CN112501150B

CN112501150B - Chitin deacetylase, coding gene thereof, recombinant vector, recombinant strain, leavening agent, enzyme preparation and application of chitin deacetylase, recombinant strain, leavening agent and enzyme preparation

Info

Publication number: CN112501150B
Application number: CN202011448924.XA
Authority: CN
Inventors: 臧传刚; 佟毅; 张媛; 沈雪梅; 王小艳; 赵国淼; 陈博; 李义; 周勇; 卢宗梅; 张钊; 商谈
Original assignee: Cofco Biochemical Energy Yushu Co ltd; Cofco Nutrition and Health Research Institute Co Ltd; Cofco Jilin Bio Chemical Technology Co Ltd; Cofco Biotechnology Co Ltd
Current assignee: Cofco Biochemical Energy Yushu Co ltd; Cofco Nutrition and Health Research Institute Co Ltd; Cofco Jilin Bio Chemical Technology Co Ltd; Cofco Biotechnology Co Ltd
Priority date: 2020-12-09
Filing date: 2020-12-09
Publication date: 2023-04-11
Anticipated expiration: 2040-12-09
Also published as: CN112501150A

Abstract

The invention relates to the field of genetic engineering, in particular to chitin deacetylase, and a coding gene, a recombinant vector, a recombinant strain, a fermentation agent, an enzyme preparation and application thereof. The chitin deacetylase has relatively low optimal catalytic temperature (about 40 ℃), shows high catalytic activity in a wide pH range (pH value of 6-10), can save energy, auxiliary materials and other costs in industrial application, and has wide application prospect. The preferred recombinant strain is Bacillus licheniformis (Bacillus licheniformis) Bl22/pMA5-bli with the preservation number of CGMCCNO.20933.

Description

Chitin deacetylase, coding gene thereof, recombinant vector, recombinant strain, leavening agent, enzyme preparation and application of chitin deacetylase, recombinant strain, leavening agent and enzyme preparation

Technical Field

The invention relates to the field of genetic engineering, in particular to chitin deacetylase and a coding gene thereof, a recombinant vector, a recombinant strain, a starter, a preparation method of the chitin deacetylase, an enzyme preparation, a method for removing acetyl on chitin, application of the chitin deacetylase in production of the chitin and application of the chitin deacetylase in removal of acetyl on the chitin.

Background

Chitin (chitin), also known as chitin and chitin, is a polysaccharide formed by connecting N-acetamido-D-glucose monomers through beta-1,4-glycosidic bonds. It is the second largest natural macromolecular organic compound with a second content of cellulose in nature, and is widely found in exoskeletons of invertebrates (such as shrimps, crabs, insects, etc.) and cell walls of fungi and algae. Chitin is insoluble in water, dilute acid, dilute alkali, organic solvent and the like due to poor solubility, so that the utilization value of the chitin is greatly limited. For example, chitosan (chitosan) with greatly improved solubility can be obtained by deacetylating chitin (deacetylation degree is more than 55%). And the chitosan has multiple physiological functions of biodegradability, biocompatibility, nontoxicity, bacteriostasis, cancer resistance, lipid reduction, immunity enhancement and the like, can be widely applied to the fields of medicine, food, textile, agriculture, environmental protection, cosmetics and the like, and has high application value and development prospect.

At present, the industrial production method of chitosan is mainly a chemical method. Chitosan is typically prepared by high temperature treatment of chitin with 40% -60% concentrated sodium hydroxide. The method has the defects of high production cost, large environmental pollution, poor product stability and uniformity and the like. Chitin deacetylase (e.c. 3.5.1.41) can remove acetyl groups from chitin to produce chitosan products with stable deacetylation degree and narrow molecular mass distribution range. In addition, the enzymatic method for producing chitosan has mild reaction conditions, low energy consumption value and environmental protection, provides a new way for solving the problems existing in the chemical method for preparing chitosan, and is a future development direction of the chitosan production industry.

Since the first report in 1974 of Mucor rouxii (Mucorrouxii) chitin deacetylase, researchers have isolated a variety of chitin deacetylases from fungi, bacteria and insects. However, the chitin deacetylases reported at present generally have the problems of long fermentation time, low enzyme production activity, high catalysis temperature, poor deacetylation effect and the like, and most of the chitin deacetylases are enzymes derived from fungi and few of the chitin deacetylases are derived from bacteria. The bacteria have more advantages than fungi in the aspect of fermentation and enzyme production, the strain is easier to realize large-scale fermentation culture, and the produced enzyme is easier to separate and purify. Therefore, the engineering strain with high enzyme yield is constructed by developing and genetically engineering the microbial resource for producing the chitin deacetylase, and is expected to meet the industrial production requirement of preparing the chitosan by the enzyme method.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provides chitin deacetylase and a coding gene thereof, a recombinant vector, a recombinant strain, a leavening agent, a preparation method of the chitin deacetylase, an enzyme preparation, a method for removing acetyl on chitin, application of the chitin deacetylase in production of the chitin deacetylase and application of the chitin deacetylase in removal of acetyl on the chitin, wherein the chitin deacetylase can be expressed by bacteria and has the advantages of low catalytic temperature and wide applicable pH.

