High-activity endoglucanase expression optimization strain

Abstract

The invention discloses a high-activity endoglucanase expression optimized strain and application thereof, and belongs to the technical field of gene engineering and enzyme engineering. According to the present invention, PCR amplification and homologous recombination are adopted to carry out strain construction and expression optimization on endoglucanase tCel5A, and screening is performed to obtain the sAOX1-pSUC2-tCel5A strain; an enzymatic property research finds that the recombinant pPICZA-tCel5A has the optimal temperature of 50 DEG C and the optimal reaction pH of 4.5, and has good thermal stability and low pH stability. The enzyme activity determination result shows that the enzyme activity of the sAOX1-pSUC2-tCel5A after expression optimization is 4632.81 U / mg prot, which indicates that the tCel5A after expression optimization can efficiently degrade cellulose, and has a great application prospect in the fields of agriculture, brewing industry and the like.

Description

Technical Field

[0001] The invention relates to an endoglucanase expression optimized strain with high activity and application thereof, belonging to the technical field of gene engineering and enzyme engineering. Background Art

[0002] Endoglucanase acts on the non-crystalline region of cellulose and degrades cellulose into oligosaccharide chains with different degrees of polymerization. It can hydrolyze cellulose with exoglucanase and glucosidase by specifically acting on β-1,4-glucosidic bonds. Endoglucanase has two main limitations in industrial application. On the one hand, the enzyme-producing strains reported so far generally have low enzyme production, harsh growth conditions, and unstable yields, resulting in excessively high production costs. On the other hand, wild-type endoglucanase is difficult to maintain high enzyme activity under extreme conditions. Although some strains screened have better stress resistance in some aspects, their natural evolutionary characteristics are relatively simple and difficult to adapt to complex industrial production conditions. At the same time, since endoglucanase is also widely used in biomedicine, pollution control and other fields, how to construct strains that efficiently and stably produce endoglucanase has become a key issue in research. Summary of the invention

[0003] In view of the shortcomings of the above-mentioned prior art, the present invention provides a high-activity expression optimized endoglucanase strain and its application, the purpose of which is to select endoglucanase EG II (Cel5A) from Trichoderma reesei, construct a recombinant strain for production using a Pichia pastoris expression system, and screen promoters and signal peptides of different sources and characteristics for replacement and combination, so as to achieve efficient expression of endoglucanase and apply it to aspects such as lees degradation.

[0004] The first technical solution provided by the present invention is an endoglucanase containing a signal peptide SUC2, and the amino acid sequence of the endoglucanase is shown as SEQ ID NO.1.

[0005] SEQ ID NO.1:

[0006] MLLQAFLFLLAGFAAKISAQQTVWGQCGGIGWSGPTNCAPGSACSTLNPYYAQCIPGATTITTSTRPPSGPTTTTRATSTSSSSTPPTSSGVRFAGVNIAGFDFG CTTDGTCVTSKVYPPLKNFTGSNNYPDGIGQMQHFVNEDGMTIFRLPVGWQYLVNNNLGGNLDSTSISKYDQLVQGCLSLGAYCIVDIHNYARWNGGIIGQGGP TNAQFTSLWSQLASKYASQSRVWFGIMNEPHDVNINTWAATVQEVVTAIRNAGATSQFISLPGNDWQSAGAFISDGSAAALSQVTNPDGSTTNLIFDVHKYLDS DNSGTHAECTTNNIDGAFSPLATWLRQNNRQAILTETGGGNVQSCIQDMCQQIQYLNQNSDVYLGYVGWGAGSFDSTYVLTETPTGSGNSWTDTSLVSSCLARK

[0007] S

[0008] The second technical solution provided by the present invention is a gene encoding the endoglucanase described in the first technical solution.

[0009] The gene encoding the endoglucanase is shown in SEQ ID NO.2. SEQ ID NO.2:

[0010]

[0011] The third technical solution provided by the present invention is a recombinant plasmid carrying the gene described in the second technical solution.

[0012] In certain embodiments, in the recombinant plasmid, the gene described in the second technical solution is driven for expression by the promoter AOX1.

