Studies on the Production, Purification and Characterization of Laccases by the Novel Isolates of Basidiomycetes

Abstract

Lignin degradation is in a central position in the earth's carbon cycle. The most renewable carbon present in the nature is either present in lignin or in compounds, which are protected by lignin from enzymatic degradation (cellulose and hemicellulose). Lignin biodegradation is responsible for much of the natural destruction of wood in use, and it may have an important role in plant pathogenesis. Lignin degrading enzymes are becoming an effective tool in industrial processes, from crude applications such as bioremediation to fine processes such as biotransformation processes. Recently, there has been a growing interest in studying the lignin modifying enzymes of a wide array of white-rot fungi, not only from the standpoint of comparative biology but also with the expectation of finding better lignin-degrading system for use in various biotechnological applications. They have received considerable attention for industrial application due to both their broad substrate range and their ability to degrade the most recalcitrant natural polymer, lignin. They may provide environmentally friendly technologies for the pulp and paper industry and for the treatment of many xenobiotic compounds, stains, and dyes. This group of enzymes was therefore taken as target enzyme for the present study. Lignin biodegradation was considered an unusual biological process involving extracellular oxidations. The organisms principally responsible for lignocellulose degradation are aerobic filamentous fungi, and the most rapid degraders in this group are basidiomycetes. These fungi are unique in their ability to degrade most components of wood due to their capability to synthesize the relevant hydrolytic and oxidative extracellular enzymes. Wood-rotting basidiomycetous fungi are usually divided into white-rot, brown-rot and litter decomposing fungi. White rot fungi and related litter degrading fungi produce various isoforms of extracellular Lignin Modifying Enzymes (LMEs): lignin peroxidase, manganese dependent peroxidase and laccase. Some wood degrading fungi contain all three classes of ligninolytic enzymes, while other contains only one or two of these enzymes.Present research is undertaken with the aim of isolating and screening novel autochthonal fungal strains for ligninolytic enzymes production. The study also deals with the optimization and formulation of an inexpensive medium for acquiring high titre of the enzyme by submerged fermentation process. Latter use of cheap agro-industrial substrates for enzyme production by solid state fermentation is carried out. Further, purification of the enzyme is carried out by using simple and economical method such as three phase partitioning (TPP). The purified enzyme is characterized with respect to its activity and stability at various pH and temperature ranges, molecular weights determinations, kinetic constants and effects of compounds on the activity of the enzyme. In the present study, forty five basidiomycetous fungi were isolated from a village forest of Mangalore, Karnataka, India. These fungi were screened using solid agar medium containing indicator compounds for ligninolytic enzymes production. For LiP’s and MnP’s activities, screening was performed based on the decolorization ability of the dye azure-B. For screening laccase activity, guaiacol oxidation was used. Further confirmation and selection of predominant ligninolytic enzymes producers was done by growing the isolates in liquid cultures in defined medium for 14 days. Among forty five isolates, 12 strains exhibited dye decolorization and 24 strains showed the oxidation of guaiacol on solid agar plates within 20 days of incubation period. However, 5 strains were found to be laccase hyperproducers. Screened strains were further cultivated in specific media for the production of laccase and peroxidases (LiPs and MnPs). Only 6 out of 17 strains were able to produce significant amounts of laccase, MnP and LiP. Moreover, two isolates such as Peniophora sp. hpF-04 and Phellinus noxius hpF-17 were obtained as efficient laccase producers. Hence, further work is focused on the production of laccase by these two novel isolates. The effect of different carbon, nitrogen and inducer sources on laccase production by these fungi was carried out to select suitable carbon, nitrogen and inducer sources, respectively. Six different carbon sources such as glucose, carboxy methyl cellulose (CMC), sucrose, cellobiose, xylan, and starch were selected to study the effect of carbon onlaccase production. Similarly, seven different nitrogen sources such as KNO3, NH4Cl, NH4NO3, ammonium tartarate, aspargine, urea and yeast extract were used for studying the effect of nitrogen on laccase production. Compounds, such as copper sulphate (0.5, 1.0 and 2.0 g/l) veratryl alcohol (0.1, 0.3, and 1 ml/l), Tween 80 (0.1, 0.5, and 1.0 %), 2,5-xylidine (0.5, 1.0 and 2 mM) and guaiacol (0.1, 0.5 and 1.0 % ) were selected to study their inducing effect on laccase production by both the isolates. The time course production of laccase in basal medium showed similar enzyme production curves. Both fungi showed