Consensus mutation and structure-guided directed evolution of endocellulase mutants

Abstract

The present application relates to the sustained endo-cellulase mutant with significantly improved enzyme activity and its construction method, and belongs to the field of genetic engineering. The present application adopts single-point saturated mutation technology, adopts consensus site mutation and structure-guided screening method to obtain the sustained endo-cellulase mutant. In the amino acid sequence shown in SEQ ID NO:1, one or more of the 91st, 198th, 237th and 240th sites are mutated, wherein the optimal mutant is K91I / A198T / Q237D / V240P. Compared with the original enzyme, the catalytic efficiency is greatly improved, which is of great significance for efficient degradation of cellulose substrate and reduction of production cost.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of genetic engineering, and particularly relates to a persistent endoglucanase mutant with improved enzyme activity and application thereof. BACKGROUND

[0002] Cellulose is the most widely distributed and most abundant renewable resource on earth, and its degradation into biofuels and chemicals by enzymes is a major strategic demand of the state. The resource utilization of cellulose is of great significance to solve the energy crisis and promote the sustainable development of society. However, the structure of cellulose is complex and highly dense, and its efficient degradation must be completed under the synergistic action of multiple hydrolytic enzymes. The enzymes required in the hydrolysis process are of many types and in large quantities, which leads to high enzyme application cost and seriously restricts the process of cellulose biorefining. It is a bottleneck factor for the commercialization of cellulosic ethanol and high-value chemicals.

[0003] Precessive endoglucanase has both endoglucanase and exoglucanase functions, and has higher thermal stability and lower product inhibition than exoglucanase. It has great application value in reducing enzyme cost of cellulose bioconversion and promoting large-scale application of cellulose hydrolysis technology. However, the existing precessive endoglucanase still has problems such as weak exoglucanase activity and poor sustained hydrolysis capacity. Molecular modification cannot effectively enhance the function due to unclear relationship between structure and function, which seriously affects the application of the enzyme in low-cost and efficient degradation of cellulose. Therefore, developing precessive endoglucanase with high sustained hydrolysis capacity and high catalytic activity has become an urgent industrial technology demand.

[0004] Compared with traditional methods for improving enzyme catalytic performance, target mutagenesis guided by sequence or structure information has become a new approach to improving enzyme catalytic activity, which may reduce efforts in protein methods (Journal of Biotechnology, 2019, 293:8-16). In order to improve the thermal stability of decarboxylase from Lactobacillus brevis, Hua et al. (Applied Biochemistry and Biotechnology, 2020, 191:1456-1469) used consensus mutagenesis method, and screened 8 key amino acid sites by using Consensus Finder software. Subsequently, a mutant with significantly improved thermal stability was obtained by using site-directed mutagenesis technology, and at the same time, the catalytic efficiency k cat / K mThe improvement is also obtained. Liu et al. (ACS Catalysis 2020, 10: 12393-12402) performed protein engineering on D-carbamoylase based on structure guidance, determined two loop regions critical to the performance of the enzyme by comparing the RMSF values with and without substrate, and performed conservative analysis on the sequences of the regions, then obtained mutants with significantly improved catalytic efficiency through saturation mutation and combined mutation.

[0005] Although researchers at home and abroad have made very significant progress in improving the catalytic activity of cellulase through protein engineering technology. However, there is still a lack of overall systematic theoretical understanding of the persistence mechanism of persistent endoglucanase, which is difficult to meet the needs of the directed design and modification of persistent endoglucanase. Therefore, the enzyme modification method based on sequence and structure guidance not only provides theoretical guidance for protein engineering technology to modify persistent endoglucanase to improve its catalytic activity, but also helps to explain the special catalytic mechanism of this type of enzyme. SUMMARY

[0006] The present application adopts single-point mutation and saturation mutation technology, adopts the combination of consensus site mutation technology and structure-guided protein engineering technology, takes the single catalytic domain of the GH5 family of persistent endoglucanase as the sequence alignment object for consensus site analysis, then compares the RMSF values of the enzyme structure with and without substrate, finds the key loop region in the enzyme structure, performs conservative analysis on the region, screens the key sites for molecular modification, and obtains a class of persistent endoglucanase mutants with significantly improved enzyme activity. The mutant persistent endoglucanase described in the present application has high catalytic activity, providing new feasibility for the industrialized bioconversion of cellulose raw materials.

