CN103642779B - A kind of high specific activity acidic beta-mannase Man5D and gene thereof and application - Google Patents

Abstract

The present invention relates to genetically engineered field.Particularly, the present invention relates to a kind of high specific activity acidic beta-mannase Man5D and gene thereof and application, its aminoacid sequence is as shown in SEQ ID NO.1 or SEQ ID NO.2.Mannase of the present invention has following character: optimal pH is 5.0, and within the scope of pH3.5-8.0, this enzyme can maintain its enzyme activity of more than 60%, and optimal reactive temperature is 65 DEG C, and highly stable at 50 DEG C, and in addition, its Rate activity is 3133Umg ^{– 1}.This mannase has acidity, high specific activity, preferably thermotolerance, can be applied to the industry such as feed, food, medicine.The mannase utilizing the excellent applicable industrial application of genetic engineering means nature of production just can be realized according to technical scheme of the present invention.

Description

A high specific activity acidic β-mannanase Man5D and its gene and application

technical field

The invention relates to the field of genetic engineering, in particular, the invention relates to a high specific activity acidic β-mannanase Man5D and its gene and application.

Background technique

Plant cell walls are mainly composed of cellulose, hemicellulose, and lignin. Mannan is an important component of plant hemicellulose. It is a linear polymer connected by β-1,4-D-mannose. The side chains of the polysaccharide mainly contain glucosyl, acetyl and hemicellulose. Lactosyl and other substituent groups. β-mannanase (β-mannanase EC3.2.1.78) is an endohydrolase that hydrolyzes mannan. It degrades the β-1,4 glycosidic bond of the mannose backbone in an endo-cutting manner, releasing a short β- l,4 mannan oligosaccharides.

In recent years, with the discovery of the physiological functions of mannan oligosaccharides, the rise of green feed, the enhancement of people's awareness of environmental protection, and the research on the regeneration and utilization of energy, the research and utilization of β-mannanase have entered a new stage. β-mannanase has been widely used in many fields such as food, medicine, feed, papermaking, textile printing and dyeing, petroleum exploration, fine chemical industry and biotechnology. It is a new type of industrial enzyme with great potential application value.

β-mannanase widely exists in bacteria, actinomycetes, fungi, plants, animals and other organisms. Mannanases of bacterial origin are mainly acid-neutral mannanases. Most of its molecular weights are between 35kDa and 55kDa, and the optimum reaction temperature is 50°C to 70°C. At present, Bacillus is the most researched one. Except for the optimal pH value of alkalophilic Bacillus reaching above pH 9.0, most of the optimal reaction pHs are between 5.5 and 8.0. The fungal β-mannanase is generally acidic, with a molecular weight of about 45kDa to 55kDa, an optimum pH of 4.0 to 6.0, and an optimum temperature of 55°C to 75°C. Compared with bacteria, fungal-derived β-mannanase has a lower optimum reaction pH value and lower pH stability, and its heat resistance is worse than that of bacteria. At present, at home and abroad, although many β-mannanases have been cloned and isolated and their properties are determined, there are some defects in the properties and characteristics of these enzymes, for example, the pH range is not suitable, the thermal stability is poor, and the expression level is low. meet the needs of practical applications. Therefore, people hope to find a new β-mannanase that can meet the needs of practical applications, so as to further promote the application of the β-mannanase in feed, food, medicine and other industries.

The present invention obtains a new β-mannanase gene from the Staphylotrichum coccosporum NBRC31817 strain, and the mannanase encoded by it has the following advantages: acidity, better thermostability, high specific activity, strong protease Resistant, easy to ferment and produce. All these advantages mean that the newly invented β-mannanase will have more application value than previously reported β-mannanase in feed, food, medicine and other industries.

Contents of the invention

The purpose of the present invention is to provide a β-mannanase with acidity, good thermal stability and high specific activity.

Still another object of the present invention is to provide the above-mentioned β-mannanase gene.

Another object of the present invention is to provide a recombinant vector comprising the above-mentioned β-mannanase.

Another object of the present invention is to provide a recombinant strain comprising the above-mentioned β-mannanase gene.

Another object of the present invention is to provide a method for preparing β-mannanase.

Another object of the present invention is to provide the application of the above-mentioned β-mannanase.

