阅读说明：本技术 吡咯伯克霍尔德氏菌内切葡聚糖酶及其重组表达方法和应用 (Burkholderia pyrrocinia endoglucanase, recombinant expression method and application thereof ) 是由 范光森 孙宝国 胡晓晴 于 2020-06-18 设计创作，主要内容包括：本发明公开从吡咯伯克霍尔德氏菌B1213克隆的内切葡聚糖酶基因,采用含有可溶性融合标签的pCold TF载体(使用TF)和cspA启动子成功得以表达。通过酶活性,SDS-PAGE和酶谱分析成功地表达了带有或不带有信号肽的所述酶,其中切除信号肽更好。通过优化培养条件后,酶活性提高至未优化前的11.5倍。同时对该酶的酶学性质研究表明,该酶最适pH为6.0,最适反应温度为45℃；该酶在20-35℃下保温半小时后残余酶活可达80％以上,且在pH5.5-11.0环境中保温半小时后残余酶活保持在95％以上,具有极强的耐碱性,该酶具有很好的工业应用潜力。(The invention discloses an endoglucanase gene cloned from Burkholderia pyrrocinia B1213, which is successfully expressed by a pCold TF vector (using TF) containing a soluble fusion tag and a cspA promoter. The enzyme with or without signal peptide was successfully expressed by enzymatic activity, SDS-PAGE and zymogram analysis, with better signal peptide cleavage. After optimization of the culture conditions, the enzyme activity increased to 11.5 times before optimization. Meanwhile, the research on the enzymology property of the enzyme shows that the optimum pH value of the enzyme is 6.0, and the optimum reaction temperature is 45 ℃; the enzyme has the advantages that the residual enzyme activity can reach more than 80% after the enzyme is kept at the temperature of 20-35 ℃ for half an hour, the residual enzyme activity can be kept more than 95% after the enzyme is kept at the pH value of 5.5-11.0 for half an hour, the strong alkali resistance is realized, and the enzyme has good industrial application potential.)

1. The amino acid sequence of the Burkholderia pyrrocinia endoglucanase is shown in SEQ ID NO: 2 or 4.

2. The gene encoding a Burkholderia pyrrocinia endoglucanase according to claim 1, preferably having a nucleotide sequence as set forth in SEQ ID NO: 1 or 3.

3. A recombinant vector comprising the gene encoding the Burkholderia pyrrocinia endoglucanase according to claim 2.

4. The recombinant vector according to claim 3, wherein the starting vector is a pCold TF vector, preferably using TF and cspA promoters, further preferably excluding the signal peptide coding sequence.

5. A recombinant cell comprising the recombinant vector of claim 3 or 4.

6. A method for expressing the gene encoding the Burkholderia pyrrocinia endoglucanase of claim 2, comprising the steps of: cloning the coding gene without signal peptide to pCold TF carrier, transforming to obtain recombinant expression cell, culturing the cell, inducing expression and collecting the endoglucanase of Burkholderia pyrrocinia produced by expression.

7. The method of claim 6, wherein the recombinant expression cell is E.

8. The method according to claim 7, wherein the culturing is preceded by a strain activation step in which positive transformants are activated overnight in LB medium containing ampicillin with shaking at 200 rpm.

9. The method as claimed in claim 8, wherein the activated strain is transferred to LB medium with pH 5.6-6.6 at 180-220rpm and 35-39 ℃ for 5-8 hours with shaking, IPTG is added to the culture to induce EG expression for 10-14 hours, preferably at 18-22 ℃ for 12 hours; the preferred inoculum size of the activated strain is 0.8-1.2%.

10. The method of claim 9, wherein the activated strain is inoculated into LB medium at pH 5.9 at 200rpm and 37 ℃ for 6 hours with shaking, and 500. mu. mol/L IPTG is added to the culture to induce EG expression. The induction temperature was 20 ℃ and the protein expression was induced for 12 hours with an inoculum size of 1%.

Technical Field

The invention belongs to the field of genetic engineering, and particularly relates to glucanase, a recombinant expression method of a coding gene of the glucanase and application of the glucanase in degradation of a fibrous material.

