Violuric Acid Research Articles

Study of suitable media for laccase synthesis of basidiomycete fungi from Bidoup - Nuiba park and its reactive dye decolorization ability

A fungal strain FBD154 with high laccase production was isolated from Bidoup-Nui Ba Parks, Lam Đong, Vietnam. FBD154 was identified as Polyporus sp. FBD154 based on traditional method and ITS sequence analysis. Polyporus sp. FBD154 synthesized laccase with high activity 74925 U/l on modified TSH1 (200 g/l containing potato extract, 1 g/l casein, 1 g/l rice bran, 5 g/l soybean meal, 10 g/l manose, 3 g/l NH4Cl, 0.3 mM CuSO4, pH 4). Crude laccase of Polyporus sp. FBD154 was applied to decolor several synthetic and commercial dyes. After 30 min, laccase from Polyporus sp. FBD154 could decolorize synthetic dyes at 100 mg/l concentration with efficiency of 60% acid red (NY1), 18% acid red 299 (NY7), 52% acid blue 281 (NY5) and 83% Remazol Brilliant Blue R (RBBR) without mediator. For commercial dyes at concentration of 100 mg/l, color removal efficiency reached to 62% megafix black CLS (CLS) and 72% everzol red LF2B (LF-2B) without mediator. Efficiency of synthetic dye removal by Polyporus sp. FBD154 crude laccase with 200 µM mediator obtained 90% RBBR with violuric acid (VIO) after 30 min; 80% NY5 with hydroxybenzotriazole (HBT) for 24 hours; 87% NY1 with acetosyringone (Ace) for 5 min; 92% NY7, 91% LF-2Band 73% CLS with syringaldehyde (Syr) for 5 min. The obtained evidences show that laccase was synthesized by Polyporus sp. FBD154 with high potential for application in wastewater treatment of textile plants in particular as well a sin detoxification of polycyclic aromatic compounds in general.

Impacts of redox-mediator type on trace organic contaminants degradation by laccase: Degradation efficiency, laccase stability and effluent toxicity

This study compares the effectiveness of seven redox-mediating compounds namely, 1-hydrozybenzotriazole (HBT), N-hydroxyphthalimide (HPI), 2,2,6,6-Tetramethyl-1-piperidinyloxy (TEMPO), violuric acid (VA), syringaldehyde (SA), vanillin (VA), and 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS), which follow distinct oxidation pathways, for the degradation of trace organic contaminants (TrOCs). These redox-mediators were investigated for improved degradation of four TrOCs showing resistance to degradation by crude laccase from the white-rot fungus Pleurotus ostreatus. ABTS and VA achieved the highest degradation of the phenolic compounds (i.e., oxybenzone and pentachlorophenol), whereas the non-phenolic compounds (i.e., naproxen and atrazine) were best removed using VA or HBT. This implies that the non-phenolic compounds are more effectively removed by the radical species generated by the NOH type mediators (i.e., VA and HBT), while removal of the phenolic compounds may depend more on the stability and the redox potential of the radicals generated from the mediator, irrespective of the type. Notably, enzyme stability was greatly affected by the NOH type mediators but it was compensated by their rapid degradation capacity. Overall, VA and HBT (NOH type) appear to be the best mediators for enhanced degradation of the selected compounds without causing significant toxicity in the effluent.

A Pycnoporus sanguineus laccase for denim bleaching and its comparison with an enzymatic commercial formulation

A laccase from the basidiomycete Pycnoporus sanguineus strain RVAN5 was evaluated for its ability to decolorize synthetic dyes and denim bleaching. Dye color reduction and denim bleaching were monitored at different dye concentrations and incubation times. Dye decolorization by Pycnoporus sanguineus fungal crude extract (FCE) ranged from 80 to 96% within 2–4 h at 25–65 °C. Comparable results were obtained when violuric acid (VA) was added as mediator to the FCE, however, the number of decolorized dyes increased significantly. Dye decolorization rates with VA varied of initial and final optical density (595 nm) values of 2.5–3.0 and 0.2–0.02, respectively. P. sanguineus FCE had no substantial effect on denim bleaching when used alone, notwithstanding, the mixture of FCE with VA (10 mM) showed significant denim color reduction values and considerably higher than those obtained with a bleaching enzyme from a commercial formulation; CIElab values obtained with FCE/VA mixture were of ΔL = 6.4, versus a ΔL 1.4 value obtained with an enzyme from commercial formulation.

