CN100351379C - Transglutaminase Gene and Its Encoded Transglutaminase from Streptomyces ladaka - Google Patents

Abstract

The invention provides a DNA molecule encoding transglutaminase from streptoverticillium ladakanum (Streptoverteillium ladakanum), the encoded transglutaminase and the use of the transglutaminase in industrial engineering.

Description

Transglutaminase Gene and Its Encoded Transglutaminase from Streptomyces ladaka

technical field

The present invention relates to a novel DNA molecule encoding transglutaminase and the encoded transglutaminase.

Background technique

Transglutaminase is a Ca ²⁺ -dependent enzyme that catalyzes the formation of an isopeptide bond (isopeptide bond) between the glutamine side chain γ-carbamoyl group and the lysine side chain ε-amino group in proteins. Transglutaminase can be used in food processing, such as producing gelatinous cross-linked gels, inducing surface cross-linking of fiber bundles and producing cheese products. Transglutaminase is also useful in the treatment of chronic wound conditions and as a bioglue.

Transglutaminase is found both extracellularly and intracellularly. A variety of transglutaminases have been identified and characterized from many animal and some plant species. Unfortunately, transglutaminases derived from animals such as guinea pigs are not available industrially because of the difficulty in obtaining such animal-derived transglutaminases at low cost in large quantities. Only a few microbial transglutaminases have been disclosed so far, namely from the species Streptoverticillium mobaraense, Streptoverticillium cinnamoneum and Streptoverticillium griseocarneum (see US Patent 5,156,956) and transglutaminase from a species predicted to be Streptomyces lavendulae (see US Patent 5,252,469). According to Wu et al., among the strains screened, the transglutaminase of Streptoverticillium ladakanum has the highest activity (Wu et al., 1996, Chinese Agric.Chem.Soc.34 (2): 228-40).

The gene encoding transglutaminase has been obtained from Streptothrix spp. S-8112 (Washizu et al., 1994, Biosci.Biotechnol.Biochem.58 (1): 82-7), Streptothrix cinnamomi (Pasternack et al., 1998, Eur.J.Biochem.257(3):570-6), Streptomyces lydicus (WO9606931) and Bacillus subtilis (Kobayashi et al., 1998, J.Gen.Appl.Microbiol.44:85 -91) cloned.

In addition, the current genetic engineering technology has made it relatively easy to obtain a large number of enzymes, which can be achieved by isolating the gene encoding the enzyme, determining the base sequence of the enzyme gene, producing a recombinant DNA containing the gene encoding the enzyme, and incorporating the recombinant DNA into microorganisms or animals or plant cells, and culturing the obtained transformants.

Contents of the invention

An object of the present invention is to provide isolated and purified DNA molecules comprising a sequence encoding transglutaminase, wherein said nucleic acid hybridizes to the sequence shown in SEQ ID NO: 1 or its complementary sequence under highly stringent conditions.

Another object of the present invention is to provide expression vectors comprising the DNA molecules of the present invention.

Another object of the present invention is to provide a host cell comprising the expression vector of the present invention.

Yet another object of the present invention is to provide a polypeptide comprising the amino acid sequence encoded by the DNA molecule of the present invention.

Description of drawings

Figure 1 shows a partial fragment (644bp) of the transglutaminase gene of Streptothrix mobara CCRC 12165.

Figure 2 shows the 3241 nucleotide sequence of the KprI fragment and the genes located around nucleotides 700-1900 of the second open reading frame of the KpnI fragment.

Figure 3 shows restriction maps of plasmids pAE051 and pAE052.

Figure 4 shows the expression activity of plasmid pAE051 in Streptomyces lividans (S. lividans) JT46.

Detailed ways

The invention features a novel DNA molecule encoding a transglutaminase from Streptoverticillium ladakanum. In addition, the present invention provides a construct expressing the encoded transglutaminase with high activity.

definition

The term "isolated and purified" as used herein refers to the in vitro separation of a DNA molecule or protein from its natural cellular environment and association with other coding regions of the bacterial genome so that it can be sequenced, replicated and/or expressed. Preferably, the isolated and purified DNA molecules of the invention comprise a single coding region. Although a DNA molecule contains a single coding region, it may contain additional nucleotides that do not negatively affect its function. For example, the 5' and 3' noncoding regions may contain different numbers of nucleotides. Preferably, the additional nucleotides are located outside the single coding region.

