β-mannanase mutant m-q7s having high enzyme activity, recombinant strain, and use of mutant

Abstract

The present invention relates to the technical field of biology, and in particular to a β-mannanase mutant M-Q7S having a high enzyme activity, a recombinant strain, and a use of the mutant. The single-site β-mannanase mutant Csman5A-Q7S is obtained by mutating into serine (S) the amino acid asparagine (Q) at position 7 of the β-mannanase having the amino acid sequence as shown in SEQ ID NO: 1. Experiments show that the specific enzyme activity of the mutant Csman5A-Q7S in the whole induction period is significantly greater than that of the original Csman5A, and after 144 h of induction, when the specific enzyme activity reaches the maximum value, the specific enzyme activity of the mutant Csman5A-Q7S is 5.4 times that of the original Csman5A.

Description

β-Mannanase high-enzyme-activity mutant M-Q7S, recombinant bacteria and their applications Technical Field

[0001] This invention relates to the field of biotechnology, specifically to the β-mannanase high-enzyme-activity mutant M-Q7S, recombinant bacteria, and their applications. Background Technology

[0002] As the world's most populous country, my country has a large demand for grain, but the competition between humans and livestock for grain is becoming increasingly prominent, leading to a severe shortage of protein resources in my country's feed industry. To alleviate this resource shortage, finding new feed ingredients is currently the most important task, with the development and utilization of agricultural by-products becoming a key focus. Among numerous agricultural by-products, palm meal has advantages such as abundant resources, low price, absence of aflatoxin, and the presence of vitamin E for antioxidant effects, making it a promising new feed ingredient. However, palm meal contains a large amount of indigestible non-starch polysaccharides, mainly mannan.

[0003] However, mannans are not easily degraded in the animal digestive tract, and their partial solubility in water easily forms viscous substances, increasing the viscosity of chyme and thus reducing the utilization rate of nutrients. β-Mannanases can randomly hydrolyze β-1,4-glycosidic bonds in the mannan backbone to produce low molecular weight mannans, making them the most important glycosidic hydrolases in the mannan-degrading enzyme system. Therefore, β-Mannanases have been widely used in the food, feed, and energy industries in recent years. Currently, many β-Mannanases have been cloned and expressed, but these β-Mannanases often suffer from low expression levels and low enzyme activity; for example, the enzyme activity mentioned in the literature is only 1.5 × 10⁻⁶. 6 U / g (Lv Xiao. Recombinant expression and functional study of two new β-mannanases in Flavobacterium saccharidogenum [D]. Northeast Normal University, 2023. DOI:10.27011 / d.cnki.gdbsu.2023.000636.), the development of novel mannanases with high enzyme activity is urgently needed. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide a β-mannanase mutant with enhanced enzyme activity. Specifically, the unit site β-mannanase mutant of the present invention, Csman5A-Q7S, is obtained by changing the 7th amino acid asparagine (Q) of the β-mannanase with the amino acid sequence SEQ ID NO: 1 to serine (S).

[0005] This invention provides a β-mannanase high-enzyme-activity mutant M-Q7S, which is obtained by changing the 7th amino acid asparagine (S) of the β-mannanase with the amino acid sequence SEQ ID NO: 1.

[0006] The present invention further provides a nucleic acid encoding the β-mannanase high-enzyme-activity mutant M-Q7S.

[0007] Further, a recombinant vector containing the nucleic acid is provided. Preferably, the nucleic acid is inserted into a suitable site on an expression vector, so that its nucleotide sequence is operatively linked to the expression regulatory sequence to obtain a recombinant expression plasmid.

[0008] More preferably, it is a yeast expression plasmid.

[0009] The present invention also provides recombinant bacteria comprising the recombinant vector described above. Preferably, the recombinant bacteria is Pichia pastoris.

[0010] This invention provides the application of the β-mannanase high-enzyme-activity mutant M-Q7S in the degradation of mannan raw materials.

[0011] Specifically, the mannan raw material is one or more of palm meal, coconut meal, soybean meal, sunflower meal, or sesame meal.

[0012] Specifically, the β-mannanase high-activity mutant M-Q7S was obtained through recombinant expression in recombinant bacteria.

[0013] The present invention yields a single-site β-mannanase mutant, Csman5A-Q7S, obtained by replacing the 7th amino acid (Q) of the β-mannanase sequence SEQ ID NO: 1 with serine (S). Experiments show that the specific enzyme activity of mutant Csman5A-Q7S is significantly higher than that of the original Csman5A throughout the entire induction period. At its peak after 144 hours of induction, the specific enzyme activity of mutant Csman5A-Q7S is 5.4 times that of the original Csman5A. Furthermore, experiments demonstrate a significant and effective degradation effect on various mannan raw materials, exhibiting clear advantages and substantial application value. Attached Figure Description

[0014] Figure 1 shows the Csman 5A-Q7S plasmid.