Synthesis of 5-(hetarylmethylidene)pyrimidine-2,4,6(1H,3H,5H)-triones

Pyrimidine-2,4,6(1H,3H,5H)-trione (barbituric acid) 1 due to the high mobility of methylene hydrogen atoms in the pyrimidine ring is known to be nitrated to 5,5-dinitrobarbituric acid and nitrosated to violuric acid [1]. Compound 1 as well as its 2-thioand 2selenoanalogs enter in the condensation with aromatic aldehydes and dialdehydes (terephthalic aldehyde), and also with heterocyclic aldehydes (2-furaldehyde, 5aryl-2-thiophenecarbaldehydes) or with diketone (6,6dimethyl-1-phenyl-6,7-dihydro-1Н-indazole-4,5-dione) to form the corresponding 5-arylmethylidene(hetarylmethylidene) derivatives [2–6]. The formation of analogous products is also the result of the reaction of pyrimidine-2,4,6(1H,3H,5H)-trione or its 2-thioanalog with 2-monosubstituted or 2,6-disubstituted γ-pyrones and 4-pyridones [7, 8].

Characterization of a solvent, surfactant and temperature-tolerant laccase from Pleurotus sp. MAK-II and its dye decolorizing property.

Laccase is industrially important but a major challenge is the production of an ideal laccase with suitable physicochemical properties to tolerate temperature, surfactants, metal ions and solvents towards its potential application in bioremediation. A laccase with a molecular mass of 43kDa was purified from Pleurotus sp. MAK-II. It was optimally active at pH 4.5 and 60°C using ABTS (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) as substrate. The laccase was susceptible to NaN3 and NaCN. Activity was strongly enhanced by Cu(2+), Mg(2+) and Ca(2+). The purified laccase showed stability towards various surfactants and solvents and decolorized, in the presence of violuric acid as redox mediator, the diazo dye Congo Red and the anthraquinone dye Remazol Brilliant Blue R to the extent of 96 and 72%, respectively. The ideal physicochemical properties of Pleurotus sp. MAK-II-derived laccase suggest that it could be effectively used in the textile dye industry.

Comparative evaluation of the action of two different endoglucanases. Part II: On a biobleached acid sulphite pulp

A TCF sulphite pulp, bleached at the laboratory scale with a laccase–violuric acid system and complemented with a pressurized hydrogen peroxide stage, was treated with two endoglucanases, one obtained from Paenibacillus barcinonensis (B) and the other one produced from Cerrena unicolor (F) to improve cellulose reactivity. The treated pulps were evaluated in terms of brightness, viscosity, α-cellulose, water retention value, fibre morphology, Fock solubility, NMR and carbohydrate composition of pulps and liquors. Results revealed that both endoglucanases improved cellulose reactivity, albeit in a different way; thus, B caused no scissions in the cellulose chain and no significant reduction in fibre length, whereas F decreased viscosity and shortened fibre length, leading to lower reactivity value. The liquor composition of soluble carbohydrates released by the enzymatic treatments revealed the B had a processive mode of action since short oligosaccharides, cellobiose and glucose, were obtained. F hydrolysed, from high to low concentration, cellobiose, glucose and cellotriose. Importantly, environmentally friendly dissolving pulp with 90 % Fock solubility was obtained, combining two enzymatic treatments: a laccase–mediator system and then a cellulase from P. barcinonensis (B). In order to improve the quality of final dissolving pulp, a pulp purification step was introduced before the B endoglucanase treatment. The cold caustic extraction lead to reduce the amount of hemicelluloses by 42 % with respect to biobleached pulp, but Fock solubility was also reduced. However, complementing the purification step with F treatment, reduced the amount of hemicelluloses but also improved Fock solubility by 17 %, although some presence of cellulose II was detected by NMR.

Synthesis and electronic properties of π-extended flavins.

