脉冲强光诱变对黑曲霉产果胶酶活力的影响

Effect of Aspergillus Niger induced by intense pulsed light on pectinase-producing activity

脉冲强光技术可应用于微生物诱变育种以及获得高产菌株。该文采用响应面法确定脉冲强光诱变黑曲霉高产果胶酶的最佳条件,同时探究突变菌株高产果胶酶的酶学性质。结果表明:脉冲强光诱变条件为脉冲电压2075 V,脉冲次数36次,脉冲距离5.4 cm。以此工艺参数进行诱变,得到了高产果胶酶的突变菌株L9,与原始菌株相比,其果胶酶活力提高了82.22%±0.18%。突变菌株L9遗传性能良好,所产果胶酶具有更高的pH稳定性和热稳定性。因此,脉冲强光可用于黑曲霉诱变,得到稳定性良好的高产果胶酶的突变菌株。

Intense pulse light(IPL)technology is a new non-toxic and environmentally friendly cold treatment technology,which can be applied to mutation breeding of microorganisms and to obtain high-producing strains.Aspergillus Niger is the main strain producing pectinase in food industry at present,but its pectinase yield is low.In order to verify the feasibility of applying intense pulse light technology to mutagenize Aspergillus Niger strains for high yield of pectinase,this experiment used intense pulse light technology to mutagenize Aspergillus Niger.With pulse voltage,pulse number and pulse distance as independent variables and ratio of transparent circle to colony diameter as dependent variables,steepest slope moving tests,response surface tests and result analysis are carried out to determine the optimum conditions for high-yield pectinase induced by the intense pulse light.At the same time,secondary screening of mutant strains was carried out and the genetic stability of the mutant strain was determined and the enzymatic properties of the mutant strain with high pectinase production were explored.The results showed that the multiple quadratic regression equation of transparent circle and colony diameter ratio(Y)against impulse voltage(A),pulse number(B)and pulse distance(C)is as follows:Y=1.59+0.068A+0.048B?0.033C?0.011AB?0.019AC?0.022BC?0.071A2?0.087B2?0.13C2.All the factors in the response surface design test were significant,and the interaction between two factors was not significant.The optimum mutagenesis conditions were when the pulse voltage was 2075 V,the pulse number 36 times and the pulse distance 5.4 cm.Under the optimum conditions,the ratio of transparent circle to colony diameter could reach 1.58,which was in good agreement with the predicted value of response surface fitting equation,indicating that the model was credible.Induce mutation to the Aspergillus Niger under such optimized condition,screen mutant strains using transparent circle for 60 mutant strains with bigger ratio of transparent circle to colony diameter.The mutant strain L9 with high pectinase production was finally selected by re-screening the above-mentioned 60 strains by determining pectinase activity,which was as high as(188.21+1.22)U/mL,which was 82.2% higher than that of the original strains.The results of genetic stability analysis showed that the mutant strain L9 had stable pectinase performance within 6 generations and no significant changes were seen in terms of pectinase activity,which indicated that the mutant strain L9 had good genetic stability.The optimum pH value and temperature for producing pectinase were 5.0 and 45℃.Compared with the original strain,the mutant strain L9 produced pectinase with better activity at the optimum pH and temperature.The range of the pH stability and thermal stability of the mutant strain were also significantly wider than that of the original strain,indicating that the mutant strain had good pH stability and thermal stability.Through the above experimental results,we can know that the application of intense pulse light technology to Aspergillus Niger mutation is feasible.After the intense pulse light induced mutation and secondary screening,a mutant strain of Aspergillus Niger with higher pectinase production with high enzymatic activity and good genetic stability can be obtained.