| With the enhancement of people's awareness of environmental protection,laccase has been applied in many fields because of its unique substrate adaptability and oxidation characteristics.In this study,the laccase-producing strains were rationally modified to obtain a mutant enzyme with significantly improved catalytic activity,which provided a theoretical basis for further application in environmental protection,biological detection and medicine.The main research contents of this topic are as follows:1.Screening of laccase-producing strains and breeding of mutant strainsThe soil and water samples of laccase-producing bacteria may be selected from the environment.The samples were collected from the Xuancheng papaya garden in Anhui,the Miaofeng Mountain in Mentougou,Beijing,and the soil with dyed leaves and dyeworks in the Maershan Mountain in Shandong Province.Water sample.The wild-type fungus Trametes versicolor with the highest laccase production rate was used as the starting strain for UV mutagenesis and ARTP mutagenesis to obtain 14 groups of mutant strains: FG-obtained by UV mutagenesis compared with the initial strain.The enzyme activity of the 18-2-1 strain was 3.96 U/m L,which was 2.22 times higher;the enzyme activity of the FG-18-4-1 strain obtained by ARTP mutagenesis was 6.48 U/m L,which was increased by 4.30 times.The genetic stability experiment showed that after 5 subcultures,the enzyme activity of FG-18-2-1 decreased by 57.79% and the enzyme activity of FG-18-4-1 decreased by 21.70% compared with the original strain;FG-18-4-1,FG-18-2-1 genetic stability was 78.30% and 42.21%,respectively.According to the strain identification,the fungus strain with the highest yield was identified as Trametes versicolor mutant strain FG-18-4-1.2.Study on the enzymatic properties of white rot fungus laccaseFG-18-2-1 and FG-18-4-1 obtained by UV mutagenesis and ARTP mutagenesis were separately purified and studied in terms of enzymatic properties.After continuous purification,ABTS was used as the bottom.The specific activity of FG-18 laccase was 165.50 U/mg,the specific activity was increased by 30.62 times compared with that before purification,and the activity recovery rate was 18.10%;the specific activity of FG-18-2-1 laccase was 203.5 U/mg.The activity was 23.71 times higher than that before purification,and the activity recovery rate was 14.69%.The specific activity of FG-18-4-1 laccase was 462.20 U/mg,which was 40.45 times higher than that before purification,and the activity recovery rate was 12.05%.3.Optimization of laccase culture conditions for white rot fungiThe temperature,p H adaptability and stability of the mutant strain FG-18-4-1 laccase were analyzed.The results showed that the optimum temperature of laccase was 40 °C,and it maintained a certain activity at 25-60 °C,and the temperature adaptability was good.The starting strain laccase was almost completely inactivated after incubation at 55 ° C for 10 minutes,while the mutant strain laccase was reduced to 54% by incubation at 55 ° C for 10 minutes,and the enzyme activity remained about 20% after 60 minutes.The temperature stability of the mutant laccase was better.The optimum p H of the mutant strain FG-18-4-1 laccase is 4.4,and the optimum p H of the initial strain laccase is 4;when the p H is 5,the laccase activity of FG-18-4-1 and the starting strain are maintained separately.97.11% and 69.95%,the p H stability has been greatly improved.The single factor fermentation optimization experiment was carried out with a shake flask and a 5 L fermenter.The test conditions were determined to be a carbon source concentration of 10.00 g/L and a nitrogen source addition concentration of 22.00 g/L.1 The carbon source single factor experimental group had glucose as the control group.The sucrose group in the experimental group had the highest laccase activity,which reached 8.37 U/m L on the 8th day of fermentation,which was 2.40 times of the control group,and reached the highest enzyme activity fermentation.The time was shortened by 1 day compared with the control group,and the selected sucrose was the best carbon source for the laccase fermentation of the mutant strain FG-18-4-1.2 In the single factor experiment of nitrogen source,ammonium tartrate was used as the control group.The laccase activity of the yeast extract group rich in organic nitrogen in the experimental group was the highest,reaching 8.18 U/m L on the 9th day of fermentation,which was 2.34 times of the control group;ammonium sulfate The laccase activity of the group was next,which reached 6.28 U/m L on the 9th day of fermentation,which was 1.80 times that of the control group.Considering the actual production and application costs,ammonium sulfate is selected as the optimal nitrogen source.3 Inducer was selected from low-cost copper sulfate.The enzyme activity of laccase increased with the addition of copper ions.The highest enzyme activity in the fermentation group with 2 m M copper sulfate was 8.67 U/m L,which was 2.48 times that of the unadded group.The best carbon source for the laccase production by the mutant strain FG-18-4-1 is sucrose,the optimum nitrogen source is ammonium sulfate,and the optimal addition amount of the inducer copper ion is 2 m M.The laccase fermentation process was optimized by the response surface method: the model P value was less than 0.0001,which was the most significant laccase fermentation process condition: the sucrose addition amount was 10.09 g/L,and the ammonium sulfate addition amount was 21.74 g/L.The addition amount of copper sulfate was 1.32 m M;the total yield of laccase was predicted to be 15.96 U/m L,while the actual total yield was 15.90 U/m L,which was 4.56 times higher than that before optimization of fermentation conditions. |
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