In order to achieve the above object, a first aspect of the present invention provides a chitin deacetylase which is the enzyme according to any one of (a) to (e):

(a) An enzyme having an amino acid sequence shown in SEQ ID NO. 2;

(b) SEQ ID NO:2 by substituting, deleting or adding one or more amino acid residues and still has the chitin deacetylase activity;

(c) And SEQ ID NO:2 has homology of more than 80 percent and has chitin deacetylase activity;

(d) An enzyme represented by an amino acid sequence wherein a tag is attached to the amino terminus and/or the carboxy terminus of the amino acid sequence of (a), (b) or (c);

(e) An enzyme represented by an amino acid sequence wherein a signal sequence is linked to the amino terminus of the amino acid sequence of (a), (b) or (c).

In a second aspect, the present invention provides a gene encoding chitin deacetylase, which has a nucleotide sequence encoding chitin deacetylase as described above.

In a third aspect, the present invention provides a recombinant vector containing the gene as described above.

In a fourth aspect, the present invention provides a recombinant strain containing the gene as described above or the recombinant vector as described above.

Preferably, the recombinant strain is a bacillus licheniformis engineering strain Bl22/pMA5-bli, and the preservation number is CGMCC NO.20933.

In a fifth aspect, the invention provides a starter culture comprising a recombinant strain as described above.

The sixth aspect of the present invention provides a method for producing chitin deacetylase, comprising: and inoculating the recombinant strain and/or the leavening agent into a fermentation medium for fermentation, and separating and purifying a fermentation product to obtain the chitin deacetylase.

The seventh aspect of the present invention provides an enzyme preparation comprising the chitin deacetylase prepared by the method described above.

The eighth aspect of the present invention provides a method for removing acetyl groups from chitin, comprising: at least one of the recombinant strain, the leavening agent, the chitin deacetylase and the enzyme preparation is contacted with chitin to deacetylate chitin.

The ninth aspect of the present invention provides the use of at least one of the chitin deacetylase described above, the gene described above, the recombinant vector described above, the recombinant strain described above, the starter culture described above, and the chitin deacetylase prepared by the method described above for producing chitin deacetylase.

In a tenth aspect, the present invention provides the use of at least one of the chitin deacetylase described above, the gene described above, the recombinant vector described above, the recombinant strain described above, the starter culture described above, the chitin deacetylase prepared by the method described above, and the enzyme preparation described above for deacetylation of chitin.

The chitin deacetylase has the advantages of relatively low optimal catalytic temperature (about 40 ℃), high catalytic activity in a wide pH range (pH value of 6-10), energy and auxiliary material cost saving in industrial application, and wide application prospect.

The invention also has the advantages that the preferred recombinant strain (the bacillus licheniformis engineering strain Bl22/pMA 5-bli) provided by the invention can utilize common carbon sources and nitrogen sources to rapidly culture and ferment to produce chitin deacetylase with high yield.

Drawings

FIG. 1 shows the result of agarose gel electrophoresis of a gene bli encoding chitin deacetylase;

FIG. 2 shows the result of polyacrylamide gel electrophoresis (SDS-PAGE) of chitin deacetylase.

Biological preservation

The Bacillus licheniformis (Bacillus licheniformis) genetic engineering strain provided by the invention is preserved in the China general microbiological culture Collection center (address: no.3 of West Lu No.1 of Beijing Korean district, microbiol research institute of Chinese academy of sciences, postal code: 100101) within 22 days 10 and 10 months 2020, wherein the preservation number is CGMCC No.20933, abbreviated as Bl22/pMA5-bli.

Detailed Description

The endpoints of the ranges and any values disclosed herein are not limited to the precise range or value, and such ranges or values should be understood to encompass values close to those ranges or values. For ranges of values, between the endpoints of each of the ranges and the individual points, and between the individual points may be combined with each other to give one or more new ranges of values, and these ranges of values should be considered as specifically disclosed herein.

In the present invention, the term "enzyme activity", i.e., the amount of enzyme content, is used in the sense that the enzyme activity unit, i.e., the enzyme unit (U), which is defined in the present invention, is not stated to the contrary: at the conditions of pH 7 and 40 ℃, the enzyme amount of 1 mu g of paranitroanilide generated by deacetylation of paranitroanilide per hour is one enzyme activity unit; "specific activity of enzyme" represents the catalytic ability per unit mass of protein, and can reflect the activity of enzyme, and the larger the value, the higher the activity of enzyme, the calculation formula of specific activity is: specific activity (U/mg) = total enzyme activity units/mg total protein; the unit M represents mol/L.

