Fourteen sphere-shaped and 30 rod-shaped lactic acid bacteria were isolated from soy sauce mash of two factories in Thailand. These strains were separated into two groups, Group A and Group B, by cell shape and DNA-DNA similarity. Group A contained 14 tetrad-forming strains, and these strains were identified as Tetragenococcus halophilus by DNA similarity. Group B contained 30 rod-shaped bacteria, and they were further divided into four Subgroups, B1, B2, B3, and B4, and three ungrouped strains by phenotypic characteristics and DNA similarity. Subgroup B1 contained 16 strains, and these strains were identified as Lactobacillus acidipiscis by DNA similarity. Subgroup B2 included two strains, and the strains were identified as Lactobacillus farciminis by DNA similarity. Subgroup B3 contained five strains. The strains had meso-diaminopimelic acid in the cell wall, and were identified as Lactobacillus pentosus by DNA similarity. The strains tested produced DL-lactic acid from D-glucose. Subgroup B4 contained four strains. The strains had meso-diaminopimelic acid in the cell wall, and they were identified as Lactobacillus plantarum by DNA similarity. Two ungrouped strains were homofermentative, and one was heterofermentative. They showed a low degree of DNA similarity with the type strains tested, and were left unnamed. The distribution of lactic acid bacteria in soy sauce mash in Thailand is discussed.

Tetragenococcus muriaticus strains 3MR10-3 and PMC-11-5 are homofer-mentative halophilic lactic acid bacteria isolated from Thai fish sauce during natural fermentation. Their draft genomes were sequenced. Our interest in these organisms is related to their impact on fish sauce flavor and their high osmotolerance. H alophilic lactic acid bacteria in the genus Tetragenococcus, including T. muriaticus and T. halophilus, have been reported to be isolated from fish sauce (1-4) and to play important roles in flavor development of the sauce (3, 4). Fish sauce is a fermented clear brownish liquid condiment with salty taste and distinct aroma and flavor (5, 6). Herein, we present the genomic sequences of two T. muriaticus strains, 3MR10-3 and PMC-11-5, which were isolated from fish sauce processed in eastern Thailand. Genomic DNA was prepared from cultures propagated in De Man, Rogosa, and Sharpe (MRS) broth containing 0.5% CaCO 3 and 5% NaCl, which had been incubated at 30°C for 48 h. The genomic DNA was prepared using the cetyltrimethylammonium bromide (CTAB) method (DOE Joint Genome Institute, CA) and subjected to genomic sequencing using 454 GS FLX Titanium paired-end sequencing platform (Life Technologies , Thermo Fisher Scientific, Inc., Waltham, MA, USA) at the Biotechnology Center, University of Wisconsin-Madison. The nucleotide sequences were assembled using the GS de novo assembler version 2.6 (Life Technologies, Thermo Fisher Scientific, Inc.). Functional annotation was performed by the Rapid Annotations using Subsystems Technology (RAST) server (7). The genome sizes of T. muriaticus strains 3MR10-3 and PMC-11-5 were 2,080,407 and 2,103,938 bp with a total of 322 and 376 contigs, respectively. The GC contents of the two strains, 3MR10-3 and PMC-11-5, were 35.96 and 36.03%, respectively, and the total numbers of predicted coding sequences (CDSs) were 2,252 and 2,626 CDSs, with 52 and 50 tRNAs and five and three rRNAs, respectively. The genomes of strains 3MR10-3 and PMC-11-5 were examined for genes potentially involved in osmotolerance or fish sauce flavor development. For osmotolerance, we focused on genes coding for transporters responsible for inorganic ion or compatible solute uptake from hypersaline environments (8, 9). This analysis resulted in the identification of genes responsible for K transport, an ABC-type proline/glycine be-taine transporter, a glycine betaine/choline transporter, and a choline/carnitine/betaine transporter. To identify genes likely involved in fish sauce flavor development, we

Expression of the ansZ gene encoding a putative L-asparaginase II (BsAII) from Bacillus subtilis in Escherichia coli was examined. No expression was detected in E. coli transformed with a plasmid containing the full-length ansZ gene. Three N-terminal truncated enzymes (BsAIIT18M, BsAIIS40M, and BsAIID49M) were prepared based on comparison with the N-terminal sequences of other type II L-asparaginases. BsAIIT18M became easily inactivated during DEAE-Toyopearl column chromatography. The purified N-terminal-truncated enzymes BsAIIS40M and BsAIID49M had tetrameric subunit structures and V max values of 45.5 and 45.8 U/mg towards L-asparagine, respectively. Their K m values were 2.06 and 7.02 mM, respectively. The enzymes differed from asparaginase II from E. coli and Erwinia carotovora in substrate specificity and affinity for L-asparagine. BsAIIS40M and BsAIID49M retained over 80% of their original activities in the presence of 15% NaCl, and thus may find application in the food industry for products in which NaCl is used. This study also revealed that BsAII is rather different from the type I enzyme (BsAI) from B. subtilis in substrate specificity and salt-tolerance.

The present study focused on cloning and expression of the ansA gene, encoding putative asparaginase I of Bacillus subtilis in Escherichia coli, and investigation of the basic properties of the recombinant enzyme for application in food processing industry. The ansA gene of B. subtilis was cloned by polymerase chain reaction and expressed in E. coli Rosseta Gami B. It consisted of an open reading frame of 987 nucleotides encoding a protein of 329 amino acids with a calculated molecular mass of 36441 Da. The recombinant enzyme showed increased expression (21.7 U mg -1 ) and was purified to homogeneity by a two-step procedure that included L-asparagine affinity column chromatography. Based on the kinetic properties and primary structure of the native enzyme, the product of the ansA gene was classified as L-asparaginase I. The enzyme showed a high specific activity compared with that of L-asparaginase II from E. coli. Results of this study will allow us to apply the enzyme to food industry.

Five glutaminase-producing lactic acid bacteria were isolated from Nham, a Thai fermented pork sausage. Weisella cibaria (MSS1, MSS2, MSS3, and MBP5) and Leuconostoc citreum (MHV2) were identified by carbohydrate metabolism tests and 16S rDNA sequence -based phylogenetic analysis. Of these, W. cibaria MSS2 had the highest glutaminase activity (0.0085 U/mg) among them. The specific activity of glutaminase (0.024 U/mg) was obtained in static culture at 20 C using the modified MRS medium in which sucrose was substituted for glucose. The enzyme retained 40% of its original activity in the presence of 15% NaCl, which is about 2 -folds higher than the activity of glutaminase from Aspergillus oryzae.