A study on increasing enzymatic stability and activity of Baliospermum montanum hydroxynitrile lyase in biocatalysis.

• Bm HNL has the longest half-life among all currently known α/β hydrolase fold HNLs at its optimal pH and temperature. • Polyol addition increases Bm HNL's stability and activity. • Glycerol addition to Bm HNL biocatalysis increases % ee of the synthesized cyanohydrins. HNL catalysis is usually carried out in a biphasic solvent and at low pH to suppress the non-enzymatic synthesis of racemic cyanohydrins. However, enzyme stability under these conditions remain a challenge. We have investigated the effect of different biocatalytic parameters, i.e., pH, temperature, buffer concentrations, presence of stabilizers, organic solvents, and chemical additives on the stability of Baliospermum montanum hydroxynitrile lyase (Bm HNL). Unexpectedly, glycerol (50 mg/mL) added Bm HNL biocatalysis had produced >99% of (S)-mandelonitrile from benzaldehyde, while without glycerol it is 54% ee. Similarly, Bm HNL had converted 3-phenoxy benzaldehyde and 3,5-dimethoxy benzaldehyde, to their corresponding cyanohydrins in the presence of glycerol. Among the different stabilizers added to Bm HNL at low pH, 400 mg/mL of sucrose had increased enzyme's half-life more than fivefold. Bm HNL's stability study showed half-lives of 554, 686, and 690 h at its optimum pH 5.5, temperature 20 °C, buffer concentration, i.e., 100 mM citrate-phosphate pH 5.5. Addition of benzaldehyde as inhibitor, chemical additives, and the presence of organic solvents have decreased both the stability and activity of Bm HNL, compared to their absence. Secondary structural study by CD-spectrophotometer showed that Bm HNL's structure is least affected in the presence of different organic solvents and temperatures. [ABSTRACT FROM AUTHOR]