The present invention provides, in a first aspect, a chitin deacetylase which is an enzyme according to any one of (a) to (e):

(a) An enzyme having an amino acid sequence shown in SEQ ID No. 2;

(d) An enzyme represented by an amino acid sequence wherein a tag is linked to the amino terminus and/or the carboxy terminus of the amino acid sequence of (a), (b) or (c);

The 20 amino acid residues constituting a protein can be classified into four types according to the side chain polarity: 1. non-polar amino acids: alanine (Ala), valine (Val), leucine (Leu), isoleucine (Ile), methionine (Met), phenylalanine (Phe), tryptophan (Trp), and proline (Pro); 2. polar uncharged amino acids: glycine (Gly), serine (Ser), threonine (Thr), cysteine (Cys), aspartic acid (Asn), glutamine (Gln) and tyrosine (Tyr); 3. positively charged amino acids: arginine (Arg), lysine (Lys), and histidine (His); 4. negatively charged amino acids: aspartic acid (Asp) and glutamic acid (Glu) (see "biochemistry" (second edition) supra, shen Tong, wang Jingyan, pages 82-83, higher education Press, 1990 month 12).

If the substitution of amino acid residues belonging to the same class, for example, substitution of Arg for Lys or Leu for Ile, occurs in the protein, the role of the residues in the protein domain (e.g., the role of providing positive charge or forming a hydrophobic pocket structure) is not changed, and thus the steric structure of the protein is not affected, and thus the function of the protein can still be achieved. The substitution of an amino acid residue of the same genus may occur at any one of the amino acid residue positions of the above-mentioned chitin deacetylase.

In addition to the above-mentioned amino acid residue substitutions, the chitin deacetylase provided by the present invention also includes proteins in which one or more amino acid residues are deleted or added or both at any position of amino acid residues 1 to 254 as compared with the amino acid sequence shown in SEQ ID NO. 2. Preferably, the amino acid sequence of the chitin deacetylase of the present invention may be a sequence in which amino acid residues at positions 2 to 7 and 251 to 254 are partially or completely deleted compared with the amino acid sequence shown in SEQ ID NO.2, or a sequence in which 1 to 5 amino acid residues are added at any position between amino acid residues at positions 2 to 7, or a sequence in which 1 to 3 amino acid residues are added at any position between amino acid residues at positions 251 to 254, or an amino acid sequence formed by any combination of the above.

As mentioned above, the chitin deacetylase provided by the invention can also be modified or mutated to obtain a derivative protein. The derived protein means a protein having an amino acid sequence different from that of chitin deacetylase having the above amino acid sequence, and may have a difference in modified form which does not affect the sequence, or both. These proteins include natural or induced genetic variants. The induced variants may be obtained by various techniques, such as random mutagenesis by irradiation or mutagenic agents, etc., or by techniques such as site-directed mutagenesis or other known molecular biology techniques. Derivatized proteins also include analogs having residues of natural L-amino acids (e.g., D-amino acids), as well as analogs having non-naturally occurring or synthetic amino acids (e.g., beta-amino acids, gamma-amino acids, etc.).

Modifications (which do not generally alter primary structure, i.e., do not alter amino acid sequence) include: chemically derivatized forms of the protein such as acetylation or carboxylation, in vivo or in vitro. Modifications also include glycosylation, such as those proteins that result from glycosylation modifications during synthesis and processing of the protein or during further processing steps. Such modification may be accomplished by exposing the protein to an enzyme that performs glycosylation, such as mammalian glycosylating or deglycosylating enzymes. Modified forms also include sequences having phosphorylated amino acid residues (e.g., phosphotyrosine, phosphoserine, phosphothreonine). Also included are proteins that have been modified to increase their resistance to proteolysis or to optimize solubility.

In the present invention, the enzyme may also be a polypeptide having an amino acid sequence substantially similar to SEQ ID NO:2 has homology of more than 80 percent and has chitin deacetylase activity. Preferably, the chitin deacetylase is a chitinase that hybridizes with SEQ ID NO:2, more preferably 90% or more, still more preferably 95% or more, still more preferably 98% or more, and most preferably 99% or more, and has chitin deacetylase activity.

For purification, additional modifications of (a), (b) or (c) may be made using tags commonly used in the art, and may be obtained, for example, by attaching the amino acid sequence of a tag (e.g., at least one of Poly-Arg, poly-His, FLAG, strep-tag II and c-myc shown in Table 1 below) to the amino terminus and/or the carboxy terminus of (a). The label does not influence the activity of the chitin deacetylase, and whether the label is added or not can be selected according to requirements in the practical application process.

