CN105886484A - Thermophilic cellulase, encoding gene thereof and application of thermophilic cellulase - Google Patents

Abstract

The invention relates to the field of genetic engineering, in particular to a thermophilic cellulase and its encoding gene and application. The amino acid sequence of the thermophilic cellulase is shown in SEQ ID NO.1 or SEQ ID NO.2. The invention provides a new cellulase, which can be used in feed, food, medicine and other industries. According to the technical scheme of the invention, the production of cellulase with excellent properties and suitable for industrial application can be realized by means of genetic engineering.

Description

A kind of thermophilic cellulase and its coding gene and application

technical field

The invention relates to the field of genetic engineering, in particular to a thermophilic cellulase and its encoding gene and application.

Background technique

Plant cell walls are mainly composed of cellulose, hemicellulose, and lignin. Cellulose is an important polysaccharide. It is the material of plant cell support material and the most abundant biomass resource in nature. The structure of cellulose is determined as β-D-glucose units connected by β-(1→4) glycosidic bonds. The resulting linear polymer has no branches in the structure, which can be degraded to glucose by cellulase.

Cellulase refers to the general term for a group of enzymes that can hydrolyze glucosidic bonds and decompose cellulose into cellobiose and glucose. The hydrolysis process of cellulose mainly includes three steps: the first step is that the endo-cellulase acts on the amorphous region inside the cellulose, and then hydrolyzes the β-(1→4) glycosidic bond to shorten the cellulose molecule, Then the exo-cellulase acts on the end of the cellulose linear molecule to hydrolyze the β-(1→4) glycosidic bond, cutting off one cellobiose molecule at a time, and finally, the glucosidase hydrolyzes the cellobiose into glucose molecular.

Cellulase 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. Cellulase widely exists in bacteria, actinomycetes, fungi, plants, animals and other organisms. The cellulase produced by different microorganisms has great differences in structure and function. Compared with normal temperature cellulase, thermophilic cellulase has many advantages, such as high catalytic efficiency, strong substrate specificity, good enzyme stability during heat treatment of lignocellulose, and thus good compatibility with the reaction system. So far, thermophilic cellulase is mostly derived from bacteria, mainly from Thermospora maritima, Thermoascus aurantiacus, etc. However, the expression level of thermophilic cellulase derived from high-temperature bacteria is low, and it is difficult to carry out industrial production.

Currently commercialized cellulase is mainly produced by fungi, such as Trichoderma, Penicillium, and Aspergillus. The optimum temperature of cellulase derived from fungi is mostly between 45 and 65°C, and it is easy to lose activity at high temperature. The present invention obtains a new cellulase gene from the thermophilic cyanobacterium Talaromyces leycettanus JCM 12802 strain, and the cellulase encoded by it has the following advantages: thermophilic, acidic, broad substrate specificity, easy fermentation Production. All these advantages mean that the newly invented cellulase will have more application value than the previously reported cellulase in feed, food, medicine and other industries.

Contents of the invention

The purpose of the present invention is to provide a cellulase which is thermophilic, acidic and has wide substrate specificity.

Another object of the present invention is to provide the above-mentioned cellulase gene.

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

Another object of the present invention is to provide a recombinant strain comprising the above cellulase gene.

Another object of the present invention is to provide a method for preparing cellulase.

Another object of the present invention is to provide the application of the above cellulase.

The technical problem to be solved by the present invention is to overcome the deficiencies in the prior art, and provide a new cellulase with excellent properties and suitable for application in feed, food, medicine and other industries, its amino acid sequence is as SEQ ID NO.1 :

Among them, the full length of the enzyme is 409 amino acids, and the N-terminal 18 amino acids are the signal peptide sequence "MKFSNVILAASASSLVLA".

Therefore, the theoretical molecular weight of mature thermophilic cellulase is 42.5kDa, and its amino acid sequence is as SEQ ID NO.2:

The optimum pH of the cellulase is 3.5, and the enzyme can maintain more than 60% of its enzyme activity in the range of pH2.5-pH5.0; the optimum temperature is 80°C, and it still has 30% of the enzyme at 85°C Activity, treatment at 70°C for 60 minutes, basically no loss of enzyme activity, treatment at 75°C for 5 minutes, can maintain more than 70% of the enzyme activity, and treatment at 80°C for 2 minutes, can maintain more than 60% of the enzyme activity, with good thermal stability.

