A mutant of cutinase and its preparation method

Abstract

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

Description

technical field

[0001] 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. Background technique

[0002] 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 th...

Examples

Embodiment 1

[0025] Example 1: Method for determining mutation sites of cutinase mutants of Thermobifida fusca.

[0026] 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, proceeding from the above two aspects, determine the mutation site and then perform site-directed mutation on the cutinase gene. 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...

Embodiment 2

[0027] Example 2: The preparation method of the cutinase mutant of Thermobifida fusca.

[0028] I218A-pet20b(+), W195 / F249-pet20b(+), Q132A / T101A-pet20b(+) mutant plasmids were constructed by rapid PCR site-directed mutagenesis.

[0029] 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 transformed Subsequence verification.

[0030] 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 amplifie...

Embodiment 3

[0053] Example 3: Method for expression and purification of cutinase mutants from Thermobifida fusca.

[0054] 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. The positive transformant after verification was 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) in 37°C liquid culture overnight, then inserted into TB fermentation liquid medium and cultured at 37°C for 3 hours, then induced with 4 mg / L IPTG (isopropylthioβD-galactoside), cooled to 25°C and incubated at constant temperature for 80 hours .

[0055] 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 activated carbon column for salting ou...

technical field

[0001] 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. Background technique

[0002] 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 th...

Examples

Embodiment 1

[0025] Example 1: Method for determining mutation sites of cutinase mutants of Thermobifida fusca.

[0026] 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, proceeding from the above two aspects, determine the mutation site and then perform site-directed mutation on the cutinase gene. 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...

Embodiment 2

[0027] Example 2: The preparation method of the cutinase mutant of Thermobifida fusca.

[0028] I218A-pet20b(+), W195 / F249-pet20b(+), Q132A / T101A-pet20b(+) mutant plasmids were constructed by rapid PCR site-directed mutagenesis.

[0029] 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 transformed Subsequence verification.

[0030] 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 amplifie...

Embodiment 3

[0053] Example 3: Method for expression and purification of cutinase mutants from Thermobifida fusca.

[0054] 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. The positive transformant after verification was 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) in 37°C liquid culture overnight, then inserted into TB fermentation liquid medium and cultured at 37°C for 3 hours, then induced with 4 mg / L IPTG (isopropylthioβD-galactoside), cooled to 25°C and incubated at constant temperature for 80 hours .

[0055] 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 activated carbon column for salting ou...

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