CN112662652A

CN112662652A - Alkaline protease mutant with reduced collagen degradation activity

Info

Publication number: CN112662652A
Application number: CN202110072841.3A
Authority: CN
Inventors: 李玉; 路福平; 刘逸寒; 李家霖; 杨子璇; 王兴吉; 李庆刚; 刘文龙
Original assignee: Shandong Lonct Enzymes Co ltd; Tianjin University of Science and Technology
Current assignee: Shandong Lonct Enzymes Co ltd; Tianjin University of Science and Technology
Priority date: 2021-01-20
Filing date: 2021-01-20
Publication date: 2021-04-16

Abstract

The invention mainly utilizes error-prone PCR technology to carry out directional transformation of the alkaline protease derived from Bacillus clausii, and obtains an alkaline protease with reduced collagen degradation activity, the amino acid sequence of which is shown in SEQ ID NO: 4. Under the same conditions, the alkaline protease activity and collagen degradation activity of the mutant of the present invention are 91.97% and 65.84% of the wild type, respectively, and the protease has high application value in the leather industry.

Description

Alkaline protease mutant with reduced collagen degradation activity

Technical Field

The invention belongs to the technical field of microorganisms and genetic engineering, and particularly relates to an alkaline protease mutant with reduced collagen degradation activity and application thereof.

Background

Protease is a hydrolase with a complex structure and function, and is capable of cleaving peptide bonds to produce short peptides or amino acids. Proteases can be classified into three types according to their optimum pH: the optimal pH value of the acidic protease is 2.0-5.0, and the acidic protease is mainly derived from fungi; neutral protease, with an optimum pH of 7.0, mainly from plant sources; alkaline protease, having an optimum pH of 8.0 or more, is mainly derived from microorganisms. Protease is one of the most important industrial enzyme preparations, and has been widely used in the fields of detergents, medicines, foods, leather, silk, photography, etc., because the sale amount of protease accounts for 60% or more of the total sale amount of all enzyme preparations.

In the leather industry, dehairing is the primary step in leather processing, i.e., the removal of hair, cuticles, non-collagenous proteins, and other binding substances from leather. Sodium sulfide is used in the traditional unhairing process, a large amount of sulfur-containing waste is generated, so that the serious pollution problem is caused, and in addition, a large amount of chemical materials are added in the preparation before tanning to cause serious environmental hidden trouble. Currently, the related applications of enzyme preparations instead of chemicals for clean production are receiving increasing attention in tanning. However, because the activity of the collagen degradation protease in the traditional protease is strong, the collagen is easy to be damaged in the application process, so that the phenomena of loose noodles, rotten noodles and the like occur, the economic benefit is greatly reduced, and the application limitation of the protease in the leather industry is caused. Therefore, the protease more suitable for leather processing is developed, so that the safety in the use process can be improved, and the application requirement and practical value are higher.

Disclosure of Invention

Aiming at the defects of the prior art, the invention mainly utilizes the error-prone PCR technology to directionally transform the alkaline protease from the Bacillus clausii, so as to obtain the alkaline protease with reduced collagen degradation activity, and the alkaline protease has higher application value in the leather industry.

One of the purposes of the invention is to provide an alkaline protease mutant, wherein the amino acid sequence of the alkaline protease mutant is shown as SEQ ID NO: 4, respectively.

Another object of the present invention is to provide a gene encoding the alkaline protease mutant.

In a specific embodiment of the invention, the nucleotide sequence of the gene is as shown in SEQ ID NO: 3, respectively.

It is a further object of the present invention to provide a vector comprising a gene of the alkaline protease mutant. The vector may be one of vectors for producing a protein by gene recombination, such as an expression vector, known to those skilled in the art.

In a specific embodiment of the invention, the vector is pWB 980.

In another aspect, the present invention provides a host cell comprising said encoding gene or vector. The host cell may be any host suitable for producing the alkaline protease mutant of the present invention from the gene or vector of the present invention, for example, Bacillus amyloliquefaciens.