[0007] To achieve the above object, the technical scheme adopted by the present application is as follows:

[0008] A class of endoglucanase mutants, the mutant has amino acid mutations at positions 91 and / or 198 and / or 237 and / or 240 based on the amino acid sequence of the endoglucanase (EG5C-1) shown in SEQ ID NO: 1. Preferably, the lysine at position 91 of the mutant amino acid is mutated to alanine, glutamic acid, aspartic acid and isoleucine, preferably isoleucine; and / or the alanine at position 198 is mutated to arginine, lysine, threonine and phenylalanine, preferably threonine; and / or the glutamine at position 237 is mutated to alanine, glutamic acid, asparagine and aspartic acid, preferably aspartic acid; and / or the valine at position 240 is mutated to alanine, leucine, proline and glycine, preferably proline.

[0009] More preferably, the mutant amino acid sequence of the present application is shown in any one of SEQ ID NO: 3 (K91I), SEQ ID NO: 4 (A198T), SEQ ID NO: 5 (Q237D), SEQ ID NO: 6 (V240P), SEQ ID NO: 7 (K91I / A198T / Q237D / V240P).

[0010] Another object of the present application is to provide an endocellulase mutant gene, a recombinant vector comprising the endocellulase mutant gene and a transformant of the recombinant vector.

[0011] Further, the present application also provides a preparation method of the recombinant vector, the endocellulase mutant gene of the present application is prepared by artificial synthesis or gene cloning, an expression vector is constructed to obtain a recombinant plasmid, and the recombinant plasmid is transformed into a host cell. Preferably, the host cell is selected from Escherichia coli, Pichia pastoris cells or Bacillus subtilis, and preferably is Escherichia coli BL21. The endocellulase mutant of the present application can be used for catalyzing cellulose biodegradation.

[0012] Compared with the prior art, the present application has the beneficial effects that: the present application adopts site-directed mutation and saturation mutation technology, and combines consensus site analysis and structure-guided protein engineering method, explores and finds the influence of key structures in enzyme structure on enzyme catalytic performance, and obtains a class of sustained endocellulase mutants with significantly improved enzyme activity. The mutant of the sustained endocellulase has high catalytic activity, and provides new feasibility for industrialized biotransformation of cellulose raw materials. BRIEF DESCRIPTION OF DRAWINGS

[0013] Figure 1 . Relative hydrolytic activity of single-point mutants based on consensus site analysis on CMC (blue) and Avciel (green) substrates.

[0014] Figure 2 . Relative hydrolytic activity of single-point saturation mutants based on consensus site analysis on CMC (A) and Avicel (B) substrates. Figure 3 . Relative hydrolytic activity of saturation mutants of key sites S232, Q237, G239, V240, F241 based on structure guidance on CMC and Avicel substrates.

[0015] Figure 4Relative hydrolysis activities of the combined mutants between the best mutants obtained by consensus site screening and the best mutants obtained based on structure guidance (M2-1: K91I / A198T / Q237A / V240P, M3-1: K91I / A198T / Q237D / V240P, M4-1: K91I / A198T / Q237N / V240P, M5-1: K91I / A198T / Q237G / V240P, M6-1: K91I / A198T / Q237S / V240P, M7-1: K91I / A198T / Q237T / V240P) on CMC and Avicel substrates.

[0016] Figure 5 . Optimal temperature and pH as well as temperature and pH stability analysis of processive endocellulase EG5C-1 and its mutant M3-1.

[0017] Figure 6 .The ratio of soluble reducing ends to insoluble reducing ends in the reaction solution of processive endocellulase EG5C-1 and the best mutant M3-1 using regenerated amorphous material PASC as substrate after reaction for 30min, 60min, 120min and 180min.

