Research On Enzyme Production Characteristics Of The Mutant Strain Of Phanerochaete Chrysosporium And The Lignin Degradation By It

White rot fungi mainly rely on the following three types of extracellular enzymes to degrade lignin and a variety of xenobiotic:lignin peroxidase,manganese peroxidase and laccase.But these three kinds of enzymes are generated in nutrition restriction conditions and belong to the typical secondary metabolites,which makes the enzyme yield low.Besides,the environmental pollution is mainly caused by eutrophy,which further results in the limited practical application of white rot fungi.In order to solve this problem,the predecessors carried out mutation breeding experiment in order to gain the mutants which could produce enzymes under eutrophy conditions and got the expected results.But whether the physiological metabolic characteristics,the characteristics of enzyme production and lignin degradation of mutant strains are the same as those of the original strain remains to be studied.For example,the original strain need a kind of common substrate to degrade lignin and can't use lignin as the sole carbon source,and its degradation of lignin can not be induced by substrate.In this paper,Phanerochaete Chrysosporium pc530 and the mutant strains pcR5324 and pcR5326 were studied in order that their differences were understood,which can provide a theoretical basis for the industrial application of the mutant strain.The research result was as follows:(1)The SEM observation showed that the hyphal length of PC530 was shorter,the interstice between the hypha was larger and the hypha distribution was sparse with many spores covering its tip.The mycelial growth of pcR5324 was exuberant,mycelium cross-section was wide and slightly flat,the mycelium wound with each other and couldn't be recognized and the mycelium surface was covered with few spores.pcR5326 had many spores distributed on the surfac of the hypha,and it was difficult to see the hypha.Besides,spores were concentrated and there was little gap between the spores.Enzyme production petri dish validation showed that basically the laccase activity of pc530 couldn't be detected while the laccase activity of both pcR5324 and pcR5326 could be detected,but the laccase activity of pcR5324 was much higher than that of pcR5326;All the three strains could produce lignin peroxidase.(2)Through the analysis on the correlation between cell growth and enzyme production,it was found that lignin peroxidase produced by pc530 was a typical secondary metabolite,and under nitrogen-rich condition the enzyme activity was significantly lower than that under nitrogen-limited condition.Besides,basically pc530 could not produce laccase,and under both the nitrogen-rich condition and the nitrogen-limited condition,the enzyme activity was very low.Besides,laccase was secreted as a typical secondary metabolite;pcR5324 could produce lignin peroxidase during the primary metabolism stage,and the enzyme activity was much higher than that of pc530.Besides,under nitrogen-rich condition the enzyme activity was much higher than that under the nitrogen-limited condition.pcR5324 could produce laccase during the primary metabolism stage,and when nutrient was limited,the laccase yieldwas obviously higher;pcR5326 could-produce lignin peroxidase and laccase resisting nutritional repression,but both the lignin peroxidase activity and the laccase activity were far lower than those of pcR5324.In the process of cultivation,the carbon consumption rate was higher than the nitrogen consumption rate,and both of the consumption trends of nitrogen and carbon accorded with the following rule:high rate at the earlier period,low rate at the latter period.As for pcR5324,at the end of the period of fast consumption of Glucose and Ammonnia nitrogen,the enzyme activity reached the highest level.Different ions had different effects on the enzyme activity,and under nitrogen-rich condition,when zinc ion,copper ion,manganese ion,and phenylalanine were separately added into the culture medium,their concentrations under which laccase reached the highest yield were 0.2g/L,0.1g/L,0.2g/L and 0.05g/L respectively while when lignin peroxidase reached the highest yield,their concentrations were 0.6g/L,0.4g/L,0.2g/L and 0.05g/L respectively.lt was showed that different metal ions and different metal ion concentrations had different effects on the enzyme activity.Different phenylalanine concentrations also had different effects on the enzyme activity.(3)Through the study on the lignin degradation characteristics of the original strain pc530 and the mutant pcR5324,it was found that the original strain need other carbon sources as the cosubstrate while pcR5324 could use lignin as the sole carbon source,but without the existance of other nutrient,the lignin degradation rate by pcR5324 was lower.The study also found that the mutant strain could be induced by substrate when degrading lignin while the original strain couldn't and even was restricted by the substrate when degrading lignin.Under nitrogen-rich condition the effect of substrate was more obvious than that under nitrogen-limited condition.