Background

Endo-beta-1, 4-glucanase (EC 3.2.1.4) (EG) is a hydrolase that catalyzes the hydrolysis of cellulose, cereal beta-D-glucans and beta-1, 4-D-glucosidic linkages in cereals. EG has received wide attention due to its potential diversity of uses, such as production of oligo-glucose oligosaccharides, reduction of viscosity of saccharified liquid and improvement of efficiency of wort separation during brewing, improvement of digestibility of feed, control of phytopathogenic fungi, and production of bioethanol [ Huang JF, Xia T, Li GH, Li XL, Li Y, Wangyt, Wang YM, Chen YY, Xie GS, Bai FW, Peng LC, Wang LQ (2019) over production of native end-1, 4-glucanes leaves to large enhancement of biomasssaving and bio-ethanol production by specific modification of cellulose in genetic rice.Biotechnology Biofuels 12.http:// hti. htg/10.1186/1301-1301 ]. Furthermore, a new generation of renewable energy utilizing lignocellulosic residues has become one of the most likely strategies to overcome environmental problems, including increased energy consumption, depletion of fossil fuel resources, and the necessity of mitigating global warming. Enzymatic hydrolysis of lignocellulose is an ideal process and is expected to be utilized in an environmentally friendly and efficient manner. EG can randomly hydrolyze glycosidic bonds to short chains, so it plays an important role in the lignocellulose hydrolysis process. EG is produced widely by a variety of bacteria, fungi and plants. Although more fungal-derived EG has been identified and analyzed for related properties, bacterial sources have been less studied, and bacterial sources generally have higher stability and tolerance to environmental extremes, and thus have a high potential for EG production [ Maki M, Leung KT, Qin WS (2009) The promoters of cell-producing bacteria for The biochemical conversion of lignocellulosic biomass. int J Biol Sci 5:500-516.http:// doi. org/doi:10.7150/ijbs.5.500 ].

EG is classified into 16 Glycosyl Hydrolase (GH) families based on amino acid sequence and three-dimensional structure [ Bernardi AV, Yonamine DK, Uyemura SA, Diamilco TM (2019) A thermostable Aspergillus fumigatus GH7 endogenous lipase over-expressed in Pichia pastoris. int J Mol Sci 20.http:// doi. org/10.3390/ijims 20092261 ]. Among them, the GH8 enzyme can act on a variety of substrates including cellulose, dextran, chitosan and even xylan, which makes EG of GH8 very attractive for a variety of industrial applications. However, only a few reports have recorded the presence of GH8 EG [ Naressiplin SM, Moreira Souza FH, Zanphorlin LM, de Almeida TS, Sade YB, Cardoso AM, Pinheiro GL, Murakami MT (2017) Structure and function of a novel GH8end restriction from the bacterial cell synthesis complex of Raoulla organic decomposition. plos One 12.http:// doi. org/10.1371/j ournal. p. 0176550 ].

Disclosure of Invention

The inventor selects a new lipase production strain Burkholderia pyrrocinia B1213(Burkholderia pyrrocinia) [ CN201610880271.X ] from a sesame-flavor liquor production environment (from a soil sample) in Shandong province; li JL, Shen WJ, Fan GS, Li XT (2018) Screening, publication and publication of Lipase from Burkholderia Pyrococcia B1213.3Biotech8.http://doi.org/10.1007/s13205-018-1414-9]The strain is preserved in China general microbiological culture Collection center (CGMCC No. 12806). The analysis of the inventor believes that the Burkholderia pyrrocinia probably plays an important role in the synthesis of ester due to high lipase yield, because the ester is an important flavor substance in white spirit and further has the potential of being used for improving the flavor in brewing. Based on this, the present inventors have conducted bioinformatics analysis, found that GH8 glucanase was not reported in the genus, and completed the present invention through further intensive studies.

The invention firstly provides a Burkholderia pyrrocinia endoglucanase, the amino acid sequence of which is shown as SEQ ID NO: 2 or 4, wherein the former is a full-length amino acid sequence containing a signal peptide and the latter is an amino acid sequence excluding the signal peptide.

The invention also provides the coding gene of the Burkholderia pyrrocinia endoglucanase, and the preferable nucleotide sequence is shown in SEQ ID NO: 1 or 3, wherein the former is a sequence encoding a full-length gene containing a signal peptide and the latter is a nucleotide sequence encoding a sequence excluding the signal peptide coding sequence.