[0013] Furthermore, the recombinant plasmid uses plasmid pPICZA (alpha) as an expression vector.

[0014] The fourth technical solution provided by the present invention is a recombinant cell expressing the endoglucanase described in the first technical solution, or containing the gene described in the second technical solution, or transformed with the recombinant plasmid described in the third technical solution.

[0015] In certain embodiments, the recombinant cell uses bacteria or fungi as host cells.

[0016] In certain embodiments, the host cell is Pichia pastoris X-33.

[0017] The fifth technical solution provided by the present invention is a recombinant yeast, wherein the recombinant yeast uses plasmid pPICZA (alpha) as an expression vector and Pichia pastoris X-33 as a host to express the endoglucanase described in the first technical solution.

[0018] The sixth technical solution provided by the present invention is a method for optimizing the expression of endoglucanase in Pichia pastoris X-33, wherein the endoglucanase described in the first technical solution is connected to the pPICZA (alpha) expression vector, and transformed into Pichia pastoris X-33 for expression by promoter AOX1.

[0019] The present invention also provides a highly active keratin-degrading product, which is an industrial product and contains the above-mentioned endoglucanase or the above-mentioned recombinant cell.

[0020] In certain embodiments, the industrial product comprises a crude fiber degradation product.

[0021] In certain embodiments, the crude fiber degrading products include, but are not limited to, composting agents and microbial agents.

[0022] The seventh technical solution provided by the present invention is a method for degrading cellulose, wherein the method utilizes the endoglucanase described in the first technical solution or the recombinant yeast described in the fifth technical solution to degrade crude cellulose.

[0023] In certain embodiments, the crude fiber-containing material includes distiller's grains, tea leaves, tobacco, straw, and rice husks.

[0024] The eighth technical solution provided by the present invention is the application of the endoglucanase described in the first technical solution, the gene described in the second technical solution, the recombinant plasmid described in the third technical solution, the recombinant cell described in the fourth technical solution, the recombinant yeast described in the fifth technical solution, the method described in the sixth technical solution or the method described in the seventh technical solution in the fields of medicine, animal husbandry, feed and brewing.

[0025] The ninth technical solution provided by the present invention is the use of the endoglucanase described in the first technical solution, the gene described in the second technical solution, the recombinant plasmid described in the third technical solution, the recombinant cell described in the fourth technical solution, the recombinant yeast described in the fifth technical solution, the method described in the sixth technical solution or the method described in the seventh technical solution in degrading crude fiber of wine grains, tea leaves, tobacco, straw, rice husks and other plant raw materials.

[0026] The technical effects of the present invention are as follows:

[0027] The present invention provides a highly active endoglucanase, wherein the promoter AOX1 and the signal peptide SUC2 are screened and replaced for expression optimization, and the enzyme is secreted and expressed extracellularly using Pichia pastoris X-33 as a chassis cell and a pPICZA (alpha) expression vector, so that the enzyme activity is increased to more than 4000U / mg prot, so that this expression system can greatly improve the application of endoglucanase in the circulation of industrial byproducts or agricultural byproducts such as wine lees, tobacco stems, and straw. After degrading plant crude fiber, the enzyme is more conducive to the recycling and reprocessing of byproducts, so the highly active endoglucanase provided by the present invention has more green and environmentally friendly application value and potential. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 Promoter replacement design, protein expression and enzyme activity under optimal conditions: A) Schematic diagram of promoter replacement design; B) Expression of tCel5A in the fermentation supernatant of the promoter replacement strain; C) The enzyme activity of the successfully expressed tCel5A under optimal conditions.

[0029] Figure 2 Signal peptide replacement design, protein expression and optimal enzyme activity: A) Schematic diagram of signal peptide replacement design; B) Expression of tCel5A in the fermentation supernatant of the signal peptide replacement strain; C) The enzyme activity of the successfully expressed tCel5A under optimal conditions.

[0030] Figure 3The degradation rate of lees by pPICZA-tCel5A and sAOX1-pSUC2-tCel5A under different solid-liquid ratios. DETAILED DESCRIPTION

[0031] The preferred embodiments of the present invention are described below. It should be understood that the embodiments are for better explaining the present invention and are not used to limit the present invention.

