CN107893060B - Thermostable salt-tolerant SGNH family hydrolase derived from marine bacteria and application thereof - Google Patents

Abstract

The invention discloses a novel thermostable salt-tolerant organic solvent-tolerant SGNH family hydrolase AlinE4 derived from marine bacteria, and a coding gene and application thereof. The invention relates to a hydrolase gene aline4 from marine bacteria Alterythromobacter indicus DSM18604, and the nucleotide sequence is shown in SEQ ID NO. 1. After the hydrolase gene is subjected to heterologous expression, the hydrolase has esterase activity, the catalytic activity is highest when a substrate is p-nitrophenol butyrate (C4), and the enzyme activity is 25.8U/mg. The optimal pH and temperature for the catalyzed hydrolysis of alinE4 are 7.5 and 40 ℃; after incubation for 60min at 40, 50 and 60 ℃, the activity can still be maintained by more than 50 percent; under the condition of organic solvent and metal ion, the enzymatic activity is higher. The hydrolase has the characteristics of thermal stability and alkalinity, and can be applied to industrial production under the conditions of high temperature, salt content and organic solvent in the fields of detergents, wastewater treatment, fine chemical engineering, pharmacy, environmental remediation and the like.

Description

Thermostable salt-tolerant SGNH family hydrolase derived from marine bacteria and application thereof

Technical Field

The invention belongs to the field of genetic engineering, and particularly relates to a novel thermostable salt-tolerant SGNH family hydrolase derived from marine bacteria, an encoding gene thereof and application thereof.

Background

The SGNH family of hydrolases is a class of hydrolases that contain 4 strictly conserved catalytic residues serine, glycine, asparagine and histidine in 4 conserved sequence regions. SGNH family hydrolase widely exists in eukaryotes and prokaryotes, has various activities such as lipase, protease, thioesterase, aromatic esterase, lysophospholipase, carbohydrate esterase, acyltransferase and the like, is a hydrolase with a wide substrate spectrum, and can participate in various physiological functions such as bacterial virulence, plant growth and development, morphogenesis, defense and the like. The wide substrate spectrum and the functional diversity enable the family hydrolase to have wide potential application value, and become a hot point for research at home and abroad.

The SGNH family hydrolase reported at present is mostly derived from eukaryotes, and the family hydrolase derived from bacteria is few and has unclear functions. The sea is a huge treasure house of microbial resources, and the hydrolases from the sea generally have excellent properties related to the marine environment, such as temperature stability, salt resistance, alkali resistance, low temperature resistance, excellent chiral selectivity and the like, so that the screening of the hydrolases with unique characteristics from the marine microorganisms becomes an important direction for developing novel industrial enzyme preparations.

The invention screens a novel SGNH family hydrolase gene from marine bacteria, and carries out recombinant expression on the gene, and the recombinase has the characteristics of thermal stability, salt tolerance, organic solvent resistance and the like, and can be used in the industrial fields of fine chemistry industry, pharmacy, washing, wastewater treatment, environmental remediation and the like.

Disclosure of Invention

The invention aims to provide a novel marine bacteria-derived hydrolase, a coding gene thereof and a preparation method thereof, wherein the hydrolase can be used for biocatalysis and transformation of ester degradation and other ester compounds under high-temperature and high-salt conditions.

The present invention relates to isolated polypeptides having hydrolase activity, selected from the group consisting of:

(a) a polypeptide having a sequence identical to that shown for the polypeptide of SEQ ID NO. 2;

(b) the polypeptide is a mutant obtained by carrying out various substitutions, additions and/or deletions of one or more amino acids at the position far away from the catalytic center of the polypeptide shown in SEQ ID NO.2, and the mutant has at least 90 percent of homology and at least 90 percent of enzyme activity with a protein sequence shown in SEQ ID NO. 2.

The polypeptide with hydrolase activity is derived from the bacterium alternanthebacter indicus of the rhizobium soil of mangrove wild rice.

