Study On Screening Of High-yield Neutral Proteiase Producing Bacterial Strain And Enzyme Characterizations

This experiment studied systematically for the first time on the screening of neutral proteinase producing strain, compound mutation of protoplast and enzyme-producing conditions, enzyme characterizations and enzyme storage. The research paper includes mainly four aspects:(1) Screening of neutral proteinase-producing bacteria and preliminary study on partial enzyme characterizations.A stable high-yield neutral-proteinase strain B_x-4 was gained from soil nearby creameries and identified as Bacillus subtilis. Skim milk medium was screened finally as identification medium by comparing casein medium, starch casein medium, yeast extract casein medium, Luria-Bertani medium (added casein) and skim milk medium due to its good observation and good growth of strain. Study of single factor (carbon resource and nitro resource) and orthogonal test discovered that the fermentation medium containing 2% corn powder ,3% bean cake powder and 3% bran is good for neutral proteinase(NPR) production.Zn²⁺, K⁺,Na⁺, Ca²⁺, Mg²⁺ can activate enzyme-producing ability at different level, especially Zn²⁺. Fe²⁺ is able to restrain NPR's production extremely. The optimal pH value for proteinase producing is 8.0.Study of enzyme characterizations found out that the optimum temperature and pH value for enzyme producing are 55℃ and 7.5 respectively. Ca²⁺ can activate enzyme-producing, and then,Fe³⁺ have a heavy inhibition to enzyme.(2)Compound mutant screening of protoplast from Bacillus subtilis B_x-4Study of growth curve illustrates that the lag phrase of Bacillus subtilis B_x-4 is 0^-2h,reach its logarithm growth telophase at 7h,the middle of logarithm growth telophase is 5h. Bacillus subtilis B_x-4 is sensitive to glycin and penicillin-Na. The optimum doses of glycin, penicillin are 2mg/mL, 1U/mL respectively. The optimum concentration and time of lysozyme is 2mg/mL and 60min respectively. The formation ratio and regeneration ratio of protoplast from Bacillus subtilis B_x-4 can reach 77.44%, 53.07% respectively at its optimal formation and regeneration conditions. A positive mutant strain UV₁₁ was obtained through ultraviolet radiation mutation on B_x-4. Positive mutant strain UN₉ was gained by compound mutation of UV+HNO₂ on UV₁₁. Finally, a high-yield, stable neutral-proteinase strain was obtained by compound mutation of UV+NTG on UN₉ and proteinase activity is 4286.09U/mL.The genetic stability of Bacillus subtilis Promax NTG₁₄ is very good.