TABLE 1

Label (R)	Number of residues	Amino acid sequence
			Poly-Arg	5-6 (typically 5)	RRRRR(SEQ ID NO：3)
Poly-His	2-10 (generally 6)	HHHHHH(SEQ ID NO：4)
			FLAG	8	DYKDDDDK(SEQ ID NO：5)
Strep-tagⅡ	8	WSHPQFEK(SEQ ID NO：6)
			c-myc	10	EQKLISEEDL(SEQ ID NO：7)

In the present invention, the amino terminus of the chitin deacetylase may also be linked to a signal sequence. The signal sequence may be from bacillus licheniformis, bacillus amyloliquefaciens, and bacillus subtilis, but is not limited thereto. The signal sequence is preferably SEQ ID NO: 8.

In the present invention, the enzyme still having the enzyme activity means that the enzyme derived from (a) still has the enzyme activity with a percentage (relative activity) between the enzyme activity and the enzyme activity of (a) of not less than 60%, preferably not less than 70% (or 80%, or 90%, or 95%, or 96%, or 97%, or 98%, or 99%, or 100%) under the same measurement conditions.

The chitin deacetylase can be obtained by artificial synthesis, or can be obtained by synthesizing the coding gene and then performing biological expression.

Accordingly, the gene may be (1) or (2) as follows:

(1) A DNA molecule having an amino acid sequence encoding the chitin deacetylase of the first aspect;

(2) And (2) the DNA molecule which is hybridized with the DNA sequence defined in the step (1) under strict conditions and does not change the enzymatic activity of the encoded chitin deacetylase. Wherein the stringent conditions may be: hybridization was carried out at 65 ℃ in a solution containing 6 XSCC, 0.5% SDS, and the SDS and 1 XSCC, 0.1% SDS were eluted once each. The enzyme activity is not changed, and means that the percentage (relative activity) between the enzyme activity of the protein encoded by (2) and the enzyme activity of the protein encoded by (1) is not less than 95% (or 96%, or 97%, or 98%, or 99%, or 100%) under the same assay conditions.

It is well known in the art that 18 amino acids, other than Met (ATG) or Trp (TGG), each encoded by a single codon, of the 20 different amino acids that make up a protein are each encoded by 2-6 codons (Sambrook et al, molecular cloning, cold spring harbor laboratory Press, new York, USA, second edition, 1989, see appendix D page 950). That is, due to the degeneracy of genetic code, there is usually more than one codon determining one amino acid, and the substitution of the third nucleotide in a triplet codon does not change the composition of the amino acid, and thus the nucleotide sequences of genes encoding the same protein may differ. From the amino acid sequences disclosed in the present invention and the amino acid sequences with no change in the chitin deacetylase activity obtained from the amino acid sequences, nucleotide sequences of genes encoding the amino acid sequences can be completely deduced by those skilled in the art from well-known codon tables, and the nucleotide sequences are obtained by biological methods (e.g., PCR method, mutation method) or chemical synthesis methods, and thus the partial nucleotide sequences should be included in the scope of the present invention. Conversely, using the DNA sequences disclosed herein, amino acid sequences consistent with the chitin deacetylase activity of the present invention can also be obtained by modifying the nucleic acid sequences provided herein by methods well known in the art, e.g., sambrook et al (molecular cloning, cold spring harbor laboratory Press, new York, U.S. Pat. No.5, second edition, 1989).

Preferably, the gene has a nucleotide sequence encoding an enzyme having an amino acid sequence shown in SEQ ID NO. 2.

As described above, the 5 'end and/or the 3' end of the nucleotide sequence may be linked with the coding sequence of the tag shown in Table 1 above, respectively.

More preferably, the gene has a nucleotide sequence shown in SEQ ID NO. 1.

The nucleotide sequence provided by the present invention can be obtained by a Polymerase Chain Reaction (PCR) amplification method, a recombinant method, or an artificial synthesis method. For example, one skilled in the art can easily obtain templates and primers based on the nucleotide sequences provided by the present invention, and obtain the relevant sequences by PCR amplification.

Once the nucleotide sequence of interest is obtained, the amino acid sequence of interest can be obtained in large quantities by recombinant methods. The nucleotide sequence obtained is usually cloned into a vector, then transferred into genetically engineered bacteria, and then separated from the proliferated host cells by a conventional method to obtain the relevant nucleotide sequence.

In addition, the nucleotide sequence can be synthesized by a known artificial chemical synthesis method.

