A cellulase mutant and its preparation method and application

Abstract

The present invention relates to a cellulase mutant and a preparation method and application thereof, belonging to the field of enzyme engineering technology. The present invention provides a cellulase mutant, whose amino acid sequence is SEQ ID NO.5, and the nucleotide sequence of the encoding gene is SEQ ID NO.6; compared with the wild-type cellulase EccslH, the cellulase mutant provided by the present invention has an optimum temperature increased from 55°C to 65°C, a significantly improved heat resistance, an optimum pH value unchanged, both of which are 8.0, and a stable alkali resistance. The cellulase mutant provided by the present invention broadens the application scope of cellulase, changes its application conditions, and has broad application prospects in the fields of pulping, papermaking, textiles, feed, etc.

Description

Technical Field

[0001] The invention relates to a cellulase mutant and a preparation method and application thereof, belonging to the technical field of enzyme engineering. Background Art

[0002] Cellulose is a type of organic compound with the general chemical formula (C6H 10 O5)n, is a linear polysaccharide composed of hundreds to thousands of D-glucose units connected by β-1,4-glycosidic bonds. It is the most abundant organic polymer on earth, the most widely distributed and abundant polysaccharide in nature, and the main component of plant cell walls. Cellulase is a general term for a class of enzymes that degrade cellulose to produce glucose. It is not a single-component enzyme, but a multi-component enzyme system that works synergistically. It is a complex enzyme, mainly composed of exo-β-glucanase, endo-β-glucanase and β-glucosidase, etc. It has a wide range of sources, but the tolerance temperature of most cellulases is between 35℃-50℃, and acidic enzymes and neutral enzymes are the main ones.

[0003] The three main structures of cellulase include the binding domain CBM, the catalytic domain CD and the bridge LINGKER connecting the two. With the development of protein engineering and genetic engineering, the analysis of protein structure has become more and more accurate and intuitive, and the method of genetic engineering to transform cellulase has become more and more mature.

[0004] For example, Chinese invention patent CN 110373402 A (application number: 201910743692.1) discloses a method for improving the thermal stability of heat-resistant cellulase, by performing site-directed mutagenesis on the conserved N-glycosylation sequence N88-E89-T90 in the heat-resistant cellulase and the conserved non-catalytic amino acid Y173 in the substrate binding region, the threonine at position 90 is mutated to alanine, and the tyrosine at position 173 is mutated to phenylalanine. The enzyme activity of the mutated heat-resistant cellulase mutant enzyme is greatly improved, and the thermal stability under high temperature conditions is significantly improved.

[0005] For example, Chinese patent document CN 104450653 A (application number: 201410783788.8) provides a cellulase mutant, wherein the amino acid sequence of the cellulase is SEQ ID NO: 1, and the 10th amino acid is changed from Cys to Trp, the 29th amino acid is changed from Ala to Arg, the 61st amino acid is changed from Thr to Ser, the 141st amino acid is changed from Asp to Lys, and the 216th amino acid is changed from Val to His. The optimal action temperature of the cellulase mutant is 65°C, and it can maintain 96% of the enzyme activity at 70°C, and it can still maintain more than 48% of the enzyme activity at 80°C; while the optimal action temperature of the wild type is 60°C, and it can only maintain 51% of the enzyme activity at 70°C, and only 11% of the enzyme activity at 80°C; thus, it is shown that the heat resistance of the cellulase mutant is significantly higher than that of the wild type. The optimum action pH of the cellulase mutant is 5.5, and the enzyme activity level can be maintained at more than 60% within the pH range of 4.5-8.0.

[0006] For example, Chinese patent document CN 105039288 A (application number: 201510560739.2) provides a cellulase mutant, wherein the amino acid sequence of the cellulase of SEQ ID NO: 1 is changed from Pro to Ser, the amino acid sequence of the 38th amino acid is changed from Leu to Ile, and the amino acid sequence of positions 213-284 is replaced with the amino acid sequence of positions 447-514 of the cellulase from Trichoderma reesei. The cellulase mutant has an optimal action temperature of 60°C, an optimal action pH of 6.5, and can maintain an enzyme activity level of more than 80% in the pH range of 6.0-7.0, while the optimal action pH of the wild-type cellulase is 5.5, and maintains an enzyme activity of more than 80% in the pH range of 5.0-6.5. Compared with the wild-type, the pH application range of the cellulase mutant is more biased towards neutral conditions. Summary of the invention

[0007] In view of the deficiencies in the prior art, the present invention provides a cellulase mutant and a preparation method and application thereof.

