Beta-1, 4-glucanase mutant and application thereof
By performing specific amino acid mutations on β-1,4-glucanase NfEG12A, the thermal stability and half-life of the enzyme are improved, and the problem of low application efficiency of existing enzymes at high temperatures is solved, and efficient cellulose degradation under high temperature conditions is achieved.
Patent Information
- Application Number
- CN202311733835.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2023-12-18
- Publication Date
- 2025-06-20
AI Technical Summary
The existing β-1,4-glucanase has a short half-life at 50°C, limiting its efficiency in application at high temperatures.
By performing site-directed mutation of β-1,4-glucanase NfEG12A derived from Aspergillus Fisher, specific amino acid mutation sites, such as N34C and N36C, L84C and V233C, the thermal stability of the enzyme is significantly improved.
The thermal stability of the mutant is significantly improved, the half-life of 50°C and 65°C is significantly extended, and the catalytic activity has not decreased significantly, making it suitable for cellulose degradation under high temperature conditions.
Smart Images
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Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of enzyme preparations, and particularly to a β-1,4-glucanase mutant and its application. Background Art
[0002] The complete hydrolysis of cellulose into monosaccharides requires the synergistic action of three types of enzymes: endoglucanase, cellobiohydrolase, and β-glucosidase. Among them, the most important is β-1,4-glucanase, which can randomly act on the β-1,4-glycosidic bonds in cellulose, causing them to break into oligosaccharide chains of different lengths. In recent years, with the increasingly prominent energy problem and the depletion of substances such as petroleum, the production of green and environmentally friendly bioethanol using renewable plant cellulose has become a research hotspot. However, low production efficiency and high production costs are the main problems in the current development of bioethanol. Since the cellulose degradation system is usually at 50°C, the short half-life of β-1,4-glucanase at this temperature greatly limits its application. Therefore, improving the thermal stability of β-1,4-glucanase is an urgent problem to be solved.
[0003] Currently, there have been some reports on improving the thermal stability of glucanase using irrational, semi-rational, and rational strategies. For example, Zhang Xiuyan et al. used random mutagenesis combined with DNA shuffling technology to perform in vitro evolution of β-glucanase from Bacillus subtilis ZLF-1A5 and established a high-throughput screening method for thermotolerant β-glucanase. The Tm values of the obtained EGs1 and EGs2 mutants were increased by 3°C and 5°C respectively compared with the wild enzyme; Gargallo et al. conducted molecular dynamics simulation studies on β-glucanase from the genus Bacillus and found that the root mean square deviation (RMSD) values of the N-terminal and C-terminal regions of β-glucanase were relatively high, and modifying the N / C terminus had a significant impact on the thermal stability of the enzyme. β-1,4-glucanase NfEG12A is a known β-1,4-glucanase with high catalytic activity. However, the research on improving the thermal stability of NfEG12A is still very limited. Therefore, it is of great significance to develop mutants of β-1,4-glucanase NfEG12A with high thermal stability. Summary of the Invention
[0004] The present invention provides a β-1,4-glucanase mutant and its application.
[0005] In previous studies, it was found that β-1,4-glucanase NfEG12A derived from Aspergillus fischeri has high catalytic activity. The present invention aims to improve the thermal stability of this enzyme. After a large number of site-directed mutagenesis attempts, mutants that can significantly improve the thermal stability of this enzyme were found.
[0006] Specifically, the present invention provides the following technical solutions:
[0007] In a first aspect, the present invention provides a β-1,4-glucanase mutant, which contains any one or more mutations of the following (1) to (5) compared to the β-1,4-glucanase NfEG12A derived from Aspergillus fischeri:
[0008] (1) N34C and N36C;
[0009] (2) L84C and V233C;
[0010] (3) A119C and H124C;
[0011] (4) A138C and P179C;
[0012] (5)P145C and W163C.
[0013] In the above amino acid mutations, N34C represents the mutation of asparagine at position 34 to cysteine, and the meanings of other amino acid mutations are similar and are well known in the art.
[0014] Preferably, the amino acid sequence of the β-1,4-glucanase NfEG12A derived from Aspergillus fischeri is shown in SEQ ID NO.2.
[0015] Preferably, the β-1,4-glucanase mutant contains N34C and N36C mutations, or L84C and V233C mutations, or A119C and H124C mutations, or A138C and P179C mutations, or P145C and W163C mutations, or L84C, V233C, P145C and W163C mutations compared to the β-1,4-glucanase NfEG12A derived from Aspergillus fischeri.
