Mutation Research With Enhanced Yield Of Lipase In Flavobacterium Sp. By Supercritical CO₂

Induced mutations by physical and chemical factors have been widely used in organism breeding due to simple procedure and high efficiency. Generally, chemical mutagens are more effective for genetic modification of microorganisms, but many of them are virulent to human beings and unsafe to environment. On the other hand, some of physical mutagens have still been restricted in application because of high cost and radiation damage. Supercritical CO₂, as an environmentally benign solvent, is capable of killing microorganisms at moderate pressure and lower temperature. In this paper, supercritical CO₂ was used as a potential mutagen to induce mutations with enhanced yield of lipase in Flavobacterium sp. strain YY25 for the first time. The purpose was to provide a new, simpler and environmentally friendly mutation method.Flavobacterium sp. strain YY25 was isolated from the fat soil samples using selective medium and was characterized using automatic microorganism identification instrument. The lipase activity of this wild strain was 16.4 u/mL after fermentation of 24 h.During treatment by supercritical CO₂, the survival rate and positive mutation rate of strain YY25 were strongly dependent on pressure, temperature, treatment time and additive （DMSO and HZH）. 8 MPa, 35°C, 30 min of supercritical CO₂ and 1% of DMSO、0.5% of HZH were believed as the optimum doses. After the seed liquid was treated under these conditions, a mutant strain （YY25-H0.5） was obtained. Its yield of lipase was 29.0 u/mL, with about 76.7% increases comparing with the wild strain.In order to optimize the lipase-production conditions of YY25-H0.5, the fermentation experiments were carried out by single factor design, Plackett-Burman design and response surface methodology. Under the acquired optimal conditions, the highest yield of lipase by YY25-H0.5 was 38.9 u/mL, with 1.34 times of primary yield.This study indicated that the novel mutation method by supercritical CO₂ was feasible and promising in microbial breeding field.