Screening Of Laccase High-yield Strains And Their Application In Tobacco Stem Pretreatment

Laccase is a kind of polyphenol oxidase produced by white-rot fungi that can efficiently degrade lignin,and plays an important role in the degradation and utilization of lignocellulose resources.However,the laccase is greatly limited used in industrial application for the engineering strains of laccase generally have low yield and application level.Therefore,it is of great significance to screen strains with excellent properties and highly-yield laccase for the degradation and reuse of biomass resources.In this study,two Pycnoporus sanguineus with strong laccase-producing ability,XYG384（242.3 U/m L）and H275（361.0U/m L）,were screened from 39 strains of Pycnoporus from white-rot fungi at the laccase producing medium.The laccases produced by XYG384 and H275 strains have similar enzymatic properties:the optimum reaction p H is 2.0,optimum reaction temperature is 60℃,and the michaelis constant K_mare 0.034 mmol/L and 0.0385 mmol/L,respectiyely.After laccases were incubated at 60℃（XYG384）and 45℃（H275）for 6 hours,the laccases activity could still be maintained above70%,and the activity of both laccases could exceed 80%in the range of p H4.0～9.0.The stabilizing agents such as fructose,galactose,pectin and casein could significantly improve the thermal stability of laccase.In addition,2 mg/m L potassium sorbate or 15 mmol/L Fe²⁺could significantly improve the storage conditions of laccase.In order to promote the utilization of tobacco stems,laccase produced in the P.sanguineus was applied to the pretreatment process of tobacco stems for improving the quality of cigarettes.Results showed that:（ⅰ）The mycelia of P.sanguineus could degrade 79.1%of lignin after 21days.（ⅱ）The degradation rate of lignin could reach 65.1%with the conditions at 5 U/m L of laccase,enzymatic hydrolysis at p H4.5 and 30℃for 48 hours.（ⅲ）The smoking test of cigarette was best,when the pretreatment conditions of laccase on tobacco stems were follows:tobacco stem 100 g,laccase 30 U,material-liquid ratio 35%,p H5enzymatic hydrolysis 48 hours at 30℃,in which the degradation rates of cellulose,lignin,pectin and hemicellulose in tobacco stems were 15.1%,20.2%,12.4%and 7.2%,respectively.（ⅳ）To achieve similar degradation effect to the results of laccase pretreatment,the degradation system of laccase complex enzyme was obtained by the response surface method as follow:cellulase 1.0 U,laccase 14.7 U,pectinase 8.5 U and hemicellulase 1.0 U per 100 g tobacco stems.The application of laccase complex enzyme decreased the amount of laccase by half,which further reduced the cost of laccase in the pretreatment process of tobacco stems.In this study,HPLC and FITR were used to analyze the degradation products of tobacco stems.It was found that laccase could efficiently degrade phenol-type lignin models,and degrade tobacco stems into products with important application value,such as p-coumaric acid,etc.This substance may be one of the important reasons leading to the improvement of tobacco quality.In conclusion,this study achieved two strains of P.sanguineus which highly produced laccase.The laccase produced by the strains showed good enzymatic properties,and the enzymes could efficiently degrade tobacco stem biomass resources,thereby improving cigarette quality.The application of laccase complex enzyme further reduces the cost of laccase pretreatment of tobacco stems,laying a foundation for the efficient utilization of waste tobacco stem biomass.