The present invention is directed to the bacterium Alterythrobacter indica DSM18604 isolated from the root nodule soil of mangrove wild rice, which strain was purchased from the German Collection of microorganisms and strains DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen). Based on the whole genome sequence, the hydrolase gene aline4 is obtained by screening, and the nucleotide sequence is shown as SEQ ID No. 1. The size of gene aline4 is 573bp, the basic group composition is 139A (24.26%), 115T (20.07%), 149C (26.00%) and 170G (29.67%), the size of the encoded protein is 190 amino acid residues, and the amino acid sequence is shown in SEQ ID No. 2. The amino acid sequence of the hydrolase AlinE4 is subjected to homologous search in a GenBank nr database, and the hydrolase is metagenome-derived aryl esterase with the highest consistency, wherein the consistency is 71 percent (the registration number of the hydrolase in the GenBank database is OJW 68931.1). The amino acid sequence analysis result shows that the sequence near the active site serine is a conserved region (the amino acid position is 11 to 14) consisting of glycine-aspartic acid-serine-leucine, and the 13 th serine, the 162 th aspartic acid and the 165 th histidine form a serine hydrolase catalytic triad together. In addition, serine at position 13, glycine at position 50 and asparagine at position 81 together constitute an oxyanion hole. The amino acid sequence characteristics of the polypeptide accord with the characteristics of SGNH hydrolase families. Taken together, AlinE4 should be a new member of the SGNH hydrolase family.

Under the premise of not influencing the activity of the AlinE4 protein, various substitutions, additions and/or deletions of one or more amino acids can be carried out on the amino acid sequence which is far away from the catalytic center amino acid position (preferably far away from the amino acid positions 11-14, 162 and 165) and is shown in SEQ ID NO.2 to obtain the derivative protein with the activity of the AlinE 4. According to the common general knowledge of the art, the biological activity of a protein is closely related to its functional domain. In general, only site mutations occurring in functional domains may have an effect on the two-and three-dimensional structure of a protein, thereby affecting its biological activity. Whereas for amino acid positions occurring away from the functional domain (preferably amino acid positions 11-14, 162 and 165), since this region is not involved in the functional conformation of the protein, individual point mutations of the amino acids do not have a substantial effect on the biological activity of the protein, thereby enabling the biological function of the original protein to be substantially retained. Preferred mutants of the hydrolase AlinE4 have at least 90% homology with the amino acid sequence shown in SEQ ID No.2, more preferably at least 95% homology, and most preferably at least 99% homology. The mutant can basically retain the biological function of the hydrolase AlinE4, and preferably has the enzymatic activity of at least 90 percent, more preferably at least 95 percent and most preferably at least 99 percent of the enzymatic activity of the hydrolase with the amino acid sequence shown in SEQ ID NO. 2.

The present invention relates to artificial variants of the mature polypeptide of SEQ ID No.2 or homologous sequences thereof comprising substitutions, deletions and/or insertions of one or more amino acids, preferably at mutation positions of less than 5, more preferably less than 3, most preferably only 1 amino acid. Examples of conservative substitutions are within the following groups: the basic amino acid group (arginine, lysine and histidine), the acidic amino acid group (glutamic acid and aspartic acid), the polar amino acid group (glutamine and asparagine), the hydrophobic amino acid group (leucine, isoleucine and valine), the aromatic amino acid group (phenylalanine, tryptophan and tyrosine) and the small amino acid group (glycine, alanine, serine, threonine and methionine). Amino acid substitutions that do not generally alter specific activity are known in the art and are described, for example, by H Neurath and R.L.Hill, 1979 in the proteins, Academic Press, New York. The most commonly occurring exchanges are Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Tyr/Phe, Ala/Pro, Lys/Arg, Asp/Asn, Leu/Ile, Leu/Val, Ala/Glu, Asp/Gly, and the like.

Known mutagenesis, recombination and/or shuffling methods can be used, followed by relevant screening procedures, as described by Reidhaar-Olson and Sauer, 1988, Science, 241: 53-57; bowie and Sauer, 1989, Proc. Natl. Acad. Sci. USA86: 2152-2156; those disclosed in WO95/17413 or WO 95/22625, in which one or more amino acid substitutions, deletions and/or insertions are made and tested. Other methods that can be used include error-prone PCR, phage display (e.g., Lowman et al, 1991, Biochemistry30: 10832-;

U.S. Pat. nos. 5,223,409; WO92/06204) and region-directed mutagenesis (Derbyshire et al, 1986, Gene46:145 and 1988, DNA7: 127).

The present invention also relates to isolated polynucleotides comprising or consisting of a nucleotide sequence encoding the hydrolase AlinE4 of the present invention, or a mutant having the activity of the hydrolase AlinE 4.

The present invention relates to isolated polynucleotides encoding polypeptides having the activity of hydrolase AlinE4, selected from the group consisting of:

(a) a polynucleotide which is identical to the sequence shown by the nucleotide sequence of SEQ ID NO. 1;

(b) the polynucleotide is a polynucleotide which encodes a mutant obtained by variously substituting, adding and/or deleting one or more amino acids in an amino acid sequence which is far away from the catalytic center and is shown in SEQ ID NO.1, and the polynucleotide has homology of at least 90 percent with the nucleotide sequence shown in SEQ ID NO. 1.