Further, the present invention provides a recombinant vector comprising a gene encoding the aforementioned Burkholderia pyrrocinia endoglucanase. In a preferred embodiment, the starting vector is a pCold TF vector, preferably using TF and cspA promoters, further preferably excluding the signal peptide coding sequence.

The present invention also provides a recombinant cell, such as E.coli, containing the above recombinant vector.

The invention further provides a method for expressing the coding gene of the Burkholderia pyrrocinia endoglucanase, which is characterized by comprising the following steps: cloning the coding gene without signal peptide to pCold TF carrier, transforming to obtain recombinant expression cell, culturing the cell, inducing expression and collecting the endoglucanase of Burkholderia pyrrocinia produced by expression.

In a preferred embodiment, the recombinant bacterium is Escherichia coli, and the culture medium used is LB medium.

In a specific embodiment, the large-scale cultivation is preceded by a strain activation step, i.e. positive transformants are activated overnight, preferably 37 ℃, in LB medium containing ampicillin, with shaking at 200 rpm.

In a preferred embodiment, the activated strain is transferred to LB medium with pH 5.6-6.6 at 180-; the preferred inoculum size of the activated strain is 0.8-1.2%.

In a more preferred embodiment, the activated bacterial species are transferred to LB medium at pH 5.9 for 6 hours with shaking at 37 ℃ at 200rpm, and 500. mu. mol/L IPTG is added to the culture to induce EG expression. The induction temperature was 20 ℃ and the protein expression was induced for 12 hours with an inoculum size of 1%.

The invention clones endoglucanase which is different from the known endoglucanase in Burkholderia pyrrocinia B1213, has special properties, can not be successfully expressed according to the conventional expression in the recombinant expression, needs special treatment, and better cuts off a signal peptide coding sequence for the recombinant expression. Meanwhile, the research on the enzymology property of the enzyme shows that the enzyme has the special characteristics, for example, the optimum pH of the enzyme is 6.0, and the optimum reaction temperature is 45 ℃; the enzyme has a residual enzyme activity of more than 80% after being kept at 20-35 ℃ for half an hour, and the residual enzyme activity is kept at more than 95% after being kept at pH5.5-11.0 for half an hour, so that the enzyme has extremely strong alkali resistance. In addition, the enzyme activity increased to 11.5 times before optimization by optimizing the culture conditions.

Drawings

FIG. 1 multiple sequence alignment and secondary structure of BpEG 01790. The alignments include EG of B.stabilias (WP _129514311.1), B.cepacian (WP _048244211.1), B.pyrrocinia (WP _114176742.1) and B.puracquae (WP _ 085040568.1). 3D crystal structure of cellulase BcsZ of Escherichia coli K12 strain (P37651; PDB: 3 QXF). The 6 glycosidase loops "hairpin" structures and active sites were determined manually after alignment with cellulase 3 QXF. The alpha helix and beta sheet are labeled alpha and beta, respectively. The blue boxes indicate conserved residues.

FIG. 2 is a phylogenetic tree of BpEG01790 based on the proximity method.

FIG. 3 predicted 3D model of BpEG01790 with signal peptide. The catalytically active centers Glu84 and Asp145 are shown as yellow rods.

FIG. 4 schematic representation of an expression vector. After restriction enzyme digestion and ligation, the target gene was inserted into pET-28a (+) (a and b) and pCold TF (c and d) vectors. The restriction sites used are shown in the figure.

