CN117187218A

CN117187218A - Low temperature resistant cellulase mutants

Info

Publication number: CN117187218A
Application number: CN202311344960.5A
Authority: CN
Inventors: 刘艳萍; 吴秀秀; 黄亦钧
Original assignee: Qingdao Vland Biotech Group Co Ltd
Current assignee: Qingdao Vland Biotech Group Co Ltd
Priority date: 2019-11-13
Filing date: 2019-11-13
Publication date: 2023-12-08
Also published as: CN112795554B; CN112795554A

Abstract

The invention relates to the technical field of genetic engineering and protein transformation, in particular to a low-temperature-resistant cellulase mutant and application thereof. The mutant provided by the invention comprises amino acid substitution at least one position selected from the group consisting of: 120, 130, 179. The relative enzyme activities of the mutant are obviously improved at the low temperature of 40 ℃, so that the mutant is favorable for the wide application of the cellulase in the textile field.

Description

Low temperature resistant cellulase mutants

Technical Field

The invention relates to the technical field of genetic engineering and protein transformation, in particular to a low-temperature-resistant cellulase mutant and application thereof.

Background

Cellulases are a generic term for a group of enzymes that catalyze the hydrolysis of degraded cellulose to glucose and low degree of polymerization fibers, including 3 major components of endoglucanases, exoglucanases and cellobiases. Cellulases are not single enzymes, but rather are a synergistic multicomponent enzyme system. Cellulases are widely found in organisms in nature and are produced by bacteria, fungi, animals, etc. Cellulases generally used in industrial production are derived from fungi, more typically Trichoderma, aspergillus and Penicillium. Cellulase is one of the most widely used enzymes in industry, can be widely applied to the industrial fields of textile, detergent, pulp and paper, feed and food, and has huge potential markets in the aspects of oil extraction, medicine, and the like.

Cellulases can be classified according to their primary sequence into various glycosyl hydrolase families, for example glycosyl hydrolase families 5, 7, 12 and 45 contain endoglucanases. Most textile acid cellulases belong to family 5, while most textile neutral cellulases belong to family 12 or 45.

At present, in the textile industry, cellulose fabrics are subjected to biological finishing, namely enzymatic degradation finishing, by using cellulase, and the finished fabrics are fluffy, plump, soft, smooth, clear in cloth cover, good in drapability and strong in hygroscopicity, and have a certain mercerizing effect. The neutral cellulase has mild corrosion effect on the fabric, little strong loss of the fabric and little staining, can obtain more plump hand feeling after treatment, can achieve satisfactory finishing effect when the dosage of the cellulase is 0.5-3%, and has the advantages of environmental protection, energy conservation and high efficiency. However, most industrial cellulases generally have higher catalytic efficiency at a temperature higher than 50 ℃, and in the textile field, in order to save heating or cooling cost, and further to improve the color fastness of fabrics and reduce shrinkage of clothes, low-temperature conditions of 30-40 ℃ are generally adopted for treatment. Thus, there is an urgent need to develop cellulases that still have higher levels of enzymatic activity at low temperature levels.

Disclosure of Invention

The invention aims to provide a low-temperature cellulase mutant and application thereof. The invention obtains mutant protein by protein engineering of cellulase. Compared with the wild type, the mutant can keep higher enzyme activity under the low temperature condition, thereby ensuring the dehairing and washing effects and being more suitable for treating textiles under the low temperature condition.

In order to achieve the above object, the present invention provides the following technical solutions:

the present invention relates to a cellulase mutant comprising an amino acid sequence having at least 90% identity to SEQ ID NO. 1 and having an amino acid substitution at least one position of 120, 130, 179 compared to SEQ ID NO. 1.

In some embodiments of the invention, the amino acid sequence of the mutant has at least 91%,92%,93%,94%,95%,96%,97%,98%, or at least 99% identity as compared to SEQ ID NO. 1.

In some more specific embodiments, the amino acid sequence of the mutant has at least 99.1%,99.2%,99.3%,99.4%,99.5%,99.6%,99.7%,99.8%, or at least 99.9% identity compared to SEQ ID No. 1.

In some embodiments of the invention, the mutant comprises a substitution of at least one amino acid in the group consisting of: H120Q, V130F/L, D179S.