The invention provides a gene AlinE4 for coding hydrolase alinE4, which is consistent with a nucleotide sequence shown in SEQ ID NO. 1; the size of gene aline4 is 573bp, the basic group composition is 139A (24.26%), 115T (20.07%), 149C (26.00%) and 170G (29.67%), the size of the encoded protein is 190 amino acid residues, and the amino acid sequence is shown in SEQ ID No. 2. The invention also provides a method for obtaining a mutant gene which codes for a mutant capable of basically keeping the biological activity of the Aline4 protein by replacing, adding and/or deleting one or more nucleotides in the nucleotide sequence shown in SEQ ID NO.1 except the nucleotides at positions 31-42, 484-495 and 493-495. Preferred hydrolase AlinE4 mutant genes have at least 90% or more homology with the nucleotide sequence shown in SEQ ID NO.1, more preferably at least 95% or more homology, and most preferably at least 99% or more homology.

The invention also relates to nucleic acid constructs comprising the isolated polynucleotides of the invention, which can be manipulated in a number of ways to provide for expression of the hydrolase. The isolated polynucleotide is operably linked to one or more control sequences that direct the expression of the coding sequence in a suitable host cell under conditions compatible with the control sequences. The control sequence may be an appropriate promoter sequence, a nucleotide sequence recognized by a host cell for expression of a polynucleotide encoding a polypeptide of the present invention. The promoter sequence contains transcriptional regulatory sequences that mediate the expression of the polypeptide. The promoter may be any nucleotide sequence which shows transcriptional activity in the host cell of choice including mutant, truncated, and hybrid promoters, and may be obtained from genes encoding extracellular or intracellular polypeptides either homologous or heterologous to the host cell.

The cloned hydrolase AlinE4 gene can be connected to a proper vector by using a gene cloning technology, and is transformed or transfected into a prokaryotic or eukaryotic host for expression to prepare the recombinant hydrolase AlinE 4. Suitable prokaryotic hosts include various bacteria such as e.coli and the like, and suitable eukaryotic hosts include yeast (e.g., methanol yeast) and mammalian cells (e.g., chinese hamster ovary cells) and the like, preferably with the prokaryotic expression system e.coli.

Suitable vectors are commercially available prokaryotic or eukaryotic expression vectors, such as pET series vectors, pQE series vectors, yeast expression vectors pPICZ- α -A, pHIL-D2, pPIC9, pHIL-S1(Invitrogen Corp. san Diego. California. USA), animal cell expression vectors pSVK3, pMSG (Amersham Pharmacia Biotech Inc. USA), and the like, which are well known to those skilled in the art.

The present invention also relates to recombinant host cells comprising the isolated polynucleotides of the present invention, which may be advantageously used in the recombinant production of the hydrolase AlinE 4. Vectors comprising a polynucleotide of the invention are introduced into host cells, the choice of which depends to a large extent on the gene encoding the polypeptide and its source. The host cell may be any cell useful in the recombinant production of the hydrolase AlinE4 of the invention, e.g., a prokaryotic or eukaryotic cell. The cloned hydrolase AlinE4 gene can be connected to a proper vector by using a gene cloning technology, and is transformed or transfected into a prokaryotic or eukaryotic host for expression to prepare the recombinant hydrolase AlinE 4. Suitable prokaryotic hosts include various bacteria such as e.coli, etc., and vectors can be transformed into prokaryotic cells by protoplast transformation or electroporation as follows. Suitable eukaryotic hosts include yeast (e.g., methylotrophic yeast), mammalian cells (e.g., chinese hamster ovary cells), and the like. Coli expression in the prokaryotic expression system E.coli is preferably adopted in the invention for producing the hydrolase AlinE 4. A preferred example is that the hydrolase gene aline4 screened by the present invention is connected to an Escherichia coli expression vector pET28a, and transformed into Escherichia coli BL21(DE3), and a high activity recombinase is induced and expressed.

The present invention also relates to a method for producing the hydrolase AlinE4 of the invention, comprising:

(a) cultivating a recombinant host cell under conditions conducive for production of the hydrolase AlinE4, wherein the host cell comprises the nucleotide sequence set forth in SEQ ID No.1 or at least one mutation site thereof;

(b) recovering the polypeptide.