[0018] Figure 7 Analysis of hydrolysis products of the processive endocellulase EG5C-1 and its optimal mutant M3-1 using CMC, Avicel, and PASC as substrates (A) and cellooligosaccharides as substrates (B). G1: glucose, G2: cellobiose, G3: cellotriose, G4: cellotetraose, and G5: cellopentaose. (A) M represents a product standard sample. Channels 1, 2, and 3 represent the hydrolysis products of EG5C-1 on CMC, Avicel, and PASC, respectively; channels 4, 5, and 6 represent the hydrolysis products of M3-1 on CMC, Avicel, and PASC, respectively. (B) M represents a product standard sample. Channels 1, 2, 3, and 4 represent the hydrolysis products of EG5C-1 on cellobiose, cellotriose, cellotetraose, and cellopentaose, respectively; channels 5, 6, 7, and 8 represent the hydrolysis products of M3-1 on cellobiose, cellotriose, cellotetraose, and cellopentaose, respectively.

[0019] Figure 8 The yield of reducing sugars produced by the processive endocellulase EG5C-1 and the best mutant M3-1 after hydrolysis of filter paper for 4h, 8h, 12h, 16h, 20h, and 24h DETAILED DESCRIPTION

[0020] In order to better illustrate the purpose, technical solutions and advantages of the present invention, further detailed description will be given below with reference to the accompanying drawings and specific embodiments.

[0021] Example 1 Screening of key amino acid residues based on consensus site analysis

[0022] Different persistent endo-cellulases from different sources have different catalytic domains (CD) and substrate binding domains (CBM), which have a significant impact on their persistence ability. Based on this, four persistent endo-cellulases from GH5 family containing only single catalytic domain were downloaded from GeneBank based on screening from Uniport database and published literature, which were Cel5 from Hahella chejuensis, Cel5H from Saccharophagus degradans, Cel5A from Cytophaga hutchinsonii, and CHU_2103 from Cytophaga hutchinsonii. Using Clustal W, the sequences were subjected to multiple sequence alignment, and 20 key consensus sites S35L, H36F, G37W, L38S, V45Y, L50V, F62V, T68V, A86V, K91I, E92A, G107H, P109A, S124A, Q189P, V195T, A198T, P223A, Q244E, Y251F were obtained through consensus site analysis. Subsequently, the corresponding mutants were obtained by site-directed mutagenesis method, and the enzyme activities of the above mutants were determined using CMC and Avciel as substrates, and the results are shown in Table 1. Figure 1

[0023] Example 2 Screening of key amino acid residues based on structure guidance

[0024] Firstly, the crystal structure of wild-type EG5C-1 was predicted by homology modeling of the catalytic module of BsCel5A (PDB ID: 3PZT), and then the substrate hexaose was docked in the substrate channel using Autodock software, and the RMSF value of the enzyme structure with and without substrate was compared. It was found that the RMSF value fluctuated sharply between positions 230-241, and through structure analysis, it was found that this position was in the loop region at the substrate entrance, which affected the entry of the substrate. Therefore, the sequence conservation analysis of the sites in this region was performed, and S232, Q237, G239, V240, F241 sites were selected for subsequent research.

[0025] Example 3 Construction of persistent endo-glucanase mutants and screening of optimal mutants

[0026] ​The mutation sites S35, H36, G37, L38, V45, L50, F62, T68, A86, K91, E92, G107, P109, S124, Q189, V195, A198, P223, Q244, Y251, S232, Q237, G239, V240, F241 obtained from Examples 1 and 2 were subjected to saturation mutation, and a recombinant plasmid containing SEQ ID NO: 2 was constructed using the method disclosed by Bin Wu (Biotechnology for Biofuels, 2018, 11:20). The sequence shown in SEQ ID NO: 2 was used as a template, and the full plasmid was amplified to obtain a site-directed mutation sequence by using the following designed mutation primers. The primers used are as follows:

[0027]

[0028]

[0029] The codons of different amino acids are as follows: TGT (Cys), GAT (Asp), GAA (Glu), TTT (Phe), GGC (Gly), CAT (His), ATT (Ile), AAA (Lys), CTG (Leu), ATG (Met), AAT (Asn), CCG (Pro), CAG (Gln), CGT (Arg), TCA (Ser), ACA (Thr), GTT (Val), TGG (Trp), TAT (Tyr).