In one embodiment of the present invention, the host cell is Bacillus amyloliquefaciens CGMCC No.11218 (biological depositary information disclosed in patent CN 105087448B).

In another embodiment of the invention, the host cell is Bacillus subtilis WB 600.

The fifth object of the present invention is to provide the use of the alkaline protease mutant of the present invention for hydrolyzing peptide bonds of proteins to produce polypeptides or amino acids, and which has reduced collagen degrading enzyme activity as compared with the wild type.

The sixth object of the present invention is to provide a method for preparing the alkaline protease mutant of the present invention by gene recombination and expression using the gene encoding the alkaline protease mutant of the present invention or the vector of the present invention. Genetic recombination methods and expression hosts known to those skilled in the art can be used, and media and culture conditions suitable for expression by the host are selected. The method may further comprise a step of recovering the alkaline protease mutant, which may involve a step of isolating or purifying the alkaline protease mutant from the culture or expression product of the host, and may be carried out using any method known to those skilled in the art.

Has the advantages that:

the alkaline protease mutant obtained by the error-prone PCR technology is recombined and expressed in a bacillus expression system by the genetic engineering technology, under the same condition, the alkaline protease activity and the collagen degradation activity of the mutant are respectively 91.97 percent and 65.84 percent of the wild type, the alkaline protease activity is equivalent to that of the wild type alkaline protease, the collagen degradation activity is obviously reduced, and the mutant has higher application value in the leather industry.

Drawings

FIG. 1: PCR amplification electropherograms of the wild-type alkaline protease gene in the examples; wherein: m is DNA Marker, 1 is alk gene.

FIG. 2: in the examples, the enzyme activities of the wild type and the mutant are compared.

FIG. 3: in the examples the temperature stability of the wild type was compared with that of the mutant.

FIG. 4: in the examples the pH stability of the wild type was compared with that of the mutant.

FIG. 5: electrophoresis of purified proteins from wild type and mutant was performed as shown in the examples.

FIG. 6: the amino acid sequence of the alkaline protease mutant of the invention.

Detailed Description

The invention is further described below by means of specific embodiments. Technical means, materials and the like to which the following embodiments refer may be known to those skilled in the art, and appropriate ones may be selected among known means and materials capable of solving the respective technical problems, unless otherwise specified. In addition, the embodiments should be considered illustrative, and not restrictive, of the scope of the invention, which is defined solely by the claims. It will be apparent to those skilled in the art that various changes or modifications in the components and amounts of the materials used in these embodiments can be made without departing from the spirit and scope of the invention.

The following definitions are used in the present invention:

1. nomenclature for amino acid and DNA nucleic acid sequences

The accepted IUPAC nomenclature for amino acid residues is used, in the form of a three letter code. DNA nucleic acid sequences employ the accepted IUPAC nomenclature.

2. Identification of alkaline protease mutants

"amino acid substituted at the original amino acid position" is used to indicate a mutated amino acid in the alkaline protease mutant. Such as Gly95Glu, indicating the substitution of the amino acid at position 95 by glycine Gly of the parent alkaline protease to glutamic acid Glu, the numbering of the positions corresponding to SEQ ID NO: 2, amino acid sequence number of the alkaline protease. For example, Gly95Glu/Gly258Met indicates that the amino acids at positions 95 and 258 have been mutated.

In the present invention, alk represents the gene sequence of wild type alkaline protease, i.e., the original sequence (shown as SEQ ID NO: 1), and alkm represents the gene sequence of alkaline protease mutant (shown as SEQ ID NO: 3); ALK represents a wild-type alkaline protease (the amino acid sequence of which is shown in SEQ ID NO: 2), and ALKM represents an alkaline protease mutant (the amino acid sequence of which is shown in SEQ ID NO: 4). Base and amino acid controls before and after mutation are as follows:

alkaline protease	Base	Amino acids
			ALK	537 + 539 bit GGG, 1026 + 1028 bit GGA	Gly95、Gly258
ALKM	537 of 539 and 1026 of 1028 as GAGs and ATG	Gly95Glu/Gly258Met