FIG. 5 SDS-PAGE and zymogram analysis of BpEG01790 expressed in E.coli BL21(DE 3). Wherein, (a) BpEG01790 expressed by pET28a (+). M is a protein marker; lanes 1,3, 5 and 7 are respectively 0.5mmoL/L IPTG-induced fermentation broth of E.coli BL21(DE3)/pET28a (+), total cells, cell-free extract and cell pellet. Channels 2,4,6 and 8 are fermentation broth of E.coli BL21(DE3)/BpEG01790-pET28a (+) induced by 0.5mmoL/L IPTG, total protein, cell-free extract and cell pellet. (b) BpEG01790 without the signal peptide DNA fragment was expressed by pET28a (+). M: protein labeling; lanes 1,3, 5 and 7 are respectively 0.5mmoL/L IPTG-induced fermentation broth of E.coli BL21(DE3)/pET28a (+), total cells, cell-free extract and cell pellet. Lanes 2,4,6 and 8 are the fermentation broth, total cells, cell-free extract and cell pellet of 0.5mmoL/L IPTG-induced E.coli BL21(DE3)/BpEG01790-pET28a (+) -r, respectively. (c) BpEG01790 was expressed via pCold TF. M: protein labeling; lanes 1,3, 5 and 7 are 0.5 mmoL/LIPTG-induced fermentation broth of E.coli BL21(DE3)/pCold TF, total cells, cell-free extract and cell pellet, respectively. Lanes 2,4,6 and 8 are 0.5mmoL/L IPTG-induced fermentation broth of E.coli BL21(DE3)/BpEG01790-pCold TF, total cells, cell-free extract and cell pellet, respectively. (d) BpEG01790 without signal peptide DNA fragment was expressed via pCold TF. M: protein labeling; lanes 1,3, 5 and 7 are 0.5mmoL/L IPTG induced fermentation broth of E.coli BL21(DE3)/pCold TF, total cells, cell-free extract and cell pellet, respectively. Lanes 2,4,6 and 8 are 0.5mmoL/L IPTG-induced fermentation broth of E.coli BL21(DE3)/BpEG01790-pCold TF-r, total cells, cell-free extract and cell pellet, respectively. (e) Zymogram analysis of BpEG01790 expressed in E.coli BL21(DE 3). Lane 1, cell-free extract of E.coli BL21(DE3)/pET28a (+) induced by 0.5mmoL/L IPTG; lane 2, cell-free extract of E.coli BL21(DE3)/BpEG01790-pET28a (+) induced by 0.5mmoL/L IPTG; lane 3, cell-free extract of E.coli BL21(DE3)/pET28a (+) -r induced by 0.5mmoL/L IPTG; lane 4, cell-free extract of E.coli BL21(DE3)/pCold TF induced by 0.5mmoL/L IPTG; lane 5, cell-free extract of E.coli BL21(DE3)/BpEG01790-pCold TF induced by 0.5mmoL/L IPTG; lane 6, cell-free extract of E.coli BL21(DE3)/BpEG01790-pCold TF-r induced by 0.5mmoL/L IPTG.

FIG. 6 is a graph showing the results of measurement of the influence of pH on EG enzyme activity and stability. (a) Influence of pH on EG enzyme activity. The highest enzyme activity group is calculated as 100 percent, and the rest is calculated by percentage. (b) Influence of pH on EG stability. The enzyme activity of the proenzyme is 100 percent, and the enzyme activity after treatment is calculated by relative enzyme activity.

FIG. 7 is a graph showing the results of measurement of the influence of temperature on EG enzyme activity and stability. (a) Influence of temperature on EG enzyme activity. The highest enzyme activity group is calculated as 100 percent, and the rest is calculated by percentage. (b) Influence of temperature on EG stability. The enzyme activity of the proenzyme is 100 percent, and the enzyme activity after treatment is relative enzyme activity.

FIG. 8 the effect of the interaction of different pH with temperature on the enzyme stability, based on 100% of untreated proenzyme, the remaining residual enzyme activity being expressed as a percentage.

FIG. 9 Effect of medium type on EG activity.

FIG. 10 Effect of IPTG concentration on EG activity.

FIG. 11 Effect of pH on EG activity.

FIG. 12 Effect of induction timing on EG activity.

FIG. 13 Effect of induction time on EG activity.

FIG. 14 response surface (3D) optimizes EG viability (U/mL). Wherein (a) the influence of pH (A) and the timing of induction (h, B); (b) influence of pH (A) and induction time (h, C); (c) influence of Induction timing (h, B) and Induction time (h, C).

Detailed Description

The invention is further illustrated by the following specific embodiments or examples in order to provide a better understanding of the invention.

The operations or methods described in the following examples are conventional in the art unless otherwise specified. Reagents and instruments are conventionally available or commercially available unless otherwise specified.

Materials and methods

1. Material

Plasmids pMD18-T, pET28a (+) and pCold TF, Taq polymerase, genomic DNA purification kit, DNA gel extraction kit, isopropyl- β -D-thiogalactopyranoside (IPTG), DNA and protein standard molecular weight Mark from Takara (Tokyo, Japan)

High fidelity DNA polymerase was purchased from nebinc.. Plus PCR kit from Novoprotein Scientific inc. Bovine Serum Albumin (BSA) was purchased from Roche, barley β -dextran was purchased from Sigma-aldrich.

