Study On Pleurotus Eryngii Var.ferulae Laccase Gene Expression And Corresponding Proteins Effected By Co-cultured Rhodotorular Mucilaginosa

Laccases(EC 1.10.3.2)are copper-containing enzymes catalyzing the oxidation of polyphenolic compounds,have widely applications in the environment,textile and other industries.In the laccase fermentation technology,co-cultuivation has become an effective way to improve the level of laccase fermentation.At present,the specific mechanism of co-culture to improve laccase activity is not very clear,our previous study has found that ?-carotene was the key factor to improve laccase activity when co-cultured Pleurotus eryngii var.ferulae with Rhodotorula mucilaginosa,the paper explored the mechanism of the promotion factor on this basis,which will provide a theoretical foundation for the future laccase fermentation in a high level.The main contents and results are as follows:(1)The electrophoretic pure laccase proteins were obtained from the mono-cultivation and co-cultivation.For the control and treatment,results of enzymatic properties showed that the optimum temperature of laccase was 60 ?,optimum pH was 4.0,as well as the Km of ABTS were 0.182 mmol·L-1 and 0.193 mmol·L-1,respectively.The effects of metal ions and inhibitors on co-cultivation laccase were also substantially same as mono-cultivation laccase,that means there were no obviously different enzymatic properties between two of them.(2)The full-length cDNA sequence and the genomic DNA sequence of the laccase were obtained.Real time fluorescence quantitative PCR was used to perform transcriptional level analysis.The results showed that the laccase reached the highest level on the 5th day after the addition of ?-carotene.The results demonstrated that the increase in production of laccase resulted in increased enzyme activity.(3)About 900 bp upstream sequence of the laccase were obtained.Analysis of the upstream sequence revealed that there were multiple stress-related potential responsive elements such as xenobiotic-responsive element(XRE),stress-responsive promoter element(STRE),oxidative stress response element(SKN7)etc,which could affect the transcription of the corresponding structural gene.The addition of ?-carotene might have a certain stress on P.ferulae,which in turn activated the responsive element to improve the transcription efficiency of the laccase gene.(3)Two-dimensional electrophoresis was used to analyze the differences of intracellular proteins between co-culture and ?-carotene addition culture.The mass spectrometry resulted in the identification of 9 different proteins related to the laccase production of P.ferulae,most of them are concerned with protein synthesis and biological energy metabolism.