CN106811449B

CN106811449B - Lipase BM19

Info

Publication number: CN106811449B
Application number: CN201510849071.3A
Authority: CN
Inventors: 曾阿娜; 冯晓琴; 牛其文
Original assignee: Wilmar Shanghai Biotechnology Research and Development Center Co Ltd
Current assignee: Wilmar Shanghai Biotechnology Research and Development Center Co Ltd
Priority date: 2015-11-27
Filing date: 2015-11-27
Publication date: 2021-04-30
Anticipated expiration: 2035-11-27
Also published as: CN106811449A

Abstract

The application provides a polypeptide with lipase activity, which comprises an amino acid sequence shown in SEQ ID NO. 1 or a sequence comprising at least one amino acid substitution, determination or addition in the sequence. The application also provides polynucleotides encoding the polypeptides, expression vectors and host cells comprising the polynucleotides, and methods for producing the polypeptides. In addition, the application of the polypeptide with lipase activity is also related.

Description

Lipase BM19

Technical Field

The present application is in the field of genetic or enzymatic engineering, and in particular relates to polypeptides having lipase activity, nucleic acids encoding the same, and expression vectors and host cells comprising the encoding nucleic acids. The application also relates to a preparation method and application of the polypeptide.

Background

Lipases are widely present in animals, plants and microorganisms, and are important sources of industrial lipases because microorganisms are not only diverse in species, fast in propagation, and prone to genetic variation, but also have broader pH, temperature range, and substrate patentability than animals. According to statistics, the microorganisms producing lipase belong to 65 genera, and are mainly concentrated in strains such as aspergillus niger, candida, rhizopus, pseudomonas, streptomyces, pseudomonas alcaligenes and the like.

The lipase has important application in the aspects of food industry, medicine and health, chemistry and chemical engineering and the like:

food industry: in the aspect of oil processing, due to the introduction of lipase, the hydrolysis of oil can be carried out at normal temperature and normal pressure, so that biological substances such as highly unsaturated fatty acid, tocopherol and the like are not denatured. In the aspect of dairy processing, the lipase is used for carrying out milk fat hydrolysis in dairy, so that the flavors of cheese, milk powder and cream can be enhanced, the maturity of cheese is promoted, and the quality of dairy products is improved. In the aspect of processing the wheaten food, lipase is added to improve the elasticity of the wheaten food, improve the taste and improve the fresh-keeping capacity of bread.

Medical treatment and health: in the medical field, lipase is used as a diagnostic tool, and diseases can be predicted, for example, lipase in serum can be used for detecting acute pancreatitis and pancreatic injury. In addition, the lipase has application in the aspects of medicine production, weight reduction and the like.

Chemical engineering: in the aspect of biodiesel synthesis, the enzyme method has the advantages of simple extraction and purification process, low equipment investment, low energy consumption, small pollution and the like, and increasingly attracts people to pay general attention, wherein the Novozym 435 and Candida sp.99-125 with fabric membrane immobilized are common enzymes for biodiesel production. In the aspect of washing industry, in 1988, Novit company firstly puts detergents which contain lipase and can effectively remove oil stains to the market, and the development of lipase for detergents by applying genetic engineering is one of the most successful applications of modern biotechnology in large-scale industrialization. In the pulp and paper industry, lipases are used to remove "consistent lipids" from pulp, and the Nippon paper industry in Japan developed a "consistent lipid" control method, i.e., hydrolysis of triglycerides with Candida rugosa lipase, with a degree of hydrolysis of 90%.

Lipases are used in dairy products to produce flavour and can therefore be used in cheese making. Conventionally, a lipase preparation derived from ruminants such as goats or calves is used. These preparations are derived from the forestomach tissue of these animals, and these lipase preparations, also known as forestomach lipases. Commercial reagents Piccantase C, L, KG and K (DSM Food Specialties, the netherlands) are available on the market. These lipases are used in a variety of italian, spanish, greek and french cheese preparations, the generation of specific flavour profiles during these types of cheese ripening being largely due to the action of the lipase on milk fat. Lipases catalyze the hydrolysis of milk fat to produce free fatty acids. The fatty acids may have short chain (C4-C6 fatty acids, i.e., butyric acid, caproic acid) and medium to long chain (C12-C18) fatty acids. The free fatty acids may then participate in chemical reactions, for example the formation of flavour compounds such as ethyl acetate, beta-keto acids, methyl ketones, esters and lactones. The conversion of fatty acids in the flavour components may be catalysed by enzymes from the microbial population in the cheese.

The lipase used may influence the type of free fatty acids released in the cheese, for example, pungent, strong aromas are mainly produced from lipases releasing short chain fatty acids (C4-C6), whereas medium-long chain fatty acids give a soapy taste. Much research has been devoted to adapting lipases to short chain preferences for use in cheese making. (CN 102016015A; Moore et al, 1995; Smith et al, 1996; Schmitt et al, incorporated).

Summary of The Invention

In a first aspect, the present application provides a polypeptide having lipase activity comprising or consisting of a sequence selected from:

(a) 1, and an amino acid sequence as shown in SEQ ID NO, and

(b) a sequence obtained by substituting, deleting or adding at least one amino acid from the sequence of (a), wherein the polypeptide variant obtained from (b) still maintains lipase activity.

In an optional embodiment, the above polypeptide is fused to a heterologous polypeptide.

In one embodiment, the polypeptide of the present application comprises the amino acid sequence shown in SEQ ID NO. 1. In a preferred embodiment, the polypeptide consists of the amino acid sequence shown in SEQ ID NO. 1.