As the "vector" used in the recombinant vector, various vectors known in the art can be used, such as various commercially available plasmids, cosmids, phages, retroviruses and the like, and a preferred expression vector of the present invention is the pMA5 plasmid. The recombinant vector construction can adopt various endonucleases (such as BamH I, mluI and the like for pMA 5) which can have cutting sites at the multiple cloning sites of the vector to carry out enzyme digestion to obtain linear plasmids, and the linear plasmids are connected with gene segments cut by the same endonucleases to obtain recombinant plasmids. The invention preferably adopts BamHI and MluI double enzyme digestion pMA5 and gene segments connected with the same to construct a recombinant vector pMA5-Bli through ligase connection.

The fourth aspect of the present invention provides a recombinant strain containing the gene as described above or the recombinant vector as described above.

The recombinant vector may be transformed, transduced or transfected into a host cell (strain) by methods conventional in the art, such as chemical transformation by calcium chloride method, high-voltage shock transformation, preferably shock transformation. The host cell may be a prokaryotic or eukaryotic cell, preferably at least one of Bacillus licheniformis (Bacillus licheniformis), bacillus amyloliquefaciens and Bacillus subtilis, more preferably the host cell is Bacillus licheniformis, such as Bacillus licheniformis Bl22.

The starter may be present in liquid form or in solid form. The leavening agent can contain auxiliary materials added in the conventional preparation of microbial inoculum in the field, and the skilled person can select the auxiliary materials according to the needs.

Preferably, the content of said recombinant strain is 10 per gram of said starter culture ⁵ -10 ¹⁰ CFU, more preferably 10 ⁷ -10 ⁹ CFU。

The sixth aspect of the present invention provides a method for preparing chitin deacetylase, comprising: and inoculating the recombinant strain and/or the leavening agent into a fermentation medium for fermentation, and separating and purifying the fermentation product to obtain the chitin deacetylase.

The method for preparing chitin deacetylase provided by the invention comprises the following steps: culturing the recombinant strain provided by the invention, and inducing the expression of a gene coding the chitin deacetylase; separating and purifying the expressed chitin deacetylase.

Wherein the culture conditions are conventional culture conditions, such as LB medium (solvent is water, solute and final concentration thereof are 5-15g/L tryptone, 1-10g/L yeast extract, and 5-15g/L NaCl), and culture is performed at 35-37 deg.C.

The recombinant strain provided by the invention contains a gene for coding the chitin deacetylase, so that the chitin deacetylase can be efficiently expressed. After culturing, the chitin deacetylase with high purity can be obtained by separation and purification.

The separation and purification can be carried out by methods known to those skilled in the art, and will not be described herein.

The enzyme preparation can exist in a solid, semi-solid or liquid form, and the enzyme preparation can contain auxiliary materials or additives for preparing the enzyme preparation, and the like, and the enzyme preparation can be selected by a person skilled in the art according to needs, and is not described herein again.

The method for removing acetyl groups from chitin may be a method for removing acetyl groups conventionally used in the art.

Wherein the contacting conditions may be conditions suitable for the chitin deacetylase, preferably the contacting conditions include: a pH value of 4-13, more preferably 7-10; the temperature is 25-55 deg.C, more preferably 30-45 deg.C.

The chitin deacetylase may be used in an amount of 10-100 μ g per gram of chitin sample.

In a tenth aspect, the present invention provides a use of at least one of the chitin deacetylase described above, the gene described above, the recombinant vector described above, the recombinant strain described above, the starter culture described above, the chitin deacetylase prepared by the method described above, and the enzyme preparation described above for deacetylation of chitin.

The present invention will be described in detail below by way of examples.

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

Chitin was purchased from Wako (japan).

Example 1

This example is for explaining the acquisition of the target gene bli.

Shanghai BioEngineers, inc. was entrusted to synthesize the gene sequence shown in SEQ ID NO.1 and the primer sequences shown in SEQ ID NO.9 and SEQ ID NO. 10. Wherein, SEQ ID NO.9 is GCGGGATCCGTGAACCATTTTTATGTGTG；SEQ ID NO.10:GCGACGCGTCTACTTTACTTCTGAAGATT. The upstream and downstream primers are respectively introduced with BamH I and Mlu I restriction enzyme cutting sites.

PCR amplification was performed using the synthesized DNA as a template. The PCR reaction system is as follows: premix (25. Mu.l), ddH ₂ O (22. Mu.l), upstream and downstream primers (1. Mu.l each), DNA template (1. Mu.l). The reaction conditions are as follows: 94 ℃ for 5min,1 cycle; 30 cycles of 94 ℃ 30s,55 ℃ 30s,72 ℃ 1min; 10min at 72 ℃ and 1 cycle. The PCR product was subjected to agarose gel electrophoresisAfter analysis (FIG. 1), the desired fragment was recovered by cutting the gel.

The fragment of interest was ligated to the pMD-19T cloning vector. The connection reaction system is as follows: mu.l vector, 5. Mu.l recovery fragment, 1. Mu.l T4 DNA ligase (NEB), 1. Mu.l 10 × buffer, ddH ₂ And O2. Mu.l. The ligation reaction conditions were 16 ℃ for 2h.