[0015] Figure 2 shows the agarose gel electrophoresis results of Csman 5A and Csman 5A-Q7S transformants.

[0016] Figure 3 shows the electrophoresis diagram of Csman 5A-Q7S protein.

[0017] Figure 4 shows the changes in enzyme activity of the original enzymes Csman5A and Csman5A-MQ7S at different induction times. Detailed Implementation

[0018] The present invention will be further illustrated below with specific embodiments in order to better understand the present invention, but this does not constitute a limitation thereof.

[0019] Example 1: Evaluation of Mannan Mutant Expression Levels in Pichia pastoris using a Model. This invention employs a rational design and protein surface charge optimization strategy. By screening amino acid sites, a directed evolution was performed on β-mannanase Csman5A (Sequence ID: HQ718590.1, amino acid sequence SEQ ID NO: 1, nucleotide sequence SEQ ID NO: 2) from Chaetomium, resulting in a library of site-specific mutants that enhance the activity of mannanase Csman5A. The heterologous expression levels of these mutants in Pichia pastoris were evaluated using the following model.

[0020] We collected Pichia pastoris proteomics data from previous studies, calculating the protein abundance of 3914 proteins. The NSAF value for each protein ranged from 0 to 1 (values ​​close to 1 indicated the highest protein expression, while values ​​close to 0 indicated lower protein expression). Based on the NSAF values, the data were divided into high expression level and low expression level groups.

[0021] Subsequently, we retrained the deep learning model MPEPE using the above data to predict the expression level of proteins in Pichia pastoris, achieving an accuracy of 78.2%.

[0022] Among them, the mutant Csman5A-Q7S was predicted to have the highest expression level. Csman5A-Q7S was then selected as the β-mannanase Csman5A mutant for heterologous expression in Pichia pastoris. The nucleotide sequences of mannanase Csman5A and mutant Csman5A-Q7S, optimized according to the dominant codons of Pichia pastoris, are SEQ ID NO: 2 and SEQ ID NO: 3, respectively.

[0023] Example 2: Construction of Recombinant Vectors for β-Mannanase Site-Directed Mutants Recombinant plasmids pPIC9k-Csman5A and pPIC9k-Csman5A-Q7S were constructed in this example: The pPIC9k plasmid (AOX promoter) was used to construct the target gene expression vector. The pPIC9k plasmid backbone (excluding the multiple cloning site MCS, containing the AOX1 terminator to α-factor secretion signal sequence) was PCR-added with homologous arms that could be linked to the target gene. The fusion plasmid was then assembled with the Csman5A fragment containing the added homologous arms using Gibson assembly, as shown in Figure 1. After transformation with E. coli, plasmid extraction, and gene sequencing, the integrative plasmids pPIC9k-Csman5A and pPIC9k-Csman5A-Q7S were finally obtained.

[0024] Using the recombinant vector pPIC9k-Csman5A containing the gene as a template, primers containing the mutation site were added. The primers were designed with homologous arms. PCR amplification was performed using a high-fidelity enzyme. The amplified PCR product was assembled by Gibson to obtain a fusion plasmid. After transformation with E. coli, positive clones were screened on LB plates containing Kanr and sequenced for verification. After successful construction of the recombinant plasmid pPIC9k-Csman5A and the mutant plasmid, a pair of primers was designed in the AOX1 promoter of the plasmid fragment. The obtained pPIC9k-Csman5A and mutant plasmid pPIC9k-Csman5A-Q7S were used as templates to prepare linearized pPIC9k-Csman5A and mutant plasmid pPIC9k-Csman5A-Q7S by PCR. After verification by electrophoresis, the linearized pPIC9k-Csman5A and mutant plasmid pPIC9k-Csman5A-Q7S were purified and recovered. The recovered linearized pPIC9k-Csman5A and mutant plasmid pPIC9k-Csman5A-Q7S were transformed into Pichia pastoris GS115 competent cells by electroporation and plated on MD plates for culture at 30℃. After the colonies have grown on MD plates, wash them off with sterile ddH2O and dilute them appropriately. Spread them on MDG plates containing 5 mg / mL LG-418 for screening. Incubate at 30°C for 2-3 days to obtain single colonies. Select single colonies with better morphology and condition and inoculate them on YPD liquid medium for expansion culture. At the same time, use glycerol to preserve the recombinant Pichia pastoris strain at -80°C.

[0025] The bacterial culture in YPD liquid medium was expanded for crude genome extraction, which was used as a PCR template. Amplification was performed using the Phanata high-fidelity enzyme PCR system, and the PCR products were verified by electrophoresis. The lanes showed the expected bright band of approximately 1300 bp in length (Figure 2), indicating that the target gene Csman5A and its mutant Csman5A-MQ7S were successfully integrated into the genome of Pichia pastoris strain GS115. Further sequencing results confirmed that the sequence matched the target gene, indicating that the recombinant Pichia pastoris strain was successfully constructed.