[0032] The culture medium involved in the following examples is as follows:

[0033] LB liquid medium (low salt): yeast powder 5g L -1 , Tryptone 10 g·L -1 、NaCl 5g·L -1 .

[0034] LB solid medium (low salt): yeast powder 5g L -1 , Tryptone 10 g·L -1 、NaCl 5g·L -1 , agar powder 20g·L -1 .

[0035] YPD liquid medium: yeast powder 10g·L -1 , Tryptone 20 g·L -1 , D-glucose 20g·L -1 .

[0036] YPDS solid medium: yeast powder 10g·L -1 , Tryptone 20 g·L -1 , D-glucose 20g·L -1 , D-sorbitol 1 mol·L -1 , agar powder 20g·L -1 .

[0037] The detection methods involved in the following embodiments are as follows:

[0038] Enzymatic properties of endoglucanase:

[0039] The optimum temperature and pH value of endoglucanase were determined by 3,5-dinitrosalicylic acid (DNS) method, with a temperature gradient of 30-80°C (one gradient for every 10°C) and a pH gradient of 3.0-8.0 (one gradient for every 1.0). The buffers with different pH values ​​used in the detection were acetic acid-sodium acetate and phosphate buffer. The sample with the highest enzyme activity was set as 100%, and a line graph of relative enzyme activity under different reaction conditions was drawn to determine the optimal reaction conditions. The temperature stability and pH stability of endoglucanase were determined by incubating at different temperatures (30-80°C) and pH values ​​(3.0-8.0) for 2 hours, and then the relative enzyme activity under the optimal reaction conditions was determined. The sample with the highest enzyme activity was set as 100%, the relative enzyme activity of the highest group was calculated, and a histogram was drawn to reflect the ability of the enzyme to maintain enzyme activity under different treatments. There were 3 parallel experimental groups, and the rest of the operations were the same as above.

[0040] Enzyme activity unit definition: the amount of enzyme (U) required to release 1 μg reducing sugar from the substrate per minute under different reaction conditions

[0041] Endoglucanase activity assay method:

[0042] The protein concentration was determined using a modified Bradford protein assay kit (purchased from Shanghai Shenggong Biotechnology Co., Ltd.), and a standard curve was constructed using bovine serum albumin (BSA). The activity of endoglucanase was determined using the DNS method. The CMC-Na substrate (purchased from McLean Biotechnology Co., Ltd. (Shanghai)) was prepared into a 1% (w / v) uniform solution using acetic acid-sodium acetate buffer (pH 4.5). 100 μL of the purified endoglucanase sample solution was prepared into a reaction system with 500 μL of 1% (w / v) CMC-Na solution, and the reaction was shaken at 900 rpm for 60 min at 50 °C. After the reaction was completed, the sample solution was immediately transferred to a boiling water bath to terminate the reaction, and DNS colorimetric solution (purchased from Krypton Road (Guangzhou) Biological Co., Ltd.) was added in a ratio of 1:1 between DNS and the reaction sample, and the solution was allowed to stand in a boiling water bath for 5 min for color development, and then taken out and cooled to room temperature. The reaction solution was diluted with ddH2O, the absorbance was measured at 540 nm, and the results were compared with the standard curve of the glucose solution. There were three parallel experiments. The blank control was inactivated by boiling the sample before adding the substrate. The rest of the operations were the same as above.

[0043] Definition of enzyme activity unit: the amount of enzyme (U) required to release 1 μg reducing sugar from the substrate per minute under the optimal reaction conditions (50°C, pH 4.5).

