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Beta-mannase mutant Man5AS11R with improved heat resistance and specific activity and encoding gene thereof

A mannanase and specific activity technology, applied in the fields of biotechnology and enzyme engineering, can solve the problems of low enzyme activity, unsatisfactory heat resistance and enzyme activity, and high price

Inactive Publication Date: 2020-01-10
TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, most of the mannanases currently on the market are not ideal in terms of heat resistance and enzyme activity, which has become a bottleneck in the application of mannanases in the feed industry and needs to be solved urgently
In addition, the relative price of β-mannanase in the domestic and foreign markets is relatively high due to its low enzyme activity and small output, which also greatly reduces the scope of application of the enzyme.

Method used

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  • Beta-mannase mutant Man5AS11R with improved heat resistance and specific activity and encoding gene thereof
  • Beta-mannase mutant Man5AS11R with improved heat resistance and specific activity and encoding gene thereof
  • Beta-mannase mutant Man5AS11R with improved heat resistance and specific activity and encoding gene thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0017] Example 1 Optimal pH and pH Stability of Thermostable Acidic β-Mannanase Mutants

[0018] The wild-type thermostable acid β-mannanase Man5A was determined and analyzed respectively for its mutants Man5AS (K16C and D296C), Man5A11R (R21V, I80D and I218E) and Man5AS11R (K16C, R21V, I80D, I218E and D296C). suitable for pH, the result is as figure 1 As shown, the optimum pH of each mutant is 3, and the change trend of its relative enzyme activity between pH 2.5-7.5 is consistent with that of Man5A. It can be seen that the mutation site of each mutant does not change the optimal activity of the enzyme. Suitable for pH.

[0019] As a control wild-type heat-resistant acidic β-mannanase Man5A, the mutants Man5AS (K16C and D296C), Man5A11R (R21V, I80D and I218E) and Man5AS11R (K16C, R21V, I80D, I218E and D296C) were analyzed at pH 2 respectively. .5-7 pH stability preserved for 1h, the results are as follows figure 2 As shown, the change trend of the residual enzyme acti...

Embodiment 2

[0020] Example 2 Optimum Action Temperature and Temperature Stability of Thermostable Acidic β-Mannanase Mutants

[0021] Control wild-type heat-resistant acid β-mannanase Man5A, respectively assay and analyze its mutants Man5AS (K16C and D296C), Man5A11R (R21V, I80D and I218E) and Man5AS11R (K16C, R21V, I80D, I218E and D296C) Optimum temperature, the results are as follows image 3 As shown, the optimum action temperature of each mutant is 60 o C, where, mutants Man5AS and Man5AS11R at 80 o The relative enzymatic activity of C was increased, while the mutant Man5A11R was consistent with Man5A. It can be seen that the introduction of intramolecular disulfide bonds can improve the relative enzymatic activity of the enzyme under high temperature conditions.

[0022] As a control wild-type heat-resistant acidic β-mannanase Man5A, the mutants Man5AS (K16C and D296C), Man5A11R (R21V, I80D and I218E) and Man5AS11R (K16C, R21V, I80D, I218E and D296C) were analyzed at 40 -80 o ...

Embodiment 3

[0023] Example 3 Increased Specific Activity of Thermostable Acidic β-Mannanase Mutants

[0024] On the basis of the mutant enzyme Man5AS, the present invention introduces three point mutations of R21V, I80D and I218E to further improve its specific activity. At the same time, in order to investigate the influence of the three mutation points on the activity of the enzyme, as shown in Table 1, each The specific activity of the mutants changed as Figure 5 As shown, the specific activities of Man5A11R and Man5AS11R in the mutant were increased by 25.6% and 22.5%, respectively, compared with the wild enzyme Man5A, but as image 3 and Figure 4 As shown, the heat resistance of Man5A11R was not significantly improved, while Man5AS11R was improved in both heat resistance and specific activity. At the same time, the present invention also found that on the basis of Man5AS, only one or two mutation sites in Man5A11R were mutated, but the specific activity was not improved. It ca...

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Abstract

The invention, which relates to the fields of biotechnology and enzyme engineering, particularly discloses a beta-mannase mutant Man5AS11R with improved heat resistance and specific activity and an encoding gene thereof. The preparation method comprises the following steps: carrying out double mutation of K16C and D296C on beta-mannase Man5A of which the amino acid sequence is shown as SEQ ID NO.1to obtain a mutant Man5AS11R that enables the heat resistance of the mutant to be improved by 5 DEG C by being compared with Man5A; for the Man5ASamino acid sequence shown by SEQ ID NO. 2, carrying out simultaneous mutation of three points of R21V, I80D and I218E continuously to obtain a mutant Man5AS11R with the amino acid sequence shown by SEQ ID NO.4. The obtained mutant Man5AS11R has the activity that is improved by 22.5% and 31.4% respectively be being compared with the Man5A and the Man5AS on the premise that the heat resistance of the mutant Man5AS11R is kept to be unchanged. The over80% of enzyme activity of the Man5AS can be left when the Man5AS is kept for 3min at a high temperature of 80 DEG C but the Man5A can only keep 80% of enzyme activity when being kept for 3min at a temperature of 75 DEG C. Therefore, the beta-mannase mutant Man5AS11R and the encoding gene thereof have the broad development and application prospects.

Description

technical field [0001] The invention belongs to the fields of biotechnology and enzyme engineering, and relates to a β-mannanase mutant with improved heat resistance and specific activity and its coding gene. Background technique [0002] At present, β-mannanase is one of the 12 kinds of feed-grade enzyme preparations approved for use in my country, and is widely used in the feed industry as a high-efficiency feed additive. Since the 1990s, enzyme preparations have been added to 90% of poultry feed in Europe and the United States, and enzyme preparations have been added to 60% of pig rations. my country started late, but it has always been the focus of feed production and application research. As an important feed enzyme, mannanase has received more and more attention in recent years. Adding mannanase to chicken (poultry) feed can effectively increase the growth of broiler chickens, increase the laying rate of laying hens, improve the utilization rate of feed, reduce the ex...

Claims

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Application Information

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IPC IPC(8): C12N9/42C12N15/56
CPCC12N9/2494C12Y302/01078
Inventor 郑宏臣宋诙徐健勇李树芳付晓平
Owner TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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