2. Method of producing a composite material

(1) The culture method comprises the following steps: for Burkholderia pyrrocinia (CGMCC, accession No.: 12806), EG in B.pyrrocinia B1213 was identified by Congo red solution after growing in Luria-Bertani (LB, 5g/L yeast extract, 10g/L tryptone and 10g/L NaCl) medium and culturing on LBC (0.4% barley beta-glucan in LB) at 37 ℃ for 24 hours. Pyrococcus B1213 was cultured in a medium containing 4% (w/v) of wheat bran as a carbon source [ Li H, Chen J, Li AN, Li D (2007) Purification and characterization of beta-1,3-glucanase from Chaetomium thermophilum. world JMicrobBiot 23:1297-1303.http:// doi. org/10.1007/s11274-007-9366-y ]. Shaking at 180rpm for 5 days at 37 ℃. The pH, protein concentration and EG activity were measured every 24 hours in each culture flask.

Coli DH 5a was used for cloning and plasmid amplification, while E.coli BL21(DE3) was used as expression host. Coli cells were grown in LB medium for gene cloning and protein overexpression. Ampicillin or kanamycin was added as needed to a final concentration of 50. mu.g/mL.

2. Expression analysis of EG: induced cells were harvested by centrifugation at 10000rpm for 10 minutes at 4 ℃, resuspended in 50mmol/L Tris-HCl buffer (pH 7.0), and sonicated in an ice water bath for 10 minutes with 2s/4s sonication. Cell debris and suspension were collected by centrifugation at 10000rpm for 10 minutes at 4 ℃ and the suspension was filtered through a 0.22 μm membrane. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), EG activity determination, and EG enzyme profile analysis were performed to assess EG expression in E.coli. Coli harboring the BpEG01790-pCold-r plasmid showed more soluble protein expression and was further optimized for maximum soluble expression.

3. EG Activity assay and protein assay

EG activity was measured according to the method reported by Mandana and Ahmad. The method comprises the following specific steps: (i) adding 25 μ L of an appropriately diluted enzyme solution to a mixture containing 225 μ L of 1.0% (w/v) barley β -glucan; (ii) the reaction mixture was reacted at 50 ℃ for 10 minutes (50mmol/L Tris-HCl buffer, pH 7.0); (iii) the enzyme reaction was stopped by adding 250. mu.L of 3, 5-dinitrosalicylic acid (DNS) reagent and boiled in a water bath for 15 minutes. The amount of free reducing sugars was determined by the 3, 5-dinitrosalicylic acid (DNS) method, with glucose as a standard [31 ]. EG activity of 1 unit (U) was defined as the amount of enzyme that released 1. mu. mol glucose per minute from the substrate under the assay conditions described above. The protein concentration was determined by the Lowry method using BSA as a standard.

4. SDS-PAGE and zymography

Proteins were visualized by SDS-PAGE using separation and stacking gels as described by Laemmli, staining with Coomassie Brilliant blue R250 [ Laemmli UK (1970) clean of structural proteins and the assembly of the head of bacterial proteins T4.Nature 227:680-685.http:// doi. org/10.1038/227680a0 ]. Add 30 μ Ι _ of protein sample to 7.5 μ Ι _ of sample buffer, then boil in water bath for 5 minutes; after cooling to room temperature, the sample was loaded onto the gel. Zymograms analysis was performed using the method of Morag et al [ Morag E, Bayer EA, Lamed R (1990) Relationship of cellular and noncelluloral xylanases of Clostridium thermocellum to cellular-deoxygenizymes J Bacteriol 172:6098-6105.http:// doi. org/10.1128/jb.172.10.6098-6105.1990 ]. Samples were analyzed by SDS-PAGE (12.5% gel and 0.2% (w/v) barley beta-glucan). After electrophoresis, the gel was washed 4 times with 25% (v/v) isopropanol at 4 ℃ for 15 minutes each to remove SDS from the gel. Then washed four times each for 15 minutes at 4 ℃ in 50mM7.0Tris-HCl buffer (pH 7.0). Further incubation in new buffer at 30 ℃ for 20 min; finally, the gel was stained with 0.5% (w/v) congo red for 15 minutes at room temperature, washed with 1mol/L NaCl until a clear area could be seen, 0.5% (v/v) acetic acid was added to the gel and the resulting area became clear.