PET. the

The cutinase-producing gene of Thermobifida fusca is very similar to the protein Tfu_0883 encoded by Thermobifida fusca in the NCBI database. The gene has a full length of 906 nucleotides and encodes 301 amino acids. 1 nucleotide difference, the coded amino acid is the same (Chen Jian, Wu Jing, Chen Sheng. A heat-resistant cutinase and its coding gene and expression. Application No. 200710026074.2). the

The cutinase mutant is 218-position amino acid isoleucine Ile is mutated into alanine Ala, named I218A. the

The cutinase mutant is a simultaneous mutation of the 132nd amino acid glutamine Gln and the 101st amino acid threonine Thr to alanine Ala, named Q132A/T101A. the

The cutinase mutant is a simultaneous mutation of the 195th amino acid tryptophan Trp and the 249th amino acid phenylalanine Phe to alanine Ala, named W195A/F249A. the

Another technical problem to be solved by the present invention is to provide a method for preparing the above-mentioned mutant. the

In order to solve the above problems, the technical scheme of the present invention is: determine the mutation site on the basis of the cutinase simulation structure of thermophilic actinomycete (Thermobifida fusca); design the mutation primer of site-directed mutation, take the carrier carrying the cutinase gene as template Conduct site-directed mutagenesis to construct mutant plasmids I218A-pet20b(+), W195/F249-pet20b(+), Q132A/T101A-pet20b(+); transform the mutant plasmids into Escherichia coli BL21(DE3) cells, and select positive cells after verification. The clones were fermented and cultured at a constant temperature of 25°C for 80 hours, and the supernatant was subjected to ammonium sulfate precipitation and ion exchange column purification of cutinase mutants I218A, Q132A/T101A and W195A/F249A. the

The carrier carrying cutinase is any one of pUC series, pET series, or pGEX. the

Beneficial effects of the present invention: the present invention provides a group of cutinase mutants with high fiber catalytic efficiency and a preparation method thereof. The catalytic efficiency of I218A, Q132A/T101A and W195A/F249A to cotton fibers has been improved, and the mutants The catalytic efficiency of I218A on cutin is 50% higher than that of original cutinase; the catalytic efficiency of Q132A/T101A on synthetic fiber polyethylene terephthalate (PET) is 3 times higher than that of original cutinase. These three mutants have important application value in the textile industry. the

Description of drawings

Figure 1 SDS-PAGE electrophoresis diagram of purified cutinase mutants from Thermobifida fusca. the

Lane 1: standard molecular weight protein;

Swimming lane 2: I218A fermentation broth;

Lane 3: I218A purified product;

Swimming lane 4: W195A/F249A fermentation broth;

Lane 5: W195A/F249A purified product;

Lane 6: Q132A/T101A fermentation broth;

Lane 7: Q132A/T101A purified preparation. the

Fig. 2 Analysis of cutinolysis activity of cutinase mutants from Thermobifida fusca. the

△: Thermobifida fusca cutinase; □: I218A; ■: W195A/F249A; ▲: Q132A/T101A

Figure 3 Analysis of PET hydrolysis activity of cutinase mutants from Thermobifida fusca. the

△: Thermobifida fusca cutinase; □: I218A; ■: W195A/F249A; ▲: Q132A/T101A

Detailed ways

Embodiment 1: the method for determining the mutation site of the cutinase mutant of thermophilic actinomycete (Thermobifida fusca). the

Polyesters such as cutin are hydrophobic macromolecules composed of a large number of fatty acids. The size of amino acids around the active center of cutinase affects the combination of polyester macromolecules and active centers. In addition, the hydrophobicity of amino acids affects the substrate binding site and substrate. Therefore, based on the cutinase mimic structure, starting from the above two aspects, the mutation site was determined and then the cutinase gene was subjected to site-directed mutation. Based on the analysis of the simulated structure of the cutinase of Thermobifida fusca, three sets of mutation series were designed: (1) The amino acid isoleucine I218 located directly above the active center of serine occupies a large space, which has a large impact on the active center and The combination of macromolecular substrates forms a large steric hindrance, so it is mutated into alanine with less steric hindrance to improve the affinity between the substrate and the active center; (2) Aromatic amino acids located on both sides of the serine active center Tryptophan W195 and phenylalanine F249 also take up a lot of space due to the presence of the benzene ring, so they are also mutated into alanine to reduce steric hindrance; (3) glutamine, a hydrophilic amino acid located near the active center Q132 and threonine T101 have poor binding ability to hydrophobic substrates, so they were mutated into hydrophobic amino acid alanine to increase the binding ability to hydrophobic substrates and improve catalytic efficiency. the

