CN102796751A - Mutant Pul 324 of pullulanibacillus naganoensis pullulanase and use thereof - Google Patents

Abstract

A pullulanase mutant from Bacillus naganoensis, Pul324, is obtained by molecularly modifying the amino acid sequence of the pullulanase derived from Bacillus naganoensis ( Pullulanibacillus naganoensis ) by using modern enzyme engineering technology, and deleting the former on the original enzyme by PCR. With 108 amino acid residues, a mutant Pul324 capable of secreting and expressing efficiently in Escherichia coli hosts was obtained. It was inserted into pET-22b(+) to construct a recombinant vector, which was introduced into Escherichia coli host for secreted expression. The mutant gene is smaller than the wild-type gene, which is beneficial to the stability of the plasmid. Compared with the original enzyme, the mutant Pul324 can be efficiently secreted and expressed in the E. coli host, the enzyme activity of the supernatant of the fermentation broth is increased by 24 times, and the intracellular enzyme activity is increased by 40%. Adopting the present invention can increase the secreted expression amount of pullulanase of the host, and the expression product can improve the hydrolysis efficiency of starch.

Description

A kind of Bacillus nagano pullulanase mutant Pul324 and its application

technical field

The invention relates to the field of biological technology, in particular to a pullulanase mutant Pul324 of Bacillus nagano and application thereof. the

Background technique

Starch raw materials generally contain 60-90% amylopectin, and 4-6% glucose residues contained in amylopectin are linked by α-1,6-glycosidic bonds. Pullulanase (Pullulanase, EC 3.2.1.41) is a debranching enzyme that can specifically cut the α-1,6-glucosidic bond of the branch point of amylopectin, thereby cutting off the entire side branch and forming amylose. It has considerable value in improving the effect of amylase on starch, increasing the utilization rate of starch, reducing grain consumption, improving product quality and developing new products. It has very important uses in food, medicine, textile, bio-energy and other industries. And good market prospects, for example, it can be used to produce high-purity glucose and fructose, high maltose syrup, fuel ethanol, and modified starch. the

In recent years, with the development of the deep processing industry of starch raw materials, the enzyme preparation industry is required to continuously update and improve the types and properties of enzymes to meet the requirements of industrial production. my country has been researching and developing pullulanase since the 1970s, but it is limited to laboratory research due to its low enzyme activity. At present, pullulanase is the only one in the starch sugar-making enzyme system that my country cannot produce independently. The product. At present, the pullulanase with the most wide application and the largest output comes from Novo Company in Denmark. The pullulanase produced by the company is produced by submerged fermentation of Bacillus acidopullulyticus . In addition, foreign pullulanase derived from Klebsiella aerogenes ( Klebsiella aerogenes ), Bacillus subtilis ( Bacillus subtilis ) and Bacillus deramificans has also been commercially produced. Therefore, there is a big gap between my country and developed countries in the research of pullulanase. The industrial pullulanase is completely dependent on imports, and the pricing power is in the hands of a few foreign companies. The monopolized market supply has led to the high price of domestic pullulanase. The sales price has greatly restricted the development of related domestic industries.

In order to change our country's dependence on imported products and find a road to localization, this research uses enzyme engineering technology to develop high-performance pullulanase with independent intellectual property rights, and uses genetic engineering technology to construct genetically engineered strains for secretion and expression. Lay a solid foundation for large-scale production of pullulanase. the

Contents of the invention

The object of the present invention is to provide a pullulanase mutant Pul324 of Bacillus nagano and application thereof. the

The new pullulanase mutant Pul324 (SEQ ID NO. 2) of the present invention is obtained by molecularly transforming the pullulanase gene of Bacillus nagano (GenBank sequence number JN872757.1), specifically by using Modern enzyme engineering technology deletes the first 324 bases of the wild-type gene and transforms it into a new pullulanase mutant Pul324. Compared with the original enzyme, this mutant can be effectively secreted and expressed in the E. coli host. The supernatant of the fermentation broth is 24 times that of the original enzyme, and the intracellular enzyme activity is increased by 40%. Its properties are conducive to reducing the pullulanase activity. Production cost, more suitable for industrial production. the

A Bacillus nagano pullulanase mutant gene pul 324, the nucleotide sequence of which is shown in SEQ ID NO. 1.

