Research On Anti-Nutritional-Repression Laccase Production Characteristics And Physiological Regulation Mechanism Of Phanerochete Chrysosporium

Phanerochaete Chrysosporium(called pc)is the type strain which has the typical nutritional repression enzyme production characteristics for the white rot fungi research.Belonging to the oxidase,Laccase(Lac)has more advantages than peroxidase(LiP and MnP),and is the most application value enzyme which can degrade lignin.The traditional research showed that P.chrysosporium only could produce Lac and MnP except Lac.Yet since 1995,some scholars had successively found a small amount and stable producing Lac from P.chrysosporium.Although some scholars were still querying about this,more evidence has proved that P.chrysosporium can produce Lac in recent years.Even so,the knowledge about Lac metabolism and physiological regulation mechanism is also less well known.According to the serious lagged research about P.chrysosporium producing Lac and the status of research gaps about the regulatory mechanism of nutritional-repression relieving,this thesis is aiming to explore the lignin synthesis regulation mechanism and enhance understanding about the lignin degradation of P.chrysosporium.We select the anti-nutritional-repression Lac production strain of pcR5305 and pcR5324 from six mutant strains which were selected and bred by ultraviolet radiation from predecessors.Then explore the growth and enzyme production characteristics,also the special Lac isozyme and physiological regulation mechanism.The results are as follows:(1)Observing and comparing the growthform and Lac activity,we can find that the strains which grow by spore have low Lac activity,and other strains which grow to a velvet-like mycelia have higher Lac activity.The mutant strains pcR5305 and pcR5324 demonstrate a significant anti-nutritional repression of Lac production characteristics under the nitrogen sufficient condition better than nitrogen limitation and the static condition better than oscillating condition.(2)Through the researching on growth and Lac-production characteristics,we find that the Lac activity of original strain pc530 is marginally detected in the late secondary metabolism period where the highest activity can be achieved 20.049U/L under C-S N-S condition at the 18th day.Different from pc530,the mutant strains pcR5305 and pcR5324 all have two Lac activity peaks with static culture,and the activity peaks appear respectively during the primary metabolism and secondary metabolism period.As the gradually enriched of the nutritional conditions,the anti-nutritional repression of Lac production characteristics is strengthened with pcR5305,and it is opposite of pcR5324 which is weaken.The highest Lac activity is the same for the two under C-L N-S condition.The Lac activity of pcR5305 can reach to 337.412U/L at 6th day and 238.222U/L at 12th day.pcR5324 can reach to 126.111U/L at 5th day and 382.417U/L at 12th day.Under oscillating condition,the two mutant strains change to one Lac activity peak which appear respectively during the primary metabolism for pcR5305 and secondary metabolism period for pcR5324.(3)Through the research on the Lac isoenzyme of mutant strains under C-L N-S condition,we find that pcR5305 contain at least three kinds of Lac isozymes,and pcR5324 contain at least two kinds.The cluster analysis shows that the Lac isozymes of pcR5305 and pcR5324 have certain kinship which considered to be the same or similar nature.The different Lac isozymes and their combination mode have a certain impact on the strains for enzyme production.(4)Researching on the carbon and nitrogen nutrient mechanism,we can find that the critical carbon concentration of original strain pc530 at the beginning enzyme production period is 1.5g/L under C-L condition and 5.8g/L under C-S condition.When reaching the activity peak,the critical carbon concentration reduce to a range from 0.108 to 0.155g/l under C-L condition and from 1.025 to 1.583g/L under C-S condition.The critical nitrogen concentration is 18mg/L under N-L condition and 100mg/L under N-S condition at that time.The C/N for enzyme production peak is always approximately 12%of the beginning production in the different nutrient conditions.For the beginning enzyme production period of pcR5305 and pcR5324,the critical carbon concentration is 5.25g/L,8.35g/L under C-L condition and 1 1.8g/L,11g/L under C-S condition.The ammonia concentration did not show great changing at the period.During the primary metabolism Lac activity peak,the critical carbon concentration is 1.8g/L,2.5g/l.When come to secondary metabolites period,the critical carbon concentration become 1.8g/L as the same,the critical ammonia concentration is 20mg/L,19mg/L in N-L condition and 145mg/L,85mg/L in N-S condition at the same time.Now the C/N is approximately 35%of the beginning Lac production period.The best C/N is 7.5 and 13 for pcR5305 and pcR5324.(5)Through the researching on physiological regulation mechanism of pcR5305 and pcR5324,we find the best culture conditions as follows:lOg/L glucose,12mmol/L Ammonium,separately adding 0.4mmol/L of Cu2+ at 6th day and 0.4/4.0mmol/L of Cu2+ at 9th day,1.0 and 0.5ml/L Tween-80.The adding of Guaiacol,oxalic acid and L-phenylalanine can significantly affect all the Lac production characteristics and activity.Through orthogonal experiment,the optimal conditions for pcR5305 culture is that:0.1mmol/L guaiacol,0.8mmol/L Cu2+,0.5mmol/L oxalic acid and 0.8mmol/L L-phenylalanine.And for pcR5324 is that:0.1mmol/L guaiacol,0.4mmol/L Cu2+,0.3mmol/L oxalic acid and 1.2mmol/L L-phenylalanine.There are different,from the numerical value between single factor research and orthogonal experiment,it indicates the comprehensive and complex physiological regulation mechanism in a multi-factor condition.