[0030] The PCR reaction system is as follows:

[0031]

[0032] The PCR program is set as follows:

[0033] 95℃, 3min;

[0034] 95℃, 15s; 60℃, 15s; 72℃, 8min; 30 cycles;

[0035] 72℃, 10min;

[0036] 4℃, Hold.

[0037] After full plasmid amplification, 2μL of PCR product was subjected to nucleic acid electrophoresis verification. After verification, DpnI digestion enzyme was used to degrade the initial template.

[0038] The digestion system is as follows:

[0039] PCR product 1μL

[0040] DpnI enzyme 1 μL

[0041] 10×QuickCut Buffer 2μL

[0042] The digestion procedure is as follows:

[0043] 37℃, 30min.

[0044] After digestion, the PCR product was transformed into competent E. coli BL21 (DE3) cells using the heat shock method and plated onto LB agar plates containing 100 μg / ml kanamycin sulfate. The plates were incubated at 37°C for 14-16 hours. Sequencing (performed by Anhui General Biotechnology Co., Ltd.) confirmed the mutation and generated the corresponding mutant.

[0045] The mutants and the original enzyme EG5C-1 constructed above were inoculated into 50 mL of LB liquid medium, and kanamycin sulfate was added to a final concentration of 100 μg / mL. The culture was carried out at 180 rpm / min and 37°C overnight. The overnight cultured seed solution was inoculated into fresh 50 mL of LB liquid medium at a 2% inoculum size and cultured at 180 rpm / min and 37°C until the OD 600 When the concentration is 0.6-1.0, add the inducer IPTG (final concentration 0.1 mmol L -1 ) and induced expression at 25°C for 24 hours. The induced fermentation broth was centrifuged at 12,000 rpm / min for 20 minutes, the supernatant discarded, and the cells resuspended in 50 mM Na₂HPO₄-KH₂PO₄ (pH 6.0) buffer and ultrasonically disrupted. The supernatant was filtered through a 0.22 μm filter to obtain a crude enzyme solution. The target protein was then isolated and purified using a nickel column (GE Healthcare, Fairfield, USA) to obtain each mutant. Electrophoresis and amino acid sequencing of each mutant revealed a distinct band at 33.4 kDa for all induced mutants. Sequencing confirmed the correct mutant sequence, indicating that the processive endoglucanase mutant was successfully induced for expression.

[0046] The changes in the enzyme activity of the mutants were determined using sodium carboxymethyl cellulose (CMC) and microcrystalline cellulose (Avicel) as substrates. The relative hydrolysis activity of the saturated mutants on CMC and Avicel substrates was shown in the following table: Figure 2 、 Figure 3 、 Figure 4 As shown. Based on the consensus site mutation analysis, it was found that among the site-directed mutations at 20 key sites, the enzyme activity of the mutants K91I and A198T was significantly improved. Compared with the original enzyme EG5C-1, their activities for CMC were increased by 1.3 and 1.7 times, respectively, and their activities for Avicel were increased by about 1.2 times (Figure 1 and Figure 2 Among the structure-guided site-saturation mutants, 6 mutants at position 237, Q237A, Q237D, Q237N, Q237G, Q237S, Q237T, and V240P at position 240 showed significantly improved activity (Table 2) while most of the other mutants showed significantly reduced activity. Figure 3

[0047] Example 4 Combination mutants between consensus site-based mutants and structure-guided mutants

[0048] According to the results of Example 3, combination mutants M2-1: K91I / A198T / Q237A / V240P, M3-1: K91I / A198T / Q237D / V240P, M4-1: K91I / A198T / Q237N / V240P, M5-1: K91I / A198T / Q237G / V240P, M6-1: K91I / A198T / Q237S / V240P and M7-1: K91I / A198T / Q237T / V240P were designed, and the corresponding mutants were obtained according to the preparation method of Example 3, and the relative hydrolysis activities on CMC and Avicel substrates were determined, and the results are shown in Table 3. Figure 4

[0049] The results show that the activities of the combination mutants are significantly improved compared with the original enzyme, among which the activity of M3-1 in the combination mutant is improved by 4.5 times for CMC substrate and 3.2 times for Avicel substrate compared with the original enzyme.