The protein encoded by the pullulanase mutant gene pul 324 consists of 818 amino acids, and its amino acid sequence is shown in SEQ ID NO. 2.

The pullulanase mutant gene pul 324 is obtained by extracting the genomic DNA of Pullulanibacillus naganoensis ATCC 53909, and designing the following primers to obtain the deletion front 324 according to the gene sequence reported in Genbank. Mutant gene pul 324 of bp base:

The upstream primer F1 is: GTA GAATTC ACCTGCTGTAAGTAACGC

The downstream primer R1 is: GTA CTCGAG TTTACCATCAGATGGGCT

The application of the protein encoded by the pullulanase mutant gene pul 324 in the starch hydrolysis process.

The present invention also relates to hosts containing the pullulanase mutant Pul324 of the present invention. the

The novel pullulanase mutant Pul324 described in the present invention has wide application in starch hydrolysis. the

The special feature is that compared with the original enzyme, the mutant Pul324 can be effectively secreted and expressed in the E. coli host, the enzyme activity of the supernatant of the fermentation broth is increased by 24 times, and the intracellular enzyme activity is increased by 40%. The mutant gene pul 324 is smaller than the wild-type gene, which is beneficial to the stability of the plasmid.

The preparation method of the new pullulanase mutant Pul324 of the present invention comprises the acquisition of the pullulanase mutant gene pul324 , the expression of the pullulanase mutant gene, and the determination of the enzyme activity of the pullulanase mutant and other steps, as follows:

(1) Acquisition of pullulanase mutant gene pul 324

According to the pullulanase gene sequence information, primers were designed from base 325 of the original gene (GenBank sequence number JN872757.1) for transformation. Using the genomic DNA of Bacillus nagano as a template, use the upstream primer F1: 5'-GTA GAATTC ACCTGCTGTAAGTAACGC-3' (SEQ ID NO. 1, containing an EcoR I restriction site) and the downstream primer R1: 5'-GTA CTCGAG TTTACCATCAGATGGGCT -3' (SEQ ID NO. 2, containing an Xho I restriction site), the pullulanase mutant gene was amplified by polymerase chain reaction (PCR).

(2) Construction and verification of recombinant plasmids

The pullulanase mutant gene was double-digested with restriction endonucleases EcoR I and Xho I, and pET-22b(+) was ligated with the same double-digested pET-22b(+) to obtain pET22b- pul 324, which was transformed into the host E. coli DH5α , extract the recombinant plasmid for enzyme digestion verification, and further conduct DNA sequencing analysis to determine the correct transformant.

(3) Construction of genetically engineered strains:

Transform the correct recombinant plasmid into E. coli BL21 (DE3) competent cells (CaCl ₂ chemical transformation), and use colony PCR to verify the positive transformant E. coli BL21 (DE3)/pET22b- pul 324. It was determined that the enzyme activity of the crude enzyme in the supernatant of the fermentation broth was 24 times that of the wild type, and the activity of the intracellular enzyme was increased by 40%.

Outstanding substantive feature and technical effect of the present invention are:

The present invention utilizes modern enzyme engineering technology to delete the first 324 bases on the pullulanase gene of bacillus nagano, and obtains a new pullulanase mutant Pul324 through transformation. The mutant gene is smaller than the wild-type gene, which is beneficial to the stability of the plasmid. Compared with the original enzyme, the pullulanase mutant Pul324 can be effectively secreted and expressed in the E. coli host, the enzyme activity of the supernatant of the fermentation broth is increased by 24 times, and the intracellular enzyme activity is increased by 40%. It is beneficial to reduce the production cost of pullulanase, and is more suitable for industrial production. the