The technical scheme of the invention is summarized as follows:

random mutation is carried out on a wild type alkaline protease alk gene sequence from parent Bacillus clausii (Bacillus clausii) through an error-prone PCR technology to obtain a mutant gene almk. The mutant gene and plasmid pWB980 are subjected to the same double enzyme digestion and are connected to obtain a recombinant vector, the recombinant vector is transferred into a Bacillus subtilis host WB600, finally the recombinant vector is electrically transferred into Bacillus amyloliquefaciens CGMCC No.11218 for expression, protein is purified, the enzyme activity of the protein is verified, and the alkaline protease mutant with the degradation activity remarkably reduced compared with that of wild type collagen is obtained.

The culture medium and the enzyme activity determination method used by the invention are as follows:

seed culture medium: 5g/L of yeast powder, 10g/L of peptone and 5g/L of sodium chloride;

fermentation medium: 64g/L of corn flour, 40g/L of bean cake powder, 4g/L of disodium hydrogen phosphate, 0.3g/L of monopotassium phosphate and 0.7g/L of high-temperature amylase.

B, preparing a culture medium by bacillus subtilis competence:

SP-A salt solution: (NH4)₂SO₄ 4g/L，K₂HPO₄·3H₂O 28g/L，KH₂PO₄12g/L, 2g/L sodium citrate;

SP-B salt solution: MgSO (MgSO)₄·7H₂O 0.4g/L；

100 × CAYE solution: casein hydrolysate 20g/L, yeast powder 100 g/L;

SPI (200 mL): 98mL of SP-A salt solution, 98mL of SP-B salt solution, 2mL of 50% glucose and 2mL of 100 xCAYE;

SPII medium (600 mL): SPI 588mL, 50mmol/L CaCl₂ 6mL，250mmol/L MgCl₂ 6mL；

100 × EGTA solution: 10mmol/L EGTA solution.

Preparing a culture medium by bacillus amyloliquefaciens in a competent manner:

LBS culture medium: 5g/L of yeast powder, 10g/L of peptone, 5g/L of sodium chloride and 9.1085g/L of sorbitol;

recovering the culture medium: 5g/L of yeast powder, 10g/L of peptone, 5g/L of sodium chloride, 9.1085g/L of sorbitol and 6.92246g/L of mannitol.

The method for measuring the enzyme activity of the alkaline protease is carried out according to a Folin phenol method in GB/T23527-2009 appendix B, namely 1 enzyme activity unit (U/mL) is defined as the enzyme quantity required by 1mL of enzyme solution to hydrolyze casein for 1min to generate 1 mu g of tyrosine under the conditions of 40 ℃ and pH 10.5.

The determination of the activity of the collagen degrading enzyme of the invention comprises the following steps:

the ninhydrin colorimetric method is adopted, i.e. type I collagen (soluble) is taken as a substrate, and glycine is taken as a standard substance.

Preparation of samples: 1mL of enzyme solution diluted by distilled water is put into a test tube, 1mL of 5mg/mL I type collagen solution is added, the mixture is uniformly mixed, after the mixture is accurately heated in a water bath at 40 ℃ for 10min, 2mL of trichloroacetic acid solution and 0.4mol/L of trichloroacetic acid solution are immediately added to stop the reaction, 2mL of reaction solution is taken, 12000 Xg is centrifuged for 1min, and 1mL of supernatant is taken to measure the glycine content (mu g/mL) in the supernatant by a ninhydrin colorimetry. Each sample was replicated 3 times and the results averaged.

Preparation of a reference substance: the same method as the sample preparation method, except that the enzyme solution in the test tube is added with trichloroacetic acid solution to inactivate the enzyme, and then the type I collagen solution is added.

Wherein, before the enzyme solution and the casein solution are mixed, both solutions are preheated in water bath at 40 ℃ for more than 2 min.

The enzyme activity of the collagenase is calculated by the formula: enzyme activity is g/t x v x n

Wherein g is the weight of glycine in the sample (μ g); t is reaction time (min); n is the dilution multiple of the enzyme solution; v is the volume of the reaction solution (mL).