[0050] Example 5 Enzymatic property analysis of persistent endoglucanase mutants

[0051] Hydrolysis activity determination method of persistent endoglucanase mutants

[0052] Definition of enzyme activity unit: one enzyme activity unit is defined as the amount of enzyme required to produce 1 mmol of reducing sugar per minute under the condition of 60°C, pH 6.0.

[0053] ​​(1) Endo-glucanase: 1 g of carboxymethylcellulose sodium (CMC-Na) was accurately weighed and dissolved in 100 mL of Na2HPO4-KH2PO4 buffer (50 mM, pH 6.0), stirred and mixed, 1.5 mL was accurately taken and added to a test tube as the enzyme reaction substrate, after preheating at 60°C for 5 min, 0.5 mL of the appropriately diluted protease solution was added, and the reaction was carried out in a 60°C water bath shaker for 10 min, 3 mL of DNS reagent was added, and after boiling water bath reaction for 5 min, it was quickly cooled to room temperature. The absorbance value at 540 nm wavelength was determined with the inactivated enzyme reaction solution as the control.

[0054] (2) Exo-glucanase: 10 g of microcrystalline cellulose (Avicel) was accurately weighed and dissolved in 100 mL of Na2HPO4-KH2PO4 buffer (50 mM, pH 6.0), stirred and mixed, 1.5 mL was accurately taken and added to a test tube as the enzyme reaction substrate, after preheating at 60°C for 5 min, 0.5 mL of the appropriately diluted protease solution was added, and the reaction was carried out in a 60°C water bath shaker for 30 min, the supernatant was centrifuged and added with 3 mL of DNS reagent, and after boiling water bath reaction for 5 min, it was quickly cooled to room temperature. The absorbance value at 540 nm wavelength was determined with the inactivated enzyme reaction solution as the control.

[0055] (3) Filter paper enzyme activity (FPase): 0.5 g of dried filter paper (FP) was accurately weighed and added with 1.5 mL of Na2HPO4-KH2PO4 buffer (50 mM, pH 6.0), after preheating at 60°C for 5 min, 0.5 mL of the appropriately diluted protease solution was added, and the reaction was carried out in a 60°C water bath shaker for 30 min, the supernatant was centrifuged and added with 3 mL of DNS reagent, and after boiling water bath reaction for 5 min, it was quickly cooled to room temperature. The absorbance value at 540 nm wavelength was determined with the inactivated enzyme reaction solution as the control.

[0056] Specific enzyme activity X = (reducing sugar content / 180 / 10(30)) / n

[0057] Where: X - specific enzyme activity, U / mg 180 - reducing sugar converted from milligrams to micromoles

[0058] 10(30) - reaction time

[0059] n - reaction protein content, mg

[0060] 1. Optimum reaction temperature and temperature stability.

[0061] The mutant enzyme solution obtained in Example 4 was diluted to a certain concentration and 500 μl was taken and added to a test tube containing 1.5 mL of CMC substrate, and was reacted at 30°C, 40°C, 50°C, 60°C, 70°C, 80°C, 90°C, respectively, for 10 min, and after the reaction was completed, 3 mL of DNS solution was added and boiled for 5 min, and the enzyme activity was measured. Taking the highest enzyme activity as 100%, the relative enzyme activity was calculated in turn, and the curve of enzyme activity change with temperature was drawn.

[0062] In order to determine the temperature stability of the mutant and the original enzyme, the diluted enzyme solution was placed in a 30°C, 40°C, 50°C, 60°C, 70°C, 80°C water bath for 2 h, and then the residual enzyme activity was determined in the manner of determining endo-cellulase activity. Taking the highest enzyme activity as 100%, the relative enzyme activity was calculated in turn, and the curve of enzyme activity change under different incubation conditions was drawn.

[0063] The results are shown in Table 1. Figure 6 As shown in Table 1, the optimal reaction temperature of the persistent endoglucanase EG5C-1 and the mutant M3-1 was 60°C, and when the temperature was 30°C-60°C, the enzyme activity of the persistent endoglucanase EG5C-1 and the mutant gradually increased, and when the temperature was higher than 60°C, the enzyme activity of EG5C-1 and the mutant decreased significantly. The persistent endoglucanase EG5C-1 and the mutant M3-1 were incubated at less than 50°C for 2 h, and still retained more than 80% of the enzyme activity, indicating that the temperature stability of the mutant enzyme was improved compared with the original enzyme.

