CN105779418B - Novel lipase - Google Patents

Abstract

The application provides a polypeptide with lipase activity, which comprises an amino acid sequence shown as SEQ ID NO. 1 or a sequence comprising at least one amino acid substitution, deletion 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

Novel lipase

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

The lipase is an enzyme with multiple catalytic capacities, can catalyze the hydrolysis of triacylglycerol into glycerol and free fatty acid, and can catalyze the hydrolysis and transesterification of other esters and the synthesis reaction of esters, and in addition, the lipase also shows enantioselectivity to a substrate, so that the lipase has wide application in industries such as food and fat processing, detergent, biodiesel, synthesis of ester bond compounds, chiral drug synthesis and the like (Abhishek Kumar Singh, Mausumimukhopadhyay. overview of Fungal L ipase: A review.2012,166(2): 486-.

For example, in the processing of fats and oils, since the hydrolysis of fats and oils can be carried out at normal temperature and pressure due to the introduction of lipase, biological substances such as highly unsaturated fatty acids and tocopherols are not denatured. 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. The lipase also has application in drug production, weight reduction, etc. 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 the Candida (Candida sp) 99-125 with fabric membrane immobilized are common enzymes for producing biodiesel. 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 catalyze the hydrolysis of milk fat to produce free fatty acids. The fatty acids may include short chains (C)₄-C₆Fatty acids, i.e. butyric acid, caproic acid) and medium to long chains (C)₁₂-C₁₈) The lipase used may influence the type of free fatty acids released in the cheese, e.g. the pungent, strong flavour mainly resulting from the release of short chain fatty acids (C)₄-C₆) While medium-long chain fatty acids give a soapy taste. Much research has been devoted to engineering lipases as short-chain preferential types for use in cheese making.

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 preferred embodiment, the host cell is E.coli B L21 (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 BM5 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 and/or one or more of the following properties: has good enzyme activity and stability in the alkaline pH8.0-8.5 range; the lipase hydrolysis activity is promoted in the presence of SDS, Tween80 and NaCl; and lipase enzyme activity is kept stable in the presence of NaCl and EDTA.

Brief description of the drawings

FIG. 1 shows a gel electrophoresis of BM5 polypeptide. Lane 1 is BM5 polypeptide, and lane 2 is a molecular weight marker.

FIG. 2 shows the catalytic hydrolytic activity of BM5 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 enzymatic activity of BM5 at different temperatures.

FIG. 4 shows the enzymatic activity of BM5 at different pH.

Figure 5 shows the stability of lipase activity at different pH of BM 5.

Figure 6 shows the effect of metal ions on the lipase activity of BM 5. The control group is prepared by adding water into the reaction system, and the stock solution of the rest groups added into the reaction system is ZnSO₄、MnCl₂、CoCl₂、CaCl₂、MgSO₄、CuSO₄、KCl、(NH4)₂SO₄、NaCl、NiSO4、FeCl₃Sodium citrate (C)₆H₅Na₃O₇) And disodium Ethylenediaminetetraacetate (EDTA).

Figure 7 shows the effect of surfactant on the lipase activity of BM 5. The control group was added with water to the reaction system, and the other groups were added with 0.5% cationic surfactant CTAB, anionic surfactant SDS, nonionic surfactant Tween80, AEO-9 and Triton X-100, respectively.

Brief description of the sequences

1, SEQ ID NO: amino acid sequence of lipase BM5

MGVQQVKGFQGDNPNKVGRHNIATSVKHYLGYGAPRTGKDRTPAYISPSDLREKFFEPYRACIEAGALTVMVNSGSINGRPVHANNELLTKWLKEDLNWDGMIVTDWADINNLYTREYVAHDKKEAIEMAINAGIDMSMEPYDLNFCTLLKELVNEGRVSQERIDDAARRVIRLKYRLGLFDTPNTYPKDYADFACDRHEQVALQAAEESEILLKNNGGILPLKKGTKILLTGPNANSMRCLNGGWSYSWQGHLTDRFASKYNTIYKALVNKFGKKNIVLEQGVTYPAEGAYHEENEPEIEKAVAAASGVDVIVACIGENSYCETPGNLSDLAVSENQRNLVKALAATGKPVILILNGGRPRIISDIEPLANAIVNILLPGNFGGDALANILAGDTNPSGKMPYTYPRHQAELTTYDYRASERMDKMKGAYDYDVVVSVQWPFRLWTQLHDIPNMNNLRCF

