Lactate Bacillus stearothermophilus

Lactate dehydrogenase (LDH) from Bacillus stearothermophilus, in its I51K D52S double mutant, exhibited a 56-fold increased specificity to NADPH over the wild-type LDH in a reaction mixture containing 15% methanol. Furthermore, the NADPH turnover number of this mutant was increased almost fourfold compared with wild-type LDH (Holmberg, 1999). 

N. Holmberg, U. Ryde, and L.Bulow, Redesign of the coenzyme specificity in L-lactate dehydrogenase from Bacillus stearothermophilus using site-directed mutagenesis and media engineering,... 

Protein engineering has been carried out to redesign substrate specificity of lactate dehydrogenase from Bacillus stearothermophilus (Wilks et al., 1988 Wilks et ah, 1990) ... 

The amino acid sequences of corresponding regions from dogfish M4 and Bacillus stearothermophilus lactate dehydrogenases are shown below ... 

Muller, K., Seifert, T., and Jaenicke, R. High pressure dissociation of lactate dehydrogenase from Bacillus stearothermophilus and reconstitution of the enzyme after denaturation in 6 M guanidine hydrochloride, Eur. Biophys.., 11, 87,1984. 

Developments in molecular biology enable us to change the substrate specificity of enzymes the enzymes can be engineered to be more suitable for the requisite substrate. For example, variations have been made to the structure of the NAD+ dependent L-lactate dehydrogenase from Bacillus stearothermophilus (LDH) 130L Two regions of LDH that border the active site (but are not involved in the catalytic... 

Table 15-7. Broadening the substrate specifity of L-lactate dehydrogenase from Bacillus stearothermophilus by rational protein engineering130.

Mutant enzymes have been produced that show a 10 -fold switch in enzyme specificity [221]. As a result, a thermally stable lactate dehydrogenase from Bacillus stearothermophilus was converted to a malate dehydrogenase with twice the activity found in the malate dehydrogenase in the same organism. The mutations were 2 ° Gln-Lys-Pro to Met-Val-Ser and 236-237 )g ja to Gly-Gly. This is an excellent example of the design of an enzyme. 

Engineered enzymes have application in the resolution of stereoisomers, especially in the pharmaceutical industry for the preparation of pure isomers of synthetically prepared drugs. For example, lactate dehydrogenase from Bacillus stearothermophilus has been engineered to accept substrates with a long branched side chain, without loss of its stereospecificity (the native enzyme reduces pyruvate to L-lactate). 

Chemical modifications of mesophilic enzymes have met with more success than protein engineering. The strategies for stabilization have been reviewed [e.g. 39, 56] and include immobilization and hydrophilization. Chemical modification of trypsin and chymotrypsin gives artificial hydrophilization of non-polar areas of the protein surface resulting in marked thermostabilization and little loss of enzyme activity [57-59]. This phenomenon has also been demonstrated for Bacillus stearothermophilus lactate dehydrogenase by Wigley et al. [60] who used site-... 

CocconceUi PS, Gasson MJ, Morelli L, Bottazzi V (1991) Single-stranded DNA plasmid vector construction and cloning of Bacillus stearothermophilus a-amylase in Lactobacillus. Res Microbial 142 643-652 Crow VL (1987) Citrate cycle intermediates in the metabolism of aspartate and lactate by Propionibacterium freudenreichii subsp. shermanii. Appl Environ Microbiol 53 2600-2602... 

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