Fermentation And Enzymatic Characteristics Of Grifola Frondosa Hyper-Producing Laccase

With the development of technology,People not only pursuing the higher standard of living,But also having a signify requirement for the environmental conservation.Lactase as a no-pollution and purify natural substance,which is being used more and more widely in our daily life.And the values are gradually accepted by people.In this research,the Grifola frondosa laccase's characteristics were initial studied by submerged fermentation.The method had great advantages,which had a shorter growth circle, lower-pollution,and the production environment could be controlled easily.The Grifola frondosa laccase's enzyme activity was up to 17990U/mL.And the result could be completely used in industry manufacture.This article mainly discussed three aspect of content,through a serial investigation and analysis,and the main results were shown as following:1.Studied on the highly stability of the preparative samples:The optimum fermentation time was 10 days for product laccase.At different temperatures,Laccase's reaction rate and endurance were different.The suitable temperature was 40℃.At different pH,they had the different change,range from 1.8 to 3.0,which had a higher enzyme activity At pH3.0,activity was the highest.The results showed that,when the reaction temperature changed from 0℃to 40℃,laccase's enzyme activity was stabilize.At 40℃,it had the highest relatively enzyme activity,was up to 89%,when the temperature over 40℃.The enzyme activity was sharp decreased.At 60℃,relatively enzyme activity was just to 40%.When pH2.8-3.4 enzyme activity was very stability,which were all above 60%.But when the pH over 3.4,the relative enzyme activity was sharp decline.At pH 5.2,it just up to 20%.Metallic ion function:Cu²⁺,Co²⁺had the obvious activation to Laccase,Fe³⁺had certain inhibitory action exactly to the enzyme activity,and Ag⁺completely inhibited the enzyme activity.Comparing with the Cu²⁺,Co²⁺Na⁺,K⁺,Mg²⁺,Zn²⁺had a lower function on the enzyme activity.In order to get a better method of precipitation to product raw laccase,the tannin process,ethanol process,acetone process and ammonium sulfate process were researched.Finally comparing with yield and purity,getting the best method was ammonium sulfate precipitation.2.Optimization research on Grifola frondosa laccase liquid fermentation conditions:Analyzed the production conditions of laccase by orthogonal test and uniform design,the result demonstrated:shaking culture were better than static.The best fermentation conditions as follows:shaking incubator raises for 10 days,culture fluid with 50mL/250mL,and the speed of rotary was 120r/min.the enzyme activity was highest.Through orthogonal test design and analysis with the SPSS software,the result obviously showed that:The best carbon source was the glucose,the best nitrogen source was the protein peptone,and the best temperature was 26℃,the optimum pH was 3.0.The optimum enzyme medium conditions included that: Glucose 10g,Protein peptone 5g,and affix ion,Na₂HPO₄ 0.475g,KH₂PO₄ 0.453g,MgSO₄ 7H₂O 0.5g,CaCl₂ 2H₂O 0.013g,Yeast extraction 1g,VB₁ 25mg,trace amount solution 1ml,constant volume to 1000mL.The maximum enzyme activity of Grifola frondosa laccase was up to 17990u/mL.