阅读说明：本技术 一种脱乙酰酶及其编码基因与应用 (A kind of deacetylase and its encoding gene and application ) 是由 赵黎明 秦臻 马佳菲 邱勇隽 于 2019-09-24 设计创作，主要内容包括：本发明提供了一种脱乙酰酶及其编码基因与应用。本发明的脱乙酰酶是如下a)或b)或c)：a)如SEQ ID NO.2所示氨基酸序列编码的蛋白质；b)如SEQ ID NO.2所示的蛋白质的N端和/或C端连接标签得到的融合蛋白质；c)将如SEQ ID NO.2所示的氨基酸序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加得到的且具有相同功能的蛋白质。本发明进一步涉及此类蛋白质在氨基葡萄糖制备中的用途。本发明还涉及与所述蛋白质相关的生物材料。本发明所述脱乙酰酶能够高效催化N-乙酰氨基葡萄糖单糖发生脱乙酰反应,可应用于工业化制备氨基葡萄糖,其反应条件温和,转化率高,无污染物产生,具有较好的工业应用前景。(The present invention provides a kind of deacetylase and its encoding gene and applications.Deacetylase of the invention be it is following a) or b) or c): a) the amino acid sequences encoded protein as shown in SEQ ID NO.2；B) fused protein that the N-terminal of the protein as shown in SEQ ID NO.2 and/or C-terminal connection label obtain；C) protein obtaining the amino acid sequence as shown in SEQ ID NO.2 by the substitution and/or deletion and/or addition of one or several amino acid residues and with the same function.The invention further relates to purposes of this proteinoid in Glucosamine preparation.The invention further relates to biomaterials relevant to the protein.Deacetylase of the present invention can efficient catalytic N-acetylglucosamine monosaccharide occur deacetylation, can be applied to preparation of industrialization Glucosamine, reaction condition is mild, high conversion rate, contamination-free generate, have preferable prospects for commercial application.)

1. a kind of protein, which is characterized in that be following protein a) or b) or c):

A) the amino acid sequences encoded protein as shown in SEQ ID NO.2；

B) N-terminal of protein shown in 1-289 and/or C-terminal connect label in the amino acid sequence as shown in SEQ ID NO.2 Obtained fused protein；

C) amino acid sequence shown in 1-289 passes through one or several ammonia in the amino acid sequence as shown in SEQ ID NO.2 Protein that the substitution and/or deletion and/or addition of base acid residue obtain and with the same function.

2. a kind of protein according to claim 1, which is characterized in that in the amino acid sequence as shown in SEQ ID NO.2 Amino acid sequence shown in 1-289 is obtained by the substitution and/or deletion and/or addition of one or several amino acid residues And protein with the same function, refer to:

At least have 90% with amino acid sequence shown in SEQ ID NO.2, at least with 95%, at least has 96%, at least has Have 97%, at least with 98% or at least with 99% similitude, and the protein with deacetylase activity.

Any one of 3. biomaterial relevant to protein described in claim 1, which is characterized in that be following B1)-B5):

B1 the nucleic acid molecules of protein described in claim 1) are encoded；

B2) contain B1) expression cassettes of the nucleic acid molecules；

B3) contain B1) recombinant vectors of the nucleic acid molecules or contain B2) recombinant vector of the expression cassette；

B4) contain B1) recombinant bacteriums of the nucleic acid molecules or contain B2) recombinant bacterium of the expression cassette or contain B3) it is described heavy The recombinant bacterium of group carrier；

B5) contain B1) cell line of the nucleic acid molecules or contain B2) cell line of the expression cassette or contain B3) it is described heavy The cell line of group carrier.

4. biomaterial according to claim 3, which is characterized in that B1) nucleic acid molecules be it is following 1) or 2) or 3) or 4) DNA molecular:

1) nucleotide sequence DNA molecular as shown in SEQ ID NO.1；

2) at least have 85% with the DNA sequence dna 1) limited, at least have 90%, at least with 95%, at least with 96%, extremely Less with 97%, at least with 98% or at least with the DNA molecular of 99% similitude；

1) or 2) 3) hybridize under strict conditions with the DNA sequence dna limited and the DNA for encoding protein described in claim 1 divides Son；

4) DNA molecular of protein amino acid sequence described in the coding claim 1 obtained after codon optimization is utilized.

