Endoglucanase mutants with acid resistance and use thereof

Abstract

The application discloses an endoglucanase mutant with acid resistance and application thereof, and belongs to the technical field of genetic engineering and enzyme engineering. The endoglucanase tCel5A is subjected to site-specific saturation mutation by PCR amplification, and n variants S45D / T55D / T59D and T88H / W255H are obtained through screening. Enzyme activity determination results show that the endoglucanase variants S45D / T55D / T59D and T88H / W255H have enzyme activities of 8828.7 U / mg prot and 5206.7 U / mg prot respectively under acidic conditions, and the degradation rates of the endoglucanase variants S45D / T55D / T59D and T88H / W255H on distillers' grains are 53.53% and 50.47% respectively under a solid-liquid ratio of 1:30, so that the endoglucanase variants have strong acid resistance and degradation capacity on plant cellulose, and have great application value in agriculture and brewing.

Description

TECHNICAL FIELD

[0001] The present application relates to an endoglucanase mutant with acid resistance and its application, and belongs to the technical field of genetic engineering and enzyme engineering. BACKGROUND

[0002] Endoglucanase is a kind of glycoside hydrolase that can catalyze crude fiber, and has a wide application in the fields of fertilizer composting, rice hull degradation, straw recycling, etc. In the brewing industry, a large amount of distiller's grains are produced during the production of white wine, which will cause serious pollution to the environment as a kind of solid waste. Endoglucanase can hydrolyze the crude fiber in distiller's grains, help the degradation and composting of distiller's grains, thereby accelerating the recycling of distiller's grains and promoting environmental protection. However, the high moisture and strong acidity of distiller's grains will limit the catalytic activity of endoglucanase, thereby affecting its catalytic activity and degradation effect.

[0003] In the existing reports, the expression amount of endoglucanase has been modified, such as Su Shaofeng et al. obtained a high-copy recombinant strain by codon optimization and erythromycin screening (reference: Su Shaofeng, Sa Chulu, Liu Hongkui, et al. Optimization of Glycoside Hydrolase and Endoglucanase Gene Codon and Expression [J]. Animal Husbandry and Feed Science); Liao Junhua optimized the endoglucanase gene Ends by bioinformatics analysis, and the expression amount was increased by 1.2 times while the same enzymatic properties were retained (reference: Liao Junhua. Expression of Optimized Endoglucanase Gene Ends in Pichia pastoris [D]. Sichuan Agricultural University, 2010.). However, in combination with the application requirements of distiller's grains environment, there is currently a lack of endoglucanase with high catalytic activity for acid substrates, and the poor acid resistance of endoglucanase limits its application in degrading distiller's grains, so that it cannot efficiently hydrolyze the crude fiber in distiller's grains. Therefore, modifying the acid resistance of endoglucanase and improving its catalytic activity for acid substrates are the keys to enhancing its application ability. SUMMARY

[0004] In view of the deficiencies of the prior art, the present application provides an endoglucanase mutant with acid resistance and its application, aiming to solve the technical problems that the catalytic activity of the existing endoglucanase for acid substrates is not high, and the poor acid resistance of endoglucanase limits its application in degrading distiller's grains, and the high acidity of the substrate makes it unable to efficiently hydrolyze the crude fiber in distiller's grains.

[0005] The first technical solution provided by the present application is an endoglucanase mutant, wherein the mutant is an endoglucanase parent with an amino acid sequence as shown in SEQ ID NO. 1, which is subjected to any one of the following mutations:

[0006] (1) the amino acid at positions 45, 55, 59, 61 and / or 260 is mutated to aspartic acid;

[0007] (2) the amino acid at position 87, 298 or 371 is mutated to histidine;

[0008] (3) the amino acid at position 85, 88 and / or 255 is mutated to histidine.

[0009] In some embodiments, the endoglucanase mutant is obtained by mutating the serine S at position 45, the threonine T at position 55 and the threonine T at position 59 of an endoglucanase parent having an amino acid sequence as shown in SEQ ID NO. 1 to aspartic acid D, or by mutating the threonine T at position 88 and the tryptophan W at position 255 of an endoglucanase parent having an amino acid sequence as shown in SEQ ID NO. 1 to histidine H.

