阅读说明：本技术 乙酰氨基葡萄糖水解酶突变体及其应用 (Acetaminoglucosohydrolase mutant and application thereof ) 是由 陈振明 贺瑾 王志国 郑晨妮 方亚煊 于 2020-07-14 设计创作，主要内容包括：本发明公开了一种乙酰氨基葡萄糖水解酶突变体及其应用,通过对现有的N-乙酰氨基葡萄糖水解酶进行定点突变得到的一种酶活明显提高的N-乙酰氨基葡萄糖水解酶,所述N-乙酰氨基葡萄糖水解酶可以高效的水解N-乙酰氨基葡萄糖,显著提高酶转化过程中氨基葡萄糖的产生效率,从而在提高酶水解法的工业价值的同时还能减少环境污染。(The invention discloses an N-acetylglucosamine hydrolase mutant and application thereof, wherein the N-acetylglucosamine hydrolase with obviously improved enzyme activity is obtained by carrying out site-directed mutagenesis on the existing N-acetylglucosamine hydrolase, and the N-acetylglucosamine hydrolase can efficiently hydrolyze N-acetylglucosamine, thereby obviously improving the generation efficiency of the glucosamine in the enzyme conversion process, improving the industrial value of the enzymatic hydrolysis method and reducing the environmental pollution.)

1. An N-acetylglucosamine hydrolase characterized in that the amino acid sequence of said enzyme is such that asparagine at position 169 of the amino acid sequence corresponding to SEQ ID NO.1 is substituted with serine.

2. An N-acetylglucosaminhydrolase according to claim 1, wherein said enzyme is a protein selected from the group consisting of:

a) protein coded by an amino acid sequence shown as SEQ ID NO. 2;

b) a fusion protein obtained by connecting a label to the N end and/or the C end of the protein shown in the 1 st to 270 th sites in the amino acid sequence shown in SEQ ID NO. 2;

c) the protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues in the amino acid sequence shown in the 1 st to 270 th positions in the amino acid sequence shown in SEQ ID NO.2 and has the same function.

3. The N-acetylglucosamine hydrolase according to claim 2, wherein the protein having the same function as that of the amino acid sequence shown in SEQ ID No.2, wherein the amino acid sequence is the amino acid sequence shown in positions 1 to 270, which is obtained by substitution and/or deletion and/or addition of one or more amino acid residues, is: a protein having at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% similarity to the amino acid sequence shown in SEQ ID No.2 and having N-acetylglucosamine hydrolysing activity.

4. A nucleic acid molecule encoding the N-acetylglucosamine hydrolase of claim 1.

5. The nucleic acid molecule according to claim 4, wherein the nucleic acid molecule is a DNA molecule of 1) or 2) or 3) as follows:

1) DNA molecule with nucleotide sequence shown in SEQ ID NO. 3;

2) a DNA molecule having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% similarity to the DNA sequence defined in 1) and encoding said protein;

3) a DNA molecule which encodes the amino acid sequence of the protein of claim 1 and the conditions of 1) or 2) above, and which is obtained by codon optimization.

6. An expression vector comprising the nucleic acid molecule of claim 4 or 5.

7. A host cell comprising the nucleic acid molecule of claim 4 or comprising the expression vector of claim 6.

8. The host cell of claim 7, wherein the host cell is E.coli.

9. A method for producing N-acetylglucosamine hydrolase, comprising: the N-acetylglucosamine hydrolase is obtained by culturing the host cell according to claim 7 or 8.

10. A method of producing glucosamine, the method comprising: catalyzing the reaction of N-acetylglucosamine with the N-acetylglucosamine hydrolase of any one of claims 1 to 3 to obtain glucosamine.

Technical Field

The invention relates to the technical field of biology, and particularly relates to an acetylglucosamine hydrolase mutant and application thereof.

Background

Glucosamine (GlcN) has the chemical formula C₆H₁₃O₅N, commonly known as aminosugar, also called Glucosamine, Glucosamine or Glucosamine, called Glucosamine for short, is a product obtained by replacing hydroxyl at the 2-position of a glucose ring by amino, has high solubility in water and hydrophilic solvents, and mainly exists in the forms of Glucosamine Hydrochloride (Glucosamine Hydrochloride), Glucosamine sulfate (Glucosamine sulfate) and N-acetylglucosamine (GlcNAc); glucosamine 6-phosphate, the natural form of glucosamine, is a biochemical precursor of all glycoses.

The glucosamine is mainly applied to the industries of medicine, food and health care products, and has wide application. In the pharmaceutical industry, exogenous supplementation of glucosamine compounds helps polymorphic cells of a human body to synthesize aminopolysaccharides and glycoproteins, stimulates chondrocyte regeneration to help repair damaged cartilage tissues, blocks the pathological process of arthritis, relieves pain of osteoarthritis patients, and improves joint function. In addition, glucosamine has immunoregulatory, antitumor and free radical scavenging effects. In the food industry, the glucosamine hydrochloride has obvious inhibition effect on common bacteria, mold and yeast in the food industry, and has no toxic effect on human bodies, so the glucosamine hydrochloride has good market prospect as a natural food preservative.