In the production methods of the invention, the cells are cultured in a nutrient medium suitable for production of the hydrolase AlinE4 using methods known in the art. For example, the cells may be cultured by shake flask culture, and small-scale or large-scale fermentation (including continuous, batch, fed-batch, or solid state fermentations) in laboratory or industrial fermentors performed in a suitable medium and under conditions allowing the hydrolase, AlinE4, to be expressed and/or isolated. The cultivation is carried out using methods known in the art in a suitable nutrient medium comprising carbon and nitrogen sources and inorganic salts. Suitable media are available from commercial suppliers or may be prepared according to published compositions. If the polypeptide is secreted into the nutrient medium, the polypeptide can be recovered directly from the medium. If the polypeptide is not secreted, it can be recovered from cell lysates.

The resulting hydrolase AlinE4 can be recovered using methods known in the art. For example, recovery from the nutrient medium may be by conventional methods including, but not limited to, centrifugation, filtration, extraction, spray drying, evaporation, or precipitation. Purification can be accomplished by a variety of methods known in the art including, but not limited to, chromatographic (e.g., ion exchange, affinity, hydrophobic, chromatofocusing, and size exclusion) or differential solubility (e.g., ammonium sulfate precipitation) methods and the like.

The invention also provides the industrial application of the hydrolase AlinE4 or the host bacterium capable of expressing the hydrolase AlinE4, for example, the host bacterium can be used for catalyzing ester hydrolysis. The esterase activity determination shows that the hydrolase AlinE4 has esterase activity. The AlinE4 or the host bacteria capable of expressing the AlinE4 can be used for hydrolyzing short-chain fatty acid esters, such as C2-C8 short-chain fatty acid esters, and has a certain degradation effect on long-chain fatty acid esters of C10-C14. The preferred short-chain fatty acid ester is p-nitrophenol ester with C2-C8 short carbon chain, such as p-nitrophenol acetate, p-nitrophenol butyrate, p-nitrophenol hexanoate, p-nitrophenol octanoate, etc., wherein the catalytic activity is the highest when the substrate is p-nitrophenol butyrate (C4), and the enzyme activity is 25.8U/mg.

The catalytic hydrolysis temperature range of the AlinE4 is 15-60 ℃, and preferably 40 ℃; the pH value of the hydrolysis is 6.0-10.5, and preferably 7.5. The activity can still be maintained by more than 80% under the conditions of incubation for 1h and 4h at the temperature of 10-60 ℃; after incubation for 0.5h and 1h at 90 ℃, the activity of the AlinE4 can be kept more than 45%; the activity of AlinE4 can be enhanced under the conditions of 0.5mol/L and 1mol/L NaCl, and more than 95% of activity can be retained under the conditions of 2mol/L and 1mol/L NaCl; at Ba²⁺、Ca²⁺、Mg²⁺And Sr²⁺More than 85% of activity can be reserved in the presence of the compound; ethanol, DMSO, glycerol, isopropanol, and methanol enhance their activities.

The invention screens and obtains a new heat-stable salt-tolerant organic solvent-tolerant hydrolase gene from Rhizobium rhizobium bacteria DSM18604 of mangrove wild rice, finds that the gene encoding protein has excellent enzymological characteristics, and can be applied to the production process of catalytic ester decomposition and enzyme method synthetic ester products. The obtained hydrolase gene can be cloned into a proper host to realize heterologous expression, so that the industrial production of the thermostable salt-tolerant organic solvent hydrolase is realized, and a low-cost thermostable salt-tolerant organic solvent hydrolase starting material is provided for subsequent industrial application. The production of the enzyme can show important economic and social values in the production processes of detergents, wastewater treatment, fine chemical engineering, pharmacy, environmental remediation and the like with high temperature, salt content and organic solvent.

Drawings

FIG. 1 is a diagram of gel electrophoresis analysis of a purified hydrolase, AlinE 4.

FIG. 2 is a substrate specificity diagram of the hydrolase AlinE 4. C2: p-nitrophenol acetate; c4: p-nitrophenol butyrate, C6: p-nitrophenol hexanoate; c8: p-nitrophenol octanoate; c10: p-nitrophenol decanoate; c12: p-nitrophenol dodecanoate; c14: p-nitrophenol myristate; c16: p-nitrophenol palmitate; the measurement was 100% when the substrate was defined as C4.

FIG. 3 is a graph showing the optimum reaction pH for the hydrolase AlinE 4.

FIG. 4 is a graph showing the optimum reaction temperature for the hydrolase AlinE 4.

FIG. 5 is a graph showing the thermal stability of the hydrolase AlinE4 at different temperatures.

FIG. 6 is a graph of the thermal stability of the hydrolase AlinE4 at elevated temperatures over time.