[0010] In some embodiments, the nucleotide sequence encoding the parent endoglucanase has the sequence as shown in SEQ ID NO. 2.

[0011] The second technical solution provided by the present application is a gene encoding the mutant of the first technical solution.

[0012] The third technical solution provided by the present application is a recombinant vector carrying the gene of the second technical solution.

[0013] In some embodiments, the recombinant vector is a plasmid pPICZA (alpha) as the expression vector.

[0014] Further, the signal peptide of the expression vector is replaced by SUC2 as the expression vector.

[0015] The fourth technical solution provided by the present application is a recombinant microbial cell expressing the mutant of the first technical solution, or containing the gene of the second technical solution, or transformed with the recombinant vector of the third technical solution.

[0016] In some embodiments, the recombinant microbial cell uses bacteria or fungi as the host cell.

[0017] In some embodiments, the host cell is Pichia pastoris X-33.

[0018] The fifth technical solution provided by the present application is a method for improving the degradation activity of endoglucanase on acidic substrates, which comprises mutating the endoglucanase parent having an amino acid sequence as shown in SEQ ID NO. 1 by any one of the mutations as shown in (a) to (j):

[0019] (a) mutating the threonine T at position 55 to aspartic acid D;

[0020] (b) the threonine T at position 59 is mutated to aspartic acid D;

[0021] (c) the alanine A at position 26 is mutated to aspartic acid D;

[0022] (d) the serine S at position 45 and the threonine T at position 55 are both mutated to aspartic acid D;

[0023] (e) the serine S at position 45 and the threonine T at position 59 are both mutated to aspartic acid D;

[0024] (f) the threonine T at position 55 and the threonine T at position 59 are both mutated to aspartic acid D;

[0025] (g) the serine S at position 45, the threonine T at position 55 and the threonine T at position 59 are all mutated to aspartic acid D;

[0026] (h) the threonine T at position 88 is mutated to histidine H;

[0027] (i) the glycine G at position 85 and the threonine T at position 88 are both mutated to histidine H;

[0028] (j) the threonine T at position 88 and the tryptophan W at position 255 are both mutated to histidine H.

[0029] The present application also provides an endoglucanase product with acid resistance, which is a humification agent or a microbial fermentation agent, and the crude fiber degradation product contains the above-mentioned endoglucanase mutant or the above-mentioned recombinant cell.

[0030] In some embodiments, the industrial degradation product includes a crude fiber degradation product.

[0031] In some embodiments, the degradation keratin product includes but is not limited to: a humification agent, a microbial fermentation agent.

[0032] The present application also provides a sixth technical solution, which is a method for degrading cellulose, using the endoglucanase variant of the first technical solution or the recombinant microbial cell of the fourth technical solution to degrade cellulose.

[0033] In some embodiments, the substance containing cellulose includes vinasse, tea leaves, tobacco, straw, rice husk, etc.

[0034] The present application provides a seventh technical solution, which is the application of the endoglucanase mutant of the first technical solution, the gene of the second technical solution, the recombinant vector of the third technical solution, the recombinant microbial cell of the fourth technical solution, the method of the fifth technical solution or the method of the sixth technical solution in the fields of medicine, animal husbandry, feed, brewing.

[0035] The eighth technical solution provided by the application is application of the endoglucanase mutant of the first technical solution, the gene of the second technical solution, the recombinant carrier of the third technical solution, the recombinant microbial cell of the fourth technical solution, the method of the fifth technical solution or the method of the sixth technical solution in degradation of crude fiber in distiller's grains, tea leaves, tobacco, straw, rice husk and other plant raw materials.

