Mannanase dcman5a mutant with improved specific activity, gene, application and method for improving activity of mannase

Abstract

The present application relates to the technical field of genetic engineering, and particularly relates to a mannanase with improved specific activity Dc Man5A mutant, gene and application thereof Dc The present application carries out single-point mutation on the 98Q site of mature mannanase Dc Man5A, and transforms the recombination vector containing the mutant coding gene into Pichia pastoris GS115, carries out large bottle induction on the mutant and wild mannanase Dc Man5A, and carries out protein concentration and purification, and carries out specific activity and substrate affinity determination and analysis Dc Man5A-Q98V is obviously higher than that of the wild mannanase under high-temperature conditions (50-60 DEG C) Dc Man5A, and is increased by about 0.8 times under 60 DEG C conditions The present application confirms the key catalytic active sites Q98 in the mannanase

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, specifically to mannanases with increased specific activity. Dc Man5A mutants, their genes, and applications. Background Technology

[0002] Mannan is a major component of plant hemicellulose, primarily found in cork plants and some special structures such as plant seeds. It is also an important plant-based feed ingredient. Mannan has a complex structure; its main chain is a linear polysaccharide linked by 1,4-β-D-mannopyranoside bonds, and the main chain or side chains contain various substituents. Due to the diversity and structural complexity of mannan, its complete hydrolysis requires the synergistic action of multiple enzymes, including endo-β-mannanase, exo-β-mannosidase, β-glucosidase, acetylmannan esterase, and α-galactosidase. Among these, endo-β-mannanase, which degrades the β-1,4-glycosidic bonds of the mannan main chain, plays the most crucial role in the degradation process.

[0003] According to the classification of glycoside hydrolases, currently discovered endo-β-mannanases can be divided into the GH5, 26, 113, or 134 families. Among these, there are numerous reports on GH5 family β-mannanases, but their poor tolerance to extreme environments, low catalytic activity, and weak substrate affinity greatly limit their applications in various fields, prompting research into the development of novel enzyme genes and enzyme molecular modification. Many factors influence the structure and properties of β-mannanases, including hydrogen bonds, salt bridges, disulfide bonds, and the thermal stability of proteins, which is related to various structural characteristics. Summary of the Invention

[0004] To address the problem of low catalytic activity in existing β-mannanases, this invention... Dc The amino acid Q at position 98 of the N-terminus of the mature Man5A protein was mutated to V, resulting in a mannanase mutant with increased specific activity.

[0005] The purpose of this invention is to provide a mannanase mutant containing a single Q98V mutation.

[0006] Another object of the present invention is to provide a gene encoding the above-mentioned mutant.

[0007] Another object of the present invention is to provide a recombinant vector containing the above-mentioned mutant gene.

[0008] Another object of the present invention is to provide a recombinant strain containing the above-mentioned mutant gene.

[0009] According to the technical solution of the present invention, wild mannanase DcThe amino acid sequence of Man5A is shown in SEQ ID NO:1.

[0010] The first 17 amino acids at the N-terminus of this enzyme form the signal peptide sequence “MRLSICLATATAALSLA”, which is a wild-type mannanase. Dc The amino acid sequence of the mature Man5A protein is shown in SEQ ID NO: 2.

[0011] Mannanase with increased specific activity according to the present invention Dc The Man5A mutant is produced by maturing mannanase. Dc Man5A was obtained by performing a point mutation at position Q98V, which involves mutating glutamine (Q) at position 98 of the N-terminus of the mature mannanase protein to valine (V), resulting in the mutant Q98V, whose amino acid sequence is shown in SEQ ID NO: 3.

[0012] The mannanase gene with increased specific activity according to the present invention encodes the aforementioned mannanase with increased specific activity. Dc Man5A mutant.

[0013] The mannanase gene with enhanced specific activity according to the present invention has the nucleotide sequence shown in SEQ ID NO: 4.

[0014] The present invention provides a recombinant expression vector containing the mannanase gene with the above-mentioned enhanced specific activity.

[0015] The present invention provides a recombinant strain containing the mannanase gene with enhanced specific activity as described above.

[0016] The method for preparing mannanase with enhanced specific activity according to the present invention comprises the following steps:

[0017] The host cells were transformed using the recombinant vector containing the mutant encoding gene to obtain recombinant strains;

[0018] Culture recombinant strains and induce recombinant mannanase Dc Man5A mutant expression;

[0019] The expressed mannanase was recovered and purified. Dc Man5A mutant.

