Identification of a L-Lactate dehydrogenase with 3,4-dihydroxyphenylpyruvic reduction activity for L-Danshensu production

Danshensu (DSS), also known as 3,4-dihydroxyphenyllactate, is an herbal preparation with prominent pharmacological activities. It has lactic acid structure and may be obtained by reducing 3,4-dihydroxyphenylpyruvate with lactate dehydrogenase. We screened a coenzyme-aspecific L-lactate dehydrogenase (LF-L-LDH0845) from lactobacillus fermentum for the bioconversion of 3,4-dihydroxyphenylpyruvate to optically pure (ee >= 99.99%) L-DSS. LF-L-LDH0845 has an approximate molecular weight of 33.65 kDa, exhibits wide substrate scope for 2-keto-carboxylic acids. Values of Km, Kcat, and Kcat/Km for LF-L-LDH0845 with 3,4-dihydroxy-phenylpyruvate substrate were 11.37 mM, 0.2931 s(-1), and 0.0258 mM -1 s(-1), respectively. LF-L-LDH0845 was most active and stable at pH 6.0, the optimum temperature was 25 degrees C, stability decreased with increasing temperature, and activity was lost completely at 50 degrees C. K+ stimulated while Fe2+ and Cu2+ inhibited the enzyme activity significantly. Glucose dehydrogenase gene was coexpressed with If-I-Idh0845 in E. coli to regenerate cofactors by oxidising glucose, which efficiently reduced 3,4-dihydroxyphenylpyruvate to L-DSS with 95.45% isolation yield.