Abstract:
A DNA molecule encoding transglutaminase, a transglutaminase, an expression vector containing the DNA molecule, and a cell containing the expression vector.

Description:
CROSS-REFERENCE TO RELATED APPLICATION 
     This application claims benefit of and priority to Taiwanese Patent Application No. 92126049 filed on Sep. 22, 2003, and which is incorporated by reference in its entirety. 
     BACKGROUND 
     The invention relates to a transglutaminase gene and a transglutaminase obtained therefrom. 
     Transglutaminase (hereafter, referred to as TGase) catalyzes intramolecular or intermolecular formation of ε-(γ-Gln)-Lys covalent bond, and the crosslink between protein molecules forms gel protein with a tertiary structure. The gel protein can be applied in the food-processing industry, including meat, fish, soybean, wheat, milk, or egg, as a new protein food or gel membrane. 
     TGases has been found in various tissues and organs of mammals and plants. The first commercialized TGase was isolated from the liver of guinea pig, however, its price is relatively high, about 80 US dollars per unit, because of the difficulties of acquisition. The high price also restricts its use in the food-processing industry. In addition, the technology of isolating TGases from fish or plants is immature. Large scale production of TGase is, therefore, an important task. 
     It has been found that many strains of microbes produce TGases. Those whose TGases have been cloned include  Streptoverticillium  S-8112 (Washizu et al., 1994),  Streptoverticillium mobaraense  (Pasternack et al., 1998),  Streptomyces lydicus  (Bech et al., 1996),  Bacillus subtilis  (Kobayashi et al., 1998). Those having exocrine TGase include S-8112 (Ando et al., 1989),  S. mobaraense  (Pasternack et al,, 1998),  S. cinnamoneum  (Duran et al., 1998), and  S. lydicus  (Bech et al., 1996). According to Wu et al. (1996), most TGases derived from  Streptoverticillium  sp. are exocrine. In the twenty strains of  Streptoverticillium  sp. tested by Wu et al., TGase derived from  Streptoverticillium ladakanum  has highest activity. The expression activity of those genes, however, are still restricted in some way, therefore, obtaining a TGase with high expression activity is still required. 
     SUMMARY 
     The inventors screened out a TGase producing strain,  S. platensis,  from more than 300 strains of  Streptomyces  stored in the Bioresources Collection and Research Center of the Food Industry Research and Development Institute. Overexpression of the cloned TGase gene from  S. platensis  produce a TGase with activity of 5.7 U/ml, which is 5.7 times that of the wild type. The invention was then achieved. 
     Accordingly, an embodiment of the invention provides a DNA molecule isolated from  Streptomyces platensis  encoding TGase, and the DNA molecule is composed of a nucleotide sequence of SEQ ID NO: 1. 
     In the DNA molecule derived from  S. platensis,  the sequence encoding TGase is composed of a nucleotide sequence of SEQ ID NO: 3. 
     Also provided is a TGase composed of an amino acid sequence of SEQ ID NO: 2. 
     Another embodiment of the invention provides an expression vector of TGase including a DNA sequence encoding TGase, composed of a nucleotide sequence of SEQ ID NO: 1. 
     Yet another embodiment of the invention provides a host cell including the expression vector of TGase. In this embodiment of the invention, the host cell is  Streptomzyces lividans.    
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
       Embodiments of the invention can be more fully understood and further advantages become apparent when reference is made to the following description and the accompanying drawings in which: 
         FIG. 1  illustrates the restriction map of the entire  S. platensis  TGase gene sequence of 2.9 Kb in an embodiment of the invention. 
         FIG. 2A–2E  illustrate the nucleotide and amino acid sequences of the  S. platensis  TGase gene in an embodiment of the invention. The frame region is the predictive ribosomal binding site, the underlined region is the mature form of the enzyme, and the bold words represent the amino acid sequence of the enzymatic active center, YGCV. 
         FIG. 3  illustrates the restriction map of pAE053. The abbreviation: tgs, TGase gene; tsr, thistrepton gene. 
         FIG. 4  illustrates the relation of TGase activity to culturing time in transformant 25-2. 
     
    
    
     DETAILED DESCRIPTION 
     More than 300 strains of  Streptomyces  stored in the Bioresource collection and research center of the Food Industry Research and Development Institute were screened and a strain M5218 was found having high activity of TGase, about 1.0 U/ml. After morphological, physiological, biochemical characteristic analysis and 16s rRNA sequence comparison, the strain was identified as  Streptomyces platensis.  The chromosomal DNA of the strain was digested with restriction enzyme Sau3AI, DNA fragments of 3–5 kb were then separated by electrophoresis and isolated from the gel. These DNA fragments were ligated with pIJ702 which is a high-copy vector and the plasmids were transformed into host  S. lividans  JT46 for overexpression. The cloned, sequenced, and analyzed TGase gene has a length of 1.25 kb and can be translated to be 418 amino acids. The transformed clone denominated as 25-2 was incubated in a 250 mL Erlenmeyer flask with 50 mL of media under 250 rpm vibration at 30° C. for 2 days. The TGase activity can be 5.7 U/mL, 5.7 times that of wild type. 
