Method for improving stability of modified neutral mannanase plm5a and mutants

Abstract

The application relates to the field of agricultural biotechnology, in particular to a method for improving the stability of a neutral mannanase PLM5A and a mutant. The neutral mannanase PLM5A is subjected to K52V, T266D, W291S, A93LK205R, F63Y, D66Y, S283L, G288S and / or L292I mutation. The mutant has improved optimum temperature, increased thermal stability and stronger acid resistance.

Description

Technical Field

[0001] This invention relates to the field of agricultural biotechnology, and more specifically to a method and mutant for improving the stability of neutral mannanase PLM5A. Background Technology

[0002] Mannanase is a hydrolytic enzyme that can degrade the backbone of mannan, glucomannan, and galactomannan. It belongs to the hemimannanase class and has important applications in the food, pharmaceutical, paper, feed, and petroleum industries.

[0003] In recent years, the application of mannanase in the feed industry has received increasing attention. Non-starch polysaccharides in feed, such as β-mannan, are anti-nutritional factors in poultry, are difficult to digest, and increase intestinal viscosity. β-mannanase can break down β-mannan to produce mannan oligosaccharides, reducing the binding of β-mannan with water molecules, promoting intestinal peristalsis, and preventing food accumulation. Mannan oligosaccharides can bind to exogenous lectins on the surface of harmful bacteria such as Escherichia coli, inhibiting the contact between harmful bacteria and receptors in the intestine, effectively preventing the proliferation of pathogens. Simultaneously, as nutrients, they promote the proliferation of beneficial bacteria such as Bifidobacteria and Lactobacilli, maintaining the acid-base balance of the intestinal environment. Studies have shown that adding β-mannanase to poultry feed can improve poultry weight and feed utilization, improve the intestinal environment, and enhance poultry immunity.

[0004] To meet the needs of the feed industry, an ideal β-mannanase should possess characteristics such as high specific activity, good thermal stability, and strong acid resistance. Most natural β-mannanases do not possess these properties.

[0005] Researchers have been working on the molecular modification of β-mannanases to enhance their stability and activity. However, commercially available mannanases generally suffer from drawbacks such as low catalytic efficiency, poor thermal stability, and rapid enzyme activity loss. Therefore, the research and development of mannanases with high thermal stability and strong acid resistance is of great significance for improving the overall performance of mannanases and reducing their usage costs. Summary of the Invention

[0006] The purpose of this invention is to provide a method for improving the stability of neutral mannanase PLM5A.

[0007] Another object of the present invention is to provide a stability-improved neutral mannanase PLM5A.

[0008] The method for improving the stability of neutral mannanase PLM5A according to the present invention includes the step of performing a K52V mutation on the amino acid sequence of the neutral mannanase PLM5A, wherein the NCBI sequence number of the neutral mannanase PLM5A is WP_125083321.1 (SEQ ID NO:1).

[0009] SEQ ID NO:1

[0010] MKNLRKKSLSICMAMAMMFSLVTLLGGQDIRAASGFYVSGTILCDSTGNPFKIRGINHAHSWF

[0011] KNDSATAMEAIAATGANTVRIVLSNGQQYAKDDANTVSNLLSLANQHKLIAILEVHDATGSDS

[0012] VSALDHAVDYWIEMKNVLVGKEDRVLINIANEWYGTWDSNGWADGYKSAIPKLRNAGINHT

[0013] LIVDAAGWGQYPQSIVDKGNEVFNSDPLRNTIFSIHMYEYAGGNADMVRANIDQVLNKGLAVI

[0014] IGEFGHYHTGGDVDETAIMSYTQQKGVGWLAWSWKGNGAEWLYLDLSYDWAGNHLTEWGETIVNGANGLKATSTRAPIFGN.

[0015] The method for improving the stability of neutral mannanase PLM5A according to the present invention includes a step of further performing a T266D mutation on the amino acid sequence of the neutral mannanase PLM5A.

[0016] The method for improving the stability of neutral mannanase PLM5A according to the present invention includes a step of further performing a W291S mutation on the amino acid sequence of the neutral mannanase PLM5A.

[0017] The method for improving the stability of neutral mannanase PLM5A according to the present invention includes the step of further mutagenesis of the amino acid sequence of the neutral mannanase PLM5A by A93L and / or K205R mutations.

[0018] The method for improving the stability of neutral mannanase PLM5A according to the present invention includes the steps of further mutating the amino acid sequence of the neutral mannanase PLM5A by F63Y, D66Y, S283L, G288S and L292I.

[0019] According to the present invention, a stability-improved neutral mannanase mutant is provided, wherein the sequence of the mutant is the sequence obtained by K52V mutation of the amino acid sequence of neutral mannanase PLM5A, wherein the NCBI sequence number of neutral mannanase PLM5A is WP_125083321.1.

