Laccase Production By Trametes Versicolor Sdu-4 Under Submerged And Solid-State Fermentation And Ionic Liquid Pretreatment Of Biomass

Biomass is not only the renewable energy source, provides energy to us, but also is the renewable resource, provides raw material which is needed in the production of compounds. Agricultural straw, which mainly consists of cellulose, hemicellulose, and lignin, is the most abundant renewable lignocellulosic biomass. After cellulose, lignin is the most abundant renewable carbon source on earth. The utilization of biomass is hindered by the presence of lignin which is quite resistant to degradation under natural conditions. Lignin degradation process is thought as a rate-determining step of the carbon cycle in the biosphere. White rot fungi are the most efficient lignocellulose de-graders and the only known organisms that can completely break down lignin to carbon dioxide and water. Among them, the most widely studied is Trametes versicolor. Lignin biodegradation by white rot fungi involves various enzymes and the most significant ones are laccases.Laccases can catalyze the oxidation of an array of substrates, such as mono-, di-, and polyphenols, aromatic amines, methoxyphenols, and ascorbate by one-electron transfer mechanism. Because of their high nonspecific oxidation capacity, and their use of readily available molecular oxygen as electron acceptor, laccases are useful as biocatalysts for a wide range of biotechnological applications. Laccases can be used in paper pulp bleaching, decolorization of synthetic dyes, wine clarification, fruit juice processing, bioremediation, ethanol production, biosensors, biofuel cells, organic synthesis, and drug synthesis. In this paper, I studied the submerged and solid-state fermentation by Trametes versicolor sdu-4 which could secret a high level of laccase. T. versicolor sdu-4 laccase was purified and characterized. I also studied its application in dye decolorization.The second generation bio-ethanols which are made form biomass materials, such as straw, wood residues, agricultural residues, are expected to be valuable alternatives of gasoline. A key step in production of bio-ethanol is hydrolysis of lignocellulosic biomass into monosackcharide. Enzymatic hydrolysis of cellulose by cellulase enzyme is the most promising methods. Due to the recalcitrant structure of lignocelluloses, a pretreatment step is needed prior to enzymatic hydrolysis in order to make the cellulose more accessible to cellulase enzyme. Over the past decades, ionic liquids (ILs), often referred to as 'green solvents', have been the great focus of scientists in various fields due to their unusual physical and chemical properties like high thermal stability, lack of inflammability, low volatility, chemical stability and excellent solubility with many organic compounds. Some studies have shown that lignocellulosic materials can be dissolved in some hydrophilic ionic liquids, such as 1-butyl-3-methylimidazolium chloride (BMIMC1) and 1-allyl-3-methylimidazolium chloride (AMIMC1). The pretreatment of the agricultural straw and wood powder by ILs have been studied in the present study.The main results of the present paper are as follows:1. Strain identificationDuring the previous study of our laboratory, we have screened a strain which could secret a high level of laccase by the mothed of guaiacol culture medium plate color deterioration, and the growth of this strain is rapid. The fungus was characterized as Trametes versicolor sdu-4 according to its morphology and ITS1-5.8S rrNA-ITS2 gene sequence.2. Optimization of laccase production by Trametes versicolor sdu-4 under submerged fermentationThe T. versicolor sdu-4 laccase production under submerged fermentation was optimized by the method of factorial design, central composite design, and response surface methodology. Results indicated that the concentration of carbon source (glucose), nitrogen source (yeast extract), copper sulfate (CuSO4), and nitial pH value of the culture medium play an important role in laccase production by Trametes versicolor sdu-4 under submerged fermentation. Maximum laccase production (4146 U l-1) was observed at glucose (3.31g l-1), yeast extract (8.051 g l-1), CuSO4 (8.36 mg l-1) and nitial pH (4.8). The optimization results led to reduction in culture medium cost for laccase