[0008] The cellulase mutant of the present invention replaces the 21st to 25th amino acid sequence QNGEP of the N-terminus of the wild-type cellulase EccslH with RDGKA. The optimum temperature of the modified cellulase mutant of the present invention is significantly improved, and the optimum pH value is not affected.

[0009] The technical solution of the present invention is as follows:

[0010] A cellulase mutant, whose amino acid sequence is shown as SEQ ID NO.5.

[0011] The nucleotide sequence of the gene encoding the above cellulase mutant is shown in SEQ ID NO.6.

[0012] A recombinant plasmid containing the gene encoding the cellulase mutant.

[0013] Preferably according to the present invention, the vector of the recombinant plasmid is pET-28a(+).

[0014] A recombinant bacterium containing the above-mentioned cellulase mutant encoding gene or the above-mentioned recombinant plasmid.

[0015] Preferably according to the present invention, the host of the recombinant bacteria is Escherichia coli BL21 (DE3).

[0016] The application of the above recombinant bacteria in preparing cellulase mutants.

[0017] A preferred technical solution of the present invention is a method for preparing the above-mentioned cellulase mutant, comprising the following steps:

[0018] (1) Using plasmid pET-28a(+)-Eccs1H as a template, inverse PCR amplification was performed, and the inverse PCR product was digested and purified by DpnI enzyme to obtain a recombinant plasmid containing the cellulase mutant gene;

[0019] (2) transforming the recombinant plasmid containing the cellulase mutant gene obtained in step (1) into competent Escherichia coli BL21 (DE3) cells to obtain transformants, and screening positive clones to obtain recombinant Escherichia coli engineered bacteria;

[0020] (3) The recombinant Escherichia coli engineered bacteria obtained in step (2) are inoculated into LB liquid culture medium, and cellulase mutants are obtained after inducing expression.

[0021] Preferably according to the present invention, the nucleotide sequence of EccslH in step (1) is as shown in SEQ ID NO.2.

[0022] Preferably, according to the present invention, the primer sequences for the reverse PCR amplification in step (1) are as follows:

[0023] F1:5'-CGAGACGGAAAGGCAGTCCAGCTTAAGG-3' (SEQ ID NO.9);

[0024] R1: 5'-TGCCTTTCCGTCTCGGTTAACCAATTGG-3' (SEQ ID NO. 10).

[0025] The reaction system of the reverse PCR amplification is as follows, the total system is 50 μL: 2×HiFi-PCR master 25 μL, 10 μmol / L F12 μL, 10 μmol / L R12 μL, template 2 μL, dd H2O19 μL;

[0026] The procedure of the inverse PCR amplification is as follows:

[0027] Pre-denaturation at 95°C for 5 min; denaturation at 94°C for 30 sec, annealing at 55°C for 30 sec, extension at 72°C for 30 sec, 30 cycles; extension at 72°C for 10 min, and storage at 4°C.

[0028] Preferably, according to the present invention, the system for the DpnI enzyme digestion and purification in step (1) is as follows, with a total system of 50 μL: 1 μL of DpnI enzyme, 5 μL of 5×CE II Buffer, and 44 μL of inverse PCR product;

[0029] The procedure of DpnI enzyme digestion and purification is as follows:

[0030] React at 37°C for 120 min, 75°C for 15 min, and store at 4°C.

[0031] Preferably, according to the present invention, the method for screening positive clones in step (2) is to culture the transformants at 37°C, 200r / min for 1h, then spread the transformed cells on LB solid culture medium containing 50μg / mL kanamycin, culture at 37°C overnight, pick a single colony and inoculate it into LB liquid culture medium containing 50μg / mL kanamycin, culture at 37°C until the bacterial solution becomes turbid, and then verify by reverse PCR to obtain positive clones with target gene bands, and then sequence them. The strain with correct sequencing results is the recombinant Escherichia coli engineered bacteria.