The first technical problem to be solved by the present invention is to overcome the deficiencies of the prior art and provide a new β-mannanase with excellent properties and suitable for application in feed, food, medicine and other industries. The β-mannanase Man5D has an amino acid sequence such as SEQ ID NO.1:

Among them, the full length of the enzyme is 371 amino acids, and the N-terminal 21 amino acids are the signal peptide sequence "MKSSILSPIALGLGLLSTAG A".

Therefore, the theoretical molecular weight of mature β-mannanase Man5D is 37kDa, and its amino acid sequence is as SEQID NO.2:

The β-mannanase Man5D has the characteristics of acidity, high specific activity and the like. The optimum pH is 5.0, and the enzyme can maintain more than 60% of its enzyme activity in the range of pH3.5-pH8.0; the optimum temperature is 65°C. Treated at 50°C for 60 minutes, the remaining enzyme activity is above 95%, even if the enzyme is treated at 60°C for 10 minutes, it can still maintain 30% of the enzyme activity, and has good stability; it has excellent resistance to pepsin and pancreas Protease processing ability; at the same time, high-density fermentation has high enzyme activity and is easy for industrial production.

The present invention also provides a gene encoding the above-mentioned β-mannanase. The full gene sequence of the enzyme is shown in SEQ ID NO.3:

The present invention isolates and clones the β-mannanase gene man5D by PCR method, and the DNA sequence analysis results show that the structural gene of β-mannanase Man5D is 1260bp in length, contains 2 introns, +813-876bp , +966～1045bp, its intron sequence, the cDNA length is 1116bp, and its cDNA sequence is shown in SEQ ID NO.4:

Wherein, the base sequence of the signal peptide is:

"ATGAAGTCCAGCATCCTCTCGCCCATCGCCCTCGGGCTCGGCCTCCTCTCGACCGCCGGCGCC"

Therefore, the coding sequence of the mature gene is

Shown in SEQ ID NO.5:

The theoretical molecular weight of the mature protein is 37kDa, and the enzyme belongs to the fifth family of glycosyl hydrolases. The cDNA sequence and deduced amino acid sequence of β-mannanase gene man5D were compared by BLAST in GenBank, and it was found that Man5D was a new mannanase.

The present invention also provides a recombinant vector comprising the above-mentioned β-mannanase gene, preferably pPIC9-man5D. The β-mannanase gene of the present invention is inserted between suitable restriction enzyme cutting sites of the expression vector, so that its nucleotide sequence is operably linked with the expression control sequence. As a most preferred embodiment of the present invention, it is preferred that the β-mannanase gene is inserted between EcoR I and the Not I restriction enzyme site on the plasmid pPIC9, so that the nucleotide sequence is positioned at the AOX1 promoter The downstream of the gene is regulated by it, and the recombinant yeast expression plasmid pPIC9-man5D is obtained.

The present invention also provides a recombinant strain comprising the above-mentioned β-mannanase gene, preferably the recombinant strain GS115/man5D.

The present invention also provides a method for preparing acidophilic β-mannanase, comprising the following steps:

1) Transforming host cells with the above-mentioned recombinant vectors to obtain recombinant strains;

2) Cultivate the recombinant strain to induce the expression of recombinant β-mannanase;

3) Recovering and purifying the expressed β-mannanase.

Wherein, preferably the host cell is a Pichia pastoris cell, a Saccharomyces cerevisiae cell or a Hansenula polymorpha cell, preferably the recombinant yeast expression plasmid is transformed into a Pichia pastoris cell ) GS115 to obtain the recombinant strain GS115/man5D.

The present invention also provides the application of the above-mentioned β-mannanase. The use of genetic engineering to industrialize the production of mannanase products with high temperature, acidity and high specific activity has not been reported yet.

The invention provides a new mannanase gene, the encoded mannanase has acidity, high specific activity, good heat resistance and protease resistance, and can be used in feed, food, medicine and other industries. According to the technical scheme of the invention, the production of mannanase with excellent properties and suitable for industrial application can be realized by means of genetic engineering.

Description of drawings

Figure 1 SDS-PAGE analysis of β-mannanase expressed by man5D in Pichia pastoris, 1, supernatant of expressed mannanase; 2, purified recombinant β-mannanase.

Fig. 2 The optimal pH value of the recombinant β-mannanase of the present invention.

Fig. 3 pH stability of β-mannanase of the present invention.

Fig. 4 Optimum reaction temperature of β-mannanase of the present invention.

Figure 5 shows the thermostability of β-mannanase of the present invention.