their maximum laccase activities and biomasses within 6-8 days of incubation period. Peniophora sp. hpF-04 and P.noxius hpF-17 respectively produced laccase of 1052±54 U/l and 545.5±14 U/l during 6 days of cultivations. Temperature of 30°C was observed to be optimum incubation temperature for laccase production in Peniophora sp. hpF-04 and Phellinus noxius hpF-17. Experiments on effects of different carbon and nitrogen proved that the presence of dissimilar carbon and nitrogen sources affected laccase production in both the fungi. Fungus Peniophora sp. hpF-04 showed maximum laccase activity in the medium containing sucrose (1249.0±12 U/l). Presence of CMC as a carbon source showed laccase activity of 1060.0±12.5 U/l in this culture. In Phellinus noxius hpF-17, maximum laccase activity (557±28 U/l) was found in the presence glucose supplemented media. Peniophora sp. hpF-04 showed maximum laccase production (1082.1±13.4 U/l) in case of ammonium chloride supplemented medium, whereas, Phellinus noxius hpF-17 produced maximum laccase in the presence of ammonium tartarate (557±23.1 U/l). Inducers like veratryl alcohol, guaiacol and 2,5-xylidine suppressed the production of laccase in both the fungi. On the other hand, increase in copper in the media, enhanced laccase in both Peniophora sp .hpF-04 and Phellinus noxius hpF-17. The optimization of medium components for enhancement of laccase production was carried out by statistical methods by using Placket-Burman design (PBD) and central composite design of response surface methodology (CCD-RSM). Initial screening of medium components was performed using a PBD and the variables showing significanteffects on laccase production were identified. The interactions among the screened variables were studied by CCD. Variables such as CMC, ammonium chloride and copper sulphate were found to influence the laccase production in Peniophora sp. hpF-04. Statistical optimization process resulted in optimum concentration of variables for maximized laccase production. The values obtained were, CMC (15 g/l), ammonium chloride (1.35 g/l) and copper sulphate (807 µM) with laccase yield of 1890±12 U/l. There was an approximate of 1.81 fold improvement in laccase production over the previous yield with un-optimized medium was achieved. In case of Phellinus noxius hpF-17, variables such as glucose, ammonium tartarate and Tween 80 were found to influence the laccase production significantly. Optimum values of tested variables for maximum laccase production are glucose (20 g/l), ammonium tartarate (2.25 g/l) and Tween 80 (2.08 ml/l) were obtained by CCD-RSM. By using this optimal fermentation medium, the laccase yield was increased up to 780±7.9 U/l, an approximate 1.43 fold improvement as compared to the previous yield with un-optimized medium. Present investigation was also aimed to exploit locally available, inexpensive agro-industrial wastes as a substrate for laccase production under SSF. Seven substrates viz., sugar cane bagasse (SCB), wheat bran (WB), rice bran (RB), corn stover (CS), saw dust (SD), grass powder (GP) and Jatropa seed cake (JSC) were evaluated. Solid state fermentation was carried out with and without nutrient supplements. Respective optimized laccase media of Peniophora sp. hpF-04 and Phellinus noxius hpF-17 were used as nutrient supplements. In Peniophora sp. hpF-04, no laccase activities (very negligible in SCB and RB) were found during 5th day of all the substrates, when the nutrient supplements were used in SSF. Among all the substrates, the fungus showed high level of laccase activity in case of Jatropa seed cake. Contrastingly, with the presence of nutrient supplements, detectable amounts of laccase activities were found in all the substrates during 5th day of incubation. The similar results were obtained for SSF using Phellinus noxius hpF-17 also. The present study revealed that, the presence of specific nutrients during SSF altered the laccase production in both the fungi, either by enhancing or by reducing the laccase synthesis. Among the seven agro-industrial wastesemployed for SSF, only JSC was found more potential in producing laccase in both fungi, with and without nutrient supplements. Hence, a detailed study of SSF with JSC was carried out to study time course production profile, effect of pH, effect of moisture on laccase production. During this, Phellinus noxius hpF-17 produced laccase in higher titre than Peniophora sp. hpF-04. It produced laccase of 79.92±2.3 U/g of the substrate and Peniophora sp. hpF-04 produced low level of laccase of 14.32±2.4 U/g during 10th day of incubation. Phellinus noxius hpF-17, produced maximum laccase at 70% moisture level and at pH.5.0, where as fungus Peniophora sp. hpF-04 showed maximum laccase at 80% of moisture level and at pH range between 5.0-6.0. The purification was achieved by simple method with a combination of ultrafiltration and three phase partitioning (TPP). Ultrafiltration by tangential flow filtration (TFF) was used to concentrate the culture supernatant. Later the laccase from the concentrated crude sample was separated and purified by three phase partitioning (TPP). The purity of the separated laccase was analyzed by RP-HPLC method. The purified enzyme was