[0020] The beneficial effects of this invention are:

[0021] This invention utilizes mature mannanase Dc A single-point mutation was made at the 98Q site of Man5A, and the recombinant vector containing the mutant coding gene was transformed into Pichia pastoris GS115, thus constructing the mutant yeast engineered strain. The mutant and wild-type mannanase were compared. DcMan5A was induced in via flasks, followed by protein concentration and purification. Specific activity and substrate affinity were then measured and analyzed. The experimental results indicated that the single-point mutant... Dc Man5A-Q98V exhibits significantly higher specific activity than wild-type mannanase under high-temperature conditions. Dc Man5A was increased by approximately 0.8 times. This invention confirms the presence of mannanase. Dc The key catalytic active site Q98 in Man5A was identified, and the importance of these sites for the high catalytic activity of the enzyme was demonstrated. Attached Figure Description

[0022] Figure 1 The mannanase was isolated and purified. Dc SDS-PAGE results of Man5A and its mutants;

[0023] Figure 2 Showing mannanase Dc The optimal pH for Man5A and its mutants;

[0024] Figure 3 Showing mannanase Dc The optimal temperature for Man5A and its mutants. Detailed Implementation

[0025] Experimental materials and reagents

[0026] 1. Strains and vectors: expression hosts Pichia pastoris GS115, expression plasmid vector pPIC9;

[0027] 2. Enzymes and other biochemical reagents: Point mutation kits and other biochemical reagents were purchased from biochemical reagent companies;

[0028] 3. Culture medium:

[0029] (1) LB medium: 0.5% yeast extract, 1% peptone, 1% NaCl, pH 7.0;

[0030] (2) YPD medium: 1% yeast extract, 2% peptone, 2% glucose;

[0031] (3) MD solid medium: 2% glucose, 1.5% agarose, 1.34% YNB, 0.00004% Biotin;

[0032] (4) BMGY medium: 1% yeast extract, 2% peptone, 1% glycerol (V / V), 1.34% YNB, 0.00004% Biotin;

[0033] (5) BMMY medium: 1% yeast extract, 2% peptone, 1.34% YNB, 0.00004% Biotin, 0.5% methanol (V / V).

[0034] Example 1 Construction of mannanase mutant engineered bacteria

[0035] (1) Construction of expression vector and expression in Pichia pastoris

[0036] by Diplodia corticola GH5 family mannanases from Dc Using Man5A-pPIC9 as a template, mutation primers Q98V-F / R (Q98V-F, SEQ ID NO: 5; Q98V-R, SEQ ID NO: 6) were designed at the Q98 site of the gene. PCR amplification was first performed using a point mutation kit. The PCR product was then demethylated with DMT enzyme and transformed into DMT competent cells. Finally, single clones were selected for verification. Positive transformants were selected for DNA sequencing. Transformants with correct sequences were used to prepare recombinant plasmids in large quantities.

[0037] The recombinant vector, which was correctly ligated to the expression vector pPIC9 and sequenced, was linearized with the restriction enzyme BglII and transformed into Pichia pastoris GS115 competent cells. The cells were cultured at 30°C for 2-3 days. Transformants grown on MD plates were selected for further expression experiments to obtain recombinant yeast strains.

[0038] (2) Screening of high mannanase-active transformants

[0039] Using a sterilized toothpick, pick a single colony from an MD plate containing transformants and spot it onto the plate according to its number. Incubate the MD plate at 30°C for 1-2 days until colonies grow. Pick transformants from the MD plate according to their numbers and inoculate them into centrifuge tubes containing 3 mL of BMGY medium. Incubate at 30°C and 220 rpm for 48 h on a shaker. Centrifuge the culture at 3,000×g for 15 min, discard the supernatant, and add 1 mL of BMMY medium containing 0.5% methanol to the centrifuge tube. Induce culture at 30°C and 220 rpm. After 48 h of induction culture, centrifuge at 3,000×g for 5 min, and use the supernatant for enzyme activity detection. Screen for transformants with high mannanase activity. For detailed procedures, please refer to the Pichia pastoris expression manual.

[0040] Example 2 Preparation of mannanase mutant and wild-type enzyme solution

[0041] (1) Shake-flask expression of mutant genes in Pichia pastoris

[0042] Transformants with the highest enzyme activity were selected and inoculated into 30 mL YPD medium for 48 h for seed culture amplification. Then, 1% of the transformant was inoculated into 1 L Erlenmeyer flasks containing 300 mL BMGY medium and cultured at 30℃ and 220 rpm for 48 h. The culture was centrifuged at 3000 g for 5 min, the supernatant was discarded, and the precipitate was resuspended in 200 mL of BMMY medium containing 0.5% methanol. The culture was then induced again at 30℃ and 220 rpm. 1 mL of methanol was added every 12 h to maintain a methanol concentration of 0.5% in the bacterial culture, and the supernatant was collected for enzyme activity assay.

[0043] (2) Purification of mutant mannanase

[0044] The crude enzyme solution cultured in shake flasks was concentrated using a 10 kDa membrane pack and transferred to a 5 kDa dialysis bag. It was then dialyzed against low-salt buffer (10 mM Tris-HCl, pH 8.0) at 4°C for 12 h to remove salt. An AKTA purifier 25 HiTrap Q anion exchange column was used for gradient elution with 10 mmol / L Tris-HCl (pH 8.0) containing 0–1 mol / L NaCl. The eluent was collected stepwise according to the UV280 response value. Chromatographic peaks containing the target band and exhibiting activity were collected. The purity of the purified enzyme solution was determined by SDS-PAGE electrophoresis. Figure 1 Collect and combine the enzyme solutions that have reached electrophoretic purity, and store them at 4°C for later use.

