| Aspergillus niger,a common Aspergillus fungus,is recognized as a safe strain,which can be used to produce a variety of enzyme preparations,and is well used in industrial,chemical,pharmaceutical and other industries,in the development of biotechnology and production and life play a big role.Most of the currently reported pectinase producing bacteria are Aspergillus niger.Although its pectinase components are relatively complete,its viability is not ideal.Pulsed light mutagenesis technology is a new type of mutagenesis.Its research started late and has the characteristics of high intensity and instantaneity.This technology has high safety and simple operation.In this paper,the optimal mutagenic parameters of pulsed light for mutagenesis of high-yield pectinase from Aspergillus niger will be determined by response surface experiment design,and the effects compared with ultraviolet mutagenesis will be compared.The article evaluates the mutagenic ability of pulsed light from three aspects: pectinase activity,bacterial cell performance of mutant strains,and enzymatic properties of mutant strains.The pourpose of this study is to explore the mutagenic mechanism of pulsed light and whether pluse light can be used as a new environmentally friendly mutagenic method to improve the activity of pectinase produced by Aspergillus niger.The main experimental contents and results are as follows:(1)The optimum mutagenesis conditions of pulsed light method were determined and the mutant strains with high pectinase activity were screened.In this experiment,the pectinase activity of the mutant strain was used as an indicator,and the single factor and response surface experiments were combined to determine the optimal mutagenic conditions for the pulsed light technology: pulse voltage of 2075 V,number of pulses of 36 times,and pulse distance of 5.4cm.Through screening,a genetically stable pulsed light high-yield mutant L9(188.21 ± 1.22 U / m L)was successfully obtained,which was superior to the UV mutant U39(138.10±1.01 U / m L).(2)The performance of the mutant strain was determined.Compared with the original strain,the acid resistance,heat resistance and ethyl alcohol resistance of mutant L9 were significantly improved(p <0.05).The logarithmic growth period of mutant L9 was prolonged compared with that of the original strain.(3)Evaluation of enzymatic properties of pectinase from mutant strains.Studies have shown that the pectinase activity of the mutant L9 and the original strain is maximum at 45 °C,and shows the best activity at p H 5.0.The p H stability of the mutant strain L9 pectinase was significantly higher than that of the original strain.At the same time,the thermal stability of the pectinase of the mutagenic strain was significantly improved.The experimental results show that the method of pulsed light mutagenesis will have certain effects on the enzyme-producing properties of the strain and the stability performance will be improved.(4)Preliminary study on the mechanism of mutagenesis.Through RAPD-PCR technology,it was found that the mutant L9 will have band amplification differences under the action of primers,but the difference bands are different.This may be because the formation of pyrimidine dimers(CPDs)hinders the normal pairing of bases,causing damage to the mutant L9 genomic DNA.SDS-PAGE gel electrophoresis found that the whole-cell bacterial protein bands of mutant L9 were different from those of the original strain,indicating that the gene expression of the strain was affected by pulsed light mutagenesis.The above results show that pulsed light technology can be used as an efficient,safe and simple mutagenesis method to mutagenesis breeding of pectinase mutant strain with high yield of Aspergillus niger,and it is better than ultraviolet mutagenesis in improving pectinase activity. |
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