In some embodiments of the invention, the mutant comprises the following substitutions or combinations of substitutions: H120Q, V130F, V L, D179S, H Q/V130F, H120Q/V130L, H Q/D179S, V F/D179S, V F/D179S, H Q/V130F/D179S or H120Q/V130L/D179S.

The invention also relates to a DNA molecule for encoding the cellulase mutant.

The invention also relates to a recombinant expression vector comprising the DNA molecule.

The invention also relates to a host cell comprising the recombinant expression vector.

In some embodiments of the invention, the host cell is Trichoderma reeseiTrichoderma reesei）。

The recombinant expression vector is transferred into Trichoderma reesei host cells for recombinant expression, and the obtained cellulase mutant has higher enzyme activity under the low-temperature condition.

The invention also relates to application of the cellulase mutant in the textile field.

The cellulase mutant provided by the invention has higher tolerance and enzyme activity under the low-temperature condition. Compared with the wild type, the relative enzyme activity of the cellulase mutants respectively containing the single-point mutation of H120Q, V130F, V L, D179S is generally improved by 10.0-56.7 percent under the condition of 40 ℃. Wherein, the relative enzyme activity of the V130L single-point mutant is up to 94% at 40 ℃, and unexpected technical effects are obtained.

In addition, the cellulase mutant containing any 2 or 3 mutation sites in H120Q, V130F/L and D179S provided by the invention, such as H120Q/V130F, H Q/V130L, H Q/D179S, V F/D179D S, V F/179S two-point mutant, H120Q/V130F/D179S, H Q/V130L/D179S three-point mutant, has a relative enzyme activity which is generally improved by 10.0% -78.2% compared with wild-type cellulase NT45 at 40 ℃. Therefore, compared with the wild type, the cellulase mutant is more suitable for being applied to the field of textile industry, can greatly reduce the consumption of cellulase, saves labor hour and energy, and reduces the production cost.

Detailed Description

The present invention uses conventional techniques and methods used in the fields of genetic engineering and molecular biology, such as those described in MOLECULAR CLONING: A LABORATORY MANUAL,3nd Ed (Sambrook, 2001) and CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (Ausubel, 2003). These general references provide definitions and methods known to those skilled in the art. The invention is not limited to any particular method, protocol, or reagents described.

The present invention will be described in detail with reference to the following embodiments.

EXAMPLE 1 screening of cellulase mutants

In order to improve the enzyme activity of wild-type cellulase NT45 (the amino acid sequence is SEQ ID NO:1, the encoding nucleotide sequence is SEQ ID NO: 2) under low temperature conditions, the applicant has carried out a large number of mutation screening on amino acids near the active site of the enzyme by directed evolution technology.

PCR primers NtE-F1 and NtE-R1 were designed as follows:

NtE-F1：GGCGAATTCATGCGCTCCT CCACCATTC (restriction enzyme underlined)EcoRI recognition sites);

NtE-R1：ATAGCGGCCGCTTAGGCGCACTGGTGGTAGTAGTC (lower line is limited)Preparation of endonucleasesNotI recognition site).

Using wild cellulase NT45 gene (SEQ ID NO: 2) as a template, using the primers to carry out PCR amplification by using a GeneMorph II random mutation PCR kit (Stratagene), recovering PCR products by gel,EcoRI、Notand I, connecting the obtained product with a pET21a carrier subjected to enzyme digestion, converting the obtained product into escherichia coli BL21 (DE 3), coating the obtained product on an LB+Amp plate, inversely culturing at 37 ℃, picking the obtained product to 96-well plates one by using toothpicks after the transformant appears, adding 150 mu L of LB+Amp culture medium containing 0.1 mM IPTG into each well, culturing at 220rpm at 37 ℃ for about 6h, centrifuging, discarding the supernatant, resuspending the thallus with buffer solution, and repeatedly freezing and thawing to break walls to obtain escherichia coli cell lysate containing cellulase.

50 μl of the lysate was removed to two new 96-well plates, and the cellulase enzyme activities were measured at 40℃and 50℃respectively, and the relative enzyme activities at 40℃were calculated based on 100% of the enzyme activities at 50 ℃. As a result, it was found that some mutants had no change in relative enzyme activity at 40℃as compared with wild-type cellulase NT45, some mutants had even decreased relative enzyme activity, and some mutants had increased relative enzyme activity at 40℃but the enzymatic properties of the mutated cellulases had significantly changed, which was not satisfactory. Finally, the applicant obtained mutation sites which can significantly improve the relative enzyme activity of the cellulase at 40 ℃ and do not affect the original enzymatic properties: H120Q, V130F, V130L, D179S.