[0032] Application of the above cellulase mutants in cellulose degradation.

[0033] The experimental steps not described in detail in the present invention are all carried out according to conventional operations in the art.

[0034] Beneficial effects:

[0035] The present invention provides a cellulase mutant, whose amino acid sequence is SEQ ID NO.5, and the nucleotide sequence of the encoding gene is SEQ ID NO.6; compared with the wild-type cellulase EccslH, the cellulase mutant provided by the present invention has an optimum temperature increased from 55°C to 65°C, a significantly improved heat resistance, an unchanged optimum pH value of 8.0, and a stable alkali resistance. The cellulase mutant provided by the present invention broadens the application scope of cellulase, changes its application conditions, and has broad application prospects in the fields of pulping, papermaking, textiles, feed, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 It is a line graph of the relative enzyme activities of the cellulase mutant EccslHt2 and the wild-type cellulase EccslH under different pH conditions.

[0037] Figure 2 It is a line graph of the relative enzyme activities of the cellulase mutant EccslHt2 and the wild-type cellulase EccslH under different temperature conditions.

[0038] Figure 3 It is a line graph of the relative enzyme activities of the cellulase mutant EccslHt1 and the wild-type cellulase EccslH under different pH conditions.

[0039] Figure 4 It is a line graph of the relative enzyme activities of the cellulase mutant EccslHt1 and the wild-type cellulase EccslH under different temperature conditions. DETAILED DESCRIPTION

[0040] The technical solution of the present invention is further described below in conjunction with embodiments, but the protection scope of the present invention is not limited thereto.

[0041] All matters not described in detail in the embodiments are based on the prior art in the art.

[0042] In the examples, the amino acid sequence of the wild-type cellulase Eccs1H is shown in SEQ ID NO.1, the nucleotide sequence of its encoding gene is shown in SEQ ID NO.2, and the wild-type cellulase Eccs1H is derived from the strain Bacillus sp. KSM-N252.

[0043] The amino acid sequence used for replacement is derived from the cellulase BsCel5A. The amino acid sequence of BsCel5A is shown in SEQ ID NO.3, and the nucleotide sequence of its encoding gene is shown in SEQ ID NO.4. BsCel5A is derived from Bacillus subtilis 168.

[0044] Example 1: Construction of a recombinant plasmid containing the cellulase mutant Eccs1Ht2 gene

[0045] The cellulase mutant EccslHt2 is obtained by replacing the amino acid sequence QNGEP at the 21st to 25th position of the N-terminus of the wild-type cellulase EccslH with RDGKA, wherein the replaced RDGKA is the amino acid sequence at the 47th to 51st position of the N-terminus of the cellulase BsCel5A. The gene sequence of the cellulase mutant EccslHt2 is constructed by reverse PCR, and the specific operation is as follows:

[0046] (i) using the recombinant plasmid pET-28a(+)-Eccs1H (synthesized by GenScript) containing the wild-type cellulase Eccs1H encoding gene as a template, performing inverse PCR amplification to replace the nucleotide fragment at the mutation site;

[0047] The primer sequences for the reverse PCR amplification are as follows:

[0048] F1:5'-CGAGACGGAAAGGCAGTCCAGCTTAAGG-3' (SEQ ID NO.9);

[0049] R1: 5'-TGCCTTTCCGTCTCGGTTAACCAATTGG-3' (SEQ ID NO. 10).

[0050] The reaction system of the reverse PCR amplification is shown in Table 1:

[0051] Table 1. Reaction system for inverse PCR amplification

[0052]

[0053] The procedure of the inverse PCR amplification is as follows:

[0054] Pre-denaturation at 95°C for 5 min; denaturation at 94°C for 30 sec, annealing at 55°C for 30 sec, extension at 72°C for 30 sec, 30 cycles; extension at 72°C for 10 min, storage at 4°C;

[0055] The inverse PCR product was tested by agarose gel electrophoresis, and the length was about 7300 bp. The product was recovered by gel recovery using SanPrep column DNA gel recovery kit (Shanghai Bioengineering Co., Ltd.), and the recovered product was stored at -20°C for future use.