In a second aspect, there is provided a polynucleotide encoding a polypeptide of the first aspect comprising or consisting of a sequence selected from:

(a) a nucleotide sequence encoding the amino acid sequence shown as SEQ ID NO. 1 or a sequence comprising at least one amino acid substitution, deletion or addition in the above sequence; and

(b) a nucleotide sequence that hybridizes under stringent conditions to the nucleotide sequence in a).

In one embodiment, the polynucleotide of the invention comprises the nucleotide sequence set forth in SEQ ID NO. 2. In a preferred embodiment, the polynucleotide consists of the nucleotide sequence shown in SEQ ID NO. 2.

In one embodiment, the polynucleotides of the present application are produced synthetically or recombinantly.

In a third aspect, there is provided an expression vector comprising at least one polynucleotide as described above.

In certain embodiments, the expression vectors of the present application further comprise a control sequence that regulates expression of the polynucleotide, wherein the polynucleotide is operably linked to the control sequence. In a preferred embodiment, the expression vector is pCold-TF.

In a fourth aspect, there is provided a host cell comprising a polynucleotide or expression vector of the present application. In a preferred embodiment, the host cell is E.coli BL21(DE 3).

In a fifth aspect, there is provided a method of making a polypeptide of the present application, comprising:

1) cloning the above-mentioned polynucleotide on an expression vector,

2) the expression vector is transferred into a suitable host cell,

3) culturing said host cell in a suitable medium,

4) isolating and purifying the polypeptide from the host cell or culture medium.

In a preferred embodiment, there is provided a method of preparing a polypeptide consisting of the amino acid sequence shown in SEQ ID NO. 1, comprising: cloning a nucleotide sequence which codes for an amino acid sequence shown as SEQ ID NO. 1 into a plasmid expression vector, transforming the plasmid expression vector with the polynucleotide sequence into escherichia coli for induced expression, and then separating and purifying the BM19 polypeptide from the escherichia coli.

In a sixth aspect, a lipase prepared according to the method of the fifth aspect is provided.

In a seventh aspect, there is provided a use of the above polypeptide, polynucleotide, expression vector or host cell in the preparation of a lipase.

In an eighth aspect, there is provided the use of the polypeptide, lipase, polynucleotide, expression vector or host cell described above in the manufacture of a food product. In a preferred embodiment, the polypeptide, lipase, polynucleotide, expression vector or host cell described above in the present application is used in the manufacture of dairy products or pasta. In a particular embodiment, the dairy product is a cheese.

In a ninth aspect, there is provided a food product made using the polypeptide, lipase, polynucleotide, expression vector or host cell described above. In a preferred embodiment, the food product is a dairy product or pasta.

The polypeptides of the present application have medium-short chain fatty acid specificity.

Brief description of the drawings

FIG. 1 shows a gel electrophoresis of BM19 polypeptide. Lane 1 is a molecular weight marker, and lane 2 is a BM19 polypeptide.

FIG. 2 shows the catalytic hydrolytic activity of BM19 as a lipase when 4-nitrophenylbutyrate (pNPB), 4-nitrophenyloctanoate (pNPO), 4-nitrophenyllaurate (pNPD) and 4-nitrophenylpalmitate (pNPP) were used as substrates, respectively.

FIG. 3 shows the results of enzyme activity measurements of BM19 at different temperatures.

FIG. 4 shows the results of enzyme activity measurements of BM19 at different pH values.

Figure 5 shows the effect of different metal ions on the enzymatic activity of BM 19.

Figure 6 shows the effect of different surfactants on the enzymatic activity of BM 19.

Detailed Description

Polypeptides

The present application provides a polypeptide having lipase activity comprising or consisting of a sequence selected from:

(a) 1, and an amino acid sequence as shown in SEQ ID NO, and

In some embodiments, the number of amino acid substitutions, deletions or additions is 1 to 30, preferably 1 to 20, more preferably 1 to 10, wherein the resulting polypeptide variant substantially retains lipase activity. In preferred embodiments, the above polypeptide variants differ from the amino acid sequence shown in SEQ ID NO. 1 by substitutions, deletions and/or additions of about 1, 2,3, 4, 5, 6, 7, 8, 9, or 10 amino acids. In a more preferred embodiment, the above polypeptide variant differs from the amino acid sequence shown in SEQ ID NO. 1 by substitutions, deletions or additions of about 1, 2,3, 4 or 5 amino acids.

In some embodiments, the polypeptide of the present application comprises the amino acid sequence set forth in SEQ ID NO 1. In a preferred embodiment, the polypeptide consists of the amino acid sequence shown in SEQ ID NO. 1.

Thus, the present application provides a lipase comprising or consisting of a polypeptide having lipase activity as disclosed herein or a variant thereof. Herein, the polypeptide consisting of the amino acid sequence shown in SEQ ID NO. 1 is designated as lipase BM 19.

In an optional embodiment, the above polypeptide is fused to a heterologous polypeptide to form a fusion protein.