Flavin derivatives with an extended π-conjugation were synthesized in moderate to good yields from aryl bromides via a Buchwald-Hartwig palladium catalyzed amination protocol, followed by condensation of the corresponding aromatic amines with violuric acid. The electronic properties of the new compounds were investigated by absorption and emission spectroscopy, cyclic voltammetry, density functional theory (DFT) and time dependent density functional theory (TDDFT). The compounds absorb up to 550 nm and show strong luminescence. The photoluminescence quantum yields ϕPL measured in dichloromethane reach 80% and in PMMA (poly(methyl methacrylate)) 77%, respectively, at ambient temperature. The electrochemical redox behaviour of π-extended flavins follows the mechanism previously described for the parent flavin.

Studying the effects of laccase treatment in a softwood dissolving pulp: Cellulose reactivity and crystallinity

An enzymatic biobleaching sequence (LVAQPO) using a laccase from Trametes villosa in combination with violuric acid (VA) and then followed by a pressurized hydrogen peroxide treatment (PO) was developed and found to give high bleaching properties and meet dissolving pulp requirements: high brightness, low content of hemicellulose, satisfactory pulp reactivity, no significant cellulose degradation manifested by α-cellulose and HPLC, and brightness stability against moist heat ageing. The incorporation of a laccase-mediator system (LMS) to bleach sulphite pulps can be a good alternative to traditional bleaching processes since thermogravimetric analysis (TGA) showed that the laccase treatment prevented the adverse effect of hydrogen peroxide on fibre surface as observed during a conventional hydrogen peroxide bleaching treatment (PO). Although VA exhibited the best results in terms of bleaching properties, the performance of natural mediators, such as p-coumaric acid and syringaldehyde, was discussed in relation to changes in cellulose surface detected by TGA.

Enhancing laccase production by a newly-isolated strain of Pycnoporus sanguineus with high potential for dye decolouration

Response surface methodology (RSM) was applied to optimise laccase production by a new strain of Pycnoporus sanguineus, isolated from the Uruguayan forests, grown on bark shavings under semi-solid-state fermentation conditions. Enhancement of laccase production was studied, with a full 24 factorial design with two replicates and four central points. Operating under the optimal conditions obtained (pH 3.0, yeast extract 10 g L−1, glucose 5 g L−1 and bark shavings 3 g), a laccase activity of nearly 3000 U L−1 was achieved. Subsequently, the effect of different potential inducers on laccase production (Cu+2, Mn+2 and lignin) was tested using the one-factor-at-a-time approach. It was found that the addition of 0.5 mM Cu+2 into the cultures considerably increased laccase production and, thus, maximum activity values of about 16000 U L−1 were achieved. Further, the optimised culture broth (containing mainly laccase) showed high capacity to decolourise different recalcitrant synthetic dyes such as Acid Red 88 (AR88), Reactive Black 5 (RB5) and Lanaset Grey G (LG). After selecting enzyme concentration and time of reaction, almost 70% of AR88 could be decolourised in the absence of redox mediators. However, mediators were necessary to bleach RB5 and LG. Methyl syringate was the most effective mediator for AR88 (reaching a decolouration degree over 90%) and RB5 (nearly 70%). Around 80% of LG could be decolourised in the presence of violuric acid. These promising results make this enzyme a very useful tool for developing wastewater treatments.

Supramolecular complexes obtained from the interaction of violuric acid with manganese ion and nitrogenous ligands

This work describes the synthesis, spectroscopic characterization (Raman and infrared) and structural arrangement of three new supramolecular complexes named [Mn(H2Vi)2(H2O)4)](bpy)2(1), [Mn(bpa)2(H2O)4](H2Vi)2(2) and [Mn(bpp)2(H2Vi)2]·(bpp)2(H2O)2(3); these compounds have been obtained making use of different building blocks such as 4,4′-bipyridyne (bpy), 1,2-bis(4-pyridyl)ethane (bpa) and 4,4′-trimethylene-dipyridine (bpp) acting as spacers with violuric acid and manganese ion, presenting behavior related to processes of molecular self-assembling and self-organization, very common in studies of supramolecular systems. In all these compounds the violurate anion appears in the crystalline arrangement as monodentate, anionic and chelate forms for 1, 2 and 3, respectively. The important to note is that monodentate coordination in 1 and chelate in 3 through O2 and O3 oxygen atoms from the oxime group can be considered the first example in literature involving violuric acid, both in coordination or interaction with manganese ion. Moreover, it can be seen a good agreement between the structural results and the spectroscopic data; for instance the presence of an intense band in the Raman spectrum around 1603 and 1012cm−1 in all obtained compounds, assigned to the ν(CC)/ν(CN) and ν(ring)modes of the pyridyl ligand, respectively. Other important band can be observed in 1031cm−1 only for compound 3, assigned to the ν(NO) mode of the violurate ligand; the band at 1284cm−1 referring to the ν(NO) mode, very characteristic of violurate species is not seen in the spectrum, thus confirming the coordination of this building block by the oxime moiety.