[0056] (ii) purifying and digesting the gel recovery product obtained in step (i) to obtain a recombinant plasmid pET-28a(+)-EccslHt2 containing the cellulase mutant EccslHt2 gene, the nucleotide sequence of the cellulase mutant EccslHt2 gene is shown in SEQ ID NO.6, and the amino acid sequence encoded by the gene is shown in SEQ ID NO.5;

[0057] The digestion system is shown in Table 2:

[0058] Table 2. Digestion system of gum recovery products

[0059]

[0060] The digestion procedure is as follows:

[0061] React at 37°C for 120 min, 75°C for 15 min, and store at 4°C;

[0062] The digestion product was examined by agarose gel electrophoresis, and the length was about 7300 bp. The DNA was recovered by gel recovery using SanPrep column DNA gel recovery kit (Shanghai Bioengineering). The recovered product was stored at -20°C for future use.

[0063] Example 2: Preparation of competent E. coli

[0064] (i) Pick a single colony of Escherichia coli BL21 (DE3) and inoculate it into LB liquid medium, and culture it at 200 r / min and 37°C overnight;

[0065] (ii) Pipette 0.1 mL of the cultured bacterial solution into 10 mL of LB liquid medium and culture at 300 rpm and 37°C until OD 600 Reach 0.6~0.8;

[0066] (iii) Pipette 1 mL OD 600 The bacterial solution with a volume of 0.6-0.8 was transferred into a 1.5 mL sterile centrifuge tube, and centrifuged at 12000 rpm for 2 min to completely remove the supernatant.

[0067] (iv) Add 100 μL of ice-cold SSCS (one-step rapid preparation of competent cells kit, Shanghai Sangon Biotechnology Co., Ltd.) and gently suspend the cells to prepare competent cells;

[0068] (v) Aliquot the prepared competent cells into 100 μL tubes and store at -80°C for later use.

[0069] Example 3: Preparation of recombinant Escherichia coli BL21 (DE3)

[0070] First, the concentration of plasmid pET-28a(+)-EccslHt2 was determined using a nucleic acid ultra-micro spectrophotometer. When the concentration reached 300 μg / mL, it was transformed into the Escherichia coli BL21 (DE3) competent cells prepared in Example 2 by chemical transformation. The obtained cells were resuscitated and cultured at 37°C for 1 h using a recovery medium, and 100 μL was spread on LB solid culture medium containing 50 μg / mL kanamycin and cultured overnight at 37°C to screen for positive recombinant colonies with kanamycin resistance.

[0071] The components of the recovery medium per liter are as follows:

[0072] Peptone 10g, yeast powder 5g, sodium chloride 10g, sorbitol 91g, mannitol 69.4g, and the balance water.

[0073] Cultivation and identification of positive recombinant bacteria:

[0074] The above positive recombinant colonies were picked and inoculated into LB liquid medium containing 50 μg / mL kanamycin resistance, and cultured at 37°C overnight. After the culture was completed, the genome of the recombinant bacteria was extracted using a kit provided by Shanghai Bioengineering Co., Ltd., and the obtained genome was used as a template, and F1 and R2 were used as primers for reverse PCR amplification. The amplified products were verified by agarose gel electrophoresis;

[0075] The primer sequences for the reverse PCR amplification are as follows:

[0076] F1:5'-CGAGACGGAAAGGCAGTCCAGCTTAAGG-3' (SEQ ID NO.9);

[0077] R1: 5'-TGCCTTTCCGTCTCGGTTAACCAATTGG-3' (SEQ ID NO. 10).

[0078] The amplification system of the reverse PCR amplification is 20 μL, see Table 3:

[0079] Table 3. Amplification system of inverse PCR

[0080]

[0081] The reaction procedure of the inverse PCR amplification is as follows:

[0082] Pre-denaturation at 95°C for 5 min; denaturation at 94°C for 30 sec, annealing at 57°C for 30 sec, extension at 72°C for 4 min, 30 cycles; extension at 72°C for 10 min, storage at 4°C;

[0083] The reverse PCR product was tested by agarose gel electrophoresis. The results showed that a specific gene band with a size of about 7300bp could be amplified using primers F1 and R1, which was close to the theoretical value of 7306bp. After correct sequencing, it was shown that the recombinant plasmid pET-28a(+)-EccslHt2 containing the cellulase mutant EccslHt2 gene had been successfully transferred into Escherichia coli cells, and a recombinant Escherichia coli engineered bacterium containing the cellulase mutant EccslHt2 gene was obtained.