[0016] The present invention has been experimentally verified to find that compared with the non-mutated NfEG12A, the thermal stability of the above-mentioned β-1,4-glucanase mutant is significantly improved, the half-life under high temperature conditions is significantly prolonged, and the catalytic activity does not show a significant decrease, which is of great significance for its application under high temperature conditions.
[0017] In a second aspect, the present invention provides a nucleic acid molecule encoding the above-mentioned β-1,4-glucanase mutant.
[0018] Based on the amino acid sequence of the β-1,4-glucanase mutant and the codon rules described above, those skilled in the art can obtain the nucleotide sequence of the nucleic acid molecule encoding the β-1,4-glucanase mutant. Due to the degeneracy of codons, the nucleotide sequence of the nucleic acid molecule is not unique, and all nucleic acid molecules capable of encoding the β-1,4-glucanase mutant are within the protection scope of the present invention.
[0019] In some embodiments of the present invention, the nucleotide sequence of the nucleic acid molecule encoding β-1,4-glucanase NfEG12A derived from Aspergillus fischeri is shown in SEQ ID NO.1. This sequence is designed according to the codon preference of Pichia Pastoris. The nucleic acid molecule encoding the β-1,4-glucanase mutant can be obtained by performing the above mutations on the basis of the sequence shown in SEQ ID NO.1.
[0020] In a third aspect, the present invention provides a biological material comprising the nucleic acid molecule described above; the biological material is an expression cassette, a vector or a host cell.
[0021] Wherein, the expression cassette is a recombinant nucleic acid molecule obtained by operably linking the nucleic acid molecule with transcriptional and / or translational regulatory elements.
[0022] The vector comprises the nucleic acid molecule or comprises the expression cassette. The vector includes but is not limited to plasmid vectors, viral vectors, transposons, etc. The plasmid vectors include expression vectors and cloning vectors.
[0023] In some embodiments of the present invention, the vector is a pUC57 or pPIC9K vector comprising the nucleic acid molecule.
[0024] The host cell comprises the nucleic acid molecule or comprises the expression cassette or comprises the vector. The host cell includes microbial cells, preferably bacteria of the genus Escherichia or yeast, more preferably Escherichia coli or Pichia pastoris.
[0025] In a fourth aspect, the present invention provides a recombinant genetically engineered bacterium that expresses the β-1,4-glucanase mutant described above or comprises the nucleic acid molecule described above.
[0026] Preferably, the recombinant genetically engineered bacterium is a recombinant Escherichia coli genetically engineered bacterium or a recombinant yeast genetically engineered bacterium.
[0027] In a fifth aspect, the present invention provides any one of the following applications of the β-1,4-glucanase mutant, the nucleic acid molecule, the biological material or the recombinant genetically engineered bacterium described above:
[0028] (1) Use in the preparation of β-1,4-glucanase;
[0029] (2) Use in constructing microorganisms for β-1,4-glucanase production;
[0030] (3) Use in degrading cellulose or cellulose-containing biomass;
[0031] (4) Use in the preparation of food or feed.
[0032] The β-1,4-glucanase mutant provided by the present invention can be used to degrade cellulose or cellulose-containing biomass, and can also be used as raw materials such as additives in food or feed.
[0033] In the sixth aspect, the present invention provides an enzyme composition, which comprises the above-mentioned β-1,4-glucanase mutant.
[0034] Preferably, the enzyme composition further comprises cellobiohydrolase and / or β-glucosidase.
[0035] In the seventh aspect, the present invention provides a method for degrading cellulose or cellulose-containing biomass, the method comprising: subjecting cellulose or cellulose-containing biomass to enzymatic hydrolysis treatment using the above-mentioned β-1,4-glucanase mutant or the enzyme composition.
[0036] In the eighth aspect, the present invention provides a method for preparing β-1,4-glucanase, the method comprising: culturing the above-mentioned recombinant genetic engineering bacterium, collecting the culture, and obtaining β-1,4-glucanase through purification.
[0037] In the ninth aspect, the present invention provides a method for improving the thermal stability of β-1,4-glucanase, the method comprising: performing any one or more groups of mutations of the following (1)-(5) on β-1,4-glucanase NfEG12A derived from Aspergillus fischeri:
[0038] (1) N34C and N36C;
[0039] (2) L84C and V233C;
[0040] (3) A119C and H124C;
[0041] (4) A138C and P179C;
[0042] (5) P145C and W163C.