As used herein, the term "amino acid" refers to naturally occurring and non-naturally occurring amino acids as well as amino acid analogs and mimetics. Naturally occurring amino acids include the 20 (L) -amino acids used in protein biosynthesis, as well as other amino acids, such as 4-hydroxyproline, hydroxylysine, desmosine, isodesmosine, homocysteine, citrulline, and ornithine. Non-naturally occurring amino acids include, for example, (D) -amino acids, norleucine, norvaline, p-fluorophenylalanine, ethylmethionine, and the like, as known to those skilled in the art. Amino acid analogs include modified forms of naturally and non-naturally occurring amino acids. Such modifications may include, for example, substitution of chemical groups and moieties on the amino acids, or derivatization of the amino acids. Amino acid mimetics include, for example, organic structures that exhibit functionally similar properties, such as the charge and charge space characteristics of an amino acid. For example, the organic structure that mimics arginine (Arg or R) has a positively charged moiety that is located in a similar molecular space and has the same degree of mobility as the e-amino group of the side chain of the naturally occurring Arg amino acid. The mimetic also includes a constraining structure to maintain optimal steric and charge interactions of the amino acid or amino acid functional group. One skilled in the art can determine what structures constitute functionally equivalent amino acid analogs and amino acid mimetics.

In some embodiments, variants of the amino acid sequence set forth in SEQ ID NO. 1 have at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more homology with SEQ ID NO. 1. In a preferred embodiment, the polypeptide variant has more than 99% homology with the sequence shown in SEQ ID NO. 1.

"homology" as used herein is defined as the percentage of residues in an amino acid or nucleotide sequence variant that are identical, if necessary to the maximum percentage, after alignment and introduction of gaps in the sequence. Methods and computer programs for alignment are well known in the art.

The terms "polypeptide" and "protein" are used interchangeably herein to refer to polymers of amino acid residues and variants and synthetic and naturally occurring analogs thereof. Thus, these terms apply to naturally occurring amino acid polymers and naturally occurring chemical derivatives thereof, as well as amino acid polymers in which one or more amino acid residues are synthetic non-naturally occurring amino acids, such as chemical analogs of corresponding naturally occurring amino acids. Such derivatives include, for example, post-translational modifications and degradation products, including phosphorylated, glycosylated, oxidized, isomerized, and deaminated variants of the polypeptide fragment shown in SEQ ID NO: 1.

In a preferred embodiment, the sequence of the BM19 polypeptide variant is a sequence comprising one or several conservative amino acid substitutions in the amino acid sequence shown in SEQ ID No. 1, wherein the substituted sequence still retains lipase catalytic activity.

Certain amino acid substitutions, known as "conservative amino acid substitutions," can occur frequently in proteins without changing the conformation or function of the protein, a well-established rule in protein chemistry.

Conservative amino acid substitutions in the present invention include, but are not limited to, substitution of any one of glycine (G), alanine (a), isoleucine (I), valine (V), and leucine (L) for any one of these aliphatic amino acids; substitution of threonine (T) with serine (S) and vice versa; substitution of aspartic acid (D) for glutamic acid (E)) And vice versa; (ii) substitution of asparagine (N) with glutamine (Q), and vice versa; substitution of arginine (R) with lysine (K), and vice versa; substitution of any one of these aromatic amino acids with phenylalanine (F), tyrosine (Y) and tryptophan (W); and substitution of cysteine (C) with methionine (M) and vice versa. Other substitutions may also be considered conservative, depending on the particular amino acid environment and its role in the three-dimensional structure of the protein. For example, glycine (G) and alanine (A) are often interchangeable, as are alanine (A) and valine (V). Methionine (M), which is relatively hydrophobic, can often be exchanged for leucine and isoleucine, and sometimes for valine. Lysine (K) and arginine (R) are often interchanged at the following positions: the important characteristics of the amino acid residues are their charge and the different pKs of the two amino acid residues are not significant. Still other changes may be considered "conservative" under certain circumstances (see, e.g., BIOCHEMISTRY at pp. 13-15, 2^nd ed. Lubert Stryer ed. (Stanford University); Henikoff et al., Proc. Nat’l Acad. Sci. USA (1992) 89:10915-10919; Lei et al., J. Biol. Chem. (1995) 270(20):11882-11886)。

In the following, amino acid residues are exemplified by the group of substitutable residues, but the substitutable amino acid residues are not limited to the residues described below:

group A: leucine, isoleucine, norleucine, valine, norvaline, alanine, 2-aminobutyric acid, methionine, O-methylserine, tert-butylglycine and cyclohexylalanine;

group B: aspartic acid, glutamic acid, isoaspartic acid, isoglutamic acid, 2-aminoadipic acid, and 2-aminosuberic acid;

group C: asparagine and glutamine;

group D: lysine, arginine, ornithine, 2, 4-diaminobutyric acid, i.e., 2, 3-diaminopropionic acid;

group E: proline, 3-hydroxyproline and 4-hydroxyproline;

and F group: serine, threonine, and homoserine;

group G: phenylalanine and tyrosine.

For example, the inventors have found that one or more conservative substitutions in the first amino acid at position 132N-terminal of the BM19 polypeptide do not substantially affect lipase activity.

In other specific embodiments, the C-terminal or N-terminal region of the BM19 polypeptide may also be truncated by about 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or more amino acids while still having the lipase activity of BM 19.

In further embodiments, 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or more amino acids may also be added to the C-terminal or N-terminal region of the BM19 polypeptide, the resulting BM19 variant still having lipase catalytic activity.

Furthermore, 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or more amino acids may also be added or deleted in regions other than the C-or N-terminus of the BM19 polypeptide, as long as the altered polypeptide substantially retains the lipase activity of BM 19.

In certain embodiments, a polypeptide of the invention, e.g., a BM19 polypeptide or variant thereof, is fused to a heterologous polypeptide. In some embodiments, the BM19 fusion protein substantially retains the lipase activity of BM 19. In certain embodiments, the heterologous polypeptide is linked to the N-terminus of the BM19 polypeptide. In certain embodiments, the heterologous polypeptide is linked to the C-terminus of the BM19 polypeptide. In these embodiments, the heterologous polypeptide can be selected from a purification tag (e.g., can include but is not limited to: GST, MBP), an epitope tag (e.g., can include but is not limited to: Myc, FLAG), a targeting sequence, a signal peptide, and the like.