Detailed ways

The Bacillus nagano strain used in the present invention can be purchased from a strain preservation center, or can be collected in the field or obtained by other means. The microbial cultivation, gene cloning, expression technology and related technical solutions adopted in the present invention are mature technologies commonly used in this field, and the professional terms involved are also common professional terms in this field, so the text of the present invention is important for those skilled in the art. is obvious. The technical content of the present invention will be further described below in conjunction with the examples. The examples are illustrative, not restrictive, and cannot be construed as limiting the protection scope of the present invention. the

The present invention will be described in detail below by embodiment:

(1) Acquisition of pullulanase mutant genes pul 234 and pul 324

Bacillus Nagano ( Pululanibacillus naganoensis ) ATCC 53909 was purchased from the German Culture Collection of Microorganisms. According to the pullulanase gene sequence information of the strain, primers were designed from the 235th and 325th bases of the original gene:

Upstream primer F2: 5'–GTA GAATTC CATCGATTTAAGCAAAGG–3'

Upstream primer F1: 5'–GTA GAATTC ACCTGCTGTAAGTAACGC–3'

Downstream primer R1: 5'–GTA CTCGAG TTTACCATCAGATGGGCT–3'

The upstream primer contains an EcoR I restriction site (underlined), and the downstream primer contains an Xho I restriction site (underlined). PCR amplification was performed using the genome of Pullulanibacillus naganoensis ATCC 53909 as a template. The reaction system is: ddH ₂ O 41.5 μL, 10×buffer 5 μL, dNTP (2.5 mmol/L) 1 μL, upstream and downstream primers (20 μmol/L) 0.5 μL each, DNA template 1 μL, high-fidelity DNA polymerase 0.5 μL. PCR amplification conditions were as follows: denaturation at 94°C for 5 min, denaturation at 94°C for 20 s, annealing at 61°C for 40 s, extension at 72°C for 3 min, 30 cycles, and finally extension at 72°C for 10 min. The pul 234 gene can be amplified by using the upstream primer F2 and the downstream primer R1, and the pul 324 gene can be amplified by using the upstream primer F1 and the downstream primer R1.

The obtained amplified product was detected by agarose gel electrophoresis, and it was detected that the size of the amplified product was completely consistent with the size of the target gene fragment. PCR products were purified using a DNA purification kit (Tiangen Company). For specific steps, refer to the instructions of the DNA product purification kit. The purified pullulanase mutant gene was double-digested with restriction endonucleases EcoR I and Xho I, and ligated with the pET-22b (+) that had also been double-digested to obtain pET22b-pul234 and pET22b-pul324, and transformed The host E.coli DH5α, the recombinant plasmid was extracted for enzyme digestion verification, and further DNA sequencing analysis was performed to confirm the correct transformant. the

Transform the correct recombinant plasmid into E. coli BL21(DE3) competent cells (CaCl ₂ chemical transformation), and use colony PCR to verify the positive transformants E. coli BL21(DE3)/pET22b- pul 234 and E. coli BL21(DE3)/pET22b- pul 324. The successfully constructed genetically engineered strains were inoculated into 5 mL liquid LB medium (containing 100 μg/mL ampicillin), and cultured at 37°C for 10 h. Take 0.5 mL of liquid seeds and inoculate 50 mL of liquid LB medium (containing 100 μg/mL ampicillin), culture at 37°C for 2-3 h, when the _OD620 is about 0.4, add 1 mM IPTG, and then place at 37°C C was induced for 12 h.

The invention adopts DNS reagent chromogenic method to measure pullulanase activity. The principle is that pullulanase hydrolyzes the pullulan sugar substrate to obtain reducing sugar, and the reducing sugar and 3,5-dinitrosalicylic acid contained in the DNS reagent will produce a brown-red complex after co-heating. The depth of its color is directly proportional to the amount of reducing sugar. The activity of pullulanase can be calculated by measuring the absorbance value by spectrophotometry. the