The term "amino acid sequence" as used herein refers to the amino acid sequence of a naturally occurring protein molecule, and "amino acid sequence" and similar terms such as "polypeptide" or "protein" do not limit the amino acid sequence to the complete amino acid sequence associated with the referenced protein molecule . Amino acid sequences include oligopeptide, peptide, polypeptide or protein sequences, and fragments or portions thereof, as well as naturally occurring or synthetic molecules.

The term "deletion" as used herein refers to an amino acid or nucleotide change wherein one or more amino acid or nucleotide residues are deleted.

The term "insertion" or "addition" as used herein refers to a change in amino acid or nucleotide sequence that results in the addition of one or more amino acid or nucleotide residues compared to a naturally occurring molecule.

The term "vector" as used herein refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. A preferred vector is an episome, ie a nucleic acid capable of extrachromosomal replication. Preferred vectors are those capable of autonomous replication and/or expression of nucleic acids to which they are linked. A vector capable of directing the expression of a gene to which it is operably linked is referred to herein as an "expression vector." In general, expression vectors of practical use in recombinant DNA techniques are usually in the form of "plasmids", which generally refer to circular double-stranded DNA loops which, in vector form, are not chromosomally bound.

The term "host cell" as used herein refers to a cell of a host that can be infected by a vector such as a plasmid. Hosts suitable for the present invention include hosts commonly and routinely used in the art.

nucleic acid

An object of the present invention is to provide isolated and purified DNA molecules comprising a sequence encoding transglutaminase, wherein said nucleic acid hybridizes to the sequence shown in SEQ ID NO: 1 or its complementary sequence under highly stringent conditions. More preferably, said DNA molecule encoding has the transglutaminase of aminoacid sequence shown in SEQ ID NO:2. Most preferably, the DNA molecule shown is represented by the complete nucleotide sequence shown in SEQ ID NO:1.

Nucleic acids with sequence similarity are detected by hybridization under highly stringent conditions. Highly stringent hybridization conditions refer to, for example, containing 0.25M Na ₂ HPO ₄ (pH 7.4), 7% sodium dodecyl sulfate (SDS), 1% bovine serum albumin (BSA), 1.0 mM ethylenediaminetetra Hybridization was carried out at 65°C in acetic acid (EDTA, pH8) buffer, followed by washing three times at 65°C with 0.1% SDS and 0.1 times SSC (0.1 times SSC contained 0.015M sodium chloride and 0.0015M trisodium citrate, pH7. 0).

The sequence encoding the transglutaminase of the present invention can be mutated in various ways known in the art to produce directional changes in promoter strength, sequence of the encoded protein, and the like. Such mutated DNA sequences or products are substantially similar to the sequences provided herein, ie, they will differ by at least one nucleotide or amino acid, and may differ by at least two, but not more than about ten nucleotides or amino acids. The changes may be substitutions, insertions or deletions.

There are currently several different methods for isolating the DNA of the invention. These methods include, for example, purification of enzyme proteins followed by amino acid microsequencing directly or after restriction cleavage. The obtained partial amino acid sequences can be used to design degenerate oligonucleotide probes or primers for generating unique non-degenerate nucleotide sequences via polymerase chain reaction (PCR), which can then be used for probe screening Sequence of the DNA library. DNA clones can also be isolated from DNA expression libraries using antibodies raised against purified proteins. Alternatively, the sequence of the DNA molecule of the relevant enzyme can be used as a starting point for a cloning strategy. This method is often referred to as "homologous cloning". Another way of using sequence information from different species is to narrow down regions of high sequence homology in related DNA molecules from different species and perform the polymerase chain reaction sequencing amplification method (PCR) to obtain "species-specific "Non-degenerate nucleotide sequence. This sequence can then be used for DNA library screening or even for direct PCR-based DNA cloning.

Oligonucleotide probes can be used to detect and amplify the DNA molecules of the invention in a variety of samples using biochemical procedures well known in the art. For example, Southern or Northern blot hybridization techniques using labeled probes can be used. Alternatively, PCR technology can be used, and nucleic acid sequencing of the amplified PCR products can be used to detect mutations in the DNA.

