[Function of nitric oxide in initiating production of lignin degrading peroxidases by Phanerochaete chrysosporium].

OBJECTIVE: By analyzing the function and mechanism of nitric oxide in initiating producing lignin peroxidases by phanerochaete chrysosporium, we studied the regulation mechanism triggering the secondary metabolism of white-rot fungi. METHODS: Mutant (pcR5305) and wild-type (pc530) strains of phanerochaete chrysosporium were respectively cultured under both the conditions of nitrogen limitation and nitrogen sufficiency. To compare their lignin peroxidases (LiP)-production and nitric oxide(NO)-production kinetics and their different influences on producing LiP after the NO donor Sodium Nitroprusside (SNP) and scavenger cPTIO were respectively added to the nitrogen limitation or sufficiency culture medium to show the function and mechanism of nitric oxide in initiating production of lignin peroxidases by white-rot fungi. RESULTS: Both strains produced nitric oxide (NO) under the two opposite nutritional conditions, but the levels of NO produced were related with the type of strain and the nutritional conditions. Strain pc530 produced NO requiring nutrition depletion and producing of NO was strongly delayed and reduced when it was cultured under nitrogen sufficiency condition. On the contrary, pcR5305 did not require nitrogen depletion to trigger and the levels of NO were higher than that of pc530. The results indicate that LiP content had positive correlation with NO value except the occurrence time of LiP peak value was later than that of NO. The ability of producing LiP was promoted after the NO donor SNP added, but SNP affected more on pc530 than pcR5305 in promoting producing LiP. 15mM cPTIO would greatly repress producing LiP, but could not completely restrain the synthesis of LiP for both strains. CONCLUSION: By producing NO, Phanerochaete chrysosporium triggers LiP synthesis. However, the evidences do not indicate that NO participates or effect directly in LiP synthesis. It is more likely that NO is reacting as an upstream signal molecule. Besides NO, there are other signal molecules that have a positive effect on NO levels also involving in the regulation producing LiP. The mechanism of the resistance to nutritional repression of pcR5305 in synthesizing lignin degrading peroxidases may be the answer to the different NO production mechanism of pcR5305 from pc530.

[Carbon-nitrogen regulation of a laccase-producing mutant of Phanerochaete chrysosporium resisting carbon and nitrogen nutritional repression].

OBJECTIVE: Comparing the effects of different carbon-nitrogen nutrition and their consumption on laccase production, we studied the ecophysiological characteristics of Phanerochaete chrysosporium resisting nutritional repression, and the carbon-nitrogen physiological regulation mechanism of the white-rot fungi. METHODS: The mutant and the wild-type strains were respectively cultured under the conditions of: carbon and nitrogen limitation, carbon limitation and nitrogen sufficiency, carbon sufficiency and nitrogen limitation, carbon and nitrogen sufficiency, to compare their laccase-production kinetics, cell growth and glucose and ammonia nitrogen consumption to show the characteristics and the regulation pathway of carbon-nitrogen nutrition on laccase production. RESULTS: The wild-type strain produced 0.107 U/L, 0.029 U/L,12.84 U/L and 18.05 U/L of laccase respectively on 11th,14th, 19th and 19th day when glucose or ammonia nitrogen was consumed to the lowest value; the mutant produced laccase throughout the whole process with two peaks respectively on 8th, 7th, 12th and 12th day with laccase of 298.83 U/L, 343.14U/L, 271.22 U/L and 251.49 U/L and on 12th, 13th, 19th and 19th day with laccase of 257.69 U/L, 298.78 U/L, 213.81 U/L and 216.93 U/L. The enzyme-production kinetics trends were similar between the two strains on the condition of the same initial carbon concentration but were different on the same initial nitrogen concentration, which showed that carbon source had more effect on laccase production. CONCLUSION: The laccase production of the wild-type strain was regulated by carbon or nitrogen starvation. Under different conditions, it was regulated by different nutrient. For example, under carbon limitation condition it was started by the glucose starvation, however under carbon sufficient condition the ammonia nitrogen starvation aroused it. The laccase production of the mutant didn't repress by carbon and nitrogen nutrition. Maybe it referred to a global regulation change which relieved nutritional repression on the laccase production.

[Breeding and characterization of laccase-producing Phanerochaete chrysosporium mutant resistant to nutritional repression].

OBJECTIVE: To screen Phanerochaete chrysosporium mutants resisting nutritional repression and to characterize laccase produced by the mutants. METHODS: We used repeated UV mutagenesis and screened the mutant strains by using the guaiacol nitrogen sufficient differential medium. We characterized enzymes production mechanism of the nutritional regulation through comparing the differences of cell growth and enzyme-production kinetics under different nutritional conditions; We validated production of laccase by Phanerochaete chrysosporium through measurements of the heat treatment, removal of manganese ion and addition of the catalase. RESULTS: Three different methods were validated that both strains of pcR5305 and pcR5324 can produce laccase under the nitrogen limitation (N-L) and nitrogen sufficient (N-S) conditions. Under the N-L conditions, pcR5305 can produce 203.5 U/L laccase and pcR5324 can produce 187.6 U/L laccase; Under the N-S conditions, pcR5305 can produce 220.6 U/L laccase and pcR5324 can produce 183.9 U/L laccase. The original strain pc530 only can produce very little laccase under either conditions. The laccase-production regulation mechanisms of the two strains are different: Production of laccase and the cell growth by pcR5305 are in synchronism. However production of the laccase by pcR5324 is repressed by nutrition. Both strains have the capacity of resisting nutritional repression and produce lignin peroxidase and manganese peroxidase with high yield. (LiP 1343.2, MnP 252.2 U/L and LiP 1169.5, MnP 172.4 U/L respectively). CONCLUSION: The mutants of Phanerochaete chrysosporium can produce laccase. At same time they showed the capacity of resisting nutritional repression and production of laccase, lignin peroxidase and manganese peroxidase. Our results possess high value for production, application and fundamental research. We provided new strains and established a very good foundation for the further research of metabolic regulation of ligninolytic enzymes production.