Definition of enzyme activity: an amount of enzyme (U/mL) that hydrolyzes type I collagen per minute at 40 ℃ per mL of enzyme solution to produce 1. mu.g glycine.

Example 1: construction of wild type alkaline protease alk recombinant Strain

1.1 Synthesis and amplification of the wild-type alkaline protease Gene alk

According to GenBank: FJ940727.1 obtains the wild type sequence (shown as SEQ ID NO: 1) of alkaline protease gene alk derived from Bacillus clausii, and entrusts the organism company to synthesize the sequence and amplify it by PCR, wherein the primer sequences are as follows:

primer P1: F5'-CCCAAGCTTATGAGGAGGGAACCGAATGAAG-3'

Primer P2: R5'-CGCGGATCCTTATTGATTAGCGTGTTGCCGC-3'

Taking P1 and P2 as upstream and downstream primers, and taking the wild type gene of alk as a template for amplification;

the reaction system for amplification is as follows:

upstream primer P1	1.5μL
		Downstream primer P2	1.5μL
DNA template	2.0μL
		Primerstar enzyme	25μL
ddH₂O	20μL

The setting of the amplification program is as follows: pre-denaturation: 5min at 95 ℃; denaturation: 30s at 95 ℃; annealing: 45s at 56 ℃; extension: 2min at 72 ℃; reacting for 30 cycles; extension: 10min at 72 ℃.

The PCR product is subjected to agarose gel electrophoresis, a band of the wild type alkaline protease gene can be seen, which is 1184bp (shown in figure 1), and then the PCR product is recovered by a small amount of DNA recovery kit, so that the wild type alkaline protease gene, namely alk, is obtained.

1.2 linearization of expression vectors

The plasmid pWB980 was extracted, and the extraction process was carried out according to the manual of the kit. The product is recovered by a DNA gel recovery kit after agarose gel electrophoresis after HindIII and BamHI double enzyme digestion, and a linearized vector sequence is obtained.

1.3, a target fragment (alk) subjected to double enzyme digestion by HindIII and BamHI is connected with a vector fragment to form a recombinant plasmid pWB980-alk, the recombinant plasmid is transformed into Bacillus subtilis WB600, and the sequence is shown as SEQ ID NO: 1.

example 2: method for obtaining alkaline protease mutant by error-prone PCR (polymerase chain reaction) method

2.1 random mutation is carried out based on error-prone PCR technology to construct novel alkaline protease, and primers are designed as follows:

primer P1: F5'-CCCAAGCTTATGAGGAGGGAACCGAATGAAG-3'

Primer P2: R5'-CGCGGATCCTTATTGATTAGCGTGTTGCCGC-3'

In the error-prone PCR reaction system, error-prone PCR was performed using P1 and P2 as upstream and downstream primers and the wild-type alkaline protease gene alk as a template.

The reaction system for amplification is as follows:

10 XPCR buffer (Mg-free)²⁺)	10μL
		dATP	0.2μL
dGTP	0.2μL
		dCTP	1.0μL
dTTP	1.0μL
		Primer P1	1.5μL
Primer P2	1.5μL
		Wild type alkaline protease gene	1.0μL
Taq DNA polymerase	1.0μL
		Mg²⁺(7mM)	28μL
Mn²⁺(0.15mM)	2μL
		ddH₂O	52.6μL

The amplification conditions were: pre-denaturation at 95 ℃ for 10 min; denaturation at 94 ℃ for 30s, annealing at 56 ℃ for 45s, and extension at 72 ℃ for 1min for 30 cycles; extension at 72 ℃ for 10 min.

2.2 cloning the obtained alkaline protease mutant genes into an expression vector pWB980 respectively to obtain a plurality of recombinant plasmids pWB980-alkmx, transforming the recombinant plasmids pWB980-alk and pWB980-alkmx into Bacillus subtilis WB600, transforming the recombinant plasmids pWB980-alk and pWB980-alkmx into Bacillus amyloliquefaciens to obtain the recombinant strains capable of expressing the alkaline protease mutants.