[0036] Beneficial effects

[0037] The application obtains two optimal endoglucanase variants S45D / T55D / T59D and T88H / W255H through screening. The collagen hydrolysis activity determination result shows that the keratinase mutants S45D / T55D / T59D and T88H / W255H are resistant to acidic environment, and the catalytic activities of the mutants on acidic substrates are 8828.68 U / mg prot and 5206.67 U / mg prot respectively. Therefore, the mutant can greatly improve the application value of endoglucanase in acidic high-fiber substrates such as distiller's grains. The variant can maintain the relative stability of the protein catalytic center structure in the acidic environment, so it will not lose the catalytic effect on the acidic substrate, and it is more efficient in the application process. Therefore, the endoglucanase variants S45D / T55D / T59D and T88H / W255H provided by the application have more application value and potential. BRIEF DESCRIPTION OF DRAWINGS

[0038] Figure 1 Structural changes of tCel5A before and after Rosetta Supercharging design and specific activities of surface mutants. A) original protein conformation charge before tCel5A mutation; B) protein conformation charge after Rosetta Supercharging mutation design; C) specific enzyme activity of single-point mutant under optimal reaction conditions and D) specific enzyme activity of single-point mutant under pH 3.0 conditions; E) specific enzyme activity of combined mutant under optimal reaction conditions and F) specific enzyme activity of combined mutant under pH 3.0 conditions.

[0039] Figure 2 Selection of tCel5A catalytic center mutation site and specific enzyme activity of single-point or double-point mutant; A) relative position of cellotetraose and tCel5A catalytic active center; B) relative position of cellotetraose and catalytic site residues; C) specific enzyme activity of single-point mutant under optimal reaction conditions; D) specific enzyme activity of single-point mutant under pH 3.0 conditions; E) specific enzyme activity of double-point mutant under optimal reaction conditions; F) specific enzyme activity of double-point mutant under pH 3.0 conditions.

[0040] Figure 3 Degradation effect of S45D / T55D / T59D and T88H / W255H on liquor vinasse: A) enzyme activity of the fermentation supernatant of the parent enzyme WT and mutant strains of tCel5A under pH 3.0 and optimal reaction conditions; B) degradation rate of the two mutants on liquor vinasse under different solid-liquid ratios. DETAILED DESCRIPTION

[0041] The following describes preferred embodiments of the present application, and it should be understood that the embodiments are for better explaining the present application and are not used to limit the present application.

[0042] The culture media involved in the following examples are as follows:

[0043] LB liquid medium (low salt): yeast powder 5 g·L -1 , tryptone 10 g·L -1 , NaCl 5 g·L -1 .

[0044] LB solid medium (low salt): yeast powder 5 g·L -1 , tryptone 10 g·L -1 , NaCl 5 g·L -1 , agar powder 20 g·L -1 .

[0045] YPD liquid medium: yeast powder 10 g·L -1 , tryptone 20 g·L -1 , D-glucose 20 g·L -1 .

[0046] YPDS solid medium: yeast powder 10 g·L -1 , tryptone 20 g·L -1 , D-glucose 20 g·L -1 , D-sorbitol 1 mol·L -1 , agar powder 20 g·L -1 .

[0047] The detection methods involved in the following examples are as follows:

[0048] Endoglucanase activity determination method:

[0049] Protein concentration was determined using a modified Bradford protein assay kit (purchased from Shanghai Biotech 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 (purchased from Macklin Biotechnology Co., Ltd. (Shanghai)) substrate was prepared into a 1% (w / v) uniform solution using acetic acid-sodium acetate buffer (pH 3.0 and pH 4.5). 100 μL of purified endoglucanase sample solution was mixed with 500 μL of 1% (w / v) CMC-Na solution to form a reaction system, which was shaken at 900 rpm at 50°C for 60 min. After the reaction was completed, the sample solution was immediately transferred to a boiling water bath to terminate the reaction. DNS color developing solution (purchased from Keda (Guangzhou) Biological Co., Ltd.) was added to the reaction sample at a ratio of 1:1, and the color was developed in a boiling water bath for 5 min. The reaction solution was cooled to room temperature. The reaction solution was diluted with ddH2O, and the absorbance was measured at 540 nm. The results were compared with the standard curve of glucose solution. Three parallel experiments were performed in the experimental group. The blank control was boiled to inactivate the sample before adding the substrate, and the rest of the operation was the same as above.