In a specific embodiment, the fusion protein comprises a BM19 polypeptide and a tag, typically a peptide tag, attached to the C-terminus or N-terminus of the BM19 polypeptide. The tag is typically a peptide or amino acid sequence that can be used to isolate and purify the fusion protein. Thus, the tag is capable of binding to one or more ligands, e.g. one or more ligands of an affinity matrix such as a chromatography support or high affinity magnetic beads. Of said labelExamples are the ability to bind nickel (Ni) with high affinity²⁺) Column or cobalt (Co)²⁺) A column-bound histidine tag (His-tag or HT), for example a tag comprising 6 histidine residues (His6 or H6). Other exemplary tags for isolation or purification of the fusion protein include Arg-tag, FLAG-tag, Strep-tag, and the like.

Polynucleotide

The present application provides polynucleotides encoding the above polypeptides comprising or consisting of a sequence selected from:

In certain specific embodiments, the polynucleotides of the invention encode BM19 polypeptides and functionally equivalent variants thereof. In one embodiment, the polynucleotide of the invention has at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% homology to a polynucleotide encoding BM19 and functionally equivalent variants thereof.

In certain embodiments, the polynucleotide of the invention comprises a nucleotide sequence that is at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% homologous to the nucleotide sequence set forth in SEQ ID NO. 2. In a preferred embodiment, the polynucleotide of the invention comprises the nucleotide sequence shown in SEQ ID NO. 2.

In a preferred embodiment, the polynucleotide of the invention consists of a nucleotide sequence which has 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% homology with the nucleotide sequence shown in SEQ ID NO. 2. In a more preferred embodiment, the polynucleotide of the invention consists of the nucleotide sequence shown in SEQ ID NO. 2.

The term "polynucleotide" or "nucleic acid" as used herein refers to mRNA, RNA, cRNA, cDNA, or DNA, including DNA in single-and double-stranded form. The term generally refers to a polymeric form of nucleotides of at least 10 bases in length, which are ribonucleotides or deoxynucleotides or modified forms of either type of nucleotide.

In certain embodiments, the polynucleotides of the invention comprise or consist of a nucleotide sequence that hybridizes specifically to a nucleotide sequence encoding a BM19 polypeptide and functionally equivalent variants thereof under stringent conditions and encodes a polypeptide functionally equivalent to a BM19 polypeptide.

Stringent conditions for DNA hybridization can be routinely selected by those skilled in the art. Generally, longer probes require higher temperatures for proper annealing, while shorter probes require lower temperatures. Hybridization generally depends on the ability of denatured DNA to reanneal when the complementary strand is in an environment below its melting temperature. The higher the degree of homology between the probe and hybridizable sequence, the higher the relative temperature that can be used. Thus, higher relative temperatures tend to make the reaction conditions more stringent, while at lower temperatures the stringency is lower. For a detailed description of the stringent conditions for hybridization reactions, see Ausubel et al, Current Protocols in Molecular Biology, Wiley Interscience Publishers (1995).

In certain embodiments, the stringent conditions employed for DNA hybridization include: 1) washing with low ionic strength and high temperature, e.g. 0.015M sodium chloride/0.0015M sodium citrate/0.1% sodium lauryl sulfate at 50 ℃; 2) denaturing agents such as formamide are used for hybridization, for example 50% (v/v) formamide at 42 ℃ plus 0.1% bovine serum albumin/0.1% Ficoll/0.1% polydiallylpyrrolidone/50 mM sodium phosphate buffer pH6.5, 750mM sodium chloride, 75mM sodium citrate; or (3) overnight hybridization at 42 ℃ in a hybridization solution containing 50% formamide, 5 XSSC (0.75M sodium chloride, 0.075M rotten 1 sodium citrate), 50mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5 XDenhardt's solution, sonicated salmon sperm DNA (50.mu.g/ml), 0.1% SDS, and 10% dextran sulfate, followed by 10 minutes of washing in 0.2 XSSC (sodium chloride/sodium citrate) at 42 ℃ and high stringency washing in 0.1 XSSC with EDTA at 55 ℃. Moderately stringent conditions can be determined by Sambrook et al, Molecular Cloning: a Laboratory Manual, New York: determined as described in Cold Spring Harbor Press, 1989. Moderately stringent conditions include those that employ wash solutions and hybridization conditions (e.g., temperature, ionic strength, and percentage SDS) that are less stringent than those described above. For example, moderately stringent conditions include hybridization with at least about 16% v/v to at least about 30% v/v formamide and at least about 0.5M to at least about 0.9M salt at 42 ℃ and a wash with at least about 0.1M to at least about 0.2M salt at 55 ℃. Moderately stringent conditions may also include hybridization with 1% Bovine Serum Albumin (BSA), 1mM EDTA, 0.5M NaHPO4(pH7.2), 7% SDS at 65 deg.C, and hybridization with (i)2 XSSC, 0.1% SDS; or (ii)0.5% BSA, 1mM EDTA, 40mM NaHPO4(pH 47.2), 5% SDS at 60-65 ℃. The practitioner will adjust the temperature, ionic strength, etc. depending on factors such as probe length. The stringency at which nucleic acids are hybridized depends on the length of the nucleic acid molecules and the degree of complementarity, as well as other variables well known in the art. The greater the similarity or homology between two nucleotide sequences, the greater the Tm for hybrids of nucleic acids containing those sequences. The relative stability of nucleic acid hybridization (corresponding to higher Tm) decreases in the following order: RNA, DNA, RNA, DNA. Preferably, the minimum length of the hybridizable nucleic acid is at least about 12 nucleotides, preferably at least about 16, more preferably at least about 24, and most preferably at least about 36 nucleotides.