The ligation solution was transformed into E.coli DH 5. Alpha. Competent cells. The method comprises the following steps: melting 50ul of competent cells on ice bath, and adding connecting liquid; standing in ice bath for 30min; heating in 42 deg.C water bath for 45sec, rapidly transferring to ice, standing for 2min, adding 500ul sterile LB culture medium (without antibiotic), culturing at 37 deg.C and 200rpm for 1h; after centrifugation at 6000rpm for 3min, a portion of the supernatant was removed, the cells were resuspended, 100. Mu.l of the supernatant was spread on LB medium solid plates containing 100. Mu.g/mL ampicillin, and cultured overnight at 37 ℃.

And (5) carrying out colony PCR verification. The PCR reaction system is as follows: premix (25. Mu.l), ddH ₂ O (23. Mu.l), upstream and downstream primers (1. Mu.l each), DNA template (single colony on LB plate). The reaction conditions are as follows: 94 ℃ for 5min,1 cycle; 30 cycles of 30s at 94 ℃, 30s at 55 ℃, 1min at 72 ℃;10 min at 72 ℃ and 1 cycle. Positive clones with correct amplification product sizes were sequenced by Shanghai Bioengineering Co., ltd.

The sequencing result shows that the target gene coding region is 765bp long, and the nucleotide sequence is shown as SEQ ID NO. 1. The target gene totally encodes 254 amino acids and a stop codon, the amino acid sequence of the target gene is shown as SEQ ID NO.2, and the predicted protein molecular weight is 28kDa.

Example 2

This example illustrates the acquisition of the genetically engineered strain of Bacillus licheniformis Bl22/pMA5-bli.

Double digestion of target gene

The objective gene obtained in example 1 was excised from the cloning vector. The enzyme digestion reaction system is as follows: vector pMD-19T-bli (5. Mu.l), restriction enzyme BamH I (1. Mu.l), restriction enzyme Mlu I (1. Mu.l), 10. Mu.buffer (1. Mu.l), ddH ₂ O (2. Mu.l); the reaction conditions are as follows: 37 ℃ and 2h. After the enzyme digestion product is subjected to 1% agarose gel electrophoresis analysis, the gel is cut to recover the target gene fragment.

Expression vector double digestion

Double enzyme digestion is carried out on the expression vector. The enzyme digestion reaction system is as follows: vector pMA5 (5. Mu.l), restriction enzyme BamH I (1. Mu.l), restriction enzyme Mlu I (1. Mu.l), 10. Mu.buffer (1. Mu.l), ddH ₂ O (2. Mu.l); the reaction conditions are as follows: 37 ℃ for 2h. And (4) carrying out 1% agarose gel electrophoresis analysis on the enzyme digestion product, cutting the gel and recovering the target gene fragment.

Connection of target gene and expression vector

The gene of interest was ligated into the pMA5 expression vector. The connection reaction system is as follows: vector 1. Mu.l, target gene 5. Mu.l, T4 DNA ligase (NEB) 1. Mu.l, 10. Mu.Buffer 1. Mu.l, ddH ₂ O2. Mu.l. The ligation reaction conditions were 16 ℃ for 2h.

Transformation of recombinant expression vectors

The ligation solution was transformed into competent cells of Bacillus licheniformis Bl22. Thawing 50ul of competent cells on ice, and adding the ligation solution; standing for 30min; transferring the mixed solution into a precooled electric rotor; setting electric shock parameters, and carrying out electric shock conversion according to the conditions of electric shock voltage of 2.5kV and electric shock time of 5 ms; immediately after the electric shock, 1ml of LB medium preheated at 42 ℃ was added to the electric rotor; repeatedly beating and uniformly mixing the mixture by using a pipettor, and then transferring the liquid in the electric shock cup into a centrifuge tube; placing into 42 deg.C water bath, thermally shocking for 6min, and rapidly transferring to ice and standing for 2min; incubating at 37 ℃ for 2h with 200rpm oscillation; the cells were resuspended by centrifugation at 6000rpm for 3min, and 100. Mu.l of the supernatant was plated on LB medium solid plates containing 5. Mu.g/mL chloramphenicol and cultured at 37 ℃ for 48h.

Recombinant strain screening

And (5) carrying out colony PCR verification. The PCR reaction system is as follows: premix (25. Mu.l), ddH ₂ O (23. Mu.l), upstream and downstream primers (1. Mu.l each), DNA template (single colony on LB plate). The reaction conditions are as follows: 94 ℃ for 5min,1 cycle; 30 cycles of 94 ℃ 30s,55 ℃ 30s,72 ℃ 1min; 10min at 72 ℃ and 1 cycle. And (4) performing glycerol tube preservation on the positive recombinant strain with the correct PCR amplification product size, and storing in a refrigerator at the temperature of-80 ℃.