Example 3: screening of alkaline protease mutants

3.196 hole deep hole plate primary screen

The recombinant strain obtained in example 2 was inoculated on a kanamycin-resistant plate, cultured at 37 ℃ for 12 hours, a single colony was selected and inoculated on 5mL of LB medium (50. mu.g/mL kanamycin-resistant), shake-cultured at 37 ℃ and 220r/min for 12 hours, then inoculated on a 96-well deep-well plate in an inoculum size of 2%, and shake-cultured at 37 ℃ and 600r/min for 48 hours, to prepare an alkaline protease enzyme solution.

Respectively measuring the alkaline protease activity and the collagen degradation activity of the enzyme solution, comparing the enzyme activity of all mutants with that of wild alkaline protease, and finally screening 1 strain of which the collagen degradation activity is obviously lower than that of the wild strain.

3.2 Shake flask rescreening, purification and enzyme Activity Studies

The recombinant strains were inoculated into 5mL of LB liquid medium (containing kanamycin, 50. mu.g/mL), cultured overnight at 37 ℃ at 220r/min, transferred to 50mL of fresh fermentation medium at an inoculum size of 2%, cultured further at 37 ℃ at 220r/min for 48h, and the recombinant Bacillus amyloliquefaciens strains containing the alk and alk m genes, respectively, were cultured in 50mL of LB medium supplemented with 50. mu.g/mL of kanamycin at 220rpm and 37 ℃ for 48 h. The culture solution was centrifuged, and the supernatant was salted out with 70% saturated ammonium sulfate. The precipitate formed was dialyzed against MES buffer (20mmol/L, pH 7.0; buffer A), the retentate was subjected to ion exchange chromatography on a CM-Sephadex column (2.5X 20CM) pre-equilibrated with buffer A, and the protein was eluted with a linear gradient using buffer A containing 0 to 1mol/L NaCl. The eluate containing protease activity was applied to a Superdex G-75 gel column (1.6X 80cm) pre-equilibrated with buffer A. The purified protein was then eluted with buffer A (0.5ml min-1). The alkaline protease activity and the collagen degradation activity of the enzyme solution after ultrafiltration purification are measured to obtain a mutant with the alkaline protease activity and the collagen degradation activity respectively being 91.97 percent and 65.84 percent of the wild type (the enzyme activity ratio is shown in figure 2). Incubating the enzyme solution after ultrafiltration purification for 20h at different temperatures and pH values, and measuring the residual alkaline protease activity, wherein the results are respectively shown in FIG. 3 and FIG. 4, the optimum temperature of the alkaline protease mutant is 40 ℃, and the optimum pH value is 10.

Example 4: determination of sequences of alkaline protease mutants

The alkaline protease gene sequence is extracted from the strain and sequenced (Beijing Hua big bioengineering company), the result shows that the nucleotide sequence of the alkaline protease mutant gene obtained by amplification is shown as SEQ ID NO.3, and the coding gene is named as alkkm.

Respectively comparing the amino acid sequence of the alkaline protease alk obtained above with the amino acid sequence of the wild type alkaline protease alk of SEQ ID NO: 1, comparative analysis is carried out, and the results show that: compared with the wild alkaline protease alk, the alkaline protease alk has the mutation from Gly to Glu at the 95 th amino acid and from Gly to Met at the 258 th amino acid (as shown in figure 6).

Although the present invention has been disclosed in the form of preferred embodiments, it is not intended to limit the present invention, and those skilled in the art may make various changes, modifications, substitutions and alterations in form and detail without departing from the spirit and principle of the present invention, the scope of which is defined by the appended claims and their equivalents.