2, SEQ ID NO: nucleotide sequence of lipase BM5

ATGGGCGTTCAGCAGGTCAAAGGCTTTCAGGGCGATAACCCGAATAAGGTAGGCAGACATAATATCGCTACCTCGGTTAAGCATTATCTCGGCTATGGCGCTCCCCGTACAGGTAAAGACCGCACGCCGGCATATATTTCACCGTCTGATCTCCGCGAGAAGTTTTTTGAGCCTTACCGGGCCTGTATCGAGGCGGGTGCGCTGACCGTAATGGTCAACAGCGGTTCAATCAACGGTCGCCCCGTTCACGCCAACAATGAACTGCTCACAAAGTGGCTCAAGGAGGACCTGAACTGGGACGGAATGATTGTGACTGACTGGGCCGACATAAACAACCTCTATACCCGCGAATATGTGGCCCACGACAAAAAAGAGGCTATTGAAATGGCTATCAATGCCGGAATCGATATGTCAATGGAACCATACGATCTCAATTTCTGCACCTTGCTGAAGGAGCTTGTAAATGAGGGTCGTGTATCGCAGGAGCGTATCGACGATGCAGCACGTCGTGTGATTCGTCTGAAATATCGTCTCGGGCTGTTTGACACTCCCAATACATATCCCAAAGACTATGCCGACTTCGCCTGCGACCGTCACGAGCAAGTCGCTCTTCAGGCCGCGGAAGAATCGGAGATACTGCTCAAAAACAACGGCGGCATTCTTCCGTTGAAGAAAGGTACAAAGATTCTGCTGACAGGTCCTAATGCCAACTCGATGCGCTGTCTCAATGGCGGCTGGAGCTATTCATGGCAAGGTCATCTGACTGACCGTTTCGCATCGAAATACAACACCATCTACAAGGCATTGGTCAATAAGTTTGGCAAAAAGAATATCGTTCTTGAACAAGGTGTCACTTATCCTGCCGAAGGAGCCTATCACGAGGAAAATGAGCCGGAGATTGAAAAAGCTGTGGCAGCGGCATCAGGTGTAGATGTCATTGTCGCCTGTATCGGTGAAAACTCCTACTGCGAGACACCGGGCAATCTGTCAGACCTAGCTGTTTCTGAGAACCAGCGCAATCTTGTGAAGGCTCTTGCTGCGACAGGTAAACCAGTTATATTGATCCTCAACGGCGGTCGCCCGCGTATCATCAGTGATATAGAGCCATTGGCAAATGCGATAGTCAACATTCTGCTTCCCGGTAACTTCGGTGGTGACGCACTTGCCAACATTCTTGCCGGTGACACCAATCCTAGCGGTAAAATGCCCTACACCTATCCACGCCATCAGGCAGAACTCACCACCTACGATTATCGTGCGAGCGAAAGAATGGATAAAATGAAAGGTGCTTACGACTACGATGTCGTTGTGTCTGTGCAGTGGCCTTTTCGGCTATGGACTCAGTTACACGACATTCCAAATATGAACAATCTCCGCTGCTTC

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

(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 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 5.

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, which are known to those skilled in the art.

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 BM5 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 other of these aliphatic amino acids, substitution of serine (S) for threonine (T) and vice versa, substitution of aspartic acid (D) for glutamic acid (E) and vice versa, and substitution of glutamic acid (D) for glutamic acid (E) and vice versaThe amino amide (Q) replaces asparagine (N), 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)^nded.Lubert Stryered.(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 BM5 polypeptide do not substantially affect lipase activity.

In other specific embodiments, the C-terminal or N-terminal region of the BM5 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 5.

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 BM5 polypeptide, the resulting BM5 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 BM5 polypeptide, as long as the altered polypeptide substantially retains the lipase activity of BM 5.