5. application of the protein described in claim 1 as deacetylase.

6. application of the protein described in claim 1 as N-acetylglucosamine deacetylase.

7. a kind of preparation method of deacetylase, which comprises the steps of:

1) place of recombinant bacterium described in claim 3 is cultivated under conditions of being beneficial to generate protein as described in claim 1 Chief cell；

2) albumen, as deacetylase are recycled.

8. a kind of method for producing Glucosamine, which comprises the steps of: with albumen described in claim 1 Matter is catalyzed the deacetylation of N-acetylglucosamine, obtains Glucosamine product.

Technical field

The invention belongs to field of biotechnology, and in particular to the deacetylase and its encoding gene of a kind of bacterial origin with answer With.

Background technique

Glucosamine (C₆H₁₃NO₅) it is also known as ammonia sugar, gucosamine or aminoglucose, it is a kind of amino-containing natural list Sugar.Glucosamine is one of the most abundant monosaccharide of nature content, is in human body to be present in articular cartilage, connective tissue more In cell membrane.Glucosamine is also the important as precursors in organism protein or lipid glycosylation, is to form cartilage The important nutrient of cell is the natural tissues composition of healthy articular cartilage.

Glucosamine is a kind of important functional food and a kind of adjuvant therapy medicaments, can mainly releive because Pain caused by arthritis, stiff and swelling；Pain caused by releiving because of arthritis, stiff and swelling；Lubricating joint and maintenance Function of joint.Glucosamine is the indispensable substance of human body, and domestic and international market demand is also growing in recent years, especially Demand in fields such as medical product, functional food, cosmetics is continuously increased.

The traditional preparation methods of Glucosamine are mainly chitin Hydrolyze method, i.e., handle ocean chitin money by soda acid Source (shrimp and crab shells) obtains chitin raw material, then final glucosamine product is obtained by sour water solution and deacetylation.It passes The a large amount of acid of chemical hydrolysis consumption, the alkali of system, environmental pollution is serious, and industry faces upgrading and transformation.In addition, traditional chemical hydrolyzes Method is serious to Yu Haiyang's chitin Resource Dependence, and industry size is extremely restricted.In recent years, microbe fermentation method prepares amino Glucose becomes the trend of industry development, prepares aminoglucose using the synthetic biology means building direct transforming glucose of engineering The technology of sugar is also increasingly mature.But since microorganism is limited to aminoglucose sugared tolerance in fermentation process, fermentation method production Glucosamine exists generally in the form of N-acetylglucosamine, subsequent that further deacetylation is also needed to obtain final ammonia Base glucose product.N-acetylglucosamine is deacetylated at present generallys use Hydrochloric Acid Hydrolysis Method, that is, uses concentrated hydrochloric acid high-temperature water N-acetylglucosamine is solved, generates Glucosamine after deacetylate.The Hydrochloric Acid Hydrolysis Method production cycle is longer, generates a large amount of Spent acid and by-product, to glucosamine product quality and environmental protection adversely affect.

Chitin deacetylase class single-minded can be catalyzed glycan, oligosaccharides or the list formed by N-acetylglucosamine monomer The deacetylation of sugar, reaction condition is mild, easy to operate, low in the pollution of the environment, is a kind of de- second of potential Glucosamine Acyl preparation method.However, catalytic activity, the reasons such as cost there is no commercialization specificity to prepare amino at present due to enzyme stability The deacetylase product of glucose.In addition, deacetylated work of most of reported deacetylated enzyme for chitosan, oligosaccharides Property is higher, and lower to the deacetylase activity of N-acetylglucosamine monosaccharide, is unsuitable for extensive N- acetamido glucose Sugared deacetylation industrial application.

Currently, have the deacetylase report of higher substrate adaptability also less N-acetylglucosamine monosaccharide, A kind of deacetylase and its encoding gene of high-glucosamine-yield as disclosed in Chinese patent CN107022538A.Therefore, it sends out The deacetylase gene with greater activity is dug, by the method for genetic recombination, constructs genetic engineering bacterium, efficient heterogenous expression is de- Acetyl enzyme explores the enzymolysis process that deacetylase prepares Glucosamine, has important industrial application value and potentiality.

Summary of the invention

Mesh of the invention is to provide a kind of deacetylase and its encoding gene and application.

The purpose of the present invention can be achieved through the following technical solutions:

It is an object of the present invention to provide a kind of protein.