On the basis of the cellulase NT45, the invention provides cellulase mutants containing single-point mutations of H120Q, V130F, V L, D179S respectively, and also provides cellulase mutants containing any 2 or 3 mutation sites in H120Q, V F/L, D179S in combination, such as H120Q/V130F, H Q/V130L, H Q/D179S, V F/D179S, V F/D179S two-point mutants, H120Q/V130F/D179S, H Q/V130L/D179S three-point mutants,

EXAMPLE 2 expression of cellulase mutants in Trichoderma reesei

2.1 Gene Synthesis and plasmid construction

Applicants have identified the nucleotide sequence SEQ ID NO:2 and the mutant described in example 1, and two restriction sites KpnI and XbaI are added to the 5 'and 3' ends of the synthetic sequence, respectively. The above-mentioned gene synthesis work was performed by Shanghai Biotechnology Co., ltd.

The synthesized plasmid is respectively subjected to enzyme digestion by restriction enzymes KpnI (Fermentas) and XbaI; simultaneously, restriction enzymes KpnI (Fermentas) and XbaI are used for carrying out enzyme digestion on plasmid pTGII; purifying the cleavage products by using a gel purification kit, and connecting the two cleavage products by using T4 DNA ligase (Fermentas); the ligation products were transformed into Trans5α E.coli (Transgen), selected with ampicillin, and several clones were sequenced (Invitrogen) to ensure accuracy. Sequencing results showed that the sequencing results were consistent for multiple clones.

Plasmid was purified from E.coli clones with correct sequencing results using the plasmid midvolume preparation kit (Axygen).

2.2 protoplast preparation

Taking a host bacterium Trichoderma reesei U4 spore suspension of cellulase gene defect type, inoculating on a PDA plate, and culturing at 30 DEG C

For 6 days; after the spore is produced in a rich way, cutting a colony with the length of about 1cm multiplied by 1cm, placing the colony in a liquid culture medium containing 120mL of YEG+U (0.5% yeast powder, 1% glucose and 0.1% uridine), and carrying out shaking culture at 30 ℃ and 220rpm for 14-16 h; filtering and collecting mycelium with sterile gauze, and cleaning with sterile water once; mycelium was placed in a triangular flask containing 20mL 10mg/mL of lyase solution (Sigma L1412), at 30℃and 90rpm for 1-2 h; protoplast transformation progression was examined by microscopic observation.

Precooling 20mL of 1.2M sorbitol (1.2M sorbitol, 50mM Tris-Cl,50mM CaCl) ₂ ) Adding into the above triangular flask, shaking gently, filtering with sterile Miracloth filter cloth, collecting filtrate, centrifuging at 3000rpm at 4deg.C for 10min; removing the supernatant, adding precooled 5mL 1.2M sorbitol solution to suspend the thalli, centrifuging at 3000rpm and 4 ℃ for 10min; removing supernatant, adding appropriate amount of precooled 1.2M sorbitol, suspending, and packaging (200 μl +.Tube with protoplast concentration of 10 ⁸ and/mL).

2.3 expression vector transformation and Strain verification

The following operations were all performed on ice, 10. Mu.g of recombinant plasmid was added to a sterile 7mL centrifuge tube containing 200. Mu.L of protoplast solution, followed by 50. Mu.L of 25% PEG (25% PEG,50mM Tris-Cl,50mM CaCl) ₂ ) Mixing the bottom of the flick pipe uniformly, and placing on ice for 20min; 2mL of 25% PEG is added, and the mixture is placed for 5min at room temperature after uniform mixing; 4mL of 1.2M sorbitol was added, gently mixed and poured into the upper medium (0.1% MgSO) which melted and maintained at 55 ℃ ₄ ，1％KH ₂ PO ₄ ，0.6％(NH4) ₂ SO ₄ 1% glucose, 18.3% sorbitol, 0.35% agarose); gently mixing and spreading on a prepared lower culture medium plate (2% glucose, 0.5% (NH) ₄ ) ₂ SO ₄ ，1.5％KH ₂ PO ₄ ，0.06％MgSO ₄ ，0.06％CaCl ₂ 1.5% agar), and culturing at 30℃for 5-7 d until transformants grow out. Picking the transformant to a lower layer culture medium plate, and culturing for 2d at 30 ℃; a proper amount of mycelium is taken and placed in a 2mL centrifuge tube, and 100mg of sterile quartz sand and 400 mu L of extraction buffer (100 mM Tris-HCl,100mM EDTA,250mM NaCl,1%SDS) are added; shaking vigorously with a bead beating instrument for 2min; after 20min of water bath at 65 ℃,200 mu L of 10M NH is added ₄ AC, ice bath for 10min; centrifuging at 13000rpm for 10min; taking the supernatant, adding 2 times of absolute ethyl alcohol, and standing at-20 ℃ for 30min; centrifuging at 13000rpm for 10min, and discarding supernatant; washing with 70% ethanol for 2 times; air drying, dissolving in water, and storing at-20deg.C.