FIG. 7 is a photograph of the activity of NaCl on the hydrolase AlinE 4.

FIG. 8 is a graph showing the effect of divalent cations on the activity of the hydrolase AlinE 4.

FIG. 9 is a graph showing the effect of organic solvent on the activity of the hydrolase AlinE 4.

Detailed Description

EXAMPLE 1 acquisition of hydrolase Gene aline4

Based on the whole genome, open reading frame prediction and gene annotation results of the bacterium Alterythobacter indica DSM18604 isolated from the root nodule soil of mangrove wild rice, genes related to the lipid hydrolase were screened. The homology of the sequences with known hydrolase gene sequences in the database was aligned by means of Blastp (http:// blast. ncbi. nlm. nih. gov. /). The aline4 gene is obtained by database alignment analysis, the size is 573bp, the base composition is 139A (24.26%), 115T (20.07%), 149C (26.00%) and 170G (29.67%), and the nucleotide sequence is shown as SEQ ID No. 1. The size of the encoded protein is 190 amino acid residues, and the amino acid sequence of the encoded protein is shown as SEQ ID No. 2. The AlinE4 protein sequence was subjected to homology search in GenBank, and the esterase of non-cultured origin with the highest amino acid sequence identity was found to have 71% identity, which is registered with the GenBank database under accession No. OJW 68931.1.

Phylogenetic analysis shows that the hydrolase AlinE4 belongs to esterase II family and also belongs to SGNH hydrolase family. The amino acid sequence analysis result shows that the sequence near the serine of the active site is a conserved region (the amino acid positions are 11 to 14) consisting of glycine-aspartic acid-serine-leucine, and the serine at the 13 position, the aspartic acid at the 162 position and the histidine at the 165 position jointly form a catalytic center of the serine hydrolase. The 13 th serine, the 50 th glycine and the 81 th asparagine together form an oxygen anion hole. The amino acid sequence characteristics of the polypeptide accord with the characteristics of SGNH hydrolase families.

In conclusion, AlinE4 should be a new member of the esterase family and the SGNH hydrolase family.

EXAMPLE 2 construction of recombinant expression plasmid and recombinant Strain of Gene aline4

The gene aline4 obtained by the invention is cloned to an expression vector to construct a recombinant expression strain. Based on the gene open reading frame sequence obtained by ORF analysis of NCBIORF Finder, an upstream primer aline4F (5' -TCGC) for amplifying the whole gene is designedGGATCCATGGGCGAATCGCGC-3 ', BamHI) and a reverse primer aline4R (5' -TCCG)CTCGAGTCACTTCTTCGCAGGCAGCGCC-3', XhoI), PCR amplification confirmed the full-length sequence of the gene. Constructing expression plasmid by enzyme cutting cloning method, namely, using BamHI and XhoI double-enzyme cutting PCR product, connecting the purified fragment with BamHI and XhoI double-enzyme cutting plasmid pSMT3, and using CaCl₂Extracting plasmid of the positive clone by adopting a plasmid extraction kit (Omega, USA), obtaining a DNA fragment of about 600bp through BamHI and XhoI double enzyme digestion identification, identifying the DNA fragment as a gene aline4 through sequencing, transforming the recombinant expression plasmid into an E.coli Rosetta (DE3) expression strain, and constructing the expression recombinant strain.

Example 3 expression of the recombinant Gene aline4 Using recombinant expression strains

The constructed 3ml recombinant expression strain was transferred to 100ml LB liquid medium containing 20. mu.g/ml kanamycin and 34. mu.g/ml chloramphenicol, and cultured with shaking at 37 ℃ to OD₆₀₀The content of the organic acid is up to 0.6,the final concentration of IPTG was 0.5mM was added for induction expression, and the mixture was transferred to 20 ℃ and cultured with shaking at 150r/min for 16 hours. The cells were collected by low-temperature centrifugation, resuspended in NTA-10 solution (500mM sodium chloride, 10mM imidazole, 20mM Tris-HCl, pH 8.0), and sonicated on ice. Centrifuging at low temperature to collect supernatant, and adopting NTA-Ni²⁺And purifying the expressed protein by affinity column chromatography. The expressed recombinant protein contains 6 × His tag at the N end, can be adsorbed on a chromatography column in an affinity manner, and is subjected to gradient elution by imidazole solutions with different concentrations, and eluent is collected. The distribution of the target protein in the eluate was examined by SDS-PAGE. Excising ubiquitin-like SUMO at the N-terminus of recombinant protein in dialysis bag using ULP1 enzyme and using NTA-Ni²⁺And removing the SUMO protein by affinity column chromatography, and collecting a sample for SDS-PAGE detection. The electrophoretically pure recombinant protein AlinE4 was obtained, having a molecular weight of about 20kDa (FIG. 1). Protein concentration was determined by Brandford method.