production.3. Laccase purification and characterizationThe laccase was purified and then biochemical properties of the enzyme was studied. It has all the characteristics of a typical blue laccase:(1) its molecular mass was 60 kDa; (2) a peak at 600 nm due to type I blue copper atom; (3) a shoulder at 320 nm indicating the presence of the type 3 binuclear copper pair; (4) four copper atoms per enzyme molecule. Therefore, We classified the laccase from T. versicolor sdu-4 as a typical blue fungal laccase. The N-terminal amino acid sequence of the laccase was AIGPAASLWANA. The optimum pH values of T. versicolor sdu-4 laccase were 2.2,3.7, and 7 for the oxidations of ABTS, DMP, and syringaldazine, respectively. The enzyme was stable at pH 3.0～10.0. The optimum temperature for ABTS oxidation was 75℃. The laccase was relatively stable at 70℃. Moreover, the half-life of the purified laccase at 70℃was 2.2 h. When the temperature was at 80℃, laccase activity decreased rapidly and was completely inactivated after 1.5 h. The most noticeable characteristic of laccase was that it oxidized methyl red, 2-bromophenol, and 4-bromophenol without mediators. The laccase showed good decolorization of the triphenylmethane and azo dyes.4. Study of solid state fermentation by T. versicolor sdu-4Rice Straw, wheat straw, corn stover, sugarcane bagasse, and wood powder were used as the lignocellulosic substrates to study solid-state fermentation by T. versicolor sdu-4. Results showed that the laccase production was highest when corn stover was used as solid medium. The solid-state fermentation conditions of T. versicolor sdu-4 were optimized by the method of factorial design, central composite design, and response surface methodology. The result indicated that laccase, cellulase (CMCase) and xylanase were produced during solid state fermentation. While lignin peroxide enzyme (LiP) and manganese peroxide enzyme (MnP) were not detected. Maximum laccase production (45.1U g-1 CS) was observed at glucose (9 mg g-1 CS), CuSCO4 (4.5μM g-1 CS) and moisture level (80%). Maximal lignin (34.8%) was degraded at glucose (9.2 mg g-1 CS), CuSO4 (4.3μM g-1 CS) and moisture level (82%).5. The stability of laccace in organic reagents and ILs and the oxidation of CEL by laccase in ionic liquidRecently, ionic liquids (ILs) are considered as an alternative to organic solvents for biocatalysis and biotransformations in view of sustainable and ultimately "green" processes, not only because enzymes displayed high level of activity and stereoselectivity of many different chemical transformations, but mainly because of an overstabilization effect on biocatalysts. In this work the stability of T. versicolor sdu-4 laccace in different concentrations of organic solvents and ILs was studied. The results indicated that:laccase was stable in organic solvent and water, IL and water composed homogeneous solution. However, the laccase activity was decreased rapidly with increasing volume percent of the organic solvent and ILs. In 30% ILs ([Bmim]Cl and [Emim][CH3C00]) solution (w/w), the remaining activity of laccase was still above 60% after 48 h. When the concentration of IL was abave 70%, laccase activity decreased rapidly.In the present paper, oxidation of CEL by laccase in 30% [Emim][CH3C00] was also studied. Infrared spectrum,1H-NMR,13C-NMR methods were used to characterize the treated CEL. The results showed that:T. versicolor sdu-4 laccase break down methoxy group, the guaiacyl and syringyl structures of CEL in [Emim][CH3COO].6. ILs pretreatment of agricultural straw and wood powder100 mesh wood powder was dissolved in ionic liquid [Pmim][CH3C00], then acetone/water (1:1 v/v) was added and stirred at room temperature for 2 h. Cellulose material was regenerated by vacuum filtration. After evaporating the acetone, lignin was regenerated by vacuum filtration. Infrared spectrum analysis, 1H-NMR and 13C-NMR methods were used to characterize the Ionic liquid-processed lignin.Rice straw, wheat straw and corn stover were pretreated by [Bmim]CI, [Emim][CH3C00] and [Pmim][CH3COO], respectively. Infrared spectrum analysis and scanning electron microscopy (SEM) were used to characterize the treated straw. The results indicated that [Emim][CH3COO] is the best IL when pretreating rice straw and [Pmim][CH3COO] is the best IL when pretreating wheat straw and corn stover.