Detailed ways

Test materials and reagents

1. Strains and vectors: Pichia pastoris GS115 was preserved in our laboratory; Pichia pastoris expression vector pPIC9 and strain GS115 were purchased from Invitrogen.

2. Enzymes and other biochemical reagents: endonucleases were purchased from TaKaRa Company, and ligases were purchased from Invitrogen Company. Oat xylan was purchased from Sigma, and the others were domestic reagents (all of which can be purchased from common biochemical reagent companies).

3. Medium:

(I) Enzyme production medium: 30g/L wheat bran, 30g/L corn cob flour, 30g/L soybean meal, 5g/L konjac flour, 5g/L (NH ₄ )SO ₄ , 1g/L KH ₂ PO ₄ , 0.5g/L MgSO ₄ 7H ₂ O, 0.01g/L FeSO ₄ 7H ₂ O, 0.2g/LCaCl ₂ in 1L deionized water, 121℃, 15 lbs, sterilized for 20min

(2) Escherichia coli medium LB (126 peptone, 0.5% yeast extract, 126NaCI, pH7.0).

(3) BMGY medium; 1% yeast extract, 2% peptone, 1.34% YNB, 0.000049<Biotin, 1% glycerol (v/v).

(4) BMMY medium: replace glycerol with 0.5% methanol, and the rest of the ingredients are the same as BMGY, pH 4.0.

Note: For the molecular biology experiment methods not specifically described in the following examples, all refer to the specific methods listed in the book "Molecular Cloning Experiment Guide" (Third Edition) J. Sambrook, or follow the kit and product manual.

Cloning of embodiment 1β-mannanase encoding gene man5D

Extraction of Staphylotrichum coccosporum genomic DNA

Staphylotrichum coccosporum NBRC31817 was purchased from NBRC (NITE biological resource center) in Japan.

The bacteria cultured in the enzyme-producing medium for 3 days were centrifuged at 12,000 rpm for 10 minutes, and the collected mycelium was added to a high-temperature sterilized mortar, and quickly ground to powder with liquid nitrogen, and then the ground bacteria were transferred to a new 50mL centrifuge tube filled with 15ml CTAB lysate, gently invert up and down to mix, place in a 70°C water bath for 3 hours, every 20min, invert up and down and gently mix once to fully lyse the bacteria. Centrifuge at 12,000 rpm at 4°C for 10 min, pipette the supernatant into a new centrifuge tube, add an equal volume of chloroform for extraction, and place at room temperature for 5 min. Centrifuge at 12,000 rpm for 10 min at 4°C. Take the supernatant and add an equal volume of phenol/chloroform for extraction, and place it at room temperature for 5 minutes. Centrifuge at 12,000 rpm for 10 min at 4°C. In order to remove foreign proteins as much as possible, the supernatant was added to an equal volume of isopropanol, and after standing at room temperature for 5 minutes, centrifuged at 10000 rpm for 10 minutes at 4°C. The supernatant was discarded, the precipitate was washed twice with 70% ethanol, dried in vacuo, dissolved by adding an appropriate amount of TE, and stored at -20°C for later use.

The degenerate primers P1 and P2 were designed and synthesized according to the published conserved sequence of β-mannanase gene (see Table 1). PCR amplification was carried out using Staphylotrichum coccosporum genomic DNA as a template. The PCR reaction parameters are: 95°C for 5min; 94°C for 30sec, 50 to 45°C for 30sec, 72°C for 30sec, 12 cycles (in which the annealing temperature drops by 1°C after each cycle); 94°C for 30min, 45°C for 30sec, and 72°C 30sec, 30 cycles; 10min at 72°C. A fragment of about 180bp was obtained, which was recovered and sent to Sanbo Biotechnology Co., Ltd. for sequencing. TAIL-PCR primers uspl, usp2, usp3; dspl, dsp2, dsp3 were designed according to the nucleotide sequence obtained by sequencing (see Table 1). The flanking sequence of the known gene sequence was obtained by TAIL-PCR, and the amplified product was recovered and sent to Sanbo Biotechnology Co., Ltd. for sequencing. The correctly sequenced fragments were spliced to obtain the full-length gene.

Table 1 Primers required for this experiment

Example 2 Obtaining of β-mannanase cDNA

Extract the total RNA of Staphylotrichum coccosporum, use Oligo(dT) ₂₀ and reverse transcriptase to obtain a strand of cDNA, then design primers F and R to amplify the open reading frame (see Table 1), and amplify the single-stranded cDNA to obtain The cDNA sequence of mannanase, the amplified product was recovered and sent to Sanbo Biotechnology Co., Ltd. for sequencing.