[0064] 2. Optimum pH and pH stability

[0065] Different pH buffers and substrates were prepared: citric acid-sodium citrate (pH 3.0-6.0), Na2HPO4-KH2PO4 (pH 6.0-8.0), glycine-sodium hydroxide (pH 8.0-9.0), and 1% CMC substrate reaction solution was prepared under different pH conditions.

[0066] The enzyme solution obtained by purification in Example 4 was diluted to a certain concentration and 500 μl was taken and added to a test tube containing 1.5 mL of CMC substrate reaction solution prepared with different pH buffers, and was reacted at 60°C for 10 min, and after the reaction was completed, 3 mL of DNS solution was added and boiled for 5 min, and the enzyme activity was measured. Taking the highest enzyme activity as 100%, the relative enzyme activity was calculated in turn, and the curve of enzyme activity change with substrate pH change was drawn.

[0067] The diluted enzyme solution was diluted with different pH buffers, incubated at 4°C for 2 h on ice, and then the enzyme activity was determined in the manner of determining endo-cellulase activity. Taking the highest enzyme activity as 100%, the relative enzyme activity was calculated in turn, and the curve of enzyme activity change under different incubation conditions was drawn.

[0068] The results, as shown in Figure 5 Figure 6, show that the optimum reaction pH of both the persistent endoglucanase EG5C-1 and the mutant M3-1 is pH 6.0. The persistent endoglucanase EG5C-1 and its mutant have the highest hydrolysis activity at pH 6.0. As the pH increases or decreases, the enzyme activity of both the persistent endoglucanase EG5C-1 and the mutant M3-1 decreases. The stability of the persistent endoglucanase EG5C-1 and the mutant M3-1 is relatively stable between pH 5.0 and 9.0. After 2 h of incubation under each condition, more than 70% of the enzyme activity is retained. Between pH 3.0 and 5.0, the stability of the persistent endoglucanase and its mutant decreases. The enzyme activity retained by EG5C-1 is lower than that of the mutant M3-1 under each pH condition, indicating that the pH stability of the mutant is improved.

[0069] The persistence of the persistent endoglucanase is determined by detecting the ratio of soluble reducing ends and insoluble reducing ends produced by degrading the regenerated amorphous cellulose (PASC). The determination steps are as follows:

[0070] (1) The enzyme solution obtained in Example 4 is diluted to a certain concentration and 500 μl is added to a test tube containing 1.5 mL of 1% PASC substrate (w / v) and mixed well;

[0071] (2) The test tube is removed after 30 min, 60 min, 120 min and 180 min of reaction at 60°C, and immediately boiled in boiling water for 5 min to terminate the reaction;

[0072] (3) The reaction is centrifuged at 12000 rpm for 10 min;

[0073] (4) The precipitate is repeatedly washed and centrifuged with Na2HPO4-KH2PO4 buffer for at least three times, and then resuspended with buffer in the same volume as the supernatant, which is the insoluble reducing sugar;

[0074] (5) The content of reducing sugar in the supernatant and the precipitate is determined according to the DNS method, and the ratio of reducing ends in different reaction time periods is calculated.

[0075] The results, as shown in Figure 6 Figure 7, show that when the reaction time is extended from 0.5 h to 3 h, the ratio of soluble reducing sugar and insoluble reducing sugar produced by the original enzyme EG5C-1 increases from 2.25 to 3.31. At the same time, the ratio of soluble reducing sugar and insoluble reducing sugar produced by the best mutant M3-1 enzyme PASC increases continuously with the extension of time, from 2.30 at 0.5 h to 4.39 after 3 h. The results show that the best mutant M3-1 has improved persistence ability while the endoglucanase and exoglucanase activities are improved.