further characterized with respect to its activity and stability at various pH and temperature ranges. SDS-PAGE profiling and determination of enzyme’s Km and Vmax parameters were also carried out. The effect of metal ions and certain compounds such as EDTA, TEMED, SDS, β-mercaptoethanol and organic acids was investigated. A one-step TPP was employed for the purification of laccase using organic solvent tert-butanol. Further, the effect of different TPP conditions such as salt saturation levels (w/v), aqueous phase to tert-butanol ratio (v/v), pH and temperature to optimize laccase separation was carried out. Concentration by ultrafiltration achieved 94.41% yield of laccase with a 2.28-fold purification in Peniophora sp. hpF-04. By single step TPP process, crude enzyme solution of pH 4.0 saturated to 55% (w/v) ammonium sulphate with a crude extract to tert-butanol ratio of 1:1.5(v/v) at 45oC resulted in 96.4% recovery of laccase with 2.61fold purification of laccase produced by this fungus. On characterization, HPLC study resulted in a single peak in TPP purified sample whichrevealed the purity of the separation. Optimum pH and temperature for maximum activity were determined as pH 4.0 and temperature range of 45-50oC. This laccase was found to be stable at 50oC and 60oC for more than 15 h. The kinetic constants (Vmax and Km) measured using Lineweaver–Burk double reciprocal (1/ [v] vs 1/[S]) plots obtained were, 30.3 mM and 0.06 mM/min for ABTS, 8.77 mM and 0.27 mM/min for 2, 4-dimethoxy phenol and 1.49 mM and 0.28 mM/min for guaiacol, respectively. SDS-PAGE profiling revealed a single band of laccase with molecular weight of 67KDa. The laccase contained 3.12±0.2 Cu with an atypical spectrum lacking peak around 600 nm. According to these features, we classified Peniophora sp. hpF-04 with so-called yellow laccases, recently found in basidiomycetes. This laccase enzyme was completely inhibited by β- mercaptoethanol, oxalic acid and almost by sodium azide. The laccase produced from Phellinus noxius hpF-17 also separated and purified by a combination of ultrafiltration and TPP. However, one step combination method of TPP resulted in poor laccase yield and purity in this fungal laccase. Hence, optimization of TPP variables had to be carried out for effective separation of laccase from the crude extract. The optimization was achieved by using Box-Behnken design (BBD) of statistical experiments. BBD was employed with experimental factors of ammonium sulphate saturation; 20-80% (w/v), ratio of crude extract to tert-butanol; 1.0:0.5 – 1.0:2.5 (v/v) and temperature; 20–60°C to optimize TPP process parameters. The second-order regression equation provided the levels of laccase yield and purity as a function of (NH4)2SO4 saturation, ratio of crude extract to tert-butanol and temperature was generated. Ultrafiltration by TFF resulted in 64% laccase yield with purity fold of 1.58 during Phellinus noxius hpF-17 laccase concentration. The combination of 57% (w/v) of ammonium sulfate with 1:1.7 (v/v) ratio of crude extract to tert-butanol at pH 5.0 and incubation temperature of 44oC obtained through optimization experiments was found to be suitable combination for maximum recovery of Phellinus noxius hpF-17 laccase. When compared to commercial purification procedure, which generally includes salt precipitation and chromatography techniques, etc., the extraction of laccase by this process was proved to be much easier and eco-friendly.Isolated Phellinus noxius hpF-17 laccase did not show any blue coloration which was further confirmed by the lack of absorption peak at 610 nm under spectral scanning. This laccase found to be bigger in size than Peniophora sp. hpF-04 laccase with molecular weight of 75KDa as it determined by SDS-PAGE analysis. It showed high stability at acidic pH and elevated temperatures. The optimum pH of purified laccase produced by Phellinus noxius hpF-17 was found to be 3.0 for ABTS oxidation. When purified laccase was incubated for 20 h at room temperature at pH 3.0, it remained quiet stable. The laccase was found to be highly thermostable with broad temperature range of 65-85oC with an optimum temperature being 75oC. At 75oC, the enzyme was very stable even after 72 h of incubation. But at 85oC, the enzyme retained only 4% of its original activity after 24 h of incubation. The catalytic properties of this laccase were similar to the corresponding enzymes of other related group. Like Peniophora sp. hpF-04 laccase, laccase form this fungus also showed high affinity towards ABTS substrates. Compounds like β-mercaptoethanol, TEMED and sodium azide were very effective as inhibitor of Phellinus noxius hpF-04 laccase. However, In case of CuCl, 8-fold increase in laccase activity was observed. Overall, the laccases produced by both isolates were found to be acid stable and thermostable, unlike most of the other fungal enzymes listed in the literature. The longer stability and higher temperature and acidic pH, lack of inhibition makes these enzyme suitable for industrial purpose/or bioremediation practices dealing with harsh process. However, further studies of structural and catalytic properties of these enzymes would be necessary for elucidation of novelty of these laccases