After comparing the genome sequence and cDNA sequence of mannanase, it was found that the gene contained 2 introns, the cDNA was 1116 bp long, encoded 371 amino acids and a stop codon, and the N-terminal 21 amino acids were its signal peptide sequence. The comparison proves that the gene encoding mannanase isolated and cloned from Staphylotrichum coccosporum is a new gene.

The construction of embodiment 3 β-mannanase engineering strains

(1) Construction of expression vector and expression in yeast

Using the correctly sequenced mannanase Man5D cDNA as a template, the expression primers F and R with EcoR I and Not I restriction sites were designed and synthesized (see Table 1) for the coding region of the mature protein of Man5D Amplify. And use EcoR I and Not I to digest the PCR product, connect it into the expression vector pPIC9 (Invitrogen, San Diego), and insert the sequence of the mature protein of β-mannanase Man5D into the downstream of the signal peptide sequence of the above expression vector, and the signal peptide The correct reading frame was formed, and the yeast expression vector pPIC9-man5D was constructed to transform Escherichia coli competent cell JM109. The positive transformants were subjected to DNA sequencing, and the transformants with the correct sequence were used for large-scale preparation of recombinant plasmids. Use the restriction endonuclease Bgl II to linearize the expression plasmid vector DNA, transform yeast GS115 competent cells by electric shock, spread on the histidine-deficient RDB plate, culture at 30°C for 2-3 days, and pick on the RDB plate The grown transformants were further used for expression experiments. For specific operations, please refer to the Pichia expression manual.

In the same way, an expression vector containing cDNA of Man5D signal peptide sequence was constructed and transformed.

(2) Screening of transformants with high mannanase activity

Use a sterilized toothpick to pick a single colony from the RDB plate with transformants, and then spot it on the MM plate according to the number, and then spot it on the MD plate with the corresponding number. Spot 100 single colonies on each plate, a total of 200 transformants; place the MM and MD plates with the transformants in a 30°C incubator for 1-2 days until colonies grow. Pick the transformant from the MD plate according to the number and inoculate it into a centrifuge tube containing 3mL of BMGY medium, culture it on a shaker at 30°C and 220rpm for 48h; centrifuge the bacterial solution cultured on a shaker for 48h at 3,000×g for 15min, remove the supernatant, Add 1 mL of BMMY medium containing 0.5% methanol to the centrifuge tube, and induce culture at 30°C and 220 rpm; after 48 hours of induction culture, centrifuge at 3,000×g for 5 minutes, take the supernatant for enzyme activity detection, and screen high mannan from it Transformants with enzyme activity, please refer to the Pichia pastoris expression manual for specific operations.

Preparation of embodiment 4 recombinant β-mannanase

(1) Mass expression of β-mannanase gene Man5D in shake flask level in Pichia pastoris

The transformant with high enzyme activity was screened out, inoculated in a 1L Erlenmeyer flask with 300mL of BMGY liquid medium, cultured on a shaking table at 30°C and 220rpm for 48h; centrifuged at 5,000rpm for 5min, discarded the supernatant gently, and then added 100mL containing 0.5% methanol BMMY liquid medium, 30 ℃, 220rpm induced culture for 72h. During the induction culture period, add methanol solution every 24 hours to compensate for the loss of methanol, and keep the methanol concentration at about 0.5%; (3) Centrifuge at 12,000×g for 10 minutes, collect the supernatant fermentation liquid, detect the enzyme activity and perform SDS-PAGE protein Electrophoretic analysis.

(2) Purification of recombinant β-mannanase

The supernatant of the recombinant β-mannanase expressed in the shake flask was collected, concentrated through a 10kDa membrane bag, and at the same time the medium was replaced with a low-salt buffer, and then further concentrated with a 10kDa ultrafiltration tube. Concentrate the recombinant Man5D that can be diluted to a certain factor, and purify it by ion exchange chromatography. Specifically, take 2.0 mL of the Man5D concentrated solution and pass it through the HiTrap Q Sepharose XL anion column equilibrated with 20 mM Tris-HCl (pH 8.0) in advance, and then perform linear gradient elution with 0-1 mol/L NaCl, and collect The eluate was used to detect the enzyme activity and determine the protein concentration, and the purity of the protein was analyzed by SDS-PAGE electrophoresis (Figure 1).