     Practical examples are described herein. 
     EXAMPLES 
     Above all, the sources of the materials used in the examples are illustrated herein. Restriction enzymes and T4 DNA ligase were purchased from Boehringer Mannheim and New England Biolab and the protocol is according to the instruction therewith. AmpliTaq Gold™ DNA polymerase was purchase from PE Applied Biosystems, Geneclean III from Bio101, thiostrepton from Sigma, and agarose from Gibco BRL. In addition, the carbon source of the medium includes 1% glucose, 1% glycerol, 1% starch, 0.1% sucrose, 1% fructose; the nitrogen source and salts include 0.5% glycine, 0.05% casein, 0.5% yeast extract, 0.05% terptone peptone, 0.05% (NH 4 ) 2 SO 4 , 0.05% polypeptone, 0.2% MgSO 4 .7H 2 O, 0.2% K 2 HPO 4 . 
     Example 1 
     Cloning of TGase Gene with High Productivity 
     300 strains of  Streptomyces  stored in the Bioresource collection and research center of the Food Industry Research and Development Institute were recovered and cultured by loop streak method in ISP3 medium at 30° C. for 3–4 days. Single colonies were selected and TGase producing clones were screened by qualitative analysis. 
     The qualitative analysis is as follows. The enzyme substrates including 1M Tris-HCl, pH6.0, 40 μl, 0.15M CBZ-Q-G, pH6.0, 20 μl, and 4M hydroxylamine, pH6.0, 20 μl were added in each well of a 96-well microplaate. The colonies were seeded to each well and incubated at 37° C. for 8 to 16 hours. Eighty μl of developing agent containing 15% TCA, 5% FeCl 3  in 2.5N HCl and 5% FeCl 3  in 0.1N HCl of volume ratio 1:1:1 was added to terminate the reaction. TGase activity was determined by the naked eye, and red-brown color represents TGase activity. 
     Five clones: M5218, M5802, M6701, PT7-1, and HTII11-2, were found having TGase activity, and M5218 has the hightest TGase activity of 1.0 U/mL. After morphological, physiological, biochemical characteristic analysis and 16s rRNA sequence comparison, the strain was identified as  Streptomyces platensis.    
     Example 2 
     Cloning of TGase gene from  Streptomycese platensis    
     The isolation of chromosomal DNA of  Streptomyces platensis  and plasmid DNA, and preparation and transformation of protoplast are according to Hopwood et al. (1985). Chromosomal DNA of  S. platensis  was digested by Sau3AI and separated by electrophoresis. DNA fragments with a size of 3–5 kb were purified and ligated into pIJ702 (obtained from the Bioresource collection and research center of the Food Industry Research and Development Institute) with BglII site. The ligation reactant was transformed into host  Streptomyces lividans  JT46 (provided by Carton W.-S. Chen). The transformants were screened in R2YE plate by thiostrepton (purchased from Sigma chemical). The host  Streptomyces lividans  JT46 was chosen for the recombinant DNA since it does not have TGase activity. Hundreds of the transformants were cultured at 30° C. for 2 days and one transformant 25-2 was screened having TGase activity. The transformant 25-2 has been deposited in the Bioresource collection and research center of the Food Industry Research and Development Institute on Sep. 2, 2003 numbered as BCRC 940430 and in the American type culture collection on Sep. 29, 2003 numbered as PTA-5442. The recombinant plasmid containing TGase gene from  S. platensis  was restriction analyzed and hybridized with DNA. It was found that TGase gene is located in a 2.9 kb KpnI fragment as shown in  FIG. 1 . The fragment was cloned and ligated into pMTL23 (obtained from the Bioresource collection and research center of the Food Industry Research and Development Institute) with KpnI cutting site and the resulting plasmid was denominated as pAE023. DNA sequencing was performed to confirm the insertion. The DNA fragment was replicated under  E. coli  and the reaction is performed with Bigdye™terminator RR mix (PE Applied Biosystems) by autosequencer ABI PRISM™ Model 310. The nucleotide sequence is shown as  FIG. 2A–2C . The whole KpnI fragment has 2910 nucleotides. Sequence analysis was performed by Wisconsin Sequence Analysis Package (version 8.0, Genetics Computing Group) to analyze codon preference and sequence similarity comparison. The GCG codon preference analysis predicts that one reading frame from nucleotides 1119 to 2375 has a gene, as shown in  FIG. 2A–2E . The nucleotide sequence of the gene was analyzed by BLASTN as similar to TGase of  Streptoverticillium  S-8112. The predicted amino acid sequence is shown in  FIG. 2A–2C  and has 418 amino acids with a molecular weight of 46511.30 Daltons. The result was compared with the mature form of TGase from  S. ladaksnum  by Kanai. et al. (1993) and the predicted mature form of TGase. from  S. platensis  starts at nucleotide 88 and has 330 amino acids with a molecular weight of 37,468.21 Daltons and an isoelectric point of 7.17. Nucleotides −12˜–15 from the starting amino acid of TGase from  S. platensis  are GGAG sequence, which is a ribosome binding site as shown in  FIG. 2B , frame region. An AT-rich region was found at 5′ untranslated region of the gene, nucleotides 1066–1117, as shown in  FIG. 2B . This region is predicted as a promoter region, however, no sequence similar to CAAT box or TATA box of  E. coli  promoter was found with sequence comparison. Other researchers also found that the promoter regions of  Streptomyces  species are not consensus as that of  E. coli  (Gilber et al., 1995). 