Example 4 Activity assay of recombinant Gene aline4

The activity of the purified recombinant hydrolase AlinE4 was determined by the p-nitrophenol butyrate method. The method comprises the following specific operations: 1ml of a reaction system containing 1mM of p-nitrophenol butyrate, 100mM of Tris-HCl buffer (pH7.5) and 185ng of pure enzyme protein was subjected to continuous measurement of the absorbance A at 40 ℃ using an ultraviolet-visible spectrophotometer (Beckman DU800, USA)₄₀₅For 2min, the inactivated enzyme solution was used as a control for zeroing. One unit of enzyme activity is defined as the amount of enzyme required to catalyze the production of l. mu. mol of p-nitrophenol from p-nitrophenol ester per minute. The esterase activity was measured to be 25.8U/mg.

Example 5 hydrolase AlinE4 substrate specificity assay

Substrate specificity analysis of the hydrolase AlinE4 Using the system (1 ml): 100mM Tris-HCl buffer (pH7.5), 1mM substrate, 185ng pure enzyme protein was added, and absorbance A was continuously measured at 25 ℃₄₀₅And 2 min. The substrates used for the assay were: p-nitrophenol acetate (C2), p-nitrophenol butyrate (C4), p-nitrophenol hexanoate (C6), p-nitrophenol octanoate (C8), p-nitrophenol decanoate (C10), p-nitrophenol dodecanoate (C12), p-nitrophenol tetradecanoate (C14), p-nitrophenol hexadecanoate (C16). Through determination tableIt is clear that AlinE4 has high catalytic activity for p-nitrophenol esters with short acyl carbon chains (C4, C6 and C8), wherein the catalytic activity is highest when the substrate is p-nitrophenol butyrate (C4), and has certain catalytic activity for p-nitrophenol esters with long acyl carbon chains (C10, C12 and C14) (fig. 2). The results show that the hydrolase AlinE4 has better catalytic activity on acyl carbon chain shorter lipid substances and hydrolysis activity on short-chain lipids is better than that on long-chain lipids.

EXAMPLE 6 analysis of the optimum reaction conditions for the hydrolase AlinE4

The optimum reaction pH of the hydrolase AlinE4 is determined within the range of 3.0-10.5. The specific operation is as follows: the absorbance A was continuously measured at 40 ℃ by adding 1mM p-nitrophenol butyrate and 185ng pure enzyme protein to buffers of different pH₃₄₈And 2 min. The buffers used for the assay were: 100mM citric acid-sodium citrate buffer (pH 3.0-6.0), 100mM potassium dihydrogen phosphate-sodium hydroxide buffer (pH 6.0-7.5), 100mM Tris hydrochloric acid buffer (pH 7.5-9.0) and 100mM 2-cyclohexylaminoethanesulfonic acid-sodium hydroxide buffer (pH 9.0-10.5). The measurement result shows that the optimal reaction pH of the AlinE4 is 7.5, and the AlinE4 has activity in the pH range of 6.0-10.5 (FIG. 3).

The optimum reaction temperature of the hydrolase AlinE4 is measured within the range of 15-60 ℃. The specific operation is as follows: 1ml of the reaction system was charged with 1mM of p-nitrophenol butyrate, 100mM of Tris-HCl buffer (pH7.5) and 185ng of pure enzyme protein, and absorbance A was continuously measured at 15, 20, 25, 30, 35, 40, 45, 50, 55 and 60 ℃ respectively₄₀₅And 2 min. The measurement results show that the reaction temperature range of the AlinE4 is 15-60 ℃, and the optimal reaction temperature is 40 ℃ (FIG. 3).

EXAMPLE 7 enzymatic stability analysis of the hydrolase AlinE4

The thermal stability analysis of the hydrolase AlinE4 was specifically performed by: (1) incubating the enzyme solution at 10, 20, 30, 40, 50, 60, 70, 80, 90 and 100 ℃ for 1h and 4h respectively, and measuring the activity of the enzyme; (2) the enzyme solution was incubated at 90, 95 and 100 ℃ for 0.5, 1, 1.5, 2 and 2.5h, respectively, to determine the activity of the enzyme. The living body measuring system comprises the following steps: 1ml of the reaction was added 1mM p-nitrophenol butyrate, 100mM Tris-HCl buffer (pH7.5) and 185ng pure enzyme protein at 40 deg.CContinuous measurement of Absorbance A₄₀₅And 2 min. The results show that the AlinE4 can still keep more than 80% of activity under the conditions of incubation for 4 hours at 10-60 ℃ and incubation for 1 hour at 10-70 ℃ (figure 5); the AlinE4 maintained 50% and 45% activity by incubation at 90 ℃ for 0.5h and 1h (fig. 6); after incubation at 100 ℃ for 0.5h and 1h, the activity of AlinE4 was maintained at 30% and 20% or more (fig. 6), indicating that AlinE4 has good thermostability.