Green Catalytic Approach for the Synthesis of Platform Chemicals from Palm Tree Lignin

Lignin is the second most abundant naturally occurring macromolecule found in plant cell-wall, vascular components and woody stems. It is the largest renewable source of aromatic biopolymer. However, lignin is recalcitrant to be broken down by most chemicals. This is because of its complicated heterogeneous molecular structure. However, lignin depolymerization has huge potentials for the synthesis of a number of useful chemicals, perfumes and pharmaceuticals and toiletries. The oxidation products of lignin are important precursors for pulp/paper and food industries, synthetic thin films. Vanillin, veratryl aldehyde and para-benzoquinone are the oxidation products of lignin. These chemicals are the precursors of optically active alcohol, ketone, violuric acid and benzaldehyde. However, the oxidation of biolignin has been remaining a challenging task. Green catalytic approaches might be an interesting solution for the selective depolymerization of lignin into various platform chemicals. Metal oxide/silica supported nanoporous gold has received strong attention as green catalyst for the transformation of various natural polymers. Mesoporous metal oxide/silica provide enlarged surfaces for the breakdown of C-C, C-H and C-OH bonds. This paper has reviewed various green catalytic approaches for the control depolymerization of biolignin into platform chemicals.

Degradation of a Phenolic β-O-4 Lignin Model Dimer by Electrochemically Mediated Delignification System with Violuric Acid

The β-O-4 type model compound guaiacylglycerol-β-guaiacyl ether was treated with a Violuric acid (VIO) mediated electrochemical delignification system. GC-MS analysis were used to investigate the changes of chemical structure of β-O-4 type model compound during reaction. Mechanism of this electrochemical delignification was also explored. Results indicated that the electrochemical oxidation by VIO as a mediator had strong oxidation capability on degradation of β-O-4 type model compound. After degradation, a series of aromatic compounds containing single benzene ring, furan compounds and aliphatic compounds with small molecule were produced. From their structures, the following reactions were caused by electrochemical oxidation: ether-bond cleavage, Cα-Cβ cleavage and benzene ring cleavage. Corrsequently, this work provides a comprehensive information on electrochemical oxidation delignification system that could be applied to industrial nonpollution bleaching.

Kinetics of Electrochemical Delignification Mediated with Violuric Acid

The violuric acid mediated chemical delignification system has a high delignification efficiency for pulp fibers and high delignification selectivity. The process conditions and effectiveness were carried out.The kinetics of electrochemical delignification has been investigated using the violuric acid (VIO) as the mediator. The reaction orders with respect to delignification, concentration of VIO and voltage of electrolytic cell were determined experimentally, which were dependent on the reaction conditions. The kinetic equation of electrochemical delignification and estimation function of kappa number were obtained respectively as follows: - dK/dt = A e-[VI1.031[2.197K6.25 , E=16.2KJ/mol, K(t)={2.89×10-7+5.25k [VI1.031[2.197t}-0.19.

New colorimetric screening assays for the directed evolution of fungal laccases to improve the conversion of plant biomass