PCR amplification verification is carried out by using the extracted genome DNA of the transformant as a template and using primers M6-F and M6-R.

M6-F：ATGCGCTCCT CCACCATTC；

M6-R：TTAGGCGCACTGGTGGTAGTAGTC。

PCR amplification conditions were 94℃for 4min;94 ℃ for 40s;58 ℃ 40s,72 ℃ 1min,30 cycles; 7min at 72℃and 16 ℃; and (3) recovering PCR amplification products by using a gel recovery kit and carrying out sequencing analysis to construct the Trichoderma reesei engineering bacteria containing the cellulase genes.

2.4 Fermentation verification

Inoculating Trichoderma reesei engineering bacteria to PDA plate, culturing at 30deg.C for 6d, collecting two mycelia with diameter of 1cm, inoculating into 250 mL triangular flask containing 50 mL fermentation medium (1.5% glucose, 1.7% lactose, 2.5% corn steep liquor, 0.44% (NH) ₄ ) ₂ SO ₄ ，0.09% MgSO ₄ ，2% KH ₂ PO ₄ ，0.04% CaCl ₂ 0.018% tween-80,0.018% trace elements), at 30 ℃ for 48 hours, then at 25 ℃ for 48 hours, and taking the fermentation broth for enzyme activity determination and enzyme property detection.

Example 3 enzyme Activity assay and enzymatic Property analysis

3.1 method for measuring enzyme activity of cellulase

The amount of enzyme required to degrade and release 1. Mu. Mol of reducing sugar per minute from a sodium hydroxymethyl cellulose solution with a concentration of 5 mg/ml at 50 ℃ and a pH value of 6.0 is one enzyme activity unit U, and the reducing sugar is glucose equivalent.

Three test tubes were each added with 0.5 mL CMC substrate and preheated in a water bath at 50℃for 5min with the enzyme solution to be tested. And adding 0.5. 0.5 mL to-be-detected liquid into each of the first test tube and the second test tube, timing, and reacting for 15 min in a water bath at 50 ℃. After the reaction was completed, 1.5 mL of DNS reagent was added to each of the three test tubes, and the third test tube was supplemented with 0.5. 0.5 mL of enzyme solution to be tested. After taking out and shaking three test tubes, the reaction was carried out in a boiling water bath for 5 min. Cool rapidly to room temperature and set with water to 5.0. 5.0 mL. The absorbance of the first test tube and the second test tube is preferably 0.25-0.35 under the condition of 540 and nm wavelength by taking the third test tube as a control. The absolute value of the difference between the absorbance of the enzyme liquid reaction liquid to be detected and the absorbance of the enzyme liquid reaction liquid is controlled to be not more than 0.015.

Enzyme activity x= (glucose equivalent/180/15/0.5) ×n

Wherein: x is enzyme activity unit, IU/g (mL);

180—glucose converted from micrograms to micromolar;

15-reaction time of the test solution with the substrate;

0.5-adding the amount of enzyme to be detected in the reaction;

n-dilution factor.

3.2 enzyme Activity assay

The enzyme activity detection is carried out according to the method, and the result shows that: the enzyme activity of the Trichoderma reesei fermentation supernatant of the recombinant expression wild cellulase NT45 and the mutant thereof obtained in the embodiment 2 of the invention is 50-200U/mL.

3.3 Analysis of low temperature resistance effect

The enzyme activities of the above recombinant expression wild-type cellulase NT45 and its mutants were measured at 40℃and 50℃and pH6.0, respectively, and the relative enzyme activities of the wild-type cellulase NT45 and its mutants at 40℃were calculated as 100% of the enzyme activities at 50℃respectively, and the results are shown in Table 1.