[0043] The beneficial effects of the present invention at least include: The present invention develops mutants with improved thermal stability for β-1,4-glucanase NfEG12A derived from Aspergillus fischeri. The obtained β-1,4-glucanase mutants have high thermal stability, and the half-lives at 50 °C and 65 °C are significantly prolonged, which is suitable for cellulose degradation under high-temperature conditions. It is of great significance for improving the degradation efficiency of cellulose-containing biomass and reducing production costs, and has good application prospects in the utilization of cellulose-containing biomass. BRIEF DESCRIPTION OF THE DRAWINGS
[0044] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the drawings in the following description are some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.
[0045] Figure 1 It is a schematic structural diagram of the recombinant plasmid pPIC9K / NfEG12A in Example 1 of the present invention.
[0046] Figure 2 It is the detection results of the optimum reaction temperature and thermal stability of NfEG12A and its mutants in Example 2 of the present invention. Among them, A is the detection result of the optimum reaction temperature, and B is the detection result of thermal stability (half-life). The light gray is the half-life at 50 °C, and the dark gray is the half-life at 65 °C. DETAILED DESCRIPTION OF THE EMBODIMENTS
[0047] To make the objectives, technical solutions, and advantages of the present invention clearer, the following will clearly and completely describe the technical solutions in the present invention with reference to the drawings in the present invention. Obviously, the described embodiments are some, but not all, of the embodiments of the present invention. Based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts belong to the scope of protection of the present invention.
[0048] The present invention performs site-directed mutagenesis screening on β-1,4-glucanase NfEG12A from Aspergillus fischeri (the complete amino acid sequence after removing the signal peptide is shown in SEQ ID NO.2), and preliminarily determines 7 groups of mutation sites, including S27C / T32C, N34C / N36C, T49C / T63C, L84C / V233C, A119C / H124C, A138C / P179C and P145C / W163C (in the mutants of the present invention, S27C / T32C represents that it contains both S27C and T32C mutations, and the meanings of other mutants are similar).
[0049] Among them, the coding gene of β-1,4-glucanase NfEG12A (sequence shown in SEQ ID NO.1) was chemically synthesized by GENEWIZ according to the codon preference of Pichia pastoris, and connected to the basic vector pUC57 to obtain the recombinant vector pUC57 / NfEG12A.
[0050] In the present invention, the expression of the β-1,4-glucanase mutant and the enzymatic property determination process are roughly as follows:
[0051] The above-mentioned target amino acids were mutated by site-directed mutagenesis technology, and NfEG12A mutants S27C / T32C, N34C / N36C, T49C / T63C, L84C / V233C, A119C / H124C, A138C / P179C and P145C / W163C were expressed in host cells, respectively, as follows:
[0052] 1. Vector construction and site-directed mutagenesis
[0053] Using the vector pUC57 / NfEG12A as a template, PCR amplification was performed by designing upstream and downstream primers F1 and R1 (Table 1). The PCR amplification product was detected by agarose gel electrophoresis, and the product was purified by column recovery. The expression vector pPIC9K was PCR amplified using primers F2 and R2 (Table 1), and the product was then detected by agarose gel electrophoresis and purified by column recovery. Finally, the above fragments were connected with a one-step cloning enzyme and transformed into Escherichia coli JM109 competent cells to obtain the recombinant expression plasmid pPIC9K / NfEG12A ( Figure 1 ). Using the above vector as a template, site-directed mutagenesis primers (Table 1) were designed to construct a vector containing the mutant.
[0054] Table 1 Primers for plasmid construction
[0055]
[0056]
[0057] 2. Expression and purification of NfEG12A and its mutants
[0058] The strain P. pastoris GS115 was used to express NfEG12A and NfEG12A mutants. Methanol was used as an inducer to enhance the transcription of promoter PAOX1, and the supernatant after centrifugation was collected after fermentation for a certain time, filtered through a filter membrane, purified by HisTrap TM FF, and desalted using a desalting column Sephadex G25.
[0059] 3. Enzymatic property determination of NfEG12A and its mutants
[0060] The enzyme activity of each obtained mutant for β-1,4-glucanase was determined, and the 3,5-dinitrosalicylic acid method (DNS) was used to determine the content of reducing sugar in the reaction system. The method is as follows: 125 μL of the enzyme solution diluted to an appropriate concentration was mixed with 375 μL of 1% barley glucan substrate (prepared using 0.2 mol / L, pH 6.0 acetic acid-sodium acetate buffer), reacted at 50 °C for 30 min, and the reaction was terminated with DNS. The reaction sample was colored in boiling water for 5 min and immediately cooled with water, and the absorbance was measured at 545 nm. The enzyme activity unit (U / mL) was defined as: under standard conditions, the amount of enzyme required to hydrolyze barley glucan to produce 1 μmol of reducing sugar per minute.