The current research on the biosynthesis pathway of glucosamine shows that in microorganisms, glucosamine can strongly inhibit the activity of a key enzyme glmS in the synthesis pathway, and if the glucosamine is accumulated in a large amount in cells, the glucosamine has toxic effect on cells, while N-acetylglucosamine (GlcNAc) has stable properties and no inhibition effect on metabolic processes. Therefore, when the glucosamine is produced by the engineering bacteria fermentation method, the glucosamine is obtained by taking the N-acetylglucosamine as a fermentation target product and then performing extracellular deacetylation hydrolysis.

Currently, N-acetylglucosamine is hydrolyzed extracellularly by acid hydrolysis, but this method causes environmental pollution, enzyme catalysis has long been considered as one of the greenest chemical synthesis technologies, and at present, there is no mature N-acetylglucosamine enzymatic hydrolysis technology. There are only sporadic reports, and none of them have been based on the development of enzymatic hydrolysis processes, nor have the activity of hydrolytic enzymes high. Therefore, the excavation of the N-acetylglucosamine hydrolase with higher activity has important industrial application value and potential.

Disclosure of Invention

The invention provides an acetylglucosamine hydrolase mutant and application thereof, and aims to solve the problems that the activity of the existing N-acetylglucosamine hydrolase is not high, and the glucosamine production efficiency in the enzymatic conversion process is low.

In order to improve the above-mentioned problems, in a first aspect, the present invention provides an N-acetylglucosamine hydrolase whose amino acid sequence is such that the asparagine at position 169 in the amino acid sequence corresponding to SEQ ID NO.1 is substituted with serine.

In one embodiment, the enzyme is a protein selected from the group consisting of:

a) protein coded by an amino acid sequence shown as SEQ ID NO. 2;

b) a fusion protein obtained by connecting a label to the N end and/or the C end of the protein shown in the 1 st to 270 th sites in the amino acid sequence shown in SEQ ID NO. 2;

c) the protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues in the amino acid sequence shown in the 1 st to 270 th positions in the amino acid sequence shown in SEQ ID NO.2 and has the same function.

Wherein, the protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues in the amino acid sequence shown in the 1 st to 270 th positions in the amino acid sequence shown in SEQ ID NO.2 and has the same function refers to:

a protein having at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% similarity to the amino acid sequence shown in SEQ ID No.2 and having hydrolase activity.

In a second aspect, the present invention provides a nucleic acid molecule encoding the gene for N-acetylglucosamine hydrolase according to the first aspect.

Wherein the nucleic acid molecule is a DNA molecule of 1) or 2) or 3) or 4) as follows:

1) DNA molecule with nucleotide sequence shown in SEQ ID NO. 3;

2) a DNA molecule having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% similarity to the DNA sequence defined in 1) and encoding said protein;

3) a DNA molecule which encodes the amino acid sequence of the protein of claim 1 and the conditions of 1) or 2) above, and which is obtained by codon optimization.

In a third aspect, the present invention provides an expression vector comprising a nucleic acid molecule according to the second aspect.

Wherein, the expression vector can be a plasmid, a cosmid, a phage or a viral vector.

In a fourth aspect, the present invention provides a host cell comprising an expression vector according to the third aspect or comprising a nucleic acid molecule according to the second aspect.

Wherein, the host cell can be selected from escherichia coli, bacillus, pichia pastoris or aspergillus niger; preferably Escherichia coli (Escherichia coli).

In a fifth aspect, the present invention also provides a method for preparing N-acetylglucosamine hydrolase, comprising: the N-acetylglucosamine hydrolase is obtained by culturing the host cell of the fourth aspect.

In a sixth aspect, the present invention also provides a method for preparing N-acetylglucosamine hydrolase, comprising the steps of: hydrolyzing N-acetylglucosamine with the N-acetylglucosamine hydrolase described in the first aspect to obtain glucosamine.

Has the advantages that: the invention provides N-acetylglucosamine hydrolase which has the capability of hydrolyzing N-acetylglucosamine, has higher catalytic activity compared with the existing N-acetylglucosamine hydrolase, can efficiently hydrolyze the N-acetylglucosamine, and obviously improves the generation efficiency of the glucosamine in the enzyme conversion process, thereby improving the industrial value of the enzymatic hydrolysis method and reducing the environmental pollution.

Drawings

In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings needed to be used in the description of the embodiments will be briefly introduced below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings based on these drawings without creative efforts.