The polynucleotides of the invention may be combined with other DNA sequences, such as promoters, polyadenylation signals, other restriction sites, multiple cloning sites, other coding segments, and the like, such that their overall lengths may vary significantly. It is therefore contemplated that polynucleotide fragments of almost any length may be utilized; the overall length is preferably limited by the ease of preparation and use in contemplated recombinant DNA protocols.

Polynucleotides and fusions thereof can be prepared, manipulated, and/or expressed using any of a variety of mature techniques known and available in the art. For example, a polynucleotide sequence encoding a polypeptide of the present invention or a variant thereof may be used in a recombinant DNA molecule to direct expression of the polypeptide in an appropriate host cell. Due to the inherent degeneracy of the genetic code, other DNA sequences encoding substantially identical or functionally equivalent amino acid sequences may also be used in the present invention, and these sequences may be used to clone and express a given polypeptide.

In certain embodiments, the polynucleotides of the invention are produced by artificial synthesis, such as direct chemical synthesis or enzymatic synthesis. In alternative embodiments, the polynucleotides described above are produced by recombinant techniques.

In certain embodiments, the sequence of the obtained polynucleotide can be determined by conventional methods, preferably, such as the dideoxy chain termination method (Sanger et al. PNAS,1977,74: 5463-. Such polynucleotide sequencing can also be accomplished using commercially available sequencing kits. Sequencing was repeated to obtain a full-length cDNA sequence. Sometimes, it is necessary to sequence the cDNA of several clones to obtain the full-length cDNA sequence.

Expression vector

The present application provides expression vectors comprising the polynucleotides of the invention.

An "expression vector" as described herein is a nucleic acid construct, produced recombinantly or synthetically, with a series of specific nucleic acid elements that permit transcription of a particular nucleic acid in a host cell. The expression vector of the present invention may be a plasmid vector such as pCold-TF, pET-24a (+), pIRES2-EGFP, pcDNA3.1, pCI-neo, pDC516, pVAC, pcDNA4.0, pGEM-T, pDC315, or a viral vector such as adenovirus, adeno-associated virus, retrovirus, semliki forest virus (sFv) vector, or other vectors well known in the art.

In certain embodiments, the polynucleotide sequence encoding the BM19 polypeptide and variants thereof is cloned into a vector to form a recombinant vector comprising the polynucleotide of the invention.

In a preferred embodiment, the expression vector used to clone the polynucleotide is a plasmid vector. In a more preferred embodiment, the plasmid vector is pCold-TF.

In a specific embodiment, the above-described expression vector further comprises a control sequence that regulates expression of a polynucleotide, wherein the polynucleotide is operably linked to the control sequence.

The term "control sequences" as used herein refers to polynucleotide sequences required to effect expression of a coding sequence to which they are ligated. The nature of such regulatory sequences varies with the host organism. In prokaryotes, such regulatory sequences typically include a promoter, a ribosome binding site, and a terminator; in eukaryotes, such regulatory sequences generally include promoters, terminators, and, in some cases, enhancers. Thus, the term "regulatory sequence" includes all sequences whose presence is minimally necessary for expression of a gene of interest, and may also include other sequences whose presence is advantageous for expression of a gene of interest, such as leader sequences.

The term "operably linked" as used herein refers to the situation wherein: the sequences involved are in a relationship that allows them to function in the desired manner. Thus, for example, a regulatory sequence "operably linked" to a coding sequence is such that expression of the coding sequence is achieved under conditions compatible with the regulatory sequences.

In certain embodiments, expression vectors comprising a nucleotide sequence encoding BM19 polypeptide and variants thereof and suitable transcription/translation regulatory elements are constructed using methods well known to those skilled in the art. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombinant techniques, etc. (Sambrook, et al. Molecular Cloning, a Laboratory Manual, cold Spring Harbor Laboratory. New York, 1989). The nucleotide sequence is operably linked to an appropriate promoter in an expression vector to direct mRNA synthesis. Representative examples of such promoters include: lac or trp promoter of E.coli; the PL promoter of lambda phage; eukaryotic promoters include CMV immediate early promoter, HSV thymidine kinase promoter, early and late SV40 promoter, LTRs of retrovirus, and other known promoters capable of controlling the expression of genes in prokaryotic or eukaryotic cells or viruses. The expression vector also includes a ribosome binding site for translation initiation, a transcription terminator, and the like. The insertion of enhancer sequences into vectors will enhance transcription in higher eukaryotic cells. Enhancers are cis-acting elements of DNA, usually about 10 to 300 base pairs, that act on a promoter to increase transcription of a gene. Examples include the SV40 enhancer on the late side of the replication origin at 100 to 270 bp, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers, among others.

In addition, the expression vector preferably contains one or more selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as dihydrofolate reductase, neomycin resistance, and Green Fluorescent Protein (GFP) for eukaryotic cell culture, or tetracycline or ampicillin resistance for E.coli, and the like.

Host cell

The present application provides host cells comprising a polynucleotide or expression vector of the invention.

In certain embodiments, a polynucleotide encoding a BM19 polypeptide or variant thereof, or an expression vector comprising the polynucleotide, is transformed or transduced into a host cell to obtain a genetically engineered host cell comprising the polynucleotide or expression vector.

