Screening And Characteristics Of Strains With Laccase Activity

Laccase,a copper-containing polyphenol oxidase,can oxidize a variety of phenolic and non-phenolic compounds and reduce molecular oxygen to water at the same time.Compared to plant and fungal laccases,bacterial laccases have the advantages of no requiring glycosylation,good thermodynamic stability,and optimum enzyme activity and a wide range of pH values.They have extensive application prospects in the paper industry,wastewater treatment,textile industry,food processing,and biosensors.With the deepening of theoretical research on bacterial laccases,it has been found that bacterial laccases can effectively treat dye wastewaters,and can adapt to the characteristics of dyestuffs which are diverse,miscellaneous,and easily changeable.Therefore,finding laccases from bacteria and extensively applying it to ecological environment become more and more significant.This study screened bacterial strains with higher laccase activity from soil and studied the characteristics of bacterial strains,the relative enzymatic properties of bacterial laccases,and the decolorization effects and methods of laccases on dyes,providing new strains and theoretical basis for dyes wastewater treatment with bacterial laccases.The main research results are as follows:(1)Screening and identification of strains with laccase activityEleven strains with laccase activity were isolated from the soil using copper ions as a screening agent.Using qualitative detection of syringalazine,two strains with higher laccase activity were selected for the study.After bacterial morphological structure analysis,physiological and biochemical characteristics and 16S rDNA sequence analysis,the strain HLS was identified as Bacillus licheniformis and the strain HL3 was identified as Bacillus sp.(2)Analysis of biological characteristics of strainsThe optimum growth temperature of strain HLS and strain HL3 were both 37?,and the optimum growth pH was 7.0.The strain HL3 grew well in medium containing 0-10%NaCl,and had strong tolerance to NaCl;HLS formed white sticky globules in a medium with a Cu2+concentration of 0.4-1.0 mmol/L,and the medium showed a clear state.When the Cu2+concentration was 2.0 mmol/L,the strain returned to a normal growth state.(3)Study on the enzymatic properties of spore laccase of strain HL3The enzyme HL3 with high laccase activity was selected for enzyme properties analysis.The laccase activity of the strain HL3 was 24.67 U/g dry weight,the optimum reaction temperature was 80?,and the optimum reaction pH was 7.0.10 mmol/L NH4+,K+,Ba2+ and Ca2+ could enhance strain HL3 spore laccase.The activity of Al3+,Fe2+,and Mn2+ significantly attenuated spore laccase activity,and Cu2+ could also inhibit laccase activity.0.1 mmol/L L-cysteine enhanced the activity of spores.When the concentration was higher than 1.0 mmol/L,spore laccase activity was essentially lost.SDS and EDTA with concentration lower than 1 mmol/L could enhance the activity of spore laccase.The concentration of SDS and EDTA was higher than 10 mmol/L,which could inhibit the activity of spore laccase.Methanol,ethanol,acetone,acetonitrile,and dimethyl sulfoxide all inhibit spore laccase activity.(4)Decolorization of dyes by strain HL3 spore laccaseAs for the decolorization rate of Congo red,crystal violet and isatin,strain HL3 spore laccase reached more than 80%after 6 hours in the non-mediated decolorization system,while the decolorization rate of reactive brilliant blue(RBBR)and reactive black 5 was only about 50%;after the addition of the mediator acetosyringone(Ace),the decolorization rate of spore laccase to Reactive Black 5 and isatin increased to more than 90%,but the mediate Ace did not have much effect on the decolorization rate of RBBR,Congo red and crystal violet.In the process of decolorization,the decolorization of isatin by the spore laccase was mainly oxidation,and the decolorization of other four dyes was mainly adsorption.When a certain amount of spore laccase is added,the removal rate of the spore laccase to the dye gradually decreases with the increase of the initial concentration of the different dyes;when the dye concentration is constant,the removal rate of the spore laccase to the dye increases with the increase of the amount of the enzyme.The strain HLS beads decolorize Congo Red,Crystal Violet and Eriochrome Red B and can be decolorized repeatedly 3 times.