Cellulase EA.GH1 and application thereof

Abstract

The invention provides cellulase derived from Enterobacter adamsii, the enzyme activity of the cellulase provided by the invention can be stably expressed within 50 minutes under the conditions that the temperature is 50 DEG C and the pH value is 5.0, a soluble cellulose substrate can be effectively degraded, and the cellulase can be used as a novel enzyme preparation, so that the enzyme activity of the cellulase is greatly improved. The method can be widely applied to the fields of feed, food, biological energy, textile, papermaking industry and the like.

Description

Technical Field

[0001] The present invention relates to the field of gene engineering technology, and more specifically, to a cellulase EA.GH1 and an application thereof. Background Art

[0002] Cellulose is the most abundant polymer substrate in nature and the main component of plant cell walls. It is widely distributed in nature, especially in wood and crop straw. The biomass of dead plants is mainly composed of cellulose and hemicellulose, in addition to small amounts of pectin polysaccharides, lignin and cell wall proteins. Therefore, the degradation of cellulose and hemicellulose is a key step in the carbon cycle. The commercialization of cellulose and hemicellulose has also attracted people's interest. Through biotechnology processing, these components can be converted into useful products such as fertilizers, feeds, raw materials, fuels and base materials through different pathways.

[0003] The presence of genes that can express cellulases in the genomes of microorganisms indicates that they have the theoretical ability to degrade cellulose, but does not provide evidence that they actually produce such proteins.

[0004] Therefore, new solutions need to be proposed to solve this problem. Summary of the invention

[0005] In view of the shortcomings of the prior art, the object of the present invention is to provide a cellulase EA.GH1 and its application.

[0006] A cellulase derived from Enterobacter asburiae, characterized in that: the amino acid sequence of the cellulase is shown in SEQ ID NO.1; We added an expression modification tag to the above cellulase, making the enzyme 611 amino acids in length. The theoretical molecular weight of the mature cellulase is 69.4 kDa, and its final amino acid sequence is shown in SEQ NO.2.

[0007] Furthermore, without changing the amino acid sequence of the cellulase, SEQ NO.1 was modified to obtain the cellulase SEQ NO.1 optimized gene EA.GH1, whose gene sequence is SEQ NO.3; The present invention also provides the above-mentioned recombinant vector as pET32a-TrxA-sumo-pA-D1 vector, the gene EA.GH1 encoding cellulase is inserted into a suitable position of the expression vector, and the gene is placed downstream of the T7 promoter and regulated by it using seamless cloning technology to obtain the recombinant plasmid pET32a-TrxA-sumo-pA-D1-EA.GH1.

[0008] The present invention also provides a recombinant BL21 (DE3) strain comprising the cellulase gene.

[0009] The present invention also provides a method for preparing cellulase EA.GH1, comprising the following steps: (1) using the above-mentioned recombinant vector to transform a host cell to obtain a recombinant strain; (2) culturing the recombinant strain and inducing the expression of the recombinant cellulase; (3) Recover and purify the expressed cellulase EA.GH1.

[0010] In summary, the present invention has the following beneficial effects: 1) The cellulase EA.GH1 provided by the present invention has an optimum temperature of 50 °C and an optimum pH of 5.0, and its enzyme activity can be stably expressed within 50 minutes, effectively degrading soluble cellulose substrates. As a new type of enzyme preparation, it can be widely used in feed, food, bioenergy, textile and papermaking industries.

[0011] 2) The present invention transforms the gene encoding cellulase without changing the amino acid sequence of cellulase, taking into account codon preference, codon adaptation index (CAI), deletion of unstable sequences, mRNA secondary structure and other factors, and transfers the EA.GH1 gene into BL21 (DE3) for expression. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 It is the purification result of the present invention; Figure 2 is the enzyme activity assay result of the present invention; Figure 3 The enzyme activity of the present invention at different times, different temperatures, and different pH conditions; Figure 4 is the enzyme activity under different fermentation conditions of the present invention; Figure 5 Design diagram for PCR primers; Figure 6 Design diagram for PCR program; Figure 7 This is the result of PCR gel electrophoresis; Figure 8 is the glucose standard curve; Fig. 9 is the enzyme activity diagram of EA.GH1 towards different substrates; Fig.10 is the pNPG standard curve. DETAILED DESCRIPTION

[0013] The present invention is described in detail below in conjunction with the accompanying drawings and embodiments.