The host cell used herein may be any host cell known to those skilled in the art, including prokaryotic cells, eukaryotic cells, such as bacterial cells, fungal cells, yeast cells, mammalian cells, insect cells, or plant cells, and the like. Exemplary bacterial cells include any of the genera Escherichia, Bacillus, Streptomyces, Salmonella, Pseudomonas, and Staphylococcus, including, for example, Escherichia coli, lactococcus lactis, Bacillus subtilis, Bacillus cereus, Salmonella typhimurium, Pseudomonas fluorescens. Exemplary fungal cells include any species of Aspergillus. Exemplary yeast cells include any of the genera Pichia, Saccharomyces, Schizosaccharomyces, or Saccharomyces, including Pichia, Saccharomyces, or Schizosaccharomyces. Exemplary insect cells include spodoptera litura or any of the drosophila species, including drosophila S2 and spodoptera Sf 9. Exemplary animal cells include CHO, COS or melanoma or any mouse or human cell line. The selection of a suitable host is within the ability of those skilled in the art.

The expression vector may be introduced into the host cell using any technique known in the art, including transformation, transduction, transfection, viral infection, gene gun, or Ti-mediated gene transfer. Specific Methods include calcium phosphate transfection, DEAE-dextran mediated transfection, lipofection, or electroporation, among others (Davis, L., Dibner, M., Battey, I., Basic Methods in Molecular Biology, (1986)). By way of example, when the host is prokaryotic, such as E.coli, the competent cells may be harvested after exponential growth phase, using CaCl as is well known in the art₂The method is used for transformation.

In a particular embodiment, the host cell used in the present invention is E.coli. In a preferred embodiment, the expression vector carrying the polynucleotide sequence of the invention is transformed into E.coli BL21(DE3) for inducible expression.

Methods of producing the polypeptides or lipases of the present application

The polypeptides of the present application may be prepared by any suitable method known to those skilled in the art, for example by recombinant techniques, or by chemical synthesis. Chemical synthesis methods for peptides are also well known to those skilled in the art, e.g., the polypeptides of the invention and variants thereof can be produced by directed peptide synthesis using solid phase techniques (Merrifield,J. Am. Chem. Soc. 85:2149-2154(1963)). Protein synthesis can be performed manually or by automation. Automated synthesis can be achieved, for example, using a 431A peptide synthesizer (Perkin Elmer) from Applied Biosystems. Alternatively, the different degrees of fragmentation can be separately chemically synthesized and chemically combined to produce the desired molecule.

In a specific embodiment, there is provided a method of making a polypeptide or lipase of the present application comprising:

1) cloning the polynucleotide encoding the polypeptide on an expression vector,

2) introducing the expression vector into a suitable host cell,

3) culturing the host cell in a suitable medium, and

Suitable host cells refer to host cells suitable for expression of the expression vector or polynucleotide of interest. Suitable medium means a medium suitable for growth of the host cell or for inducible expression thereof.

In certain embodiments, various conventional media may be selected depending on the host cell used. The culturing is performed under conditions suitable for growth of the host cell. Preferably, the engineered host cells can be cultured in conventional nutrient media modified as appropriate for activating the promoter to screen for transformants or to amplify the polynucleotides of the present application. After transformation of a suitable host cell and growth of the host cell to an appropriate cell density, the selected promoter is induced by suitable means (e.g., temperature shift or chemical induction), and the cell is cultured for an additional period of time to allow production of the polypeptide of interest or a fragment thereof.

In certain embodiments, the host cells are harvested by centrifugation, the cells are disrupted by physical or chemical means, and the resulting crude extract is retained for further purification. Microbial cells for protein expression may be disrupted by any convenient method, including freeze-thaw cycling, sonication, mechanical disruption, or use of cell lysing agents. These methods are well known to those skilled in the art.

In certain embodiments, the recombinant polypeptide produced by the host cell may be encapsulated within the cell, or expressed on the cell membrane, or secreted outside the cell. If necessary, the recombinant protein can be isolated and purified by various separation methods using its physical, chemical and other properties. For example, the expressed polypeptide or fragment thereof can be recovered and purified from recombinant cell culture by the following methods well known in the art: conventional renaturation treatment, protein precipitant treatment (salting-out method), centrifugation, cell lysis by osmosis, sonication, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, High Performance Liquid Chromatography (HPLC), and other various liquid chromatography techniques and combinations thereof. By way of illustration, affinity chromatography purification of proteins comprising a peptide tag (e.g., His-tag, etc.) at the C-terminus or N-terminus is a routine method for obtaining high purity polypeptide preparations.

In a preferred embodiment, there is provided a method of preparing a polypeptide consisting of the amino acid sequence shown in SEQ ID NO. 1, comprising: cloning a polynucleotide consisting of a nucleotide sequence shown as SEQ ID NO. 2 into an expression vector pCold-TF, transforming the pCold-TF with the polynucleotide sequence into Escherichia coli BL21(DE3) for induced expression, and then separating and purifying BM19 polypeptide from Escherichia coli BL21(DE 3).

Use of polypeptides having lipase activity

The polypeptide of the present application is a polypeptide having lipase activity, and is capable of catalyzing hydrolysis of oil and fat, particularly milk fat. More specifically, the polypeptides of the invention are capable of hydrolyzing the ester bond between a fatty acid and a glycerol hydroxyl group.

The application provides the use of the polypeptide, polynucleotide, expression vector or host cell in preparing lipase.