In certain embodiments, a polypeptide of the invention, e.g., a BM5 polypeptide or variant thereof, is fused to a heterologous polypeptide in some embodiments, the BM5 fusion protein substantially retains the lipase activity of BM 5.

In a specific embodiment, the fusion protein comprises a BM5 polypeptide and a tag, typically a peptide tag, attached to the C-terminus or N-terminus of the BM5 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. Examples of such tags are those capable of binding nickel (Ni) with high affinity²⁺) Column or cobalt (Co)²⁺) Other exemplary tags for isolating or purifying the fusion protein include Arg-tag, F L AG-tag, Strep-tag, etc.

Polynucleotide

The present application provides polynucleotides encoding the above polypeptides 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 certain specific embodiments, the polynucleotides of the invention encode BM5 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 BM5 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 BM5 polypeptide and functionally equivalent variants thereof under stringent conditions and encodes a polypeptide functionally equivalent to a BM5 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, DNA hybridization is performed using stringent conditions including 1) washing with low ionic strength and high temperature, e.g., 0.015M sodium chloride/0.0015M sodium citrate/0.1% sodium dodecyl sulfate at 50 ℃; 2) hybridization with denaturants such as formamide, e.g., 50% (v/v) formamide plus 0.1% bovine serum albumin/0.1% Ficoll/0.1% polydiallylpyrrolidone/50 mM sodium phosphate buffer pH6.5 and 750mM sodium chloride, 75mM sodium citrate at 42 ℃, or (3) overnight hybridization at 42 ℃ with hybridization solutions containing 50% formamide, 5 XSSC (0.75M sodium chloride, 0.075M sodium citrate 1), 50mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5 XDenhardt's solutions, sonicated salmon sperm DNA (50.mu.g/ml), 0.1% SDS and 10% sodium sulfate, followed by washing with a stringent conditions such as stringent conditions with at least one of a cDNA 5mM, preferably at least one of a cDNA, preferably at least one of a sequence of a length of cDNA, at least about 26% cDNA, preferably at least about cDNA, more stringent conditions including a stringent conditions of cDNA, such as stringent DNA hybridization with a cDNA, stringent conditions of at least about cDNA 5mM EDTA, more preferably at least about 10% cDNA, more stringent conditions described by stringent conditions including stringent conditions of cDNA, such as stringent conditions of cDNA, stringent conditions including stringent conditions of cDNA, stringent conditions including stringent conditions of 0.26 mM NaCl, stringent conditions of cDNA, stringent conditions of 0.8, stringent conditions of cDNA, stringent conditions including at least about 10% cDNA, stringent conditions of cDNA, stringent conditions of at least about 10, stringent conditions of at least about 10% cDNA, stringent conditions of at least about 10, stringent conditions of cDNA, more than about 10% cDNA, more preferably stringent conditions of DNA (0.8. 20.8), stringent conditions of DNA, more preferably stringent conditions of DNA, stringent conditions of at least about 10. 12. 20. 12. stringent conditions of DNA, more than about 0.8. stringent conditions of at least about 0.8. 20. stringent conditions of DNA, more than about 0.8. stringent conditions of DNA, more preferably stringent conditions of DNA, more than about 0.8. stringent conditions of DNA, more than about 0.8. stringent conditions of DNA, stringent conditions of DNA (about 0.8. stringent conditions of DNA, more preferably stringent conditions of DNA, more than about 0.8. 20. stringent conditions of DNA.