SEQUENCE LISTING

<110> Tianjin science and technology university, Shandong Longkote enzyme preparations Co., Ltd

<120> an alkaline protease mutant having reduced collagen degradation activity

<160> 6

<170> PatentIn version 3.5

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Ala Gln Ser Val Pro Trp Gly Ile Ser Arg Val Gln Ala Pro Ala Ala

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His Asn Arg Gly Leu Thr Gly Ser Gly Val Lys Val Ala Val Leu Asp

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Thr Gly Ile Ser Thr His Pro Asp Leu Asn Ile Arg Gly Gly Ala Ser

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Phe Val Pro Gly Glu Pro Ser Thr Gln Asp Gly Asn Gly His Gly Thr

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His Val Ala Gly Thr Ile Ala Ala Leu Asn Asn Ser Ile Gly Val Leu

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Gly Val Ala Pro Ser Ala Glu Leu Tyr Ala Val Lys Val Leu Gly Ala

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Ser Gly Ser Gly Ser Val Ser Ser Ile Ala Gln Gly Leu Glu Trp Ala

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Gly Asn Asn Gly Met His Val Ala Asn Leu Ser Leu Gly Ser Pro Ser

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Pro Ser Ala Thr Leu Glu Gln Ala Val Asn Ser Ala Thr Ser Arg Gly

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Ala Gln Ser Val Pro Trp Gly Ile Ser Arg Val Gln Ala Pro Ala Ala

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His Asn Arg Gly Leu Thr Gly Ser Gly Val Lys Val Ala Val Leu Asp

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50 55 60

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65 70 75 80

Gly Val Ala Pro Ser Ala Glu Leu Tyr Ala Val Lys Val Leu Glu Ala

85 90 95

Ser Gly Ser Gly Ser Val Ser Ser Ile Ala Gln Gly Leu Glu Trp Ala

100 105 110

Gly Asn Asn Gly Met His Val Ala Asn Leu Ser Leu Gly Ser Pro Ser

115 120 125

Pro Ser Ala Thr Leu Glu Gln Ala Val Asn Ser Ala Thr Ser Arg Gly

130 135 140

Val Leu Val Val Ala Ala Ser Gly Asn Ser Gly Ala Gly Ser Ile Ser

145 150 155 160

Tyr Pro Ala Arg Tyr Ala Asn Ala Met Ala Val Gly Ala Thr Asp Gln

165 170 175

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Val Ala Pro Gly Val Asn Val Gln Ser Thr Tyr Pro Gly Ser Thr Tyr

195 200 205

Ala Ser Leu Asn Gly Thr Ser Met Ala Thr Pro His Val Ala Gly Ala

210 215 220

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225 230 235 240

Arg Asn His Leu Lys Asn Thr Ala Thr Ser Leu Gly Ser Thr Asn Leu

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<210> 5

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cccaagctta tgaggaggga accgaatgaa g 31

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Claims

1 . An alkaline protease mutant, wherein the amino acid sequence of the alkaline protease mutant is shown in SEQ ID NO: 4.

2. A gene encoding the alkaline protease mutant of claim 1.

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

4 . A host cell comprising the gene of claim 2 or the recombinant vector of claim 3 . 5 .

5. The recombinant vector of claim 3, wherein the vector is pWB980.

6. The host cell of claim 4, wherein the host cell is Bacillus subtilis WB600 or Bacillus amyloliquefaciens CGMCC No. 11218.

7. Use of the alkaline protease mutant of claim 1 for hydrolyzing peptide bonds of proteins to generate polypeptides or amino acids, and the mutant has reduced collagen-degrading enzyme activity compared to wild-type.

8. The use according to claim 7, wherein the alkaline protease activity and collagen-degrading enzyme activity of the mutant are 91.97% and 65.84% of the wild type, respectively.

9. the preparation method of the described alkaline protease mutant of claim 1, comprises the steps:

(1) The alkaline protease mutant gene shown in SEQ ID NO: 3 and the linearized pWB980 vector are double digested and connected by HindIII and BamHI to obtain a recombinant vector;

(2) The recombinant vector is transformed into Bacillus amyloliquefaciens CGMCC No.11218 to obtain a recombinant strain;

(3) Expressing the recombinant strain, and purifying the expression product to obtain an alkaline protease mutant.