The application also provides the use of the above polypeptide or lipase, polynucleotide, expression vector or host cell in the manufacture of a food product. For example, in the aspect of dairy processing, the lipase is used for carrying out milk fat hydrolysis in dairy, so that the flavors of cheese, milk powder and cream can be enhanced, the maturity of cheese is promoted, and the quality of dairy products is improved. In the aspect of processing the wheaten food, lipase is added to improve the elasticity of the wheaten food, improve the taste and improve the fresh-keeping capacity of bread and the like.

In some preferred embodiments, the polypeptides of the present application are lipases with short chain specificity, which can be used to generate and/or enhance the flavour of dairy products, and thus can be applied in cheese making.

The present application also provides food products, such as dairy products and pasta, made using the above polypeptides or lipases, polynucleotides, expression vectors or host cells.

In the context of the present application, "dairy product" refers to any kind of milk-based product, including but not limited to cheese, butter, cream, dairy analogues and the like. The term "pasta" means a food product made mainly of flour.

In some embodiments of the invention, the polypeptides of the present application can be used to hydrolyze medium chain fatty acids, for example, in the production of oils.

In this specification and claims, the words "comprise", "comprising" and "contain" mean "including but not limited to", and are not intended to exclude other moieties, additives, components, or steps.

It should be understood that features, characteristics, components or steps described in a specific aspect, embodiment or example of the present application may be applied to any other aspect, embodiment or example described herein and may be arbitrarily combined and deleted as desired unless incompatible therewith.

The foregoing disclosure generally describes embodiments of the present application, which are further illustrated by the following examples. These examples are described merely to illustrate embodiments of the present application and do not limit the scope of embodiments of the present application. Although specific terms and values are employed herein, they are to be understood as exemplary and not limiting the scope of the disclosure.

Examples

Example 1: expression and purification of BM19 polypeptide

The nucleotide sequence shown in SEQ ID No.:2 (the polypeptide of which the coding sequence is shown in SEQ ID No.: 1) was synthesized by Biotechnology engineering (Shanghai) Co., Ltd and cloned on expression vector pCold-TF by the same company.

The pCold-TF with the nucleotide sequence described above was transformed into E.coli BL21(DE3) for induction expression (see, in particular, third edition of molecular cloning protocols, science publishers 2002 [ Mei ] Brooku, D.W Lassel, Huang Petang et al), under the conditions of heat shock at 42 ℃ for 50 seconds, ice bath for 2 minutes, LB plates were applied, transformants were selected and inoculated into LB medium (peptone 10g/L, yeast extract 5g/L, and sodium chloride 10g/L), overnight seed culture was carried out at 37 ℃, seed culture broth was inoculated into expression medium at 1% inoculum size, and cultured at 37 ℃ and 220 rpm to OD600= 0.6-0.8.

After cooling to 16 ℃ isopropyl-. beta. -D-thiogalactopyranoside (IPTG) was added to 0.1mM for induction and expression was carried out overnight at 190rpm at 16 ℃. After the induction expression was completed, the cells were collected by centrifugation. The cells were resuspended in lysis buffer (50 mM sodium dihydrogen phosphate, 300 mM sodium chloride, 10 mM imidazole, pH8) with 5 ml of lysis buffer per gram of cells.

The resuspended cells were sonicated at low temperature (50% voltage output, 2 sec sonication, 9 sec intervals for 20 min total) and samples were ice-cooled to preserve proteins during the procedure. After cell disruption, centrifugation was carried out at 14000rpm for 20 minutes and the supernatant was collected.

The obtained supernatant was added to a sample buffer, subjected to water bath at 100 ℃ for 10 minutes, and subjected to gel electrophoresis (10% SDS-PAGE, 100V, 2 hours), and the results are shown in FIG. 1. The results in FIG. 1 show that there was an induced band of interest and the resulting solution was purified BM19 protein.

Example 2: enzymatic Properties of Lipase BM19

Method for measuring lipase activity

The lipase activity was determined by colorimetric method. The enzyme activity was calculated from the amount of p-nitrophenol (pNP) produced by enzymatic hydrolysis of a unit volume of enzyme solution per unit time using p-nitrobenzoate (pNPB) as a substrate. The specific method comprises the following steps: preparing a substrate and a buffer solution in advance, wherein the substrate: 6 mg/mL pNPB (isopropanol dissolved), buffer: 0.05M Tris (pH 8.0, 0.1% gum arabic). A reaction mixture was prepared with the substrate and the buffer at 1:9 (v/v). Two 2 mL centrifuge tubes were taken as a control tube and a sample tube, respectively. 400 uL of each reaction mixture was added to each centrifuge tube and pre-warmed at the appropriate reaction temperature (e.g., 35 ℃) for 5 min. Adding a certain amount of diluted enzyme solution into the sample tube, mixing uniformly, and continuing to perform warm bath for 15 min. Add 1.5 mL of ethanol to both tubes to stop the reaction and add the same amount of diluted enzyme solution to the control tube. Centrifuging at 12000 rpm for 2 min, collecting supernatant, and measuring absorbance at 405 nm.

The unit of enzyme activity is defined as: 1 unit refers to the amount of enzyme required to catalytically release 1. mu. mol of pNP per minute under standard experimental conditions. The enzyme activity calculation formula obtained according to the standard curve is as follows: a = - ([ a1-a0] x 0.7885-0.0118) × V1 × n/(V2 × t). A: sample enzyme activity (U/mL), A1: OD405, a 0: OD405, V1 of control enzyme solutions: volume of total reaction solution (mL), n: dilution factor of enzyme solution, V2: volume of enzyme solution (mL), t: reaction time (min).