The steps of enzyme activity determination are as follows: take 1 mL of fermentation liquid and centrifuge at 12,000 r/min for 3 minutes to obtain supernatant enzyme liquid. Add 100 μL of sodium acetate buffer (100 mM, pH 4.75) with a concentration of 1% (w/v) pullulan to the centrifuge tube, then add 100 μL of supernatant enzyme solution, and incubate in a 60°C water bath for 15 min. Stop the reaction in an ice bath, then add excess DNS (300 μL) reagent, develop color in a boiling water bath for 5 minutes, measure OD ₅₄₀ after cooling, and then calculate the extracellular enzyme activity. The precipitated bacteria were suspended in 200 μL of sterile water, added an appropriate amount of Triton X-100 and lysozyme, bathed in water at 37°C for 30 minutes, and centrifuged at 12,000 r/min for 3 minutes, and the supernatant was analyzed for intracellular enzyme activity as described above. Compared with the original enzyme, the pullulanase mutant Pul324 can be efficiently secreted and expressed in the E. coli host, the enzyme activity of the supernatant of the fermentation broth is increased by 24 times, and the intracellular enzyme activity is increased by 40%.

During starch liquefaction, glucoamylase mainly hydrolyzes the α-1,4-glucosidic bonds of amylose. However, pullulanase can specifically cut the α-1,6-glucosidic bond of the amylopectin branch point, thereby cutting off the entire side branch, forming amylose to facilitate the effect of other amylases on starch, improving the efficiency of starch hydrolysis, It has high value in reducing energy consumption, improving product quality and developing new products. During the starch saccharification process, appropriately diluted glucoamylase Dextrozyme DX catalyzed the reaction for 0.5, 1.0, 1.5 and 2.0 hours, and produced 0.758, 1.502, 2.219 and 2.628 g/L reducing sugars, respectively. When the pullulanase mutant Pul324 was assisted and other conditions remained unchanged, the concentration of reducing sugars produced increased to 1.071, 2.307, 3.145 and 3.902 g/L, and the hydrolysis efficiency increased by more than 40%. It can be seen that the auxiliary addition of the pullulanase mutant Pul324 can indeed effectively improve the starch hydrolysis efficiency. the

The invention has important economic benefits, and the special properties of the mutant are conducive to reducing the production cost of the pullulanase, and are more suitable for industrial production. It should be understood that those skilled in the art can improve or change according to the above description, for example, the wild-type pullulanase can also be derived from Klebsiella aerogenes in addition to being derived from Bacillus nagano Bacillus, Bacillus subtilis, Bacillus megaterium, Bacillus pumilus, Bacillus licheniformis, Bacillus cereus, Bacillus deramificans , Bacillus acidopullulyticus and other strains. The vector is suitable for expression in hosts such as Bacillus subtilis, Bacillus licheniformis and Pichia pastoris. The pullulanase mutant of the present invention can also be transferred into a prokaryotic or eukaryotic host by electroporation, protoplast transformation, etc., so as to realize the expression of the pullulanase mutant. Or modify the amino acid sequence of the mutant by enzyme engineering technology. And all these improvements and transformations should belong to the protection scope of the appended claims of the present invention.

the

sequence listing

<110> Guangxi Academy of Sciences

<120> A pullulanase mutant Pul324 from Bacillus nagano and its application

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<170> PatentIn version 3.5

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Claims (4)

1. A Bacillus nagano pullulanase mutant gene pul 324, characterized in that its nucleotide sequence is as shown in SEQ ID NO. 1. 2. The protein encoded by the pullulanase mutant gene pul 324 according to claim 1, characterized in that it consists of 818 amino acids, and its amino acid sequence is shown in SEQ ID NO. 2. 3. the pullulanase mutant gene pul 324 according to claim 1, is characterized in that, the mutant is obtained by extracting Bacillus Nagano ( Pullulanibacillus naganoensis ) ATCC 53909 genomic DNA, according to the gene sequence reported in Genbank as Based on this, the following primers were designed to obtain the mutant gene pul 324 with the deletion of the first 324 bp bases: The upstream primer F1 is: GTA GAATTC ACCTGCTGTAAGTAACGC The downstream primer R1 is: GTA CTCGAG TTTACCATCAGATGGGCT. 4. the application of the protein encoded by the pullulanase mutant gene pul 324 according to claim 1 in the starch hydrolysis process.