Expression Vectors and Host Systems

Another object of the present invention is to provide an expression vector containing the DNA molecule shown in SEQ ID NO:1. In order to express the biologically active transglutaminase, the nucleic acid sequence encoding the transglutaminase can be inserted into a suitable expression vector, that is, a vector containing elements necessary for transcription and translation of the inserted coding sequence. According to the present invention, methods well known in the art can be used to construct expression vectors containing the DNA molecule shown in Figure 1 and appropriate transcriptional and translational control elements. These methods include in vitro recombinant DNA techniques, synthetic techniques and in vivo genetic recombination.

Another object of the present invention is to provide a host cell containing an expression vector containing the DNA molecule shown in SEQ ID NO: 1. According to the present invention, various host systems can be used to contain and express the sequence encoding transglutaminase, and these host systems include but are not limited to microorganisms transformed with recombinant phage, plasmid or cosmid DNA expression vectors, such as bacteria; A vector transformed with an expression vector; a plant cell system transformed with a viral expression vector or a bacterial expression vector; or an animal cell system. Preferably, Streptomyces lividans is transformed with the vector to express transglutaminase.

polypeptide

Another object of the present invention is to provide a polypeptide comprising the amino acid sequence encoded by the DNA molecule shown in SEQ ID NO:1. Preferably, the polypeptide comprises the amino acid sequence shown in SEQ ID NO: 2.

The polypeptides of the invention can be provided in large quantities. By using an expression host, proteins can be isolated and purified according to conventional methods. Lysates of expression hosts can be prepared and purified using HPLC, size exclusion chromatography, gel electrophoresis, affinity chromatography, or other purification techniques. Purified proteins are generally at least 80%, preferably at least 90%, and up to 100% pure. Purified means free from other proteins and cellular debris.

use

The DNA molecules of the present invention can be used to encode transglutaminases. The DNA molecule is expressed in large quantities to produce a large amount of transglutaminase. The resulting transglutaminase has various industrial uses including gelling proteins, improving the baking quality of flour, producing pasty food or food ingredients from protein, fat and water, producing cheese from concentrated milk, binding ground meat Or fish products, improve the taste and texture of food protein, fix casein in leather processing, shine shoes, etc.

The following examples are illustrative and not limiting.

The transglutaminase gene of embodiment 1 Ladaka sp.

A. Cloning of the transglutaminase gene of Streptomyces ladaka

(1) PCR amplification and purification of the transglutaminase gene fragment of Streptomyces ladaka, degenerate primers were designed according to two amino acid fragments FDEEKGF and KVKQGWP, which were cloned by Washizu et al. in 1994 The least degenerate region in the transglutaminase amino acid sequence of Streptomyces sp. S-8112 (Washizu K. et al., Biosci. Biotechnol. Biochem. 58(1):82-7). The two degenerate primers are:

5'-aaaaacctgaaaccctt(ct)ga(ct)ga(ag)ga(ag)aa(ag)gg(gact)tt-3', and

5'-cttatcaacggatacggcca(gatc)cc(tc)tg(tc)tt(gact)ac(tc)tt-3'.

The two nested primers are:

5'-aaaaacctgaaaccc-3', and

5'cttatcaacggatac-3'.

A partial fragment of the transglutaminase of Streptomyces mobara CCRC 12165 was amplified by nested PCR, and the result showed a DNA band of about 650bp, which corresponded to the calculated length of the PCR product (644bp). The resulting DNA sequence is shown in Figure 1 (SEQ ID NO: 3). For S. mobara CCRC12165, see the catalog of the Culture Deposit and Research Center of the Food Industry Development Institute (FIRDI), Hsinchu, Taiwan.

(2) Using the partial transglutaminase gene fragment of Streptotrichum mobara as a probe to detect the complete transglutaminase gene of Streptotrichum ladaka

Southern blot experiments were performed using the above 644bp DNA as a probe. Signals were detected in the following restriction fragments of the S. ladaka genome: 8.4 kb BamHI DNA, 6 kb BclI DNA, 9 kb NcoI DNA and 7.5 kb PstI DNA. For S. ladaka, see the catalog of the Culture Depository and Research Center of the Food Industry Development Institute (FIRDI), Hsinchu, Taiwan (Accession No. CCRC12422).