BackgroundFungal laccases are multicopper oxidases with huge applicability in different sectors. Here, we describe the development of a set of high-throughput colorimetric assays for screening laccase libraries in directed evolution studies.ResultsFirstly, we designed three colorimetric assays based on the oxidation of sinapic acid, acetosyringone and syringaldehyde with λmax of 512, 520 and 370 nm, respectively. These syringyl-type phenolic compounds are released during the degradation of lignocellulose and can act as laccase redox mediators. The oxidation of the three compounds by low and high-redox potential laccases evolved in Saccharomyces cerevisiae produced quantifiable and linear responses, with detection limits around 1 mU/mL and CV values below 16%. The phenolic substrates were also suitable for pre-screening mutant libraries on solid phase format. Intense colored-halos were developed around the yeast colonies secreting laccase. Furthermore, the oxidation of violuric acid to its iminoxyl radical (λmax of 515 nm and CV below 15%) was devised as reporter assay for laccase redox potential during the screening of mutant libraries from high-redox potential laccases. Finally, we developed three dye-decolorizing assays based on the enzymatic oxidation of Methyl Orange (470 nm), Evans Blue (605 nm) and Remazol Brilliant Blue (640 nm) giving up to 40% decolorization yields and CV values below 18%. The assays were reliable for direct measurement of laccase activity or to indirectly explore the oxidation of mediators that do not render colored products (but promote dye decolorization). Every single assay reported in this work was tested by exploring mutant libraries created by error prone PCR of fungal laccases secreted by yeast.ConclusionsThe high-throughput screening methods reported in this work could be useful for engineering laccases for different purposes. The assays based on the oxidation of syringyl-compounds might be valuable tools for tailoring laccases precisely enhanced to aid biomass conversion processes. The violuric assay might be useful to preserve the redox potential of laccase whilst evolving towards new functions. The dye-decolorizing assays are useful for engineering ad hoc laccases for detoxification of textile wastewaters, or as indirect assays to explore laccase activity on other natural mediators.

An enzyme-catalysed bleaching treatment to meet dissolving pulp characteristics for cellulose derivatives applications

Bleached cellulose with good end-properties (≈ 90% ISO brightness and 62% cellulose preservation) was obtained by using a totally chlorine-free biobleaching process (TCF). Unbleached sulphite cellulose was treated with Trametes villosa laccase in combination with violuric acid. This enzymatic stage (L) was followed by a chelating stage (Q) and then by a hydrogen peroxide stage reinforced with pressurized oxygen (Po), resulting to an overall LQPo sequence. The use of violuric acid was dictated by the results of a preliminary study, where the bleaching efficiency of various natural (syringaldehyde and p-coumaric acid) and synthetic mediators (violuric acid and 1-hydroxybenzotriazole) were assessed. The outstanding results obtained with laccase-violuric acid system fulfil most of the characteristics of commercial dissolving pulp, totally acceptable for viscose manufacturing or CMC derivatives, with the added advantage that the enzymatic treatment saved 2h of reaction time and about 70% of hydrogen peroxide consumption, relative to a conventional sequence (Po).

Enzymatic strategies to improve removal of hexenuronic acids and lignin from cellulosic fibers

Abstract Different enzymatic strategies were applied to improve lignin and hexenuronic acid (HexA) removal. Three laccases (L) with different redox potentials were applied in combination with 1-hydroxybenzotriazole or methyl syringate to softwood sulfite and hardwood kraft fibers. The enzymes with a high-redox potential from Pycnoporus cinnabarinus and Trametes villosa were found to be the most efficient. The bleaching efficiency was not significantly influenced by the presence or absence of HexA in the different types of lignocellulosic fibers. Subsequently, the lignin or HexA removal in the presence of different mediators was evaluated in eucalyptus fibers with the T. villosa laccase (TvL). Natural mediators removed only lignin, whereas the combinations of TvL with synthetic mediators removed both HexA and lignin. The mediator violuric acid (VA) was the most efficient as judged by the properties of fiber and effluent. A xylanase pretreatment stage was found to boost the access of the LVA system to HexA without affecting lignin in the pulp.

Antiproliferative effects of palladium(II) complexes of 5-nitrosopyrimidines and interactions with the proteolytic regulatory enzymes of the renin–angiotensin system in tumoral brain cells

Seventeen new palladium(II) complexes of general formulaes PdCl2L, PdCl(LH−1)(solvent) and PdCl2(PPh3)2L containing pyrimidine ligands derived from 6-amino-5-nitrosouracil and violuric acid have been prepared and characterized by elemental analysis, IR and NMR (1H and 13C) methods and, two of them, PdCl(DANUH−1)(CH3CN)]·½H2O and [PdCl(2MeOANUH−1)(CH3CN)] by X-ray single-crystal diffraction (DANU: 6-amino-1,3-dimethyl-5-nitrosouracil; 2MeOANU: 6-amino-2-methoxy-5-nitroso-3H-pyrimidin-4-one). The coordination environment around palladium is nearly square planar in the two compounds with different supramolecular arrangements. Crystallographic and spectral data are consistent with a bidentate coordination mode through N5 and O4 atoms when the ligands act in neutral form and N5 and N6 atoms in the monodeprotonated ones. The cytotoxicity of the complexes against human neuroblastoma (NB69) and human glioma (U373-MG) cell lines has been tested showing a considerable antiproliferative activity. Also, the study of the effects of palladium(II) complexes on the renin–angiotensin system (RAS) regulating proteolytic regulatory enzymes aminopeptidase A (APA), aminopeptidase N (APN) and insulin-regulated aminopeptidase (IRAP) shows a strong dependence on the compound tested and the tumoral cell type, also affecting different catalytic routes; the compounds affect in a different way the activities of enzymes of the RAS system, changing their functional roles as initiators of cell proliferation in tumors as autocrine/paracrine mediators.