[0061] The optimal reaction temperature and thermal stability of each mutant were determined. The method is as follows:
[0062] Determination of the optimal reaction temperature: The purified enzyme solution was diluted to an appropriate concentration with acetic acid-sodium acetate buffer (0.2 mol / L, pH 6.0), and the enzyme activities at 45 - 75 °C were measured respectively. Taking the highest enzyme activity as 100%, the relative enzyme activities at each temperature were calculated.
[0063] Determination of thermal stability: The above-diluted enzyme solutions were incubated at 50 °C for 0 - 200 min respectively, then stored on ice, and the enzyme activities under each condition were measured. Taking the enzyme activity at 0 min of incubation as 100%, the relative enzyme activities at each incubation time were calculated. Based on the above determination of thermal stability, the regression equation at different temperatures was fitted, and its half-life was calculated.
[0064] Through enzymatic property determination, the half-lives of 5 mutants were increased compared with wild-type NfEG12A, including mutants N34C / N36C, L84C / V233C, A119C / H124C, A138C / P179C, and P145C / W163C. Among them, the half-lives of L84C / V233C and P145C / W163C were increased by more than 20%. Compared with the half-life of NfEG12A, which was 127.6 min, the half-lives of L84C / V233C and P145C / W163C were increased by 36.4% and 43.9% respectively. The half-life of the combined mutant L84C / V233C / P145C / W163C at 50 °C was increased by 53.5% compared with NfEG12A.
[0065] Example 1 Construction of vectors and site-directed mutagenesis
[0066] According to the codon preference of P. pastoris, the coding gene sequence of β-1,4-glucanase NfEG12A was designed as shown in SEQ ID NO.1, and this gene was ligated to the basic vector pUC57 to obtain the recombinant vector pUC57 / NfEG12A. Using the vector pUC57 / NfEG12A as a template, PCR amplification was carried out by designing upstream and downstream primer pairs F1 and R1 (Table 1). The PCR program was: pre-denaturation at 98 °C for 3 min; denaturation at 98 °C for 10 s, annealing at 55 °C for 15 s, extension at 72 °C for 50 s, for 34 cycles; finally, extension at 72 °C for 5 min. The PCR amplification products were detected by agarose gel electrophoresis, and the products were purified by column recovery. The expression vector pPIC9K was PCR amplified with primers F2 and R2 (Table 1) respectively, and then its products were also detected by agarose gel electrophoresis and purified by column recovery. Finally, the above fragments were ligated with one-step cloning enzyme and transformed into E. coli JM109 competent cells to obtain the recombinant expression plasmid pPIC9K / NfEG12A( Figure 1 ). The method of one-step cloning was: mix 2 μL of the fragment, 1 μL of the vector, 2 μL of the buffer, and 1 μL of the homologous recombination enzyme, and add ddH2O to make the total volume reach 10 μL. Subsequently, the system was reacted at 37 °C for 30 min and transformed rapidly. Using the above vector pPIC9K / NfEG12A as a template, site-directed mutagenesis primers (Table 1) were designed for PCR amplification. Subsequently, the obtained fragments were transferred into E. coli JM109, and circular ligation was carried out by homologous recombination between the same sequences at both ends, so as to construct pPIC9K expression vectors containing the genes of mutants S27C / T32C, N34C / N36C, T49C / T63C, L84C / V233C, A119C / H124C, A138C / P179C, and P145C / W163C respectively.
[0067] Expression, Purification and Enzymatic Property Determination of Example 2 NfEG12A and Its Mutants
[0068] To obtain NfEG12A and NfEG12A mutants and determine their enzymatic properties, the pPIC9K vectors carrying NfEG12A and mutant genes were electrotransformed into P. pastoris GS115 respectively. The obtained single colonies were inoculated into YPD medium and cultured at 30 °C for 18 - 24 h. Subsequently, the cells were washed with normal saline and transferred to BMMY medium, and fermented at 30 °C for 120 h. To induce protein expression, 1% methanol was added to the medium every 24 h during fermentation. After the fermentation broth was centrifuged at 12000 rpm and 4 °C for 10 min, the supernatant was filtered through a 0.22 μm filter membrane and purified by HisTrap TM FF, and desalted by the desalting column Sephadex G25.