Protein provided by the invention is following protein a) or b) or c), is named as CqDac,

A) the amino acid sequences encoded protein as shown in SEQ ID NO.2；

B) N-terminal of protein shown in 1-289 and/or C-terminal connection in the amino acid sequence as shown in SEQ ID NO.2 The fused protein that label obtains；

C) amino acid sequence shown in 1-289 passes through one or several in the amino acid sequence as shown in SEQ ID NO.2 Protein that the substitution and/or deletion and/or addition of a amino acid residue obtain and with the same function.

Wherein, in the amino acid sequence as shown in SEQ ID NO.2 amino acid sequence shown in 1-289 by one or Protein that the substitution and/or deletion and/or addition of several amino acid residues obtain and with the same function, refers to: with SEQ Amino acid sequence shown in ID NO.2 at least has 90%, at least has with 95%, at least with 96%, at least 97%, at least With 98% or at least with 99% similitude, and the protein with deacetylase activity.

It, can the amino acid sequence 1-289 amino acids shown in SEQ ID NO.2 in order to make above-mentioned protein convenient for purifying The amino terminal or carboxyl terminal of the protein of residue coding connect upper label as shown in Table 1.

The sequence of table 1, label

Label	Residue	Sequence
			Poly-Arg	5-6 (usually 5)	RRRRR
Poly-His	2-10 (usually 6)	HHHHHH
			FLAG	8	DYKDDDDK
Strep-tag II	8	WSHPQFEK
			c-myc	10	EQKLISEEDL

Above-mentioned a)-c) in protein can be artificial synthesized, can also first synthesize its encoding gene, then carry out biological expression and obtain It arrives.

Above-mentioned a)-c) in the encoding gene of protein can be by will be shown in shown in SEQ ID NO.1 1-867 The codon of one or several amino acid residues is lacked in DNA sequence dna, and/or the missense of one or several base-pairs of progress is dashed forward Become, and/or is obtained in the coded sequence that its 5 ' end and/or 3 ' ends connect label shown in table 1.

It is a further object to provide biomaterials relevant to above-mentioned protein.

Any one of biomaterial provided by the invention is following B1)-B5):

B1 the nucleic acid molecules of protein described in claim 1) are encoded；

B2) contain B1) expression cassettes of the nucleic acid molecules；

B3) contain B1) recombinant vectors of the nucleic acid molecules or contain B2) recombinant vector of the expression cassette；

B4) contain B1) recombinant bacteriums of the nucleic acid molecules or contain B2) recombinant bacterium of the expression cassette or contain B3) institute State the recombinant bacterium of recombinant vector；

B5) contain B1) cell line of the nucleic acid molecules or contain B2) cell line of the expression cassette or contain B3) institute State the cell line of recombinant vector.

Wherein, B1) nucleic acid molecules are following DNA molecular 1) or 2) or 3) or 4):

1) nucleotide sequence DNA molecular as shown in SEQ ID NO.1；

2) at least have 85% with the DNA sequence dna 1) limited, at least have 90%, at least having with 95%, at least 96%, at least with 97%, at least with 98% or at least DNA molecular with 99% similitude and code for said proteins；

1) or 2) 3) hybridize under strict conditions with the DNA sequence dna limited and encode the DNA of protein described in claim 1 Molecule；

4) using the amino acid sequence of protein described in the coding claim 1 obtained after codon optimization and it is above-mentioned 1) or 2) DNA molecular of the condition.

Wherein, the nucleic acid molecules can be DNA, such as cDNA, genomic DNA or recombinant DNA；The nucleic acid molecules can also To be RNA, such as mRNA or hnRNA.

B4 the recombinant bacterium described in) is that the encoding gene of above-mentioned protein is imported recombinant bacterium obtained in host strain.

In above-mentioned recombinant bacterium, the encoding gene of the protein is to import host strain by recombinant vector；

In above-mentioned recombinant bacterium, the host strain is Escherichia coli, bacillus, Pichia pastoris or aspergillus niger.

Those of ordinary skill in the art can easily adopt by known method, such as the side of directed evolution and point mutation Method is mutated the nucleotide sequence of coding CqDac of the invention.Those are by manually modified, with coding CqDac's The nucleotide of nucleotide sequence 85% or higher similitude is derived from this as long as encoding CqDac and having identical function The nucleotide sequence of invention and it is equal to sequence of the invention.