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, for example, the dideoxy chain termination method (Sanger et. 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 BM5 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 nucleotide sequences encoding BM5 polypeptides and variants thereof and appropriate transcription/translation regulatory elements are constructed using methods well known to those skilled in the art, including in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombinant techniques, etc. (Sambrook, et al, molecular Cloning, a L anaerobic Manual, cold Spring Harbor L anaerobic. New York,1989) nucleotide sequences are operably linked to appropriate promoters in expression vectors to direct mRNA synthesis, representative examples of these promoters include the lac or trp promoter of E.coli, the P L promoter of bacteriophage lambda, eukaryotic promoters including the CMV immediate early promoter, the HSV thymidine kinase promoter, the early and late SV40 promoters, the L TRs of retroviruses, and other promoters known to control gene expression in prokaryotic cells or eukaryotic cells or viruses, expression vectors also include ribosome promoters for translation initiation, ribosome termination sites, SV terminator sites, etc. the promoter is inserted into the vector to enhance the action of the promoter on one side of the transcription enhancer sequence of the cis-acting in the vector, the enhancer from the high promoter, SV promoter, enhancer promoter on the side of the transcriptional start site, SV promoter, enhancer on the viral origin of transcription of the cis, enhancer on the viral vector, SV, enhancer on the side of the viral gene, enhancer on the viral origin of transcription factor of 10, and the promoter, SV, enhancer on the promoter, SV, etc. 300.

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 BM5 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 using any technique known in the artSpecific Methods include calcium phosphate transfection, DEAE-dextran mediated transfection, lipofection, or electroporation (Davis, L., Dibner, M., Battey, I., Basic Methods in molecular Biology, (1986)) as an example, when the host is a prokaryote such as E.coli, competent cells can be harvested after the exponential growth phase using CaCl, which is well known in the art₂The method is used for transformation.

In a preferred embodiment, the expression vector carrying the polynucleotide sequence of the invention is transformed into E.coli B L21 (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 from applied biosystems (Perkin Elmer). 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

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

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.

For example, the expressed polypeptide or a fragment thereof can be recovered and purified from recombinant cell cultures by conventional renaturation treatment, protein precipitant treatment (salting-out method), centrifugation, osmotic lysis, sonication, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HP L C) and other various liquid chromatography techniques and combinations of these methods.

In a preferred embodiment, a method for preparing a polypeptide consisting of the amino acid sequence shown in SEQ ID NO. 1 is provided, which comprises cloning a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO. 2 into an expression vector pCold-TF, and transforming the pCold-TF with the polynucleotide sequence into E.coli B L21 (DE3) for inducible expression, followed by isolating and purifying the BM5 polypeptide from E.coli B L21 (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 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 BM5 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.

pCold-TF having the above nucleotide sequence was transformed into E.coli B L21 (DE3) for induction expression (see, specifically, third edition of molecular cloning protocols, science publishers 2002[ Mei ] J. sambrook, D.W Lassel, Huang Petang et al), under the conditions of heat shock at 42 ℃ for 50 seconds, ice bath for 2 minutes, plating L B plates, selecting transformants, inoculating the transformants into L B medium (peptone 10 g/L, yeast extract 5 g/L, and sodium chloride 10 g/L), performing overnight seed culture at 37 ℃, inoculating 1% of the inoculum size of the seed culture into the expression medium, and culturing at 37 ℃ and 220rpm until OD600 is 0.6-0.8.

After cooling to 16 ℃, 1mM of isopropyl- β -D-thiogalactopyranoside (IPTG) was added for induction and expression was performed overnight at 16 ℃ at 190rpm after induction expression was completed, the cells were collected by centrifugation and resuspended in lysis buffer (50mM sodium dihydrogen phosphate, 300mM sodium chloride, 10mM 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.

Every 100 ml of supernatant was added with 100 ml of Ni-NTA resin and shaken in ice bath for 60 minutes. Transferring the cell disruption supernatant and the Ni resin to a Ni-NTA Agarose chromatography column (Qiagen, Cat. No.30210), after the cell disruption supernatant was completely passed through the Ni resin, washing with 10 column volumes of elution buffer 1(50mM sodium dihydrogen phosphate, 300mM sodium chloride, 20mM imidazole, pH8), then with 10 column volumes of elution buffer 2(50mM sodium dihydrogen phosphate, 300mM sodium chloride, 50mM imidazole, pH8), finally with 4 column volumes of elution buffer 3(50mM sodium dihydrogen phosphate, 300mM sodium chloride, 250mM imidazole, pH8), collecting the eluate, dialyzing the eluate at 4 ℃ overnight, wherein the formulation of the dialysate used was: 150mM sodium chloride, 20mM Tris-HCl, 10mM zinc sulfate, 1mM dithiothreitol, pH8. The results of detection by gel electrophoresis (10% SDS-PAGE,100V,2 hours) are shown in FIG. 1. According to the results of fig. 1, the resulting solution was a purified BM5 protein solution.