[0014] Embodiment: Enterobacter asburiae is a Gram-negative bacterium that we found in the wild and can effectively degrade cellulose, and is an important biological resource. The present invention obtains a new cellulase gene from Enterobacter asburiae, obtains recombinant acid cellulase EA.GH1 in BL21 (DE3) by heterologous expression after gene optimization, studies its optimum pH, temperature, enzyme activity stability, and its optimum storage conditions and optimum fermentation conditions, and provides a reliable theoretical basis for further application of it in more industrial fields.

[0015] Experimental materials and reagents: Strains and vectors: We collected soil samples from farmland, factories and forests near the Shaoxing Research Institute of Zhejiang Sci-Tech University and subjected them to physical and chemical treatments (such as drying and sieving) to obtain soil samples suitable for microbial cultivation. Subsequently, the enrichment culture method was used to screen cellulose-degrading bacteria. First, the samples were inoculated in a cellulose Congo red medium and cultured in a shaking incubator at 37°C and 220 r / min for 18 hours, and the color of the medium was observed to fade. Then, 10 μL of the discolored bacterial solution was spread on a plate with α-cellulose as the sole carbon source. After 24 hours of culture, the plaques were picked and expanded in 25 mL of culture medium. 10 μL was taken again and spread on a plate with α-cellulose as the sole carbon source for screening, and finally a microbial strain with cellulose degradation ability was obtained. The strain was identified by 16S rDNA technology by Qingke Biotechnology Company and finally determined to be Enterobacter asburiae by BLAST analysis. The vector pET32a-TrxA-sumo-pA-D1 was provided by our laboratory.

[0016] Culture medium LB liquid culture medium: Tryptone 10 g / L; Yeast extract 5 g / L; NaCl 10 g / L.

[0017] Congo Red culture medium: K2HPO4 2.5g / L; Na2HPO4·12H2O 6.3g / L; CMC 20g / L; Tryptone 2g / L; Yeast Extract 0.2g / L; Congo Red 0.2g / L.

[0018] α-cellulose culture medium: α-cellulose 4g / L; MgSO4 0.24g / L; CaCl2 0.01g / L; M9salts (5x) 200mL / L; Agar 15g / L.

[0019] M9 salts(5x): Na2HPO4 6.8g / L; KH2PO4 3.0g / L; NaCl 0.5g / L: NH4Cl 1.0g / L.

[0020] Enzymes and biochemical reagents: PrimeSTAR Max DNA Polymerase was purchased from TAKARA, and pNPG and other reagents were purchased from MacLean.

[0021] The molecular biology experimental methods not specifically described in the examples were all carried out with reference to the kits and product instructions.

[0022] Experimental methods: We asked Youkang Biotechnology Company to synthesize sequence SEQ NO.3, using primers such as Figure 5 As shown; PCR program design Figure 6 As shown; The expression vector pET32a was PCR-generated using trxa-f / r primers, and the gene sequence encoding cellulase EA.GH1 was PCR-generated using trxa-2f / r primers to obtain the target fragment. Figure 7 As shown, the gene fragment encoding mature cellulase was cut out and inserted into the expression vector so that the gene was located downstream of the T7 promoter and was regulated by it, thereby obtaining the recombinant vector pET32a-TrxA-sumo-pA-D1-EA.GH1.

[0023] EA.GH1 was used to transform BL21(DE3) to obtain the corresponding recombinant BL21(DE3) strain.

[0024] Recombinant cellulase protein purification and activity determination The bacterial solution was transferred to 10mL LB at a ratio of 1:100, and after 12 hours, it was transferred to 1L LB medium at a ratio of 1:100. After culturing at 37°C and 220 r / min for 4 hours, 0.6mM IPTG was added to induce the recombinant bacteria, and it was placed in a shaker at 16°C and 220 r / min for 16 hours. After that, the supernatant and bacteria were collected by centrifugation at 4°C and 8000 r / min for 20 minutes, and the bacteria were crushed by high pressure for 4 minutes (680pa pressure), and centrifuged at 4°C and 12000 r / min for 20 minutes, and the crushed supernatant and precipitate were collected respectively. The cellulase activity of the supernatant and protein of the recombinant bacteria induced by IPTG was determined. We used different enzyme activity reaction times, different pH and different temperatures, and different fermentation induction times to detect its enzyme activity.