[0084] Example 4: Fermentation test of recombinant Escherichia coli engineering bacteria

[0085] The recombinant E. coli engineered bacteria prepared in Example 3 were inoculated into 100 mL LB liquid medium (10 g / L peptone, 5 g / L yeast extract, 10 g / L NaCl, and the balance water) and cultured at 200 rpm and 37°C until the fermentation liquid OD reached 0. 600 =0.7, IPTG was added to induce for 8 h, and samples were taken.

[0086] Refer to NY / T 912-2020 Determination of cellulase activity in feed additives. Determine the cellulase activity by spectrophotometry.

[0087] Determination of optimum pH: After sample treatment, the optimum pH of cellulase in the fermentation broth was detected by 3,5-dinitrosalicylic acid (DNS) method. After treatment at 55°C and pH 6.0-11.0 for 30 min, the reaction was stopped in a boiling water bath for 10 min. The absorbance value was measured at 540 nm by UV spectrophotometer, and the relative enzyme activity of cellulase was calculated. The results are shown in Figure 1 .

[0088] Determination of the optimum temperature: The optimum temperature of cellulase in the fermentation broth was detected by the 3,5-dinitrosalicylic acid (DNS) method. After treatment at pH 6.0 and temperature 40°C-70°C (gradient of 5°C) for 30 minutes, the reaction was stopped in a boiling water bath for 10 minutes. The absorbance value was measured at 540nm by an ultraviolet spectrophotometer, and the relative enzyme activity of cellulase was calculated. The results are shown in Figure 2 .

[0089] Depend on Figure 1 and Figure 2 It can be seen that compared with the wild-type cellulase EccslH (optimum temperature of 55°C, optimal pH of 8.0, and relatively stable at pH 6.0-9.0), the optimal temperature of the cellulase mutant EccslHt2 can reach 65°C, which is 10°C higher than that of the wild-type cellulase EccslH. It is relatively stable at pH 6.0-9.0, and the optimal pH value is pH 8.0; at 55°C and pH = 9, after treatment for 30 minutes, the specific activity of the cellulase mutant EccslHt2 is 50.02U / mg, and the specific activity of the wild-type cellulase EccslH is 60.5U / mg.

[0090] Comparative example: Construction of cellulase mutant EccslHt1 and verification of its enzymatic properties

[0091] Construction of recombinant plasmid containing cellulase mutant EccslHt1 gene:

[0092] The cellulase mutant EccslHt1 is obtained by replacing the amino acid sequence DNSVVGQ at the 1st to 7th positions of the N-terminus of the wild-type cellulase EccslH with TKTPVAK, wherein the replaced TKTPVAK is the amino acid sequence at the 27th to 33rd positions of the N-terminus of the cellulase BsCel5A. The gene sequence of the cellulase mutant EccslHt1 was constructed by inverse PCR, and the specific operation is as follows:

[0093] (i) using the recombinant plasmid pET-28a(+)-Eccs1H as a template, performing inverse PCR amplification to replace the nucleotide fragment at the mutation site;

[0094] The primer sequences for the reverse PCR amplification are as follows:

[0095] F1-1:5'-ACGAAGACACCTGTAGCCAAAAATGGTCAATTG-3' (SEQ ID NO. 11);

[0096] R1-1:5'-TTTTGGCTACAGGTGTCTTCGTGCTACCACCG-3' (SEQ ID NO. 12);

[0097] The reaction system of the reverse PCR amplification is shown in Table 4:

[0098] Table 4. Reaction system for inverse PCR amplification

[0099]

[0100] The reaction procedure of the inverse PCR amplification is as follows:

[0101] Pre-denaturation at 95°C for 5 min; denaturation at 94°C for 30 sec, annealing at 55°C for 30 sec, extension at 72°C for 30 sec, 30 cycles; extension at 72°C for 10 min, storage at 4°C;

[0102] The inverse PCR product was tested by agarose gel electrophoresis, and the length was about 7300 bp. The product was recovered by gel recovery using SanPrep column DNA gel recovery kit (Shanghai Bioengineering Co., Ltd.), and the recovered product was stored at -20°C for future use.