[0050] Enzyme activity unit definition: the amount of enzyme required to release 1 μg of reducing sugar from the substrate per minute under the corresponding reaction conditions (50°C, pH 3.0 or pH 4.5) (U).

[0051] Method for detecting the degradation rate of endoglucanase on liquor lees:

[0052] Liquor lees were obtained from Anhui Gujing Group Co., Ltd. (Bozhou, Anhui, China). Fresh liquor lees were dried to constant weight in an oven at 70°C, and then crushed with a crusher. The lees were added to the fermentation supernatant (crude enzyme solution) at a solid-liquid ratio of 1:15 and 1:30. The mixture was degraded at 50°C and 900 rpm for 48 h. The degraded sample was centrifuged (2 min, 10,000 rpm, 20°C). The supernatant and the precipitate were separated to calculate the degradation rate of the lees, and the precipitate was dried to constant weight at 70°C. The degradation rate was calculated as follows:

[0053] Degradation rate = (m0-m1) / m0 x 100%

[0054] Where m0 is the dry mass of lees added to the system, and m1 is the dry mass of the precipitate after degradation. Three parallel experiments were performed for each sample.

[0055] The following examples relate to strains and plasmids:

[0056] 1. Pichia pastoris X-33: preserved in the laboratory for target protein expression.

[0057] 2. Plasmid pPICZA (alpha): preserved in the laboratory, used for target protein expression.

[0058] 3. E. coli DH5a: preserved in the laboratory, used for obtaining multiple copies of plasmids.

[0059] Example 1 Construction of endoglucanase variants

[0060] 1. Construction of mutants

[0061] (1) The construction method of the pAOX1-sSUC2-tCel5A recombinant vector is as follows:

[0062] Based on the plasmid pPICZA (alpha), the endoglucanase gene with the nucleotide sequence of SEQ ID NO. 2 was connected after the Kex2 site by chemical synthesis (Guangzhizhi Co., Ltd.), the plasmid signal peptide was replaced with SUC2 (nucleotide sequence of SEQ ID NO. 3) by whole plasmid PCR, and then it was transformed into E. coli DH5a to complete the construction and sequencing verification.

[0063] (2) Construction of mutants

[0064] In this study, the endoglucanase with the nucleotide sequence of SEQ ID NO. 2 was used as the parent enzyme, and 7 amino acid sites designed by Rosetta Supercharging on the surface and 14 amino acid sites near the catalytic active center were selected for saturation mutation modification. The forward mutation sites were combined.

[0065] According to the sequence of endoglucanase (nucleotide sequence as shown in SEQ ID NO. 2, amino acid sequence as shown in SEQ ID NO. 1), mutant primers (as shown in Table 1) were designed, and the endoglucanase gene on the pAOX1-sSUC2-tCel5A plasmid was subjected to site-directed saturation mutation.

[0066] Table 1: Primers

[0067]

[0068]

[0069] The PCR reaction system was as follows: PrimeSTAR Max Premix (2x) 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;

[0070] PCR product amplification conditions are: 98℃ pre-denaturation 3min; 98℃ denaturation 10s, 55℃ annealing 5s, 72℃ extension 2min, 30 cycles; finally 72℃ incubation 10min;