Example 2: enzymatic Properties of Lipase BM5

Method for measuring lipase activity

The method comprises the following steps of preparing a substrate and a buffer solution in advance, wherein the substrate is 6mg/M L pNPB (dissolved in isopropanol), the buffer solution is 0.05M Tris (pH8.0 and 0.1% of Arabic gum), preparing a reaction mixed solution from the substrate and the buffer solution according to a ratio of 1:9(v/v), taking two 2M L centrifugal tubes which are respectively a control tube and a sample tube, adding 400u L reaction mixed solution to the two centrifugal tubes respectively, carrying out pre-temperature bath at a proper reaction temperature (for example, 35 ℃) for 5min, adding a certain amount of diluent enzyme into the sample tube, mixing uniformly, continuing to carry out warm bath for 15min, adding 1.5M L ethanol into the two centrifugal tubes to terminate the reaction, adding the diluent enzyme with the same amount to the control tube, carrying out centrifugation at 12000rpm for 2min, and taking the supernatant with a light absorption value of 405 nm.

The enzyme activity unit is defined as 1 unit, namely the enzyme quantity required for catalyzing and releasing 1 mu mol of pNP per minute under the standard experimental conditions, the calculation formula of the enzyme activity obtained according to the standard curve is that A ═ A- ([ A1-A0] × 0.7885-0.0118) × V1 × n/(V2 × t), A is the sample enzyme activity (U/m L), A1 is the OD405 of the sample enzyme solution, A0 is the OD405 of the control enzyme solution, V1 is the volume of the total reaction solution (m L), n is the dilution multiple of the enzyme solution, V2 is the volume of the enzyme solution (m L), and t is the reaction time (min).

Example 2-1Substrate specificity

6mg/m L of 4-nitrophenyl butyrate (PNPB), 4-nitrophenyl caprylate (pNPO), 4-nitrophenyl laurate (pNPD) and 4-nitrophenyl palmitate (pNPP) are prepared, all dissolved in isopropanol, the lipase activity is detected according to the standard pNPB method (replacing the substrate with the corresponding substrate), the enzyme activity detected by using the highest substrate with enzyme activity is 100%, and the relative enzyme activity for hydrolyzing other substrates is calculated.

Examples 2 to 2Optimum temperature of action

The lipase activity is measured according to a pNPB method at different temperatures (30-50 ℃), and the relative enzyme activity (expressed in percentage) at other temperatures is calculated according to the enzyme activity when the highest enzyme activity is measured as 100%. As can be seen from FIG. 3, the enzymatic activity of BM5 has better activity in the range of 30 to 50 ℃, wherein the enzyme activity (0.30393207U/ml) is the highest at about 45 ℃, which indicates that the optimum action temperature of BM5 lipase is about 45 ℃.

Examples 2 to 3Optimum pH for action

And (3) respectively measuring the lipase activity of the enzyme solution in buffer solutions (Tris-HCl solutions) with different pH values (6.5-10.0) at 45 ℃ according to a pNPB method, wherein the enzyme activity when the activity is highest is measured to be 100%, and calculating the relative activity (expressed in percentage) of the enzyme at other pH values. As can be seen from FIG. 4, the enzyme activity of BM5 shows a tendency of rising first and then falling with the rise of pH value, wherein the enzyme activity is highest at pH8.5, therefore, the optimum pH value of BM5 is around 8.5. From this, it was found that lipase BM5 is an alkaline lipase.

Examples 2 to 4Stability of pH

Respectively mixing BM5 enzyme solution in buffer systems with different pH values (4.0-10.0) (wherein, Tris-HCl buffer solution is used at pH4-9, and Gly-NaOH buffer solution is used at pH 9.5-10) at a ratio of 1:1, keeping the temperature at 4 ℃ for 24 hours, and determining the lipase activity by a pNPB method at 45 ℃ and pH 8.0. The relative enzyme activities at other pHs were calculated with the enzyme activity (0.2933788U/ml) measured in the buffer with the highest enzyme activity as 100%. As can be seen from FIG. 5, the BM5 lipase maintains stable enzyme activity in (8.0-8.5), the lipase activity decreases rapidly at pH > 8.0, and the residual lipase activity is 0 at pH 10.0. .