[0020] According to the present invention, a stability-improved neutral mannanase mutant is provided, wherein the sequence of the mutant is the sequence obtained by further T266D of the amino acid sequence of neutral mannanase PLM5A.

[0021] According to the present invention, a stability-improved neutral mannanase mutant is provided, wherein the sequence of the mutant is the sequence obtained by further performing W291S on the amino acid sequence of neutral mannanase PLM5A.

[0022] According to the present invention, a stability-improved neutral mannanase mutant is provided, wherein the sequence of the mutant is the sequence obtained by further performing A93L and / or K205R on the amino acid sequence of neutral mannanase PLM5A.

[0023] According to the present invention, a stability-improved neutral mannanase mutant is provided, wherein the sequence of the mutant is the amino acid sequence of neutral mannanase PLM5A further mutated by F63Y, D66Y, S283L, G288S and L292I.

[0024] After treatment at 80℃ for 5 min, the experimental results showed that the thermal stability of 17 single-point mutant proteins was significantly improved compared with the wild-type PLM5A protein, while the thermal stability of the other 13 single-point mutants was not improved or even worsened compared with the wild-type protein.

[0025] The three multi-point mutant proteins PLM5AMut1, PLM5AMut2, and PLM5AMut3 showed significantly improved thermal stability compared to the wild-type PLM5A protein, with mutant PLM5AMut3 exhibiting the best thermal stability.

[0026] The optimal temperature for PLM5A is 60℃, while the optimal temperature for PLM5AMut3 is increased to 75℃. After treating both PLM5A and PLM5AMut3 at 80℃ for 5 min, the residual enzyme activity of PLM5A was only about 15%, and after 10 min of treatment at 80℃, PLM5A was completely inactivated. However, after treating PLM5AMut3 at 80℃ for 5 min, the residual enzyme activity was still above 95%, and after 60 min of treatment at 80℃, the residual enzyme activity was still above 50%, demonstrating excellent thermal stability.

[0027] PLM5AMut3 was found to have a similar optimal pH value to PLM5A. However, PLM5AMut3 exhibits stronger acid resistance compared to the wild-type PLM5A protein. After treatment in a pH 2.0 buffer for 1 hour, the enzyme activity of the wild-type PLM5A protein was completely lost, and after treatment in a pH 3.0 buffer for 1 hour, only about 30% of the enzyme activity remained. In contrast, PLM5AMut3 retained more than 30% of its enzyme activity after treatment in a pH 2.0 buffer for 1 hour, and more than 50% of its enzyme activity after treatment in a pH 3.0 buffer for 1 hour. Attached Figure Description

[0028] Figure 1 Residual enzyme activity of PLM5A and its mutants after treatment at 80℃ for 5 min;

[0029] Figure 2 Enzymatic properties of recombinant PLM5A and its mutant PLM5AMut3: A: Effect of temperature on enzyme activity, B: Effect of pH on enzyme activity, C: Thermal stability, D: pH stability;

[0030] Figure 3 SDS-PAGE detection of PLM5AMut3 expression, M: molecular weight standard, 1-7: fermentation supernatant of PLM5AMut3 expression strain. Detailed Implementation

[0031] Example 1: Construction, Expression, and Thermostability Detection of PLM5A Single-Point Mutant

[0032] Based on the codon bias of the Pichia pastoris expression system, the coding sequence of the wild-type PLM5A protein was optimized and synthesized, and then constructed into the EcoRI / NotI site of the expression vector pPICZα-A. Single-point mutants of PLM5A were constructed by mutating amino acids at corresponding positions using overlap PCR based on a single-point mutation design. The expression plasmids of wild-type PLM5A protein and single-point mutants were transformed into Pichia pastoris strain GS115 for induced expression. The induced protein was purified using a Ni column. The thermostability of the recombinant PLM5A protein and all its single-point mutants was determined. After treatment at 80℃ for 5 min, the experimental results showed that the thermostability of 17 single-point mutant proteins (K52V, F63Y, D66Y, S67T, T77W, A93L, H132E, D149P, I153Y, L179I, K205R, N238Y, T266D, S283L, G288S, W291S, L292I) was significantly improved compared to the wild-type PLM5A protein. However, the thermostability of the other 13 single-point mutants (F51V, M71L, N108I, I113Y, V118W, S126P, L144Y, R150Y, N181A, N185D, Y227A, A237N, N287K) was not improved compared to the wild-type protein, and may even have worsened. Figure 1 ).