Example 2-1,Substrate specificity

Preparing 6 mg/mL of 4-nitrophenyl butyrate (PNPB), 4-nitrophenyl caprylate (pNPO), 4-nitrophenyl laurate (pNPD) and 4-nitrophenyl palmitate (pNPP), wherein the pNPP is dissolved by isopropanol, the PNPB, the pNPO and the pNPD are dissolved by isopropanol, detecting the activity of lipase according to the standard pNPB method (replacing the substrate with the corresponding substrate), calculating the relative enzyme activity for hydrolyzing other substrates, wherein the relative enzyme activity is 100% as measured by measuring the highest substrate by using the enzyme activity, and the result is shown in FIG. 2. As can be seen from FIG. 2, BM19 only hydrolyzed pNPO, and the hydrolase activity was 0.0246U/ml. This indicates that the esterolytic activity of BM19 is specific for medium-short chain fatty acids with substrate specificity.

This indicates that the polypeptides having lipase activity of the present application are suitable for use in the production of dairy products, such as cheese, and in the hydrolysis of medium chain fatty acids in the oiling-off.

Examples 2-2,Optimum temperature of action

The lipase activity was measured at different temperatures (25-65 ℃) according to the pNPB method (substrate replacement is pNPO), and the relative enzyme activity (%) at other temperatures was calculated with the enzyme activity measured at the highest enzyme activity being 100%, as shown in fig. 3.

According to the result of figure 3, the enzymology activity of BM19 shows a trend of increasing firstly and then decreasing with the increase of temperature, and has better enzyme activity at 40-55 ℃, wherein the enzyme activity at 50 ℃ is the highest, and the enzyme activity is 0.029818U/ml, which is the optimal action temperature.

Examples 2 to 3,Optimum pH for action

The lipase activities of the enzyme solutions were measured by the pNPB method (substrate replacement: pNPO) at 50 ℃ in buffers of different pH values (3.0-9.0) respectively (50 mM MES buffer at pH3.0-6.0 and 50mM TrisHCl buffer at pH 6.5-9.0), and the relative activities (%) of the enzymes at other pH values were calculated as shown in FIG. 4, with the enzyme activity (0.038857U/ml) at the time of the highest activity being 100%.

According to the results of FIG. 4, the enzyme activity of BM19 shows a trend of increasing first and then decreasing with the increase of pH value, wherein the enzyme activity is highest at pH8, and therefore, the optimum pH value of BM19 is 8. It is inferred that the lipase of the present invention is an alkaline lipase.

Examples 2 to 4,Effect of Metal ions on Lipase Activity

Preparation of ZnSO₄、MnCl₂、CoCl₂、CaCl₂、MgSO₄、CuSO₄、KCl、(NH₄)₂SO₄、NaCl、NiSO₄、FeCl₃And inorganic salt stock solutions such as sodium citrate and EDTA. According to the pNPB (substrate for pNPO) method, a reaction mixture was prepared first, 400 uL of the mixture was packed into each reaction tube, and an inorganic salt stock solution was added to a final concentration of 5 mmol/L, and the activity was measured according to a standard method. Relative activities of other groups were calculated by taking lipase activity measured by adding water (enzyme activity of 0.02565U/ml) as a control (recorded as 100%), and the results are shown in FIG. 5.

According to the results in FIG. 5, in MnCl₂、CaCl₂、MgSO₄、KCl、(NH₄)₂SO₄、NaCl、NiSO₄The lipase activity in the storage liquid is kept better, but in ZnSO₄In the storage liquid, the lipase activity is completely lost.

Examples 2 to 6,Effect of surfactants on Lipase Activity

Stock solutions of 10% of cationic surfactant CTAB, anionic surfactant SDS, nonionic surfactant Tween 80, Triton X-100 and the like are prepared. The lipase activities were measured by adding the above surfactants to the reaction system at a final concentration of 0.5% according to the pNPB (pNPO as a substrate) standard method, and the relative activities of the other groups were calculated using the lipase activity measured by adding water (0.02565U/ml) as a control (100%), and the results are shown in fig. 6.

According to the results of FIG. 6, the enzyme was completely inactivated in CTAB, and was reduced to various degrees by other surfactants.

It is to be understood that while the application is illustrated in certain forms, it is not limited to what has been shown and described herein. It will be apparent to those skilled in the art that various changes can be made without departing from the scope of the application. Such variations are within the scope of the claims of this application.

Claims

1. The amino acid sequence of the polypeptide with lipase activity is shown as SEQ ID NO. 1.

2. A polynucleotide encoding the polypeptide of claim 1.

3. The polynucleotide according to claim 2, which consists of the nucleotide sequence shown in SEQ ID NO. 2.

4. An expression vector comprising at least one polynucleotide of claim 2 or 3.

5. The expression vector of claim 4, further comprising a control sequence that regulates expression of the polynucleotide, wherein the polynucleotide is operably linked to the control sequence.

6. A host cell comprising the polypeptide of claim 1, the polynucleotide of claim 2 or 3, or the expression vector of claim 4 or 5.

7. Use of the polypeptide of claim 1, the polynucleotide of claim 2 or 3, the expression vector of claim 4 or 5, or the host cell of claim 6 in the preparation of a lipase.

8. Use of the polypeptide of claim 1, the polynucleotide of claim 2 or 3, the expression vector of claim 4 or 5, the host cell of claim 6 in the manufacture of a food product.

9. Use according to claim 8, wherein the use is in the manufacture of a dairy product.