In this study for the first

In this study, for the first instant we report the characterization, production and in silico structural analysis of BADH1 gene in B. haloduranss SMBPL06 isolated from the salt pan. Moreover, the sequence analysis of betaine aldehyde dehydrogenase from B. haloduranss SMBPL06 displayed several base substitutions with that of reported sequences in GenBank, resulting in the altered amino SJ 172550 sequences of the translated protein sequences.
Materials and methods
Results
Discussion In this study, for the first instance we have cloned and characterized the BADH1 gene cassette from B. halodurans SMBPL06 under the control of inducible promoter. Evaluation of the deduced amino acid sequence of BADH1 gene with the reported betaine aldehyde dehydrogenase sequences in the database revealed a maximum similarity. However, the sequence analysis of BADH1 gene of B. halodurans SMBPL06 revealed several base substitutions with that of reported sequences, resulting in the altered amino acid sequences of the translated protein. The activity of the enzyme using choline as the substrate and phenazine methosulfate as the electron acceptor revealed the expressed enzyme from BADH1 gene cassette was betaine aldehyde dehydrogenase. The ability of the betaine dehydrogenase to catalyze both oxidative reactions in the conversion of choline to glycine betaine has been previously reported in choline dehydrogenase from Halomonas elongata (Gadda and Wilkins, 2003), rat liver mitochondria (Tsuge et al., 1980) and in choline oxidase from Arthrobacter globiformis (Ikuta et al., 1977). Betaine aldehyde dehydrogenase is capable of using choline as a substrate (Tsuge et al., 1980). In this study, we used choline as the substrate and the enzyme activity rate was measured by the rate of oxygen consumption in the presence of 4.5mM phenazine methosulfate as the primary electron acceptor. The enzyme activity of betaine aldehyde dehydrogenase from recombinant E. coli with pQE30-BADH1 cassette showed a sixfold-enhanced activity over the control strain. Moreover, the catalytic rate of the enzyme was in the declined manner with that of the concentration of substrate by 10mM to 2.5mM. These observations signify that the enzyme activity is maximum, only in the presence of appropriate concentration of the substrate. These results are consistent with previously reported data (Gadda and Wilkins, 2003) that, the specific activity of the enzyme was 9.4μmol of O2/min/mg with 10mM choline and 3.2μmol of O2/min/mg with 2.5mM choline. The secondary structure prediction results also revealed considerable similarity with the un reported betaine aldehyde dehydrogenase from B. endophyticus. Upon structural analysis, the predicted alpha helix and beta strand structures of betaine dehydrogenase revealed a maximum similarity, suggesting the active domains of the enzyme from B. halodurans SMBPL06 has the considerable identity with the database reports. In conclusion, based on the expression and enzymatic analysis, the enzyme encoded by BADH1 gene in B. halodurans SMBPL06 is betaine dehydrogenase. To our knowledge, this study represents the first instance in which the betaine aldehyde dehydrogenase from the halotolerant B. halodurans SMBPL06 has been cloned and characterized in detail. Moreover, the determination of protein structure modification due to the nucleotide substitutions will certainly provide the basis for performing site-directed mutagenesis to improve the production and configuration of the osmolytes of biotechnological interest. This, in turn, has great potential for biotechnological applications aimed at genetically engineering stress tolerance in crop plants of economic interest.
Introduction Ovarian cancer is recognized as the most lethal gynecological malignancy in developed countries, with 238,700 estimated new cases and 151,900 estimated deaths per year, worldwide [1]. Due to ineffective available screening strategies and to its underhand clinical manifestation, this neoplasm is diagnosed at advanced stage in 70% of cases, with a 5-year overall survival (OS) rate below 40% [2].