Application Of Protoplast Technology Screening Test Of Yielding Alkali Lipase Strain

This article provides a good way to get a strain with high ability of producing lipase. Firstly, yeasts are made a genetical marker, which is necessary for screening later. Secondly, the factors that will influence protoplasts of yeasts and streptomycetes and their distant related fusion are investigated. Finally, the determination of growth curve, the observation of colonial morphology and the assay of lipase activity are conducted after gaining the fusant. A lysine-defective strain is gained by mutagenesis in the experiment for imparting genetical marker to yeast. The experiment adopts mycostatin inspissaation to enrich auxotrophs. The optimal mutagenic conditions are DES consistency (3%) and 90mm acting time or DES consistency (3%) with 60mm acting time and UV radiation(2min). The optimal condition for mycostatin enrichment is mycostatin consistency (lOOuIg) and 60mm acting time. In the procedure of preparing for protoplasts of yeasts and streptomycetes, the sorbitol (0.8mol/L) act as the stabilizer when cell walls of yeasts are broken, while saccharose (0.SmolJL) are used at cell regeneration; the enzymes used for breaking cell walls are helicase (1%) and cellulase (1%) with the right amount of lysozyme, the acting time is ranging from 210 mm to 240 mm; pretreatment agent is mercaptoethanol (0.1%) which get from EDTA solution (0.1%), the acting time is 30 mi Saccharose acts as the stabilizer for Streptomycete and lysozyme (2%, 10-1 5mm acting time) is used to break cell wall, there is no pretreatment agent here. The factors that will influence fusion rate in the procedure of fusont are listed in sequence of their effectivity: the concentration of CaCI2>the acting time of PEG> the concentration of PEG, the concentration of calcium ion influence the fuson rate obviously. The optimal fusion condition is the following: PEG consistency (35%), CaCl2 consistency (SOmmol/L), the acting time of PEG (30mm), the stabilizer of saccharose (0.Smol/L), pH 7.0. In the procedure of identifying fusants, there is no mycelium found from the colonial morphology of the selected fusant, the appearance of the selected fusant looks like the parent yeast with larger sizes colony and cell. Logarithmic growth phase of the fusant appear at 32-36h, it between 24h of the yeast and 48h of the Streptomyce. The activities of enzymes from fusants YS1 and YS8 are determinedand the peak values, which are 789u/ml and 836.7u/ml, appear at pH8.6. The fusantsare more alkali-resistant than the parent yeast, but activity of the enzyme it producedis loweL The peak value of the activity of enZyme produced from YS6 is l94.5u/ml,which appears at pH l0 and is higher than that of the parent streptomycetes strain.