Embodiment 2: the preparation method of thermophilic actinomycete (Thermobifida fusca) cutinase mutant. the

I218A-pet20b(+), W195/F249-pet20b(+), Q132A/T101A-pet20b(+) mutant plasmids were constructed by rapid PCR site-directed mutagenesis. the

Construction of I218A-pet20b(+), W195-pet20b(+), Q132A-pet20b(+) mutant plasmids using the CUT-pet20b(+) plasmid as a template (Chen Jian, Wu Jing, Chen Sheng. A heat-resistant cutinase And its coding gene and expression. Application No. 200710026074.2), use the mutant primers of I218A, W195A and Q132A to obtain the product with a size of about 4500bp by PCR respectively, transform the Escherichia coli JM109 competent cells after the PCR product is treated with Dpn I, and select the Subsequence verification. the

The W195/F249-pet20b(+), Q132A/T101A-pet20b(+) mutant plasmids were constructed using the verified correct W195-pet20b(+), Q132A-pet20b(+) plasmids as templates, and F249A and T101A as mutation primers , PCR amplified to obtain a product with a size of about 4500bp, the PCR product was treated with Dpn I and transformed into Escherichia coli JM109 competent cells, and the transformants were selected for sequencing verification. the

Sequencing verification results showed that there were no random mutations except the required mutation sites, so the mutant plasmids I218A-pet20b(+), W195/F249-pet20b(+), and Q132A/T101A-pet20b(+) were successfully constructed. the

The mutation primers are as follows (the box is the mutation site):

1. I218A mutation primer pair:

GCGCCGTCGCCACG-3’  P I218A1: 5'-GCCGACCTCGACACG

GCGCCGTCGCCACG-3'

CGTGTCGAGGTC-3’  P I218A2: 5'-CGTGGCGACCGGCGC

CGTGTCGAGGTC-3'

2. W195A mutation primer pair:

CACCTCAACA AGAA C-3’  P W195A1: 5'-ATCCCGCTCACCCCG

CACCTCAACA AGAA C-3'

P W195A 2:

CGGGGTGAGCGGGATGG-3’  5'-GTTCTTGTTGAGGTG

CGGGGTGAGCGGGATGG-3'

3. F249A mutation primer pair:

P F249A1: 5'-gACGGCGCAACCCAC GCCCCGAACATCC-3'

GTGGGTTGCGCCGTC-3’  P F249A2: 5'-GGATGTTCGGGGC

GTGGGTTGCGCCGTC-3'

4. Q132A mutation primer pair:

CCGGACAGCCGGGC Ag-3’  P Q132A1: 5'-CATCACCACCCTCGAC

CCGGACAGCCGGGC Ag-3'

GTCGAGGGTGGTGATG-3’  P Q132A2: 5'-CTGCCCGGCTGTCCGG

GTCGAGGGTGGTGATG-3'

5. T101A mutation primer pair:

GGCACTGAGGCTTC-3’  P T101A1: 5'-GATCTCCCCCGGCTAC

GGCACTGAGGCTTC-3'

GTAGCCGGGGAGATC-3’  P T101A2: 5'-GAAGCCTCAGTGCC

GTAGCCGGGGAGATC-3'

The above PCR systems are:

The above PCR conditions are:

Pre-denaturation at 94°C for 4 minutes, followed by the following cycles: denaturation at 98°C for 10 seconds, annealing at 60°C for 5 seconds, extension at 72°C for 4 minutes; 30 cycles; extension at 72°C for 10 minutes, and incubation at 4°C. the

Embodiment 3: the expression purification method of thermophilic actinomycete (Thermobifida fusca) cutinase mutant. the

The mutant plasmids I218A-pet20b(+), W195/F249-pet20b(+), Q132A/T101A-pet20b(+) were transformed into E. coli BL21(DE3) cells, and the transformants were selected for sequencing verification. Put the verified positive transformants in TB medium (glycerol 5g/L, peptone 12g/L, yeast extract 24g/L, K ₂ HPO ₄ 12.54g/L, KH ₂ PO ₄ 2.31g/L) at 37°C Cultivate overnight, then insert into TB fermentation liquid medium and culture at 37°C for 3h, then induce with 4mg/L IPTG (isopropylthioβD-galactoside), cool down to 25°C and cultivate at constant temperature for 80h.