[0026] Example 3: Expression verification of β-mannanase and its mutant in Pichia pastoris. To test the ability of the mutant with enhanced enzyme activity in Example 2 to produce highly active enzyme solution under high-density fermentation conditions, we cultured Csman5A and the mutant strain using a 5L fed-batch fermentation method and induced enzyme expression.

[0027] The seed culture medium and cultivation conditions were as follows: YPD composite medium was selected as the seed culture medium: 1% yeast extract, 2% peptone, 2% glucose, pH natural. Cultivation conditions: 50 mL of seed culture medium was added to a 250 mL shake flask, with an inoculation amount of 1% Pichia pastoris, and the mixture was incubated at 30℃ and 220 rpm for 24 h on a shaker.

[0028] The fermentation medium and conditions for a 5L fermenter were as follows: 42 g / L glycerol, 1.2 g / L KH₂PO₄, 18 g / L NH₄H₂PO₄, and 6.5 g / L MgSO₄·7H₂O. The fermentation conditions were: 3L fermenter volume, 1% Pichia pastoris inoculum, 30℃, pH 4.5–5.0, air flow rate 4–8 m³ / h, glycerol added at 0.5–1.0 rpm / 30 min / time to maintain DO ≥ 20%; after 24 h of continuous fermentation, methanol was added at 0.5% at 0.1 rpm / h / time to maintain DO ≥ 25%. Samples were taken every 24 h after methanol addition, and SDS-PAGE analysis of the enzyme solution was performed. As shown in Figure 3, MQ7S was efficiently expressed. The enzyme activities of the original enzymes Csman5A and Csman5A-MQ7S were measured using locust bean gum as a substrate. The results are shown in Figure 4. The specific enzyme activity of the mutant M-Q7S was significantly higher than that of the original Csman5A throughout the induction period. After 144 h of induction, the specific enzyme activity reached its highest value, reaching 1.9 × 10⁻⁶. 7 U / g, the original Csman5A specific enzyme activity is 3.5 × 10 U / g. 6 U / g, the specific enzyme activity of mutant M-Q7S is 5.4 times that of the original Csman5A.

[0029] Example 4: Application of β-Mannanase Mutant in the Enzymatic Hydrolysis of Mannan Raw Materials To test the effectiveness of the produced mannan variant M-Q7S in degrading mannan raw materials in actual production, we prepared a substrate mixture by diluting powders obtained from palm meal, coconut meal, soybean meal, sunflower meal, and sesame meal at a material-to-liquid ratio of 1:3. Specifically, 10g of the above powder and 30ml of pH X phosphate buffer were added to a 250mL shake flask, followed by 0.02ml, 0.1ml, and 0.5ml of fermented β-mannanase mutant M-Q7S enzyme solution (specific enzyme activity 1.9 × 10⁻⁶). 7 The volume difference was made up with phosphate buffer at pH X. After reacting at 37℃ for 48 h, the mannose content was determined by liquid chromatography. The results are shown in Table 1.

[0030] Table 1

[0031] As shown in Table 1, the mannan degradation rates were 65.2%, 73.5%, and 86.4% for enzyme solutions added at proportions of 0.02 ml, 0.1 ml, and 0.5 ml, respectively.

Claims

1. A β-mannanase high-activity mutant M-Q7S, characterized in that, It is obtained by changing the 7th amino acid, asparagine, of β-mannanase with the amino acid sequence SEQ ID NO: 1 to serine.

2. The nucleic acid encoding the β-mannanase high-activity mutant M-Q7S as described in claim 1.

3. A recombinant vector comprising the nucleic acid as described in claim 2.

4. The recombinant vector as described in claim 3, characterized in that, The nucleic acid is inserted into a suitable site on the expression vector, so that its nucleotide sequence can be operatively linked to the expression regulatory sequence to obtain a recombinant expression plasmid.

5. The recombinant vector as described in claim 4, characterized in that, It is a yeast expression plasmid.

6. A recombinant strain comprising the recombinant vector as described in claim 4 or 5.

7. The recombinant bacteria as described in claim 6, characterized in that, The recombinant strain is Pichia pastoris.

8. The application of the β-mannanase high-activity mutant M-Q7S as described in claim 1 in the degradation of mannan raw materials.

9. The application as described in claim 8, characterized in that, The mannan raw material is one or more of palm meal, coconut meal, soybean meal, sunflower meal, or sesame meal.

10. The application as described in claim 8 or 9, characterized in that, The β-mannanase high-activity mutant M-Q7S was obtained through recombinant expression in recombinant bacteria.

Images