[0103] The above-mentioned gum recovery product was purified and digested according to the method described in Example 1 to obtain the recombinant plasmid pET-28a(+)-EccslHt1 containing the cellulase mutant EccslHt1 gene. The nucleotide sequence of the cellulase mutant EccslHt1 gene is shown in SEQ ID NO.8, and the amino acid sequence encoded by it is shown in SEQ ID NO.7.

[0104] Referring to the method of Example 3, a recombinant E. coli engineered bacteria containing the cellulase mutant EccslHt1 gene was prepared. The recombinant E. coli engineered bacteria were inoculated into 100 mL LB liquid medium (10 g / L peptone, 5 g / L yeast extract, 10 g / L NaCl, and the balance water) and cultured at 200 rpm and 37°C until the fermentation liquid OD reached 0. 600 =0.7, IPTG was added to induce for 8 h, and samples were taken.

[0105] Refer to NY / T 912-2020 Determination of cellulase activity in feed additives. Determine the cellulase activity by spectrophotometry.

[0106] Determination of optimum pH: After sample treatment, the optimum pH of cellulase in the fermentation broth was detected by 3,5-dinitrosalicylic acid (DNS) method. After treatment at 55°C and pH 6.0-11.0 for 30 min, the reaction was stopped in boiling water bath for 5 min. The absorbance value was measured at 540 nm by UV spectrophotometer, and the relative enzyme activity of cellulase was calculated. The results are shown in Figure 3 .

[0107] Determination of the optimum temperature: The optimum temperature of cellulase in the fermentation broth was detected by the 3,5-dinitrosalicylic acid (DNS) method. After treatment at pH 6.0 and temperature 55°C-90°C (gradient of 5°C) for 30 minutes, the reaction was stopped in a boiling water bath for 5 minutes. The absorbance value at 540nm was measured by an ultraviolet spectrophotometer to calculate the relative enzyme activity of cellulase. The results are shown in Figure 4 .

[0108] Depend on Figure 3 and Figure 4 It can be seen that compared with the wild-type cellulase EccslH (optimum temperature of 55°C, optimum pH of 8.0, relatively stable at pH 6.0-9.0), the optimum temperature of the cellulase mutant EccslHt1 is still 55°C, which is unchanged compared with the wild-type cellulase EccslH, and the optimum pH is 8.0, which is relatively stable at pH 6.0-9.0. Under the conditions of 55°C and pH = 9, the specific activity of the cellulase mutant EccslHt1 is 35.6U / mg, and the specific activity of the wild-type cellulase EccslH is 60.5U / mg after treatment for 30 minutes.

[0109] In summary, the cellulase mutants EccslHt2 and EccslHt1 respectively selected the sequences of 5 amino acids and 7 amino acids in the N-terminus of the wild-type cellulase EccslH for replacement, and the sequences used for replacement were derived from the cellulase BsCel5A, where the sequence used to replace 5 amino acids was RDGKA, and the sequence used to replace 7 amino acids was TKTPVAK. Both RDGKA and TKTPVAK sequences belong to the front row of the N-terminus in the cellulase BsCel5A, and from the secondary structure, they are irregular curls, avoiding the substrate binding site, and have poor similarity with EccslH, and have mutation potential.

[0110] The enzymatic properties of the cellulase mutants and wild-type cellulase constructed above are summarized in the following table:

[0111] Table 5. Enzymatic properties of wild-type cellulase and cellulase mutants

[0112] Cellulase Optimum temperature (℃) Optimum pH Enzyme activity (U / mL), 65℃, pH 6.0 Number of replaced amino acids E 55℃ 8.0 19.2 - EccslHt 55℃ 8.0 17.8 7 EccslHt2 65℃ 8.0 25.1 5

[0113] As shown in Table 5, although both cellulase mutants EccslHt1 and EccslHt2 mutated the N-terminal sequence of the wild-type cellulase EccslH, and the amino acid sequence used to replace them was derived from the same cellulase BsCel5A, the mutation sites and sequences of the two were different, resulting in differences in enzymatic properties between the cellulase mutants EccslHt1 and EccslHt2, especially the difference in the optimum temperature. Compared with the wild-type cellulase EccslH, the optimum temperature of the cellulase mutant EccslHt1 did not change, but the optimum temperature of the cellulase mutant EccslHt2 was 65°C, which was higher than that of the wild-type cellulase EccslH and the cellulase mutant EccslHt1. In addition, the enzyme activity of the cellulase mutant EccslHt2 was also improved compared with that of the wild-type cellulase EccslH, while the enzyme activity of the cellulase mutant EccslHt1 was lower than that of the wild-type cellulase EccslH.