The specific operation of the assay of the effect of NaCl on the activity of the hydrolase AlinE4 was: 0, 0.5, 1, 2, 3,4 and 5mol/L NaCl was added to the reaction system, respectively, to measure the activity of the enzyme. The living body measuring system comprises the following steps: 1ml of the reaction system was charged with 1mM of p-nitrophenol butyrate, 100mM of Tris-HCl buffer (pH7.5) and 185ng of pure enzyme protein, and the absorbance A was continuously measured at 40 ℃₄₀₅And 2 min. The result shows that NaCl has no influence on the activity of the AlinE4 basically (the activity is kept more than 95%) under the condition of 0.5-3 mol/L NaCl, wherein the activity of the AlinE4 can be enhanced under the conditions of 0.5mol/L and 1mol/L NaCl; when the NaCl concentration reaches 5mol/L, the AlinE4 can still retain more than 40% of activity, which shows that the AlinE4 has good salt resistance (figure 7).

The specific operation of the determination of the effect of divalent cations on the activity of the hydrolase AlinE4 was: 10mM Ba was added to the reaction system²⁺、Ca²⁺、Cd²⁺、Co²⁺、Cu²⁺、Mg²⁺、Mn²⁺、Ni²⁺、Sr²⁺、Zn²⁺And ethylenediaminetetraacetic acid (EDTA), and measuring the enzyme activity. The enzyme activity measuring system comprises: 1ml of the reaction system was added with 1mM of p-nitrophenol butyrate, 100mM of Tris-HCl buffer (pH7.5) and 185ng of pure enzyme protein, and the absorbance A was continuously measured at 40 ℃₄₀₅And 2 min. The determination result shows that the activity of AlinE4 can be measured by Cd²⁺、Cu²⁺、Ni²⁺And Zn²⁺Ion suppression evident at Ba²⁺、Ca²⁺、Mg²⁺And Sr²⁺EDTA enhanced the activity of the enzyme in the presence of less effect (more than 85% of activity was retained) (FIG. 8).

The specific operation of the determination of the influence of the organic solvent on the activity of the hydrolase AlinE4 is as follows: adding 15% (v/v) of organic solvent into the reaction system respectively: acetone (Acetone), acetonitrile (A)cetonitrile), Ethanol (Ethanol), Dimethylformamide (DMF), dimethyl sulfoxide (DMSO), Glycerol (Glycerol), Isopropanol (isopopapanol) and Methanol (Methanol), and the activity of the enzyme was measured. The living body measuring system comprises the following steps: 1ml of the reaction system was charged with 1mM of p-nitrophenol butyrate, 100mM of Tris-HCl buffer (pH7.5) and 185ng of pure enzyme protein, and the absorbance A was continuously measured at 40 ℃₄₀₅And 2 min. The results of the assay showed that AlinE4 activity was completely inhibited by acetonitrile, and that ethanol, DMSO, glycerol, isopropanol, and methanol enhanced its activity, especially AlinE4 activity increased more than two-fold in the presence of ethanol and glycerol (fig. 9).

Sequence listing

<110> second oceanographic institute of national oceanographic administration

<120> thermostable salt-tolerant SGNH family hydrolase derived from marine bacteria and application thereof