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

The present invention isolates and clones the cellulase gene by the method of PCR, and the DNA sequence analysis results show that the cellulase structural gene is 1464bp in length and contains 4 introns, +96～162, +374～429, +495 ～551, +650～7.3 are its intron sequences, the cDNA is 1230bp long, and its cDNA sequence is shown in SEQ ID NO.4:

Wherein, the base sequence of the signal peptide is:

"ATGAAGTTTT CCAACGTGAT TCTTGCGGCC AGCGCGTCGA GCCTGGTGCT CGCT"

Therefore, the coding sequence of the mature gene is

Shown in SEQ ID NO.5:

This enzyme belongs to the 5th family of glycosyl hydrolases. The cellulase gene cDNA sequence and deduced amino acid sequence were compared by BLAST in GenBank to confirm that the cellulase was a new cellulase.

The present invention also provides a recombinant vector comprising the above cellulase gene, preferably a Pichia pastoris expression vector. The cellulase gene of the present invention is inserted between suitable restriction 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 cellulase gene cDNA is inserted between SnaBI and the NotI restriction enzyme site on the plasmid pPIC9, so that the nucleotide sequence is positioned at the downstream of the AOX1 promoter and Under its regulation, recombinant yeast expression plasmids are obtained.

The present invention also provides a recombinant strain comprising the above cellulase gene, preferably a recombinant Pichia pastoris strain.

The present invention also provides a method for preparing cellulase, comprising the following steps:

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

2) cultivating the recombinant strain to induce the expression of the recombinant cellulase; and

3) Recovering and purifying the expressed cellulase.

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

The present invention also provides the application of the above cellulase. Using genetic engineering means to industrialize the production of cellulase. The invention provides a new cellulase, which can be used in feed, food, medicine and other industries. According to the technical scheme of the invention, the production of cellulase 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 cellulase expressed in Pichia pastoris.

Fig. 2 Optimum pH value of the recombinant cellulase of the present invention.

Figure 3 pH stability of the cellulase of the present invention.

Fig. 4 Optimum reaction temperature of cellulase of the present invention.

Figure 5 shows the thermostability of the cellulase of the present invention.

detailed description

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, ligases were purchased from Invitrogen Company, 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 powder, 30g/L soybean meal, 5g/L barley dextran, 5g/L (NH ₄ )SO ₄ , 1g/L KH ₂ PO ₄ , 0.5g/LMgSO ₄ 7H ₂ O, 0.01g/L FeSO ₄ 7H ₂ O, 0.2g/L CaCl ₂ in 1L deionized water, sterilized at 121℃, 15 pounds for 20min

(2) Escherichia coli medium LB (126 peptone, 0.5% yeast extract, 126NaCI, pH 7.0).

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

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

Explanation: For the molecular biology experimental 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.

Example 1 Cloning of cellulase-encoding gene

The genomic DNA of Talaromyces leycettanus JCM 12802 was extracted, the cloning primers F: atgaagttttccaacgtgattcttgcggc and R: ctacaggcactggtagtaataggggttcag were designed, and the total DNA of Talaromycesleycettanus JCM 12802 was used as a template for PCR amplification. The PCR reaction parameters are: 95°C for 5min; 30 cycles of 94°C for 30sec, 60°C for 30sec, 72°C for 60sec, and 72°C for 10min. A fragment of about 1.5k bp was obtained, and the full-length gene was obtained after correct sequencing.

Extract the total RNA of Talaromyces leycettanus JCM 12802, 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, amplify the single-stranded cDNA, and obtain the cellulase cDNA sequence, the amplified product was recovered and sent for sequencing.

After comparing the genome sequence and cDNA sequence of cellulase, it was found that the cellulase structural gene is 1464bp in length, contains 4 introns, and the cDNA is 1230bp long, encoding 409 amino acids and a stop codon, with 18 N-terminal The amino acid is its signal peptide sequence, and the comparison proves that the cellulase-encoding gene isolated and cloned from Talaromyces leycettanus JCM12802 is a new gene.

Example 2 Construction of Cellulase Engineering Strain

(1) Construction of expression vector and expression in yeast

Using the correctly sequenced cellulase cDNA as a template, the primers cdna-sF:act tacgta gctcccaagagcaagaccaagcgcacatc and cdnaR:ata gcggccgc ctacaggcactggtagtaataggggttcag with SnaB I and Not I restriction sites were designed and synthesized, and the maturation of cellulase The coding region of the protein is amplified. And use SnaB I and Not I to digest the PCR product, connect it into the expression vector pPIC9 (Invitrogen, SanDiego), the sequence of the cellulase mature protein is inserted into the downstream of the signal peptide sequence of the above expression vector, and form a correct reading frame with the signal peptide , constructed into a yeast expression vector, and transformed into Escherichia coli competent cells Trans1. The positive transformants were subjected to DNA sequencing, and the transformants with the correct sequence were used for large-scale preparation of recombinant plasmids. Linearize expression plasmid vector DNA with restriction endonuclease Bgl II, transform yeast GS115 competent cells by electroporation, culture at 30°C for 2-3 days, pick transformants grown on MD plates for further expression experiments, specific operations Please refer to the Pichia expression manual.