Relative enzyme activity = enzyme activity at 40 ℃ per enzyme activity at 50 ℃ x 100%.

TABLE 1 relative enzyme Activity of cellulases at 40℃

Cellulase enzymes	Relative enzyme Activity at 40 ℃
		Wild type NT45	60%
H120Q single point mutant	66%
		V130F single point mutant	88%
V130L single point mutant	94%
		d179S sheetPoint mutant	67%

As can be seen from the data in Table 1, compared with the wild type cellulase NT45, the single-point mutant provided by the invention has the general improvement of relative enzyme activity at 40 ℃ of 10.0% -56.7%, so that the single-point mutant provided by the invention has the remarkably improved enzyme activity level at 40 ℃ of low temperature. Wherein, the relative enzyme activity of the V130L single-point mutant is up to 94% at 40 ℃, and unexpected technical effects are obtained.

In addition, the cellulase mutant containing any 2 or 3 mutation sites in H120Q, V130F/L and D179S provided by the invention, such as H120Q/V130F, H Q/V130L, H120Q/D179S, V F/D179S, V F/D179S two-point mutant, H120Q/V130F/D179S, H Q/V130L/D179S three-point mutant, has the relative enzyme activity increased by 10.0% -78.2% compared with wild type cellulase NT45 at 40 ℃ generally, and has unexpected technical effects.

In conclusion, the cellulase mutant provided by the invention has higher enzyme activity under the low-temperature condition, is more suitable for application in textile industry, and has wide prospect.

EXAMPLE 4 use of cellulase mutants in the textile sector

4.1 dehairing and dyeing one bath process for knitted fabric and woven fabric

The application temperature is 35-55 ℃;

the treatment time is 30-150 min;

the pH range is 4.0-8.5;

the process conditions are particularly suitable for the condition of dyeing in the same bath; the applicable bath ratio is in the range of 1:5-1:30, the type of equipment used is overflow dyeing machine, jig dyeing machine, washing machine and the like, and the dosage of the cellulase mutant is 300-900U/L.

The cellulase mutant provided by the invention is clean in hair removal, has small strength loss on fabrics, and can realize the integrated one-bath of dyeing and hair removal processes.

4.2 Application of jean fabric in flower forming and hair removal

The application temperature is 35-55 ℃;

the treatment time is 10-60 min;

the pH range is 4.0-8.5;

the process conditions can be applied to the dehairing and flower forming process under the condition of desizing and independent stone milling; the applicable bath ratio is in the range of 1:5-1:30, the type of equipment used is industrial washing machine, and the like, and the dosage of the cellulase mutant is 300-900U/L.

The cellulase mutant provided by the invention has the advantages of clean hair removal, uniform flower formation, smaller flower points and small strength loss to fabrics.

The experimental result shows that the low-temperature cellulase mutant can be widely applied to the textile processing field, can be applied in the low-temperature condition of 35-55 ℃ and the pH range of 4.0-8.5, can be directly used without acid regulation, and has good effect; the hair is removed cleanly, and the strength loss of the fabric is small; the jean is washed with water, the flower is small, the flower point is small, and the batch difference is stable; the salt tolerance is good, and the polishing and dyeing one-bath process which can be used for neutralization and deoxidization can be directly used, so that the working hours can be greatly saved, and the production cost can be reduced.

And compared with wild cellulase NT45, the dosage of the cellulase mutant required for achieving the same treatment effect is reduced by 18.5-43.4%, so that the enzyme cost in the processing process is obviously reduced, and the production cost is further improved.

Claims

1. A cellulase mutant, wherein the mutant is a cellulase having the amino acid sequence of SEQ ID No. 1 comprising a substitution at amino acid 130: V130F or V130L.

2. The mutant of claim 1, wherein the mutant comprises a substitution or combination of substitutions selected from the group consisting of:

V130F；

V130L；

H120Q/V130F；

H120Q/V130L；

V130F/D179S；

H120Q/V130F/D179S；

H120Q/V130L/D179S。

3. a DNA molecule encoding the cellulase mutant of claim 1 or 2.

4. A vector having the DNA molecule of claim 3.

5. A host cell comprising the vector of claim 4.

6. The host cell of claim 5, wherein the host cell is Trichoderma reeseiTrichoderma reesei）。

7. Use of the cellulase mutants according to claim 1 or 2 in the textile field.