Example 5 Partial property analysis of recombinant β-mannanase

The activity of the mannanase of the present invention was analyzed by DNS method. The specific method is as follows: under the conditions of pH 5.0 and 65°C, 1 mL of reaction system includes 100 μL of appropriate diluted enzyme solution, 900 μL of substrate, reacted for 10 min, added 1.5 mL of DNS to terminate the reaction, and boiled for 5 min. After cooling, the OD value was measured at 540 nm. Definition of mannanase activity unit: Under certain conditions, the amount of enzyme required to decompose mannan to generate 1 μmol reducing sugar per minute is 1 activity unit (U).

(1) Optimum pH and pH stability of mannanase Man5D

The purified mannanase Man5D expressed in Example 3 was subjected to enzymatic reactions at different pHs to determine its optimum pH. The buffers used are citric acid monobasic sodium phosphate series buffer solution with pH 3.0-8.0 and Tris-HCl series buffer solution with pH 8.0-10.0. The pH suitability results of the purified mannanase Man5D in the buffer system of different pH. It can maintain more than 60% of its enzyme activity.

The enzyme solution was treated at 37°C for 60 min in buffer solutions with different pH values, and then the enzyme activity was measured to study the pH stability of the enzyme. The results showed (Figure 3). The analysis results showed that more than 80% of the enzyme activity could be maintained between pH4.0-pH9.0, indicating that the enzyme had excellent pH stability.

(2) Optimum temperature and thermal stability of mannanase Man5D reaction

The enzyme activity of the purified mannanase was measured at different temperatures (30-80°C) at pH 5.0, and the analysis results showed that the optimum reaction temperature of the enzyme was 65°C (Figure 4). The temperature resistance was measured by treating mannanase at different temperatures for different times, and then measuring the enzyme activity at 65°C. The thermostability test shows that the enzyme is treated at 60°C for 60 minutes, and the remaining enzyme activity is above 95%. Even if the enzyme is treated at 50°C for 10 minutes, it can still maintain 30% of the enzyme activity, which shows that the enzyme has good stability. sex (Figure 5).

(3) Determination of kinetic parameters of recombinant β-mannanase

Using different concentrations of carob gum (0.25–5mg mL ^-1 ) as the substrate, in the citric acid-disodium hydrogen phosphate buffer (pH5.0) buffer system, the enzyme activity was measured at 65°C, and its K _m value. The K _m value and Vmax were determined to be 2.94mg ml ^–1 and 8780μmol min ^–1 mg ^–1 when carob gum was used as the substrate. The protein content in the purified enzyme solution was determined to be 0.22mg mL ^-1 , and the enzyme activity was measured to be 1934U/mL by the enzyme activity assay method, and finally the specific activity of β-mannanase was 3133U mg ^-1 .

Claims (8)

1. A high specific activity acidic β-mannanase Man5D, characterized in that its amino acid sequence is as shown in SEQ ID NO.1 or SEQ ID NO.2. 2. A high specific activity acidic β-mannanase gene, characterized in that it encodes a high specific activity acidic β-mannanase Man5D according to claim 1. 3. the high specific activity acidic β-mannanase gene according to claim 2, is characterized in that its nucleotide sequence is as shown in SEQ ID NO.3, SEQ ID NO.4 or SEQ ID NO.5 . 4. A recombinant expression vector comprising the high specific activity acidic β-mannanase gene of claim 2. 5. comprise the recombinant expression vector pPIC9-man5D of high specific activity acid β-mannanase gene described in claim 2, wherein, the β-mannanase gene of sequence as shown in SEQ ID NO.5 is inserted into plasmid Between the EcoR I and the Not I restriction enzyme site on pPIC9, make this nucleotide sequence be positioned at the downstream of AOX1 promotor and be regulated by it, obtain recombinant expression vector pPIC9-man5D. 6. A recombinant bacterial strain comprising the high specific activity acidic β-mannanase gene of claim 2. 7. A method for preparing high specific activity acidic β-mannanase Man5D, comprising the following steps: (1) transforming host cells with the recombinant expression vector according to claim 4; (2) cultivating host cells; (3) Separation and purification to obtain β-mannanase Man5D. 8. The application of the high specific activity acidic β-mannanase Man5D according to claim 1 for hydrolyzing mannan.