[0025] Determination of reducing sugar by DNS method Prepare DNS reagent and use cellobiose, lactose and dextran as substrates. First, by measuring the glucose standard curve, take 2.0, 1.5, 1.0, 0.5, 0.25 and 0.125 mg / mL glucose solutions respectively, and use the concentration as the horizontal axis (x) and the corresponding absorbance change value (ΔA) as the vertical axis (y) to draw the standard curve, and obtain the linear regression equation y = kx +b as shown in Figure 8 Substitute the measured ΔA value into the regression equation to calculate the glucose concentration (x, mg / mL) in the sample to be tested.

[0026] Take 500 μL of purified protease solution, add 500 μL of substrate (cellobiose, lactose or dextran concentration), and then add 1 mL of DNS reagent. Heat the mixture in a 100°C metal bath for 10 minutes. After the reaction, the color of the solution gradually changes from yellow to orange-red or reddish brown. After the reaction is completed, cool the mixture to room temperature and use a spectrophotometer to measure the absorbance of the solution at a wavelength of 540 nm. The results are as follows Fig. 9 shown.

[0027] The enzyme activity unit is defined as the amount of enzyme that catalyzes the production of 1 μg of glucose per minute, expressed as enzyme activity units per milliliter of liquid sample (U / mL).

[0028] p-Nitrophenol (pNP) assay for enzyme activity First, the standard curve of p-nitrophenol (pNP) was determined as Fig.10 As shown. 0, 20, 40, 60, 80, 100 μmol / L pNP solutions were prepared, and their absorbance was measured at a wavelength of 420 nm. The concentration was used as the abscissa (x) and the absorbance change value (ΔA) was used as the ordinate (y). A standard curve was drawn to obtain the linear regression equation y = kx + b.

[0029] Take 0.75 mL of the purified protease solution, add 0.75 mL of substrate solution (1 mg / mL pNPG), mix well, place the reaction system in a 50°C metal bath and heat for 30 minutes, then add 0.5 mL of sodium carbonate (Na2CO3) solution to terminate the reaction. After the reaction is completed, cool the mixture to room temperature and measure the absorbance of the solution at a wavelength of 420 nm using a spectrophotometer.

[0030] The enzyme activity unit of β-glucosidase is defined as the amount of enzyme required to catalyze the hydrolysis of pNPG to produce 1 μmol pNP per minute at 50°C and pH 5.0, expressed as one enzyme activity unit (U).

[0031] Y——absorbance of enzymatic reaction; K——optical density of pNP; b——intercept of the pnp optical density standard curve; T——reaction time; V1——the total volume of the reaction solution; V2——is the amount of enzyme liquid in the reaction system.

[0032] The above is only a preferred embodiment of the present invention, and the protection scope of the present invention is not limited to the above embodiments. All technical solutions under the concept of the present invention belong to the protection scope of the present invention. It should be pointed out that for ordinary technicians in this technical field, some improvements and modifications without departing from the principle of the present invention should also be regarded as the protection scope of the present invention.

Claims

1. A cellulase derived from Enterobacter asburiae, characterized in that: The amino acid sequence of the cellulase is shown in SEQ ID NO.

1.

2. A mature cellulase according to claim 1, characterized in that: The mature cellulase is obtained by adding an expression modification tag to the cellulase according to claim 1, and the amino acid sequence of the mature cellulase is shown in SEQ ID NO.

2.

3. A gene encoding the cellulase according to claim 1.

4. A recombinant vector containing the gene according to claim 3.

5. The recombinant BL21 (DE3) strain of the cellulase gene as claimed in claim 4.

6. A method for preparing cellulase EA.GH1 as claimed in claim 5, characterized in that: The following steps are involved: S1, using the above-mentioned recombinant vector to transform host cells to obtain a recombinant strain; S2, culture the recombinant strain and induce the expression of recombinant cellulase; S3, recover and purify the expressed cellulase EA.GH1.

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