Term " similitude " used herein refers to the sequence similarity with native sequence nucleic acid.Similitude can with the naked eye or Computer software is evaluated.Using computer software, the similitude between two or more sequences can use percentage (%) It indicates, can be used to evaluate the similitude between correlated series.

In above-mentioned biomaterial, B2) described contain B1) expression cassettes of the nucleic acid molecules, the i.e. core containing coding CqDac The expression cassette (CqDac expression casette) of acid molecule is the DNA for referring to express CqDac in host cell, and the DNA is not only It may include the promoter for starting CqDac transcription, may also include the terminator for terminating CqDac transcription.Further, the expression cassette is also It may include enhancer sequence.Promoter for use in the present invention includes but is not limited to: constitutive promoter；Tissue, organ and hair Educate special promoter and inducible promoter.

In above-mentioned biomaterial, the carrier can be plasmid, sticking grain, bacteriophage or viral vectors.

It is a further object to provide the new applications of above-mentioned protein.

Application the present invention provides above-mentioned protein as deacetylase.The present invention also provides above-mentioned protein conducts The application of N-acetylglucosamine deacetylase.

Above-mentioned enzymatic activity refers in particular to the deacetylation of protein catalysis N-acetylglucosamine, generates amino Glucose products.

The present invention also provides a kind of preparation methods of deacetylase, include the following steps:

1) above-mentioned recombinant bacterium is cultivated under conditions of being beneficial to and generating protein described in first goal of the invention of the invention Host cell；

2) albumen is recycled, i.e. recombination deacetylase.

The last one purpose of the invention is to provide a kind of preparation method of Glucosamine.

The preparation method of Glucosamine provided by the invention includes the following steps:

1) above-mentioned recombinant bacterium is cultivated, N recombination deacetylase is obtained；

2) enzymatic treatment N-acetylglucosamine syrup, solution or the fermentation liquid are used, Glucosamine is obtained.

In the above method, the mass fraction of N-acetylglucosamine is 0.1- in the syrup, solution or fermentation liquid 65%.

In the above method, the deacetylated condition are as follows: pH 3.0-9.5,20-80 DEG C, reaction time 0.5-20 hour.

Compared with prior art, the beneficial effects of the present invention are:

A kind of protein with N-acetylglucosamine monosaccharide deacetylase activity provided by the present invention is (in embodiment In be specially recombinant protein c qDac) and its encoding gene；The protein can be with effectively hydrolyzing N- acetylamino as deacetylase Glucose is deacetylated to prepare Glucosamine.Compared with conventional chemical methods, spent acid alkali discharge amount is small, and energy-output ratio is few, conversion Rate is high, and the excellent catalytic performance of the enzyme makes it have better stability in industrial application.Albumen provided by the invention Matter has good N-acetylglucosamine deacetylase activity, in the industries such as Glucosamine preparation and food, medicine With good application value.

Detailed description of the invention

Fig. 1 shows purifying figure of the recombinant C qDac through Ni-IDA affinitive layer purification.

Wherein, swimming lane M represents low molecular weight standard protein, and swimming lane 1 represents crude enzyme liquid, and swimming lane 2 represents affine through Ni-IDA Destination protein after chromatography.

Fig. 2 shows CqDac to the deacetylation activity of N-acetylglucosamine.Reaction time is 5 hours, is shown in figure Show that reaction solution substrate N-acetylglucosamine largely can be converted into Glucosamine product by deacetylation.GlcNAc in figure N-acetylglucosamine is represented, GlcN represents Glucosamine.

Specific embodiment

Experimental method used in following embodiments is conventional method unless otherwise specified.

The materials, reagents and the like used in the following examples is commercially available unless otherwise specified.

The present invention analyzes a collection of genome sequence by the method that genome database is excavated, candidate a collection of in biology It is predicted to be in informatics and assumes deacetylase or the non-authentication function gene order with potential deacetylase activity.Described Method for digging carries out in ncbi database specifically, using chitin deacetylase gene disclosed in document as template probe Blast-P search, filters out the potential sequence with known array homology in 30-90%, and further progress structure domain analysis And protein family classification analysis, determine candidate sequence.

Method of the invention is described further below by specific embodiment combination attached drawing.Following embodiment is only used for It illustrates rather than for limiting the scope of the invention.