[0044] Method for determination of lees degradation rate:

[0045] Liquor lees were obtained from Anhui Gujing Group Co., Ltd. (Bozhou, Anhui, China). Fresh liquor lees were dried in a hot drying cabinet at 70°C to constant weight, accurately weighed using an analytical balance, and then crushed using a grinder. The lees were added to the fermentation supernatant separated after strain culture at a solid-liquid ratio of 1:15 and 1:30, and the mixed system was degraded at 50°C and 900rpm for 48h. The degraded samples were centrifuged at 20°C and 10,000rpm for 2min. The supernatant was separated to detect degradation products, and the precipitate was dried at 70°C to constant weight. The precipitate mass was accurately weighed, and the lees degradation rate was calculated according to the following formula:

[0046] Degradation rate = (m0-m1) / m0×100%

[0047] Where m0 is the dry mass of the lees added to the mixed system, and m1 is the dry mass of the precipitate after degradation. Three replicates were set for all sample groups.

[0048] The strains and plasmids involved in the following examples are:

[0049] 1. Pichia pastoris X-33: laboratory preserved, used for target protein expression.

[0050] 2. Plasmid pPICZA (alpha): laboratory preserved, used for target protein expression.

[0051] 3. Escherichia coli DH5α: laboratory preserved, used for obtaining multiple copies of plasmids.

[0052] Example 1 Construction of endoglucanase

[0053] 1. Construction of expression optimization vector

[0054] (1) Construction of pPICZA-tCel5A recombinant vector

[0055] The endoglucanase shown in SEQ ID NO.3 of the amino acid sequence excluding the signal peptide retained in NCBI (GenBank: ABA64553.1) was used as the parent enzyme, and GeneWeichi Biotechnology Co., Ltd. was commissioned to perform codon optimization and gene synthesis (nucleotide sequence as SEQ ID NO.4), and then connected to the Kex2 site of the pPICZA (alpha) expression vector to obtain the pPICZA-tCel5A recombinant vector.

[0056] SEQ ID NO.3:

[0057] MRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPAEAVIGYSDLEGDFDVAVLPFSNSTNNGLLFINTTIASIAAKEEGVSLEKRQQTVWGQCGGIGWSGPTNCAPGSACSTLNPYYAQC IPGATTITTSTRPPSGPTTTTRATSTSSSSTPPTSSGVRFAGVNIAGFDFGCTTDGTCVTSKVYPPLKNFTGSNNYPDGIGQMQHFVNEDGMTIFRLPVGWQYLVNNNLGGNLDSTSISKYD QLVQGCLSLGAYCIVDIHNYARWNGGIIGQGGPTNAQFTSLWSQLASKYASQSRVWFGIMNEPHDVNINTWAATVQEVVTAIRNAGATSQFISLPGNDWQSAGAFISDGSAAALSQVTNP DGSTTNLIFDVHKYLDSDNSGTHAECTTNNIDGAFSPLATWLRQNNRQAILTETGGGNVQSCIQDMCQQIQYLNQNSDVYLGYVGWGAGSFDSTYVLTETPTGSGNSWTDTSLVSSCLARK

[0058] (2) Construction of expression optimized vector

[0059] Based on the pPICZA-tCel5A recombinant vector, in addition to the original promoter and signal peptide of the pPICZA (alpha) plasmid (pAOX1 and α-factor, respectively), five other promoters (pDAS1, pFLD1, pGAP, pTEF1 and pPGK1) and signal peptides (Cel5A, KAR2, PHO1, SCW and SUC2) from different sources were selected, and the nucleotide sequences are shown in Table 1. Among them, pDAS1, pFLD1, pTEF1 and pPGK1 were cloned from the P. pastoris X-33 genome, and pGAP was cloned from the plasmid pGAPZA. The signal peptide was replaced by whole plasmid PCR.

[0060] According to the sequence of endoglucanase (nucleotide sequence as shown in SEQ ID NO.4, amino acid sequence as shown in SEQ ID NO.3) and the sequence of pPICZA-tCel5A recombinant vector, primers (as shown in Table 1) were designed to replace the promoter and signal peptide sequences on the pPICZA-tCel5A recombinant vector.