Examples 2 to 5Effect of Metal ions on Lipase Activity

Preparation of ZnSO₄、MnCl₂、CoCl₂、CaCl₂、MgSO₄、CuSO₄、KCl、(NH4)₂SO₄、NaCl、NiSO4、FeCl₃Sodium citrate (C)₆H₅Na₃O₇) According to a pNPB method, reaction mixed liquor is prepared firstly, 400U L mixed liquor is filled in each reaction tube, the salt storage liquor with the final concentration of 5 mmol/L is added, the activity is measured according to a standard method, water is added to a control group to replace the salt storage liquor, the enzyme activity (0.198126U/ml) is taken as 100%, and the relative enzyme activity of the rest groups relative to the control group is calculated.

Examples 2 to 6Effect of surfactants on Lipase Activity

Stock solutions of 10% of cationic surfactant CTAB, anionic surfactant SDS, nonionic surfactant Tween80, AEO-9, Triton X-100 and the like are prepared. And (3) according to a pNPB standard method, simultaneously adding 0.5% of the surfactant into a reaction system, and measuring the activity of the lipase. Control group supplementWater is added to replace the surfactant, the enzyme activity (0.69U/ml) is taken as 100%, and the relative enzyme activity of the rest groups relative to the control group is calculated. As shown in FIG. 7, SDS, Tween80 and NaCl promoted the hydrolytic activity of lipase, H₂O₂Completely inhibit enzyme activity, and other active agents inhibit the enzyme activity to different degrees.

From the above results, it can be seen that the polypeptides of the present application have lipase activity, and/or have at least one of the following beneficial properties:

1) the specificity of medium-short chain fatty acid, when the medium-short chain fatty acid is taken as a substrate, the enzyme activity is detected; on the other hand, when other long-chain fatty acids were used as substrates, no enzyme activity was detected. 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.

2) Has better enzyme activity and stability in alkaline pH of 8.0-8.5.

3) The lipase activity is kept stable in the presence of NaCl and EDTA.

4) SDS, Tween80 and NaCl have the function of promoting the hydrolytic activity of lipase.

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 (11)

1. A polypeptide having lipase activity, consisting of a sequence selected from the group consisting of:

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

(b) a sequence obtained by deleting an amino acid from the N-terminus of the sequence of (a),

wherein the polypeptide variant obtained in (b) retains lipase activity.

2. The polypeptide of claim 1, which consists of the amino acid sequence shown in SEQ ID NO 1.

3. A polynucleotide encoding the polypeptide of claim 1 or 2, comprising or consisting of a sequence selected from:

(a) a nucleotide sequence encoding the amino acid sequence of claim 1(a) or 1 (b); and

(b) a nucleotide sequence that hybridizes under stringent conditions to the nucleotide sequence in 3 (a).

4. The polynucleotide of claim 3, comprising the nucleotide sequence set forth in SEQ ID NO 2.

5. The polynucleotide according to claim 4, which consists of the nucleotide sequence shown in SEQ ID NO. 2.

6. An expression vector comprising at least one polynucleotide of any one of claims 3-5.

7. The expression vector of claim 6, further comprising a control sequence that regulates expression of the polynucleotide, wherein the polynucleotide is operably linked to the control sequence.

8. A host cell comprising the polypeptide of claim 1 or 2, the polynucleotide of any one of claims 3-5, or the expression vector of claim 6 or 7.

9. Use of the polypeptide of claim 1 or 2, the polynucleotide of any one of claims 3-5, the expression vector of claim 6 or 7, or the host cell of claim 8 in the preparation of a lipase.

10. Use of the polypeptide of claim 1 or 2, the polynucleotide of any one of claims 3-5, the expression vector of claim 6 or 7, the host cell of claim 8 in the manufacture of a food product.

11. Use according to claim 10, wherein the use is in the manufacture of a dairy product.

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