FIG. 1 shows the result of nucleic acid electrophoresis detection of N-acetylglucosamine hydrolase pET-28a (+) -Tk-169S;

FIG. 2 is a diagram showing the alignment of amino acid sequencing of the expression of N-acetylglucosamine hydrolase pET-28a (+) -Tk-169S provided in the examples of the present invention;

FIG. 3 is a protein electrophoresis diagram of N-acetylglucosamine hydrolase provided in the examples of the present invention.

Detailed Description

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The experimental methods used in the following examples are conventional methods unless otherwise specified, and materials, reagents and the like used in the following examples are commercially available without otherwise specified.

Definition and description:

amino acids in the present invention are represented by a single or three letter code and have the following meanings: a: ala (alanine); r: arg (arginine); n: asn (asparagine); d: asp (aspartic acid); c: cys (cysteine); q: gln (glutamine); e: glu (glutamic acid); g: gly (glycine); h: his (histidine); i: ile (isoleucine); l: leu (leucine); k: lys (lysine); m: met (methionine); f: phe (phenylalanine); p: pro (proline); s: ser (serine); t: thr (threonine); w: trp (tryptophan); y: tyr (tyrosine); v: val (valine).

In the present invention, the term "substitution" with respect to an amino acid position or residue means that the amino acid at a specific position has been replaced with another amino acid. Substitutions may be conservative or non-conservative.

The term "nucleic acid molecule" as used herein is intended to have the meaning generally understood by those of ordinary skill in the art, and a nucleic acid molecule may be a polynucleotide comprising a polynucleotide as set forth in SEQ ID NO.3, and may also comprise additional coding and/or non-coding sequences. In a particular embodiment of the invention, the nucleic acid molecule may be a DNA molecule having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% similarity to the DNA encoding the amino acid sequence shown in SEQ ID NO. 2. The nucleic acid molecules of the invention also include codon-optimized nucleic acid molecules of the polynucleotides shown in SEQ ID NO.3, for which codon optimization methods known in the art, such as the replacement of degenerate codons, can be used.

The term "expression vector" as used herein is intended to have the meaning generally understood by one of ordinary skill in the art, and can be any vector (e.g., a plasmid or virus) that can be conveniently subjected to recombinant DNA procedures and caused to express a polynucleotide. The choice of vector will typically depend on the compatibility of the vector with the host cell into which the vector is to be introduced. The vector may be a linear or circular plasmid. The vector preferably contains one or more selectable markers that allow for convenient selection of transformed, transfected, transduced, or the like cells. A selectable marker is a gene the product of which provides antibiotic or viral resistance, heavy metal resistance, prototrophy to auxotrophs, and the like.

The term "host cell" as used herein is a cell containing a nucleic acid molecule or expression vector of the invention. In other words, the present invention may utilize any host cell as long as the cell contains the nucleic acid molecule or expression vector of the present invention and is capable of producing an amino acid. Such as prokaryotic or eukaryotic cells.

The prokaryotic host cell may be any gram-positive or gram-negative bacterium, including but not limited to: bacillus, Clostridium, Lactobacillus, Streptomyces, Staphylococcus, Escherichia coli, Pseudomonas, and Paenibacillus.

Eukaryotic host cells may be mammalian, insect, plant, or fungal cells, including but not limited to: filamentous fungi (Aspergillus, Mucor, Rhizopus, Penicillium, etc.), yeasts (Pichia, Candida, Hansenula, etc.).

The term "corresponding to" as used herein is used to identify a specific position of a mutated amino acid or a mutated base in an amino acid sequence or a nucleotide sequence. Specifically, "corresponding to" means the position of one sequence corresponding to a specified position in the other sequence after alignment of the two sequences by homology or sequence identity. Thus, for example, in the case of "amino acid residue corresponding to position 40 of any one of the amino acid sequences shown in SEQ ID NO: 1", if a 6 XHis tag is added to one end of any one of the amino acid sequences shown in SEQ ID NO:1, position 40 of the resulting mutant corresponding to any one of the amino acid sequences shown in SEQ ID NO1 may be position 46. The alignment of the sequences can be done manually, by alignment software, or by an online alignment website.

The term "similarity" as used herein refers to sequence similarity to a native nucleotide sequence, for example, similarity includes a nucleotide sequence having 80% or more, or 85% or more, or 90% or more similarity to a protein nucleotide sequence consisting of the amino acid sequence shown at positions 1 to 270 shown in SEQ ID NO.2 of the present invention. Similarity can be assessed visually or by computer software. Using computer software, the similarity between two or more sequences can be expressed as a percentage (%), which can be used to assess similarity between related sequences.

"acetylglucosamine hydrolase" herein has the same meaning as "N-acetylglucosamine hydrolase (N-acetylglucosamine hydrolase)" in which the acetyl group of N-acetylglucosamine hydrolyzed by N-acetylglucosamine hydrolase is attached to the N atom. "hydrolysis" as used herein is the process of deacetylating N-acetylglucosamine.