[0069] The purified protein was detected as a single band by SDS-PAGE, and its size was the same as the theoretical value, thus obtaining NfEG12A mutants with disulfide bonds formed at different positions. The detection results of the optimal reaction temperature showed that the optimal reaction temperature of NfEG12A and its mutants was 65 °C. At 50 °C, there was no significant difference in the enzyme activity between the mutants and NfEG12A, while at 65 °C, only the activities of mutants A119C / H124C and A138C / P179C decreased by about 10%( Figure 2 A). At the same time, when the half-lives of NfEG12A and its mutants were determined at 50 °C and 65 °C, it was found that the half-lives of 5 mutants were significantly increased compared with the wild-type NfEG12A, including mutants N34C / N36C, L84C / V233C, A119C / H124C, A138C / P179C and P145C / W163C. Among them, at 50 °C, the half-lives of L84C / V233C and P145C / W163C were extended by more than 20%. Compared with the half-life of NfEG12A of 127.6 min, the half-lives of L84C / V233C and P145C / W163C were extended by 36.4% and 43.9% respectively; at 65 °C, compared with the half-life of NfEG12A of 21.2 min, the half-lives of L84C / V233C and P145C / W163C were extended by 26.4% and 26.3% respectively( Figure 2B). Based on the above results, L84C / V233C and P145C / W163C were further subjected to combinatorial mutagenesis to obtain the combinatorial mutant L84C / V233C / P145C / W163C (containing the L84C, V233C, P145C, and W163C mutations simultaneously), and its half-lives at 50 °C and 65 °C were measured. The results showed that the half-lives of the combinatorial mutant at 50 °C and 65 °C were extended by 53.5% and 31.1% respectively compared to NfEG12A ( Figure 2 B).
[0070] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements on some of the technical features; and these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.
Claims
1. A β-1,4-glucanase mutant, characterized in that The β-1,4-glucanase mutant, compared with the β-1,4-glucanase NfEG12A derived from Aspergillus fischeri, contains any one or more groups of mutations in the following (1) to (5): (1) N34C and N36C; (2) L84C and V233C; (3) A119C and H124C; (4) A138C and P179C; (5) P145C and W163C.
2. The β-1,4-glucanase mutant according to claim 1, characterized in that The amino acid sequence of the β-1,4-glucanase NfEG12A derived from Aspergillus fischeri is shown in SEQ ID NO.
2.
3. The β-1,4-glucanase mutant according to claim 1 or 2, characterized in that The β-1,4-glucanase mutant, compared with the β-1,4-glucanase NfEG12A derived from Aspergillus fischeri, contains the N34C and N36C mutations, or contains the L84C and V233C mutations, or contains the A119C and H124C mutations, or contains the A138C and P179C mutations, or contains the P145C and W163C mutations, or contains the L84C, V233C, P145C and W163C mutations.
4. A nucleic acid molecule, characterized in that The nucleic acid molecule encodes the β-1,4-glucanase mutant according to any one of claims 1 to 3.
5. A biological material, characterized in that The biological material contains the nucleic acid molecule according to claim 4; the biological material is an expression cassette, a vector or a host cell.
6. A recombinant genetically engineered bacterium, characterized in that The recombinant genetically engineered bacterium expresses the β-1,4-glucanase mutant according to any one of claims 1 to 3, or contains the nucleic acid molecule according to claim 4.
7. The recombinant genetically engineered bacterium according to claim 6, characterized in that The recombinant genetically engineered bacterium is a recombinant Escherichia coli genetically engineered bacterium or a recombinant yeast genetically engineered bacterium.
8. Any one of the following applications of the β-1,4-glucanase mutant according to any one of claims 1 to 3, the nucleic acid molecule according to claim 4, the biological material according to claim 5, or the recombinant genetically engineered bacterium according to claim 6 or 7: (1) Application in the preparation of β-1,4-glucanase; (2) Application in constructing a microorganism for β-1,4-glucanase production; (3) Application in degrading cellulose or cellulose-containing biomass; (4) Application in the preparation of food or feed.
9. An enzyme composition, characterized in that The enzyme composition contains the β-1,4-glucanase mutant according to any one of claims 1 to 3.
10. A method for degrading cellulose or cellulose-containing biomass, characterized in that The method includes: subjecting cellulose or cellulose-containing biomass to enzymatic hydrolysis treatment by using the β-1,4-glucanase mutant according to any one of claims 1 to 3 or the enzyme composition according to claim 9.