[0045] Example 3 Activity analysis and property determination of mannanase mutant and wild-type enzyme DcMan5A

[0046] The activity of the mannanase of this invention was analyzed using the DNS method, and the specific method is as follows:

[0047] Under given pH and temperature conditions, a 1 mL reaction system comprising 100 μL of appropriately diluted enzyme solution, 900 μL of substrate, and reacted for 10 min, was terminated by adding 1.5 mL of DNS and boiling for 5 min. The OD value was measured at 540 nm after cooling. One enzyme activity unit (U) is defined as the amount of enzyme required to break down carob gum to produce 1 μmol of reducing sugar per minute under given conditions.

[0048] Mutants and wild-type enzymes Dc Determination of the optimal pH value of Man5A

[0049] Enzymatic reactions were carried out in citrate-disodium hydrogen phosphate buffer systems at different pH values ​​to determine mutant and wild-type enzymes. Dc The optimal pH for Man5A.

[0050] The results show that,Figure 2 As shown, at a temperature of 60℃, mannanase... Dc The optimal pH of the Man5A single-point mutant Q98V and the wild-type enzyme remained unchanged at 5.0; at various pH values, the single-point mutant Q98V exhibited similarities to the wild-type enzyme. Dc Similar to Man5A, it can maintain more than 50% of its enzyme activity within the pH range of 4.0-6.0.

[0051] Mutants and wild-type enzymes Dc Determination of the optimal temperature of Man5A

[0052] Enzymatic reactions were carried out in a pH 5.0 citrate-disodium hydrogen phosphate buffer system at different temperatures (50, 60, and 70 °C) to determine the mutant and wild-type enzymes. Dc The optimal temperature for Man5A.

[0053] The results show that, Figure 3 As shown, under pH 5.0 conditions, mannanase... Dc The optimal temperature for both the Man5A single-point mutant Q98V and the wild-type enzyme remained unchanged at 60℃; at various temperatures, the single-point mutant Q98V exhibited similarities to the wild-type enzyme. Dc Similar patterns to Man5A.

[0054] Mutants and wild-type enzymes Dc Determination of dynamic parameters of Man5A

[0055] Determine the measurement K The reaction time for m and Vmax was 5 min. Enzyme activity was measured using different concentrations (0.25-7.5 mg / mL) of carob gum as substrate under the same conditions (60℃, pH 5.0), and the corresponding reaction rates were calculated. PrismGraphpad software was used to calculate... K The values ​​of m and Vmax are shown in Table 1.

[0056] Table 1. Mutants and wild-type enzymes Dc Dynamic parameters of Man5A

[0057] m (mg / mL) Vmax (μmol / min / mg) Specific activity (U / mg) Man5A 6.45 518 217 Man5A-Q98V 3.92 800 398 .

[0058] The results showed that mannanase Dc The specific activity of the Man5A single-point mutant Q98V is derived from that of the wild-type enzyme. Dc Man5A levels increased from 217 U / mg to 398 U / mg, an increase of approximately 0.8 times. Simultaneously, mannanase... Dc Man5A single-point mutant Q98V K The m value is determined by the wild enzyme.Dc The concentration of Man5A decreased from 6.45 mg / mL to 3.92 mg / mL, resulting in an approximately 0.4-fold increase in substrate affinity.

[0059] The above embodiments are only used to understand the technical solutions of this application and do not limit the scope of protection of this application.

Claims

1. Mannanase with increased specific activity Dc The Man5A mutant is characterized by... The amino acid sequence of the mutant is shown in SEQ ID NO:

3.

2. A mannanase gene, characterized in that, The mannanase gene encodes the mannanase with increased specific activity as described in claim 1. Dc Man5A mutant.

3. The mannanase gene according to claim 2, characterized in that, The nucleotide sequence of the mannanase gene is shown in SEQ ID NO:

4.

4. A recombinant expression vector comprising the mannanase gene of claim 2.

5. A recombinant strain containing the mannanase gene as described in claim 2.

6. A method for enhancing mannanase Dc The specific activity method of Man5A is characterized by, The method includes the following steps: wild-type mannanase Dc The glutamine at position 115 of the N-terminus of Man5A is mutated to valine, wherein the wild-type mannanase... Dc The amino acid sequence of Man5A is shown in SEQ ID NO: 1; or wild-type mannanase Dc The N-terminal 98th position of the mature Man5A protein is mutated from glutamine to valine, wherein the wild-type mannanase... Dc The amino acid sequence of the mature Man5A protein is shown in SEQ ID NO:

2.

7. The mannanase with enhanced specific activity according to claim 1 Dc The Man5A mutant is used for the application of hydrolyzed mannan.

8. A method for preparing mannanase with enhanced specific activity, characterized in that, The method includes the following steps: The host cell was transformed with the recombinant expression vector containing the mannanase gene of claim 2 to obtain the recombinant strain; The obtained recombinant strain was cultured and induced to express its contents; The mannanase with increased specific activity was recovered and purified.

Images