     Example 3 
     Expression of TGase gene of  S. platensis  in  S. lividans    
     The standard recombinant DNA manipulation is performed according to Sambrook et al. (1989). pAE023 was digested with BglII and BamHI and 2.9 kb of DNA fragment containing TGase gene was purified and ligated to the BglII restriction site of pIJ702. The ligation product dominated as pAE053 ( FIG. 3 ) was expressed in  S. lividans  JT46. 
     The TGase activity was determined by the following procedure: The spores of TGase-producing bacteria were inoculated in a 250 mL Erlenmeyer flask with 30 mL of media (carbon source: 1% glucose, 1% glycerol, 1% starch, 0.1% sucrose, 1% fructose; nitrogen source and salts: 0.5% glycine, 0.05% casein, 0.5% yeast extract, 0.05% tryptone peptone, 0.05% (NH 4 ) 2 SO 4 , 0.05% polypeptone, 0.2% MgSO 4 .7H 2 O, 0.2% K 2 HPO 4 ) with one duplicate under 220 rpm horizontal vibration at 30° C. The cultures were centrifuged under 6,000 g for 10 min and 50 μl of the supernatants were collected and mixed with 350 μl of 1M Tris-HCl (pH6.0), 80 μl of 0.15M CBZ-Gln-Gly (pH 6.0), and 20 μl of 4M hydroxylamine. After water incubation at 37° C. for 10 min, 500 μl of developer containing 1:1:1 of 15% TCA, 5% FeCl 3  in 2.5N HCl and 5% FeCl 3  in 0.1N HCl was immediately added. The absorbance of the mixture was measured by a spectrophotometer under 525 nm. Five hundred μl standard solution of L-glutamic acid-γ-monohydroxymic acid with different concentrations, 0 mM, 0.5 mM, 1.0 mM, and 2.0 mM were mixed with the developer separately and the absorbance of these standard solutions was measured by a spectrophotometer under 525 nm. A standard curve was obtained according to the standard solutions and the absorbance thereof, and the concentration of the product can be obtained with the measured absorbance and the standard curve. The TGase activity is defined as μmole amount of the reactant produced by the enzyme solution per min; the unit is μmmol/min. 
     The TGase activity in the supernatants was measured every 12 hours. The transformant 25-2 has the highest activity at 40 hours, up to 5.7 U/ml ( FIG. 4 ). The molecular weight of TGase from  S. platensis  is determined as 40.4 kD by ammonium sulfate precipitation, ion exchange, and SDS electrophoresis (data not shown), which is larger than the predicted MW of 37.5 kD. 
     Example 4 
     TGase Seqeucne Comparison of an Embodiment of the Invention and the Known Sequence 
     TGase seqeuence comparison of the gene derived from  S. platensis  and the published sequences shows that TGase of an embodiment of the invention has 78.55% similarity in amino acid sequence and 82.44% in nucleotide sequence to that derived from  Streptoverticillium mobaraense  DSMZ published by Pastermack et al. and 89.54% similarity in amino acid sequence and 82.44% in nucleotide sequence to that derived from  S. lydicus  published in U.S. Pat. No. 6,100,053 to Bech et al. Compared to the gene derived from  Streptoverticillium  species published in U.S. Pat. No. 5,420,025 to Takagi et al., it has 79.33% similarity in amino acid and 81.50% in nucleotide sequence. Only Bech et al. discloses that the gene has a determined activity of 2.4 U/ml. The activity detection of the preferred embodiment of the invention is by standard solution of L-glutamic acid-γ-monohydroxymic acid and developer, which is not more sensitive than radio-detection of Bech et al, however, the result of this embodiment of the invention (5.7 U/ml) is more than two times that of Bech et al. Therefore, it is obvious that the gene sequence of this embodiment of the invention is superior to any known sequences. The gene sequence of this embodiment of the invention can be used with suitable host cells for mass production of TGase with high activity. The cost of producing TGase can be greatly reduced. 
     While the invention has been described by way of example and in terms of preferred embodiment, it is to be understood that the invention is not limited thereto.