The fermentation broth was centrifuged at 10,000 rpm for 20 minutes at 4°C to remove bacteria. The supernatant was collected through an activated carbon column. Add 70% solid ammonium sulfate to the clear solution passing through the active carbon column for salting out overnight, centrifuge at 10000rpm for 20min at 4°C, dissolve the precipitate with an appropriate amount of buffer A (20mM Tris-HCl, pH 8), add 20% ammonium sulfate, Samples were prepared after filtration with a 0.22 μm membrane. After the Phenyl HP hydrophobic column is equilibrated with buffer A containing 20% ammonium sulfate, the loaded sample is sucked into the hydrophobic column to make it completely adsorbed, and the buffer A containing 20% ammonium sulfate and 20%-0% ammonium sulfate are used respectively. Gradient buffer A, buffer A and deionized water were used for elution, the flow rate was 1 mL/min, the detection wavelength was 280 nm, and the eluate was collected in sections. The enzyme activity fraction was further purified with a DEAE Sepharose anion exchange column equilibrated with buffer A, the elution flow rate was 1mL/min, the enzyme activity fraction was collected, and concentrated by centrifugation with a 10,000 Dalton membrane to obtain a purified cutinase mutant. After purification, the cutinase mutant is electrophoretically pure, with an apparent molecular weight of 30KDa. The purification results are shown in Figure 1. the

Example 4: Efficiency of Thermobifida fusca cutinase mutants for hydrolysis of cutin or PET. the

The hydrolysis effect of cutinase on cutin was determined using the following method: the reaction system included 1% (w/v) apple cutin, 25mM potassium phosphate buffer (pH 8.0), 1mg enzyme solution, kept at 60°C, sampled at different times, and used 0.02M NaOH solution was used to titrate the generated fatty acids. the

The hydrolysis effect of cutinase on PET is determined by the following method: the reaction system is 10ml, including 0.3g PET, 25mM phosphate buffer (pH 8.0), 0.1mg enzyme solution, 150rpm, 60℃ for more than 72h, and samples are taken during the incubation for RP - HPLC analysis. Hydrolytic activity was calculated from the sum of hydrolyzed products. Hydrolysis products Terephthalic acid (TPA), mono(2-hydroxyethyl terephthalate) (MHET), bis(2-hydroxyethyl terephthalate) (BHET), 1,2-vinyl - Monoterephthalic acid - Mono(2-hydroxyethyl terephthalate) (EMT) was quantitatively analyzed by LC-MS. the

As shown in Figure 2, the ability of I218A, W195A/F249A and Q132A/T101A mutants to hydrolyze cutin was significantly improved compared with that of the unmodified Thermobifida fusca cutinase. Among them, W195A/F249A hydrolyzed cutin at a faster rate in the early stage of the hydrolysis process, and quickly reached equilibrium; the cutin hydrolysis efficiency of I218A and Q132A/T101A increased by 50% and 20% respectively compared with the original enzyme. the

As shown in Figure 3, the ability of the Q132A/T101A mutant to hydrolyze PET was significantly improved compared with the unmodified Thermobifida fusca cutinase, and the hydrolysis efficiency was 3 times that of the original enzyme. the

Nucleotide Sequence Listing

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Claims (1)

1. the two mutants of an at; It is characterized in that at the 132nd amino acids Stimulina Gln and the 101st amino acids Threonine Thr simultaneous mutation with thermoactinomyces (Thermobifida fusca) are L-Ala Ala; Called after Q132A/T101A, the aminoacid sequence of the at of said thermoactinomyces is identical with the aminoacid sequence of Tfu_0883 genes encoding in the ncbi database.

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