[0071] PCR amplification product is detected by 1% agarose gel electrophoresis, and after determining that the size of the amplification product is correct, 0.5μL methylation template digestion enzyme (DpnI) is added to 10μL amplification product, mixed, and reacted at 37℃ for 1.5h. The amplification product treated by DpnI is transformed into E. coli DH5α competent cells, and the transformation product is coated on LB solid medium (low salt) added with Zeocin (final concentration 50μg·L -1 ) and cultured at 37℃ for 10-12h. Single colonies grown on the plate are inoculated into LB liquid medium (low salt) added with Zeocin (final concentration 50μg·L -1 ) and cultured at 37℃, 220rpm for 8-10h, and then plasmid is extracted, thereby obtaining recombinant plasmids containing different combinations of expression elements: tCel5A-mutants, including W5D (5th tryptophan mutated to aspartic acid), S45D (45th serine mutated to aspartic acid), S50D (50th serine mutated to aspartic acid), T55D (55th threonine mutated to aspartic acid), T59D (59th threonine mutated to aspartic acid), T61D (61th threonine mutated to aspartic acid), A260D (260th alanine mutated to aspartic acid), S45D / T55D, S45D / T59D, T55D / T59D, S45D / T55D / T59D, G81H (81th glycine mutated to histidine), D83H (83th aspartic acid mutated to histidine), F84H (84th phenylalanine mutated to histidine), G85H (85th glycine mutated to histidine), C86H (86th cysteine mutated to histidine), T87H (87th threonine mutated to histidine), T88H (88th threonine mutated to histidine), Y176H (176th tyrosine mutated to histidine), W255H (255th tryptophan mutated to histidine), S296H (296th serine mutated to histidine), G297H (297th glycine mutated to histidine), T298H (298th threonine mutated to histidine), F367H (367th phenylalanine mutated to histidine), Y371H (371th tyrosine mutated to histidine), G85H / T88H, G85H / W255H, G85H / G297H, T88H / W255H, T88H / G297H, W255H / G297H.

[0072] Example 2: Construction of recombinant bacteria and expression of keratinase

[0073] The specific steps are as follows:

[0074] (1) The recombinant plasmid tCel5A-mutant obtained in Example 1 was transformed into Pichia pastoris X-33 competent, respectively, and the transformation products were coated on YPDS solid medium containing Zeocin (final concentration 100 μg·L -1 ) at 30°C for 2.5-3.0 d, and then the single colonies grown on the plate medium were inoculated into YPD liquid medium containing Zeocin (final concentration 100 μg·L -1 ), and cultured at 30°C, 220 rpm for 24 h, and then seed liquid was prepared, respectively;

[0075] (2) The seed liquid obtained in step (1) was transferred to YPD liquid medium at a ratio of 1% (v / v) at 30°C, 220 rpm for 24 h. The final concentration of methanol was 1% (w / v) to induce the expression of tCel5A. The culture process lasted for 72 h, and the methanol was supplemented every 24 h.

[0076] (3) The fermentation liquid was centrifuged at 4°C, 8000 rpm for 10 min to obtain the fermentation supernatant (crude enzyme liquid).

[0077] (4) The fermentation supernatant was filtered through a 0.22 μm PES filter membrane. The filtrate was passed through a Ni-NTA gravity column (purchased from Transgen Biotech Co., Ltd.) to obtain the purified endoglucanase.

[0078] (5) SDS-PAGE (Thermo XCell Surelock Mini-Cell, NuPAGE 4-12%, Bis-Tris protein gel) was used to analyze the protein expression.

[0079] (6) The catalytic activity of endoglucanase in the purified liquid of step (4) under optimal conditions and acidic conditions and against CMC-Na was detected, and the results are shown in Tables 2, 3 and Figure 1 、 Figure 2 、 Figure 3

[0080] Table 2: Cellulose hydrolysis activity of surface-mutated endoglucanase under optimal conditions and acidic conditions

[0081]

[0082]

[0083] Table 3: Cellulose hydrolysis activity of catalytic center-mutated endoglucanase under optimal conditions and acidic conditions ​

[0084]

[0085]