The Bacillus licheniformis (Bacillus licheniformis) genetic engineering strain is preserved in China general microbiological culture Collection center (address: no.3 of West Luo No.1 of Beijing Korean district, china academy of sciences, microbiological research institute, postal code: 100101) (the abbreviation of preservation unit is CGMCC) within 10 months and 22 days of 2020, wherein the preservation number is CGMCC No.20933, which is Bl22/pMA5-bli.

Example 3

This example illustrates the acquisition of chitin deacetylase and the determination of enzymatic activity.

Recombinant strain protein expression

(1) Strain activation

The culture method comprises the following steps: taking a proper amount of the refrigerator-preserved strain Bl22/pMA5-bli at the temperature of minus 80 ℃ to streak an LB solid plate added with 5 mu g/mL chloramphenicol, and placing the plate in a constant-temperature incubator for culture at the temperature of 35 +/-2 ℃ for 20 hours.

Activating a culture medium: 5g/L of yeast extract, 10g/L, naCl g/L of tryptone, 20g/L of agar powder and the balance of water, wherein the pH value is 6.5-7.2.

(2) Seed culture

The culture method comprises the following steps: single colonies were picked from the activated plates and placed in seed medium (chloramphenicol concentration 5. Mu.g/mL) for shaking culture at a constant temperature at a stirring speed of 150rpm and a temperature of 35. + -. 2 ℃ for 9 hours.

Seed culture medium: 5g/L of yeast extract, 10g/L, naCl g/L of tryptone and the balance of water, and the pH value is 6.5-7.2.

(3) Fermentation culture

The culture method comprises the following steps: transferring the seed solution to a fermentation medium (the concentration of chloramphenicol is 5 mug/mL) according to the inoculation amount of 1%, performing constant temperature shaking culture at the stirring speed of 150rpm and the temperature of 35 +/-2 ℃ for 24h;

fermentation medium: 40g/L of glucose, 25g/L of yeast powder, 15g/L of tryptone, 3g/L of K2HPO and the balance of water, and the pH value is 6.5-7.2.

Preparation of crude enzyme solution of recombinant strain Bl22/pMA5-bli chitin deacetylase

Collecting 10mL fermentation liquor, centrifuging at 12000rpm for 5min, pouring out culture medium supernatant, taking 10mL ultrasonication buffer (50 mM Tris-cl, 20mM NaOH, pH 8.0) to suck up and down, and fully resuspending thalli; averagely dividing 10mL of the resuspended bacterial liquid into 10 parts, transferring each part of the resuspended bacterial liquid into 10 centrifugal tubes with the volume of 1mL, placing each centrifugal tube into a fixed ice-water mixed bath, and independently carrying out ultrasonic crushing, wherein an amplitude rod of an ultrasonic crusher is set to be 2, the power is set to be 20%, the ultrasonic frequency is set to be 2s/2s (namely 2s of crushing, 2s of suspension), and the total time of crushing is 5min; centrifuging all the broken solutions at 12000rpm for 5min, collecting supernatant, and transferring into a new 15ml centrifuge tube to obtain crude enzyme solution of recombinant strain chitin deacetylase.

Recombinant protein detection

The expression of recombinant chitin deacetylase was examined by polyacrylamide gel electrophoresis (SDS-PAGE) (FIG. 2).

Detection of recombinant chitin deacetylase enzymatic activity

Taking a 15ml centrifuge tube, adding 1ml 200mg/l paranitroacetanilide aqueous solution, 1ml crude enzyme solution diluted by proper times and 3ml pre-insulated phosphate buffer solution with the concentration of 0.05M and the pH value of 7.0 to ensure that the final volume of the reaction solution is 5ml, carrying out water bath reaction at 40 ℃ for 0.5h, carrying out boiling water bath for 5min, stopping enzymatic reaction, adding water to fix the volume to 10ml, uniformly mixing, centrifuging at 12000rpm for 5min, and measuring the light absorption value A400 of the supernatant at 400 nm. 1ml of the inactivated enzyme solution with the same concentration was added to the blank control system, and the absorbance A0 of the supernatant was measured as above for the rest, and each sample corresponded to one blank.

Definition of enzyme activity unit: the amount of enzyme required to produce 1. Mu.g of p-nitroaniline per hour under the above reaction conditions was defined as one unit of enzyme activity. The enzyme activity calculation formula is as follows:

enzyme activity (U/ml) = ((A400-A0) × 10 × n)/KT

A400-absorbance of the sample at 400 nm; a0-absorbance of blank; 10-volume of solution 10ml; n is the dilution multiple of enzyme solution; k-linear coefficient (0.0648); t-reaction time, h

The enzyme activity result of the obtained chitin deacetylase is 6620U by determination.