[0076] Hydrolysis product analysis of the best mutants of endoglucanase EG5C-1

[0077] The purified enzyme liquid obtained according to Example 4 was used for product hydrolysis analysis with CMC, Avicel, PASC and cellotriose as substrates, and the specific steps were as follows:

[0078] (1) The appropriate dilution of the purified enzyme liquid was mixed with the substrates CMC, Avicel and PASC at a ratio of 1:1;

[0079] (2) The reaction liquid was placed in a 45°C oven for 6h (CMC, Avicel, PASC) and 3h (cellotriose) and then boiled to inactivate the enzyme;

[0080] (3) The reaction liquid was centrifuged at 12000 rpm for 10 min, and the precipitate was discarded to collect the supernatant enzyme hydrolysis product;

[0081] (4) The hydrolysis product was precipitated by adding pre-cooled ethanol overnight, and then dried after removing impurities;

[0082] (5) The product was dissolved by adding appropriate distilled water, and the hydrolysis product was obtained.

[0083] Thin layer chromatography (TLC) detection

[0084] (1) The thin plate was activated in a 120°C oven (to remove volatile impurities and water, and to improve separation efficiency) for 1h and placed in a desiccator for standby;

[0085] (2) The developing agent (about 60mL) was prepared by mixing n-butanol: acetic acid: water = 3:2:1, and added to the developing tank about 15-30min before the experiment to prevent edge effects;

[0086] (3) Spotting: the standard sample was 1μL (glucose, cellobiose, cellotriose, cellotetraose), and each sample was 2μL;

[0087] (4) The developing tank was placed in a fume hood and developed at room temperature for about 1.5h twice;

[0088] (5) Sample coloration: the color developing agent (about 20mL) was prepared by mixing concentrated sulfuric acid: ethanol = 1:9, and evenly sprayed on the surface of the thin plate and dried with a hair dryer, and then placed in a 120°C oven for color development for 5min;

[0089] (6) The hydrolysis product of EG5C-1 and each mutant was determined according to the standard.

[0090] As Figure 7As shown, the hydrolysis products of CMC, Avicel and PASC by persistent endo- cellulase EG5C-1 and the best mutant M3-1 were mainly cellobiose and cellotriose. Meanwhile, the hydrolysis patterns of cello-oligosaccharides by EG5C-1 and mutant M3-1 were the same, which could hydrolyze cellotriose, tetraose and pentaose to cellobiose and cellotriose, but could not hydrolyze cellobiose.

[0091] Example 8 Analysis of the hydrolysis performance of the best mutant of persistent endo-glucanase on filter paper

[0092] 2.5% (w / v) of dried filter paper was mixed with Na2HPO4-KH2PO4 (50 mM, pH 6.0), and 40 μg / mL of persistent endo-cellulase EG5C-1 and its best mutant M3-1 were added respectively, and the mixture was shaken at 200 rpm and 45°C for 24 h, with no enzyme solution as a control. The DNS method was used to determine the content of reducing sugar produced by the persistent endo-cellulase after hydrolysis of filter paper for 4 h, 8 h, 12 h, 16 h, 20 h and 24 h. The results are shown in Table 2. Figure 8 As shown, the content of reducing sugar produced by the original enzyme EG5C-1 and the mutant M3-1 was similar in the initial stage, and with the extension of the reaction time, the content of reducing sugar produced by the mutant M3-1 was about 85% higher than that of the original enzyme EG5C-1 after 24 h, which indicated that the mutant M3-1 had higher efficiency in hydrolysis of filter paper.

Claims

1. A class of endocellulase mutants characterized in that The mutant is a mutation of lysine at the 91st position, alanine at the 198th position, valine at the 240th position, and glutamine at the 237th position of the amino acid sequence of the endocellulase shown in SEQ ID NO: 1 to isoleucine, threonine, proline, and alanine, asparagine, threonine, serine, glycine or aspartic acid.

2. A gene encoding the endocellulase mutant according to claim 1.

3. A recombinant vector comprising the gene according to claim 2.

4. The method of claim 3, wherein the recombinant vector is prepared by the steps of: The endocellulase mutant gene according to claim 2 is prepared by artificial synthesis or gene cloning, an expression vector is constructed to obtain a recombinant plasmid, and the recombinant plasmid is transformed into a host cell to obtain the recombinant vector.

5. Use of the endocellulase mutant according to claim 1 in cellulose biodegradation.

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