Thesis (Ph.D.)--Chulalongkorn University, 2008

In this research, improvement of Escherichia coli host cell for high production of L-alanine and screening of a novel formate dehydrogenase (FDH) were performed. For improvement of host cell, the T7 gene 1 encoding T7 RNA polymerase was introduced into chromosome of E. coli MB2795, the racemase-deficiency strain, via transduction of lambda phage. After that, the constructed clone, namely E. coli KR, was used as a host cell for transformation of pETFA containing alanine dehydrogenase gene from Aeromonas hydrophila and formate dehydrogenase gene from Mycobacterium vaccae N10. The optical purity of L-alanine produced by recombinant clone of E. coli KR was over 95%. Surprisingly, a novel type of FDH, NADP+-FDH, was found in Burkholderia cepacia complex (BCC) during the screening of formate dehydrogenase. This FDH was able to use both NAD+ and NADP+ as coenzyme, however, it preferred NADP+ over NAD+. The distribution of formate dehydrogenase gene was determined among 46 strains from 10 species of the BCC. The gene was found to be present in several or all tested strains of B. cepacia, B. multivorans, B. cenocepacia, B. stabilis and B. pyrrocinia, but potentially absent in B. ambifaria, B. vietnamiensis, B. dolosa, B. anthina and B. ubonensis. The complete coding sequence of all FDH genes from the five species consisted of 1,161 bp encoding for a polypeptide of 386 amino acids. The similarity of amino acid sequences were very high (91-96%) among the five BCC and high 65-70% when compared to other bacterial FDHs. The gene encoding formate dehydrogenase from B. stabilis was cloned into E. coli BL21 (DE3) and the enzyme was purified to homogeneity. BstFDH had a molecular mass of subunit about 42 kDa. The optimum pH was ranged from 6.0-7.5 and optimum temperature was 60 degress Celsius No loss of the enzyme activity was observed upon incubation at 45, 50 and 55 degress Celsius for 16, 10 and 10 hr, respectively and the activity retained 50% after incubation for 36, 36 and 32 hr, respectively. The enzyme was stable over a broad pH ranged from 4.0 to 12.0. The apparent K [subscript m] values for formate, NADP+ and NAD+ were 62.5, 0.16 and 1.43 mM, respectively. Interestingly, all NAD+ -dependent FDHs and other D specific 2-hydroxy acid dehydrogenases contained the conserved coenzyme binding sequence Gly(Ala)XGlyXXGlyX17Asp for NAD+ while the NADP+ -dependent FDHs from the BCC possessed the sequence GlyXGlyXXGlyX17Gln. Therefore, the Gln223Asp single mutation of BstFDH was performed. Coenzyme preference of the mutant enzyme was completely changed from NADP+ to NAD+.