[0061] Table 1: Alternative promoter and signal peptide sequences

[0062] name Serial Number pDAS1 SEQ ID NO.5 pFLD1 SEQ ID NO.6 pGAP SEQ ID NO.7 pPGK1 SEQ ID NO.8 pTEF1 SEQ ID NO.9 sCel5A SEQ ID NO.10 SSUC2 SEQ ID NO.11 sKAR2 SEQ ID NO.12 sPHO1 SEQ ID NO.13 wxya SEQ ID NO.14

[0063] Table 2: Primers

[0064]

[0065]

[0066] The PCR reaction system was as follows: PrimeSTAR Max Premix (2×) 25 μL, 10 μM forward primer 1 μL, 10 μM reverse primer 1 μL, template DNA 1 μL, and double distilled water was added to 50 μL;

[0067] The amplification conditions of PCR products were as follows: pre-denaturation at 98°C for 3 min; denaturation at 98°C for 10 s, annealing at 57°C for 5 s, extension at 72°C for 2 min, 30 cycles; and finally incubation at 72°C for 10 min.

[0068] The PCR amplification product was detected by 1% agarose gel electrophoresis. After confirming that the amplification product was of the correct size, 0.5 μL of methylated template digestion enzyme (DpnI) was added to 10 μL of the amplification product, mixed, and reacted at 37°C for 1.5 h. The amplification product treated with DpnI was transformed into Escherichia coli DH5α competent cells, and the transformation product was coated on a plate with Zeocin (final concentration 50 μg·L -1 ) in LB solid medium (low salt) at 37°C for 10-12 h, and the single colony grown on the plate was inoculated into a plate supplemented with Zeocin (final concentration 50 μg·L -1 ) in LB liquid medium (low salt), cultured at 37°C and 220rpm for 8-10h, and then the plasmids were extracted to obtain recombinant plasmids containing different expression element combinations: Promoter-SignalP-tCel5A, including promoter replacement plasmids pAOX1-sα-tCel5A, pDAS1-sα-tCel5A, pFLD1-sα-tCel5A, pGAP-sα-tCel5A , pPGK1-sα-tCel5A, pTEF1-sα-tCel5A, and the signal peptide-replaced strains pAOX1-sα-tCel5A, pAOX1-sCel5A-tCel5A, pAOX1-sSUC2-tCel5A, pAOX1-sKAR2-tCel5A, pAOX1-sPHO1-tCel5A, and pAOX1-sSCW-tCel5A.

[0069] Example 2: Construction of recombinant bacteria and expression of endoglucanase

[0070] The specific steps are as follows:

[0071] (1) The recombinant plasmid Promoter-SignalP-tCel5A obtained in Example 1 was transformed into Pichia pastoris X-33 competent cells, and the transformation products were spread on a plate containing Zeocin (final concentration 100 μg·L -1 ) on YPDS solid medium at 30°C for 2.5-3.0 days, and then the single colony grown on the plate medium was inoculated into a medium containing Zeocin (final concentration 100 μg·L -1 ) in YPD liquid medium at 30°C and 220 rpm for 24 h to prepare seed solutions;

[0072] (2) The seed solution obtained in step (1) was transferred to YPD liquid medium at a ratio of 1% (v / v), and cultured at 30°C and 220 rpm for 24 hours. Methanol was added at a final concentration of 1% (w / v) to induce the expression of tCel5A. The culture process lasted for 72 hours, and methanol was added every 24 hours.

[0073] (3) The fermentation broth was centrifuged at 8000 rpm for 10 min at 4°C to obtain the fermentation supernatant (crude enzyme solution).

[0074] (4) Filter through 0.22 μm PES filter membrane respectively. Pass the filtrate through Ni-NTA gravity column (purchased from Chuanshi Biotechnology Co., Ltd.) to obtain purified endoglucanase.

[0075] (5) SDS-PAGE (Thermo XCell Surelock Mini-Cell, NuPAGE 4-12%, Bis-Tris protein gel) was used to analyze the protein expression and catalytic activity against CMC-Na. The results are shown in Table 3 and Figure 1 , Figure 2 , Figure 3 .