Claims

1. A cellulase mutant, characterized in that: The amino acid sequence is shown in SEQ ID NO.

5.

2. The cellulase mutant encoding gene according to claim 1, characterized in that The nucleotide sequence is shown as SEQ ID NO.

6.

3. A recombinant plasmid containing the gene encoding the cellulase mutant according to claim 2.

4. The recombinant plasmid according to claim 3, characterized in that The vector of the recombinant plasmid is pET-28a(+).

5. A recombinant bacterium containing the cellulase mutant encoding gene according to claim 2 or the recombinant plasmid according to claim 3.

6. The recombinant bacterium according to claim 5, characterized in that The host of the cellulase mutant encoding gene according to claim 2 or the recombinant plasmid according to claim 3 is Escherichia coli BL21 (DE3).

7. Use of the recombinant bacteria according to claim 5 in preparing cellulase mutants.

8. The method for preparing a cellulase mutant according to claim 1, characterized in that: The steps include: (1) The recombinant plasmid pET-28a(+)- E As a template, the nucleotide sequence of the wild-type cellulase Eccs1H encoding gene is shown in SEQ ID NO.2, and reverse PCR amplification is performed. The reverse PCR product is digested with DpnI enzyme and then purified to obtain a recombinant plasmid containing the cellulase mutant gene; (2) transforming the recombinant plasmid containing the cellulase mutant gene obtained in step (1) into competent Escherichia coli BL21 (DE3) cells to obtain transformants, and screening positive clones to obtain recombinant Escherichia coli engineered bacteria; (3) The recombinant Escherichia coli obtained in step (2) is inoculated into LB liquid culture medium, and cellulase mutants are obtained after induction of expression.

9. The preparation method according to claim 8, characterized in that: Meet one or more of the following conditions: i. The primer sequences for the reverse PCR amplification in step (1) are as follows: F1: 5'-CGAGACGGAAAGGCAGTCCAGCTTAAGG-3' (SEQ ID NO. 9); R1: 5'-TGCCTTTCCGTCTCGGTTAACCAATTGG-3' (SEQ ID NO. 10); The reaction system of the inverse PCR amplification is as follows, the total system is 50 μL: 2×HiFi-PCR master 25 μL, 10 μmol / L F1 primer 2 μL, 10 μmol / L R1 primer 2 μL, template 2 μL, dd H2O 19 μL; The procedure of the inverse PCR amplification is as follows: Pre-denaturation at 95°C for 5 min; denaturation at 94°C for 30 sec, annealing at 55°C for 30 sec, extension at 72°C for 30 sec, 30 cycles; extension at 72°C for 10 min, storage at 4°C; ii. The DpnI enzyme digestion system in step (1) is as follows, with a total system of 50 μL: 1 μL DpnI enzyme, 5 μL 5 × CE II Buffer, and 44 μL inverse PCR product; The procedure of the DpnI enzyme digestion is as follows: React at 37°C for 120 min, 75°C for 15 min, and store at 4°C; iii. The method for screening positive clones in step (2) is to culture the transformants at 37°C, 200 r / min for 1 h, then spread the transformed cells on LB solid culture medium containing 50 μg / mL kanamycin, and culture them at 37°C overnight. Pick a single colony and inoculate it into LB liquid culture medium containing 50 μg / mL kanamycin, and culture it at 37°C until the bacterial solution becomes turbid. Then verify it by reverse PCR to obtain positive clones with the target gene band, and then sequence them. The strain with the correct sequencing result is the recombinant Escherichia coli engineered bacteria.

10. Use of the cellulase mutant according to claim 1 in cellulose degradation.

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