[0033] Example 2: Selection and Enzymatic Properties Detection of PLM5A Multipoint Mutants

[0034] To further improve the thermostability of PLM5A, randomized combined mutants with the aforementioned 17 effective single mutation sites were constructed and inserted into the EcoRI / NotI sites of the expression vector pPICZα-A. The PLM5A multi-site combined mutant expression plasmid was transformed into Pichia pastoris strain GS115 for induced expression. The induced protein was purified using a Ni column. After treatment at 80℃ for 5 min, thermostability tests showed that the thermostability of the three multi-point mutant proteins PLM5AMut1 (K52V, T266D, W291S), PLM5AMut2 (K52V, A93L, K205R, T266D, W291S), and PLM5AMut3 (K52V, F63Y, D66Y, A93L, K205R, T266D, S283L, G288S, W291S, L292I) was significantly improved compared to the wild-type PLM5A protein. Among them, mutant PLM5AMut3 had the best thermostability. Figure 1 ).

[0035] To assess the overall enzymatic properties of PLM5AMut3, enzyme activities of PLM5A and PLM5AMut3 were measured under different temperature conditions. The results showed that the optimal temperature for PLM5A was 60℃, while the optimal temperature for PLM5AMut3 increased to 75℃. Figure 2 PLM5A and PLM5AMut3 were incubated at 80℃ for 5 min, 10 min, 15 min, 20 min, 30 min, 45 min, and 60 min, respectively, and their enzyme activities were then measured. The results showed that after 5 min of treatment at 80℃, the residual enzyme activity of PLM5A was only about 15%, and after 10 min of treatment at 80℃, PLM5A was completely inactivated. In contrast, after 5 min of treatment at 80℃, the residual enzyme activity of PLM5AMut3 was still above 95%, and after 60 min of treatment at 80℃, the residual enzyme activity was still above 50%, demonstrating excellent thermostability. Figure 2 C).

[0036] The results of the detection of the optimal pH value and pH stability of PLM5A and PLM5AMut3 showed that the optimal pH values ​​of PLM5AMut3 and PLM5A were similar, with PLM5A having an optimal pH of 7.0 and PLM5AMut3 having an optimal pH of 7.5. Figure 2 (B). PLM5AMut3 exhibits stronger acid resistance compared to the wild-type PLM5A protein. After treatment in pH 2.0 buffer for 1 hour, the enzyme activity of the wild-type PLM5A protein was completely lost; after treatment in pH 3.0 buffer for 1 hour, only about 30% of the enzyme activity remained. In contrast, PLM5AMut3 retained more than 30% of its enzyme activity after treatment in pH 2.0 buffer for 1 hour, and more than 50% of its enzyme activity after treatment in pH 3.0 buffer for 1 hour. Figure 2 D).

[0037] Example 3: Upper fermentation of PLM5AMut3

[0038] The recombinant yeast strain PLM5AMut3 was fermented in a fermenter to investigate its protein expression at the fermenter level. Single colonies were inoculated into 50 ml of LYPD liquid medium and cultured on a shaker at 28°C for 12 h. The colonies were then transferred to 200 ml of LYPD liquid medium and cultured at 28°C and 200 rpm for 24 h. Finally, the colonies were inoculated into 3 L of fermentation medium and fermented at 28°C and 1000 rpm for 132 h with methanol induction before being transferred to the fermenter. Samples were taken periodically to detect protein expression in the fermentation broth. SDS-PAGE analysis revealed that the recombinant Pichia pastoris strain PLM5AMut3 achieved high-density expression of PLM5AMut3 under high-density culture conditions, and the expression and secretion of the target protein significantly increased with prolonged fermentation time. Figure 3 ).

[0039] The above embodiments are only used to explain the technical solutions of this application and do not limit the scope of protection of this application.

Claims

1. A method for improving the stability of a neutral mannanase PLM5A, characterized in that, The steps of the method are that the amino acid sequence of the neutral mannanase PLM5A is mutated as follows: K52V; or K52V, T266D, W291S; or K52V, A93L, K205R, T266D, W291S; or K52V, F63Y, D66Y, A93L, K205R, T266D, S283L, G288S, W291S, L292I, The NCBI sequence number of the neutral mannanase PLM5A is WP_125083321.

1.

2. A neutral mannanase mutant with improved stability, characterized in that, The sequence of the mutant is the sequence after the amino acid sequence of the neutral mannanase PLM5A is mutated as follows, K52V; or K52V, T266D, W291S; or K52V, A93L, K205R, T266D, W291S; or K52V, F63Y, D66Y, A93L, K205R, T266D, S283L, G288S, W291S, L292I, wherein the NCBI sequence number of the neutral mannanase PLM5A is WP_125083321.

1.

3. A gene encoding the stability-improved neutral mannanase mutant of claim 2.

4. Use of the stability-improved neutral mannanase mutant of claim 2 for decomposing β-mannan.

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