[0086] The results show that in the surface charge design strategy, the specific enzyme activity of S45D, T55D, T59D, T61D and A260D mutants is significantly increased under the optimum reaction conditions. Under acidic conditions (pH 3.0), the reaction activity of S45D, T55D and T59D is still higher than that of the control, indicating that the three sites are most relevant to the acid resistance of tCel5A. Further combination mutation is carried out on S45D, T55D and T59D. Under acidic and optimum reaction conditions, the enzyme activity of the double-point mutant is significantly improved compared with WT, and the enzyme activity of S45D / T55D / T59D mutant is about 3 times higher than that of WT, indicating that the acid resistance of endoglucanase is obviously improved through surface charge design. In the catalytic center design strategy, only the enzyme activity of the three single-point mutants G85H, T88H and W255H is significantly improved in the reaction under acidic conditions (pH 3.0), and the activity of G297H mutant under optimum and acidic conditions is close to WT. Although the T87H, T298H and Y371H mutants show very high catalytic activity under optimum conditions, their activities under acidic conditions are very low. Therefore, four sites G85, T88, W255 and G297 are selected for double-point His mutation. In the double-point mutation experiment, only the specific enzyme activity of T88H / W255H mutant under optimum conditions is significantly improved, about 6345.3 U / mg prot; when reacting with acidic substrate (pH 3.0), it is about 5206.7 U / mg prot, about 2400 U / mg prot higher than WT, indicating that it has better hydrolysis effect on acidic substrate. The fermentation supernatant of S45D / T55D / T59D and T88H / W255H mutants is used for degradation of vinasse, and the degradation rate is 53.53% and 50.47% respectively under the solid-liquid ratio of 1:30, which is higher than the average level (30-40%), and can meet the needs of industrial production.

[0087] The present application adopts full plasmid PCR technology to carry out site-specific saturation mutation on endoglucanase tCel5A, and the obtained endoglucanase variants S45D / T55D / T59D and T88H / W255H have good acid resistance, and have great application potential in agricultural and brewing industries. The obtained mutants S45D / T55D / T59D and T88H / W255H retain the high activity of the original enzyme under optimum conditions, and the fermentation supernatant has a vinasse degradation rate of 53.53% and 50.47% respectively under a certain solid-liquid ratio, indicating that the mutants have better acid resistance and industrial application ability than the parent enzyme.

[0088] Although the present application has been disclosed in its preferred embodiments with reference to the accompanying drawings, it is not intended to limit the present application thereto, and various modifications and alterations can be made thereto by those skilled in the art without departing from the spirit and scope of the present application, and the scope of protection of the present application should be defined by the appended claims.

Claims

1. An endoglucanase mutant, characterized in that, The endoglucanase mutant is obtained by mutating the serine S at position 45, the threonine T at position 55 and the threonine T at position 59 of the endoglucanase parent with the amino acid sequence shown in SEQ ID NO. 1 to aspartic acid D, or by mutating the threonine T at position 88 and the tryptophan W at position 255 of the endoglucanase parent with the amino acid sequence shown in SEQ ID NO. 1 to histidine H.

2. A gene encoding the mutant of claim 1.

3. A recombinant vector carrying the gene of claim 2.

4. The recombinant vector of claim 3, wherein, The recombinant vector is plasmid pPICZA (alpha) as the expression vector.

5. The recombinant vector of claim 4, wherein, The signal peptide of the expression vector is replaced by SUC2.

6. A recombinant microbial cell expressing the mutant of claim 1, or containing the gene of claim 2, or transformed with the recombinant vector of any one of claims 3-5.

7. The recombinant microbial cell of claim 6, wherein, The recombinant microbial cell is in Pichia pastoris Pichia pastoris X-33 is the host cell.

8. A method for increasing the degradation activity of an endoglucanase on an acidic substrate, characterized in that, The method is to mutate the endoglucanase parent with the amino acid sequence shown in SEQ ID NO. 1 as shown in (a)-(b): (a) mutating the serine S at position 45, the threonine T at position 55 and the threonine T at position 59 to aspartic acid D; (b) mutating the threonine T at position 88 and the tryptophan W at position 255 to histidine H.

9. A method of degrading cellulose, characterized by, The method utilizes the endoglucanase mutant of claim 1 or the recombinant microbial cell of any one of claims 6-7 to degrade cellulose.

10. Use of the endoglucanase mutant of claim 1, the gene of claim 2, the recombinant vector of any one of claims 3-5, the recombinant microbial cell of any one of claims 6-7, the method of claim 8 or the method of claim 9 in the fields of medicine, animal husbandry, feed, brewing to degrade the crude fiber in vinasse, tea, tobacco, straw, rice husk and other plant raw materials.

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