[0076] Table 3: Specific enzyme activity of purified endoglucanases from different recombinant vectors

[0077]

[0078] The results showed that in the expression optimization experiment, the inducible promoter was more effective for the expression of tCel5A, while the constitutive promoter was less effective ( Figure 1A, 1B). The inducible promoters pAOX1, pDAS1, and pFLD1 expressed active tCel5A, with pAOX1 and pDAS1 expressing particularly high levels. In contrast, the constitutive promoters expressed tCel5A at a low level, with only pGAP expressing the target protein. Further determination of specific enzyme activity showed that the enzyme activity of the pAOX1 strain was significantly higher than that of the other strains ( Figure 1 C). Different signal peptides were selected to replace α-factor ( Figure 2 A), including the original peptide (Cel5A) from T. reesei. Only Cel5A and SUC2 guide the expression of enzymes ( Figure 2 B). Both Cel5A and SUC2 peptides enhanced the secretory expression of the recombinant enzyme ( Figure 2 C). Therefore, pAOX1 and sSUC2 promoters were selected for combination, and the optimized enzyme activity was 4632.81 U / mg prot, which was about 1400 U / mg prot higher than the original enzyme activity.

[0079] Example 3: Degradation of liquor lees by recombinant endoglucanase

[0080] According to the method of Example 2, the recombinant plasmids pPICZA-tCel5A and sAOX1-pSUC2-tCel5A were transformed into Pichia pastoris X-33 and fermented to obtain fermentation supernatant.

[0081] The fermentation supernatant was used to degrade the dried and crushed liquor lees at a solid-liquid ratio of 1:15 and 1:30 for 48 hours, and the degradation rate was determined as follows: Figure 3 As shown in the figure, the degradation rate of sAOX1-pSUC2-tCel5A was higher than that of pPICZA-tCel5A at different solid-liquid ratios. At the same time, increasing the proportion of fermentation supernatant added can improve the degradation effect of lees. Under the condition of 1:30 solid-liquid ratio, the lees degradation rate of sAOX1-pSUC2-tCel5A reached 52.86%, which was significantly higher than the 30% to 40% degradation rate of endoglucanase reported in other literature, indicating that it has a high efficiency in degrading high-fiber by-products from plants, and has a significant effect only by using fermentation supernatant treatment, which reduces the complex purification process and reduces costs.

[0082] The present invention adopts whole plasmid PCR and homologous recombination technology to screen and replace the expression elements of endoglucanase from T. reesei, and the obtained sAOX1-pSUC2-tCel5A strain has good tCel5A expression ability and high efficiency degradation ability for wine lees, and has great application potential in agriculture and brewing industry.

[0083] Although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. An endoglucanase, characterized in that The amino acid sequence of the endoglucanase is shown in SEQ ID NO.

1.

2. A gene encoding the endoglucanase according to claim 1.

3. A recombinant plasmid carrying the gene according to claim 2.

4. The recombinant plasmid according to claim 3, characterized in that In the recombinant plasmid, the gene described in claim 2 is driven for expression by the promoter AOX1.

5. The recombinant plasmid according to claim 3 or 4, characterized in that The recombinant plasmid uses plasmid pPICZA (alpha) as an expression vector.

6. The endoglucanase according to claim 1, or a recombinant cell containing the gene according to claim 2, or transformed with the recombinant plasmid according to any one of claims 3 to 5.

7. The recombinant cell according to claim 6, characterized in that The recombinant cell uses bacteria or fungi as host cells; the host cell is Pichia pastoris X-33.

8. A method for optimizing the expression of endoglucanase in Pichia pastoris X-33, characterized in that: The method comprises connecting the endoglucanase according to claim 1 to a pPICZA (alpha) expression vector, and transforming the vector into Pichia pastoris X-33 for expression by promoter AOX1.

9. A method for degrading cellulose, characterized in that: The method utilizes the endoglucanase described in claim 1 to degrade crude cellulose.

10. Use of the endoglucanase according to claim 1, the gene according to claim 2, the recombinant plasmid according to any one of claims 3 to 5, the recombinant cell according to claim 6 or 7, the method according to claim 8 or the method according to claim 9 in degrading crude fiber of distiller's grains, tea, tobacco, straw, rice husk and other plant raw materials in the fields of medicine, animal husbandry, feed and brewing.

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