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

(2) Screening of transformants with high cellulase activity

Use a sterilized toothpick to pick a single colony from the MD plate with transformants, spot it on the MD plate according to the number, and place the MD plate in a 30°C incubator for 1 to 2 days until the colony grows. Pick the transformant from the MD plate according to the number and inoculate it in 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 1mL of BMMY medium containing 0.5% methanol to the centrifuge tube, induce culture at 30°C, 220rpm; after 48 hours of induction culture, centrifuge at 3,000×g for 5min, take the supernatant for enzyme activity detection, and screen out high cellulase For active transformants, please refer to the Pichia pastoris expression manual for specific operations.

Example 3 Preparation of recombinant cellulase

(1) Mass expression of cellulase gene at shake flask level in Pichia pastoris

The transformant with high enzyme activity was screened out, inoculated into a 1L Erlenmeyer flask with 300mL of BMGY liquid medium, cultured on a shaking table at 30°C at 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°C, 220rpm induction culture for 72h. During the induction culture period, add methanol solution once 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 (Figure 1).

(2) Purification of recombinant cellulase

The supernatant of the recombinant cellulase 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 cellulase that can be diluted to certain times, and purify by ion exchange chromatography. Specifically, 2.0 mL of the recombinant cellulase concentrate was passed through a HiTrap Q Sepharose XL anion column equilibrated with 20 mM Tris-HCl (pH 7.5) in advance, and then eluted with a linear gradient of 0-1 mol/L NaCl, split in half The eluate collected in the first step was used to detect enzyme activity and determine protein concentration.

Example 4 Partial Property Analysis of Recombinant Cellulase

The activity analysis of the cellulase of the present invention is carried out by DNS method. The specific method is as follows: under the conditions of pH 3.5 and 80°C, 1 mL of reaction system includes 100 μL of appropriate diluted enzyme solution, 900 μL of substrate, reacted for 10 minutes, added 1.5 mL of DNS to terminate the reaction, and boiled for 5 minutes. After cooling, the OD value was measured at 540 nm. Definition of cellulase activity unit: Under certain conditions, the amount of enzyme required to decompose carboxymethyl cellulose to generate 1 μmol reducing sugar per minute is 1 activity unit (U).

(1) Optimum pH and pH stability of cellulase

The purified cellulase expressed in Example 4 was subjected to enzymatic reactions at different pHs to determine its optimum pH. The buffers used are pH 1.0~3.0 glycine-hydrochloric acid buffer, pH 2.2~8.0 disodium citric acid monohydrogen phosphate buffer, pH 8.0~9.0 Tris-HC buffer, lpH9.0~12 glycine-NaoH series buffer. Purified cellulase in different pH buffer systems. The pH suitability results (Figure 2) measured at 80°C show that: . The optimum pH of the cellulase is 3.5, and within the range of pH 2.5-pH 5.0, the enzyme 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 ( FIG. 3 ), the analysis results showed that the enzyme was stable between pH2.0-pH11.0, and had excellent pH stability.

(2) Optimum temperature and thermal stability of cellulase reaction

Purified cellulase was tested for enzyme activity at different temperatures (40-90°C) at a pH of 3.5. The results of the analysis showed that the optimum temperature was 80°C, and 30% of the enzyme activity was still present at 85°C (Fig. 4) Treated at 70°C for 60 minutes, the enzyme activity is basically not lost, treated at 75°C for 5 minutes, can maintain more than 70% of the enzyme activity, and treated at 80°C for 2 minutes, can maintain more than 60% of the enzyme activity, has a good thermal stability (Figure 5).

Claims (7)

1. a thermophilic cellulase, it is characterised in that the aminoacid sequence of described thermophilic cellulase such as SEQ ID Shown in NO.1 or SEQ ID NO.2.

2. a thermophilic cellulase gene, it is characterised in that coding thermophilic cellulase described in claim 1.

Thermophilic cellulase gene the most according to claim 2, it is characterised in that its nucleotide sequence such as SEQ Shown in ID NO.3, SEQ ID NO.4 or SEQ ID NO.5.

4. comprise the recombinant expression carrier of fine thermophilic cellulase gene described in claim 2.

5. comprise the recombinant bacterial strain of fine thermophilic cellulase gene described in claim 2.

6. the preparation method of the thermophilic cellulase described in a claim 1, it is characterised in that the method bag Include following steps:

1) structure comprises the recombinant expression carrier of fine thermophilic cellulase gene described in claim 2；

2) with the recombinant expression carrier transformed host strain obtained；

3) express and separate the thermophilic cellulase described in claim 1.

7. the application of the thermophilic cellulase described in claim 1.