Comparison of a series of laccase mediators in the electro-oxidation reactions of non-phenolic lignin model compounds

The electro-oxidations of non-phenolic lignin model compounds in an electrolytic mediator system (EMS) have been investigated with several laccase mediators, including N-hydroxyphthalimide (NHPI), 1-hydroxybenzotriazole (HBT), violuric acid (VLA), 2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPO), and 2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonate) (ABTS), under the same reaction conditions. In the bulk electrolyses of the monomeric model compound [1-(4′-ethoxy-3′-methoxyphenyl)ethanol (1G)], oxidation with NHPI gave the corresponding CαO product (2G) in high yield, whereas the oxidations with HBT, VLA, and TEMPO afforded 2G in moderate yields. The highest reaction selectivity for the guaiacyl-units was found in the oxidation conducted in the presence of ABTS, although the yield was low. In the bulk electrolyses of the dimeric model compound [4-ethoxy-3-methoxyphenylglycerol-β-guaiacyl ether (3G)], the oxidation with NHPI gave the corresponding CαO product (4G) in high yield, whereas the oxidations with HBT, VLA, and TEMPO gave 4G in low yields. In contrast, the oxidation with ABTS gave a Cα–Cβ cleavage product (5G) in 5.5% yield. The selectivity of the mediators in the EMS reaction effectively reflected the mechanisms of their reactions, as reported for the laccase mediator system. NHPI was confirmed to be the best mediator in the present system for the selective Cα-carbonylation of the non-phenolic β-O-4 structures in lignin.

Dye Degradation by Layer‐by‐Layer Immobilised Peroxidase/Redox Mediator Systems

AbstractHorseradish peroxidase (HRP) was immobilised on Eupergit C 250 L resin coated with poly‐electrolytes, or by entrapment inside pre‐formed layer‐by‐layer (LbL) micro‐capsules of poly‐electrolytes. In these systems, namely HRP/E‐LbL, HRPm/LbL and HRPm/LbLp, the native enzyme retained its catalytic activity. Immobilised HRP showed a significant activity in the oxidation of selected azo, quinoline and fluorone dyes with H2O2 as the primary oxidant under mild experimental conditions, and HRPm/LbL was the best catalyst. A comparison between the catalytic efficiency of different redox mediators for HRP activity was made by using 1‐hydroxybenzotriazole (HOBt), violuric acid (VLA) and veratrylic alcohol (VA). As a general trend, azo dyes were degraded in higher yields, and HOBt was the best mediator for the oxidation. The degradation yield increased on increasing the reaction time and reached the highest value after 12 h, which is comparable with that observed for native HRP. Notably, HRPm/LbL retained its catalytic activity for more runs.

Thermo-molded biocomposite from cassava starch, natural fibers and lignin associated by laccase-mediator system

Treatment of cellulose fibers and lignin by laccase-mediator system was conducted to enhance the binding efficiency of natural fibers and lignin compounds into cassava starch composite matrix. Violuric acid (VA) was tested for its effect as a mediator for laccase treatment. Influence of different fiber, lignin and water contents of biocomposite was statistically investigated. The results showed that adding 15% (w/w) fibers into biocomposite at 44% (w/w) water content increased flexural strength and modulus for 4 times compared with the control. A combination of fibers+VA gave the greatest enhancement of modulus for 1140% and flexural strength for 375.8% as much as neat starch biocomposite. The presence of fibers, lignin and VA as mediator for laccase treatment substantially enhanced water resistance of starch biocomposite detected by a change in water drop contact angle on biocomposite surface.