<160>2

<170>SIPOSequenceListing 1.0

<210>1

<211>573

<212>DNA

<213>Altererythrobacter indicus

<400>1

atgggcgaat cgcgcgtgat tctcgccttc ggagacagcc tgtttgcagg ctatggcctt 60

gataaggggg agagctatcc ggcaaagctg gaaactgcgc tgcgcagcca tggcatcaat 120

gccagaatca ttaatgccgg cgtttcgggt gacaccactg cggcagggct gcagcgaatc 180

aaattcgtgc tggatagcca gccggacaag ccggaattgg ccatagtgga actgggcggg 240

aatgaccttt tacgcggcct ctcaccagcc gaagcgcggc agaacctcag cggaatcctc 300

gaagaattgc agaggcggaa aattccaatc ctgttgatgg gaatgcgagc gccgcccaat 360

ctaggggcaa aatatcagcg cgaatttgat gggatttatc cctatctggc cgaaaaatat 420

gacgccaagc tggtaccttt cttccttgag gccgtggcag atagacctga cctcattcag 480

aaggatcacg ttcaccccac tgcgcgcggt gtggaggaac tcgtgtctgc aacatcgaat 540

gcagttgcca aggcgctgcc tgcgaagaag tga 573

<210>2

<211>190

<212>PRT

<213>Altererythrobacter indicus

<400>2

Met Gly Glu Ser Arg Val Ile Leu Ala Phe Gly Asp Ser Leu Phe Ala

1 5 10 15

Gly Tyr Gly Leu Asp Lys Gly Glu Ser Tyr Pro Ala Lys Leu Glu Thr

20 25 30

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

35 40 45

Ser Gly Asp Thr Thr Ala Ala Gly Leu Gln Arg Ile Lys Phe Val Leu

50 55 60

Asp Ser Gln Pro Asp Lys Pro Glu Leu Ala Ile Val Glu Leu Gly Gly

65 70 75 80

Asn Asp Leu Leu Arg Gly Leu Ser Pro Ala Glu Ala Arg Gln Asn Leu

85 90 95

Ser Gly Ile Leu Glu Glu Leu Gln Arg Arg Lys Ile Pro Ile Leu Leu

100 105 110

Met Gly Met Arg Ala Pro Pro Asn Leu Gly Ala Lys Tyr Gln Arg Glu

115 120 125

Phe Asp Gly Ile Tyr Pro Tyr Leu Ala Glu Lys Tyr Asp Ala Lys Leu

130 135 140

Val Pro Phe Phe Leu Glu Ala Val Ala Asp Arg Pro Asp Leu Ile Gln

145 150 155 160

Lys Asp His Val His Pro Thr Ala Arg Gly Val Glu Glu Leu Val Ser

165 170 175

Ala Thr Ser Asn Ala Val Ala Lys Ala Leu Pro Ala Lys Lys

180 185 190

Claims (17)

1. An isolated polypeptide having hydrolase activity, which corresponds to the sequence shown for the polypeptide of SEQ ID NO. 2.

2. A polynucleotide encoding a polypeptide having the sequence shown in SEQ ID No. 1.

3. A nucleic acid construct comprising the polynucleotide of claim 2 operably linked to one or more control sequences that direct the production of the polypeptide in a suitable expression host.

4. A recombinant expression vector comprising the nucleic acid construct of claim 3.

5. The recombinant expression vector of claim 4, wherein the vector is prokaryotic expression vector pET series vector, pQE series vector, yeast expression vector pPICZ- α -A, pHIL-D2, pPIC9, pHIL-S1, or animal cell expression vector pSVK3, pMSG.

6. The recombinant expression vector of claim 5, wherein: the vector is an escherichia coli expression vector pET28 a.

7. A host obtained by transforming or transfecting a prokaryotic or eukaryotic host, which is a bacterial, yeast or mammalian cell, with the recombinant expression vector of any one of claims 4 to 6.

8. The host of claim 7, which is an E.

9. The host of claim 8, which is an e.

10. A method of producing the polypeptide of claim 1, comprising:

(a) culturing the recombinant host cell of claim 7 under conditions conducive for production of the hydrolase, wherein the host cell comprises the nucleotide sequence set forth in SEQ ID No. 1;

(b) and recovering the polypeptide.

11. Use of the polypeptide of claim 1 or the host capable of expressing the polypeptide of claim 7 for catalyzing hydrolysis of C2-C8 short chain fatty acid esters or C10-C14 long chain fatty acid esters.

12. The use of claim 11, wherein the C2-C8 short chain fatty acid ester is a p-nitrophenol ester having a short carbon chain of C2-C8.

13. The use of claim 12, wherein the C2-C8 short chain fatty acid ester is p-nitrophenol acetate, p-nitrophenol butyrate, p-nitrophenol hexanoate, and p-nitrophenol octanoate.

14. The use according to any one of claims 11 to 13, wherein the temperature of the catalytic hydrolysis of the hydrolase is in the range of 15 to 60 ℃.

15. The use of claim 14, wherein the temperature range of the enzymatic hydrolysis is 40 ℃.

16. The use according to any one of claims 11 to 13, wherein the hydrolase-catalyzed hydrolysis has a pH of from 6.0 to 10.5.

17. The use of claim 16, wherein the hydrolase catalyzes the hydrolysis at a pH of 7.5.

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