Cultivation Of High Ligninolytic Enzyme Of Trichoderma Harzianum WRF-2 And Study On Biodegradation Of Corn Straw

The crop straws are renewable resources wildly in nature,composed of cellulose,hemicellulose and lignin.However,the structure of heterogeneity and recalcitrance of lignin greatly retard the efficient utilization of plant biomass.Traditional ways,both physical and chemical have the side effect for the environment.In such condition, how to decompose and convert lignin by biotechnology which is a optimum choice has become a new topic.Using WRF-2 which was screemed by my our lab,studies were carried out in the following three aspects:(1)Determined the optimal culture condition producing lignin peroxidase;(2)obtained a positive mutation strain by UV treatment,analysis the MnP characteristics and purified the MnP ,finally evaluated the molecular weight;(3)Lignin degradation condition of maize straw by ligninolytic enzymes had been studied. Mainly though adding acetyl acetone,H2O2 and MnSO₄·H₂O ,we determined the lignin degradation rate by using cellulose analysis instrument. The results are illustrated as follows:Several procedures have been employed for growing lignocelluloses-degrading enzymes. The results show that carbon–limited nutrition and shallow shake cultivation favored production of LiP. The optimize LiP production medium is as follows : starch 0.60 gL-1; yeast extract 7.50 g·L^-1; KH₂PO₄ 4.000 g·L^-1; MgSO₄ 0.5 g·L^-1; CaCl₂·2H₂O 0.05 g·L^-1; veratryl alcohol 2.0 mmol·L^-1;trace element solution 40 mL·L^-1; Tween 80 2.0g·L^-1,VB12 mg·L^-1,pH 4.5.LiP activity has been increased not only from the pre-optimization of 996.72U·L^-1 to the optimized 2162.4U·L^-1 (enzyme solution also contains 1110.6 U·L^-1 of MnP activity,with no laccase activity),and so the time to reach peak activity from nine days before the optimization reduced to three days after the optimization, which greatly shortened the enzymes producing cycle.The spores suspension of initial strain (Trichoderma harizianum) were irradiated 90 s by UV treatment, and a positive mutation strain (0202) was obtained after screening. It was determined as a genetic stable high-yield strain. The MnP activity was greatly increased 169.57% more than its initial strain and the LiP activity was raised by 17.98%.The properties of MnP were researched and the results showed that the optimum temperature and pH of the MnP activity were 40℃and 3.0.Moreover,the enzyme activity was stable within pH range of 2.5³.5 under 50℃. Among different metal ions,the MnP activity was enhanced by Cu²⁺,Mn²⁺,Mg²⁺,Ca²⁺,whereas it was inhibited by the metal ions Fe²⁺,Zn²⁺.One active component(MnP)was purified by salting and DEAE-52.SDS-PAGE was evaluated the molecular weight which was about 45KDa.Through optimization studies,the optimized conditions of corn straw lignin degradation by use of crude enzyme solution are as follows:the pH 3.0 of enzyme solution,temperature of 40℃,hydrolysis time 48 h,concentration of Mn²⁺ and acetyl acetone were 10 mM and 50 mM. Under this condition,the degradation rate of lignin was 25.61%, hemicellulose loss was 2.22%, cellulose loss was 1.17% ,respectively.The results show that it had degradating effect of using LiP-MnP lignin degradation enzymes,and the raising of MnP activity was in favor of lignin degradation.