Example 4

This example illustrates the acquisition of chitin deacetylase and the determination of enzyme activity.

The procedure of examples 1-3 was followed except that the amino acid sequence of chitin deacetylase was varied, as shown in Table 1.

The results of the enzyme activities of the enzymes corresponding to the different amino acid sequences are shown in Table 1.

TABLE 1

Numbering	Distinction between	Enzyme activity U
			B1	K108R	6010
B2	Deletion of amino acids at positions 2 to 7	5880
			B3	Deletion of amino acids 251 to 254	5310
B4	Deletion of amino acids 2-7 and 251-254	4560
			B5	Inserting RLI three amino acid residues between 7 th and 8 th positions	4980
C	SEQ ID NO.11 (83% homology with SEQ ID NO. 2)	4230
			D1	The amino terminal of SEQ ID NO.2 is connected with SEQ ID NO.3	4970
D2	The carboxyl terminal of SEQ ID NO.2 is connected with SEQ ID NO.4	6480
			D3	The carboxyl terminal of SEQ ID NO.2 is connected with SEQ ID NO.5	6010
D4	The amino terminal of SEQ ID NO.2 is connected with SEQ ID NO.6	6230
			D5	The carboxyl terminal of SEQ ID NO.2 is connected with SEQ ID NO.7	5870
E	The amino terminal of SEQ ID NO.2 is connected with SEQ ID NO.8	6590

Example 5

The procedure of example 3 was followed except that the enzyme activities were measured at pH 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, 11.0, 12.0 and 13.0, respectively.

The enzyme activity results of the obtained chitin deacetylase are 2200U, 3450U, 5860U, 6620U, 6360U, 6320U, 6240U, 5090U, 3310U and 2180U in sequence.

Example 6

The procedure of example 3 was followed except that the enzyme activities were measured at temperatures of 20, 25, 30, 35, 40, 45, 50 and 55 ℃.

The enzyme activity results of the obtained chitin deacetylase are 1390U, 3130U, 4410U, 5470U, 6620U, 3980U, 1970U and 810U respectively through determination.

The results show that the chitin deacetylase produced by the bacillus licheniformis genetic engineering strain Bl22/pMA5-bli has the characteristics of wide applicable pH range and low applicable temperature, and is high in enzyme activity and suitable for industrial production.

The preferred embodiments of the present invention have been described above in detail, but the present invention is not limited thereto. Within the scope of the technical idea of the invention, many simple modifications can be made to the technical solution of the invention, including combinations of various technical features in any other suitable way, and these simple modifications and combinations should also be regarded as the disclosure of the invention, and all fall within the scope of the invention.

SEQUENCE LISTING

<110> Jilin, food biochemistry, inc.; chinese grain Nutrition and health research institute, inc.; chinese grain Biotechnology Ltd; biochemical energy resources of Chinese food grain (elm) Co Ltd

<120> chitin deacetylase, gene encoding same, recombinant vector, recombinant strain, fermentation agent, enzyme preparation, and use thereof

<130> 65859

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Claims

1. The recombinant strain is a bacillus licheniformis engineering strain Bl22/pMA5-bli, and the preservation number is CGMCC NO.20933.

2. A starter culture comprising the recombinant strain of claim 1.

3. A starter culture according to claim 2 wherein the recombinant strain is present in an amount of 10 per gram of starter culture ⁵ -10 ¹⁰ CFU。

4. A starter culture according to claim 3 wherein the recombinant strain is present in an amount of 10 per gram of starter culture ⁷ -10 ⁹ CFU。

5. A method for preparing chitin deacetylase, comprising: the recombinant strain of claim 1 and/or the leavening agent of any one of claims 2 to 4 are inoculated into a fermentation medium for fermentation, and the fermentation product is separated and purified to obtain chitin deacetylase.

6. An enzyme preparation comprising the chitin deacetylase prepared by the method of claim 5.

7. A method for removing acetyl groups from chitin, the method comprising: contacting at least one of the recombinant strain of claim 1, the starter of any one of claims 2 to 4, the chitin deacetylase produced by the process of claim 5, and the enzyme preparation of claim 6 with chitin to deacetylate the chitin.

8. The method of claim 7, wherein the conditions of the contacting comprise: the pH value is 6-10; the temperature is 30-45 ℃.

9. Use of the recombinant strain of claim 1, the starter culture of any one of claims 2 to 4, or the method of claim 5 for the production of chitin deacetylase.

10. Use of at least one of the recombinant strain of claim 1, the starter culture of any one of claims 2 to 4, the chitin deacetylase prepared by the process of claim 5, and the enzyme preparation of claim 6 for deacetylation of chitin.