In order to purify the DNA fragment containing the transglutaminase gene, the genomic DNA of Streptomyces ladaka was digested with NcoI and separated by electrophoresis. The purified 9kb DNA was inserted into the pMTL23 vector digested with NcoI and transformed into Escherichia coli DH5α to form a DNA library. Use above-mentioned 644bp DNA again as probe to carry out bacterium colony hybridization experiment, and select the recombinant vector that comprises the transglutaminase gene of S. ladaka. Since the NcoI fragment was too large to be sequenced, a 3.2 kb KpnI fragment comprising the transglutaminase gene was cloned and inserted into the KpnI restriction site on the pMT23 vector (forming pAE021), followed by sequencing. The 3241 nucleotide sequence of the KpnI fragment is shown in FIG. 2 . The sequence was analyzed with code optimization software provided by GCG, and the gene was predicted to be located at about 700-1900 nucleotides of the KpnI fragment sequence ( FIG. 2 ). The gene is the transglutaminase gene of S. ladaka. The amino acid sequence deduced from the above nucleotide sequence is also shown in Fig. 2, which consists of 410 amino acids and has a molecular weight of 45780.2 Daltons. It is predicted that the mature transglutaminase of S. ladaka starts from the 80th amino acid and consists of 331 amino acids (the underlined area in FIG. 2 ), its molecular weight is 37922.3 Daltons, and its isoelectric point is 7.07.

B. Expression of the transglutaminase gene from S. ladaka in S. lividans

pAE021 was digested with BglI and BamHI, and the resulting 3.2 kb DNA fragment containing the transglutaminase gene was purified and inserted into the BglII restriction site on pIJ702 to obtain pAE051 and pAE052 (Fig. 3A and B). pAE052 was expressed in S. lividans JT46 and the transglutaminase activity in the supernatant was measured every 24 hours. At 72 hours, the transformed clone had a maximum transglutaminase activity of 1.46 U/ml (Figure 4). Mature and unmodified or partially modified transglutaminases were detected by Streptomyces ladaka transglutaminase-specific antibody analysis of the extracellular medium with a molecular weight distribution between 45.8 kD and 38 kD . The above results indicated that Streptomyces lividans JT46 expressed a large amount of transglutaminase.

Sequence Listing

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Claims (10)

1, the dna molecular of a kind of separation and purifying, its nucleotide sequence by the coding trans-glutaminases is formed, described trans-glutaminases is formed by being no more than ten amino acid whose different aminoacid sequences by the aminoacid sequence shown in the SEQ ID NO:2 or with the aminoacid sequence shown in the SEQ ID NO:2, and described aminoacid sequence has the function of trans-glutaminases.

2, the dna molecular of a kind of separation and purifying, wherein said dna molecular is by the nucleotide sequence shown in the SEQ ID NO:1 or show the different nucleotide sequence that is no more than ten Nucleotide with the nucleotides sequence shown in the SEQ ID NO:1 and form, and described dna molecule encode trans-glutaminases.

3, the expression vector that comprises the dna molecular of claim 1.

4, the expression vector that comprises the described dna molecular of claim 2.

5, a kind of host cell, it is shallow Streptomyces glaucoviolaceus, it comprises a kind of carrier that comprises the nucleotide sequence of the trans-glutaminases of encoding, and described trans-glutaminases is by the aminoacid sequence shown in the SEQ ID NO:2 or be no more than 10 amino acid whose aminoacid sequences with the difference of the aminoacid sequence shown in the SEQ ID NO:2 and form.

6, a kind of host cell, it is shallow Streptomyces glaucoviolaceus, it comprises the expression vector of claim 3.

7, a kind of host cell, it is shallow Streptomyces glaucoviolaceus, it comprises the expression vector of claim 4.

8, a peptide species, it is made up of the coded aminoacid sequence of the different dna molecular that is no more than ten Nucleotide by the dna molecular shown in the SEQ ID NO:1 or with the dna molecular shown in the SEQ ID NO:1, and described dna molecule encode trans-glutaminases.

9, the polypeptide of claim 8, it is formed by being no more than ten amino acid whose different aminoacid sequences by the aminoacid sequence shown in the SEQ ID NO:2 or with the aminoacid sequence shown in the SEQ ID NO:2, and described polypeptide has the function of trans-glutaminases.

10, the polypeptide of claim 8 is used for gelling protein, improve the baking properties of flour, from protein, fat and aquatic product pasty food product or food ingredient, produce cheese from concentrating the Ruzhong, bonding meat mincing or fish product, improve the taste and the tissue of food protein, fixing casein in leather processing, or the purposes in the polishing-shoes.

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