Functional Characterization of Melanin Decolorizing Extracellular Peroxidase of Bjerkandera adusta

Melanin pigmentation in the human skin results from complicated cellular mechanisms that remain to be entirely understood. Uneven melanin pigmentation has been counteracted by inhibiting synthesis or transfer of melanin in the skin. Recently, an enzymatic approach has been proposed, wherein the melanin in the skin is decolorized using lignin peroxidase. However, not many enzymes are available for decolorizing melanin; the most studied one is lignin peroxidase derived from a lignin degrading fungus, Phanerochaete chrysosporium. Our current study reveals that versatile peroxidase from Bjerkandera adusta can decolorize synthetic melanin. Melanin decolorization was found to be dependent on veratryl alcohol and hydrogen peroxide, but not on Mn2+. The degree of decolorization reached over 40% in 10 min at 37 °C and a pH of 4.5. Optimized storage conditions were slightly different from those for the reaction; crude enzyme preparation was the most stable at 25 °C at pH 5.5. Since the enzyme rapidly lost its activity at 50 °C, stabilizers were screened. As a result, glycerol, a major component in several cosmetic formulations, was found to be a promising excipient. Our results suggest that B. adusta versatile peroxidase can be considered for future cosmetic applications aimed at melanin decolorization.

PURIFICATION AND CHARACTERIZATION OFESTERASE ENZYME ISOLATED FROM CONTAMINATED SOIL SAMPLE

Isolation and partial purification of esterase from contaminated soil sample was the main aim of this study. The production medium for organism was optimized by using different pH,Temperature,Carbon and Nitrogen sources.The esterase enzyme was highly active and stable from pH 5.0 to 9.0 with an optimum at pH 9.Its optimum temperature was 35°C. The best carbon and nitrogen sources were mannitol and yeast extract. Esterase was partially purified by ammonium sulphate precipitation,dialysis.The specific activity of partially purified esterase obtained from ammonium sulphate fractionation is found to be 8.6485U/mg and the fractionation is 5.4 fold purified from the crude enzyme preparation yielding 17.5U/mg from the crude protein. This result showed that Bacillus subtilis under study is a good producer of extra cellular esterase,which can be beneficial for industries

Bioproduction of Quercetin and Rutinose Catalyzed by Rutinosidase: Novel Concept of “Solid State Biocatalysis”

Quercetin is a flavonoid largely employed as a phytochemical remedy and a food or dietary supplement. We present here a novel biocatalytic methodology for the preparation of quercetin from plant-derived rutin, with both substrate and product being in mostly an undissolved state during biotransformation. This “solid-state” enzymatic conversion uses a crude enzyme preparation of recombinant rutinosidase from Aspergillus niger yielding quercetin, which precipitates from virtually insoluble rutin. The process is easily scalable and exhibits an extremely high space-time yield. The procedure has been shown to be robust and was successfully tested with rutin concentrations of up to 300 g/L (ca 0.5 M) at various scales. Using this procedure, pure quercetin is easily obtained by mere filtration of the reaction mixture, followed by washing and drying of the filter cake. Neither co-solvents nor toxic chemicals are used, thus the process can be considered environmentally friendly and the product of “bio-quality.” Moreover, rare disaccharide rutinose is obtained from the filtrate at a preparatory scale as a valuable side product. These results demonstrate for the first time the efficiency of the “Solid-State-Catalysis” concept, which is applicable virtually for any biotransformation involving substrates and products of low water solubility.

A β-mannanase from Fusarium oxysporum SS-25 via solid state fermentation on brewer spent grain: Medium optimization by statistical tools, kinetic characterization and its applications

This study concerned with the optimization of fermentation parameters for the hyper production of mannanase from Fusarium oxysporum SS-25 employing two step statistical strategy and kinetic characterization of crude enzyme preparation. The Plackett-Burman design was first used to screen out the important factors in the culture medium which were found to be: 20% (w/w) wheat bran, 2% (w/w) each of potato peels, soybean meal, malt extract, 1% tryptone, 0.14% NH4SO4, 0.2% KH2PO4, 0.0002% ZnSO4, 0.0005% FeSO4, 0.01% MnSO4, 0.012% SDS, 0.03% NH4Cl, 0.1% NaNO3 in brewer’s spent grain based medium with 50% moisture content, inoculated with 2.8×107 spores and incubated at 30oC for 6 days. Out of twenty seven factors, four variables including soybean meal, FeSO4, MnSO4 and NaNO3 were selected to study the interactive effects and optimum level of these variables in central composite design of response surface methodology. The final mannanase yield was 193 IU/g which was active at broader temperature and pH range and could result in 26.6% reduction in kappa number with 4.93% higher tear index and 1% increase in brightness when used to treat the wheat straw based kraft pulp. The hydrolytic potential of enzyme was demonstrated on both locust bean gum and guar gum.

A β-mannanase from Fusarium oxysporum SS-25 via solid state fermentation on brewer spent grain: Medium optimization by statistical tools, kinetic characterization and its applications

This study concerned with the optimization of fermentation parameters for the hyper production of mannanase from Fusarium oxysporum SS-25 employing two step statistical strategy and kinetic characterization of crude enzyme preparation. The Plackett-Burman design was first used to screen out the important factors in the culture medium which were found to be: 20% (w/w) wheat bran, 2% (w/w) each of potato peels, soybean meal, malt extract, 1% tryptone, 0.14% NH4SO4, 0.2% KH2PO4, 0.0002% ZnSO4, 0.0005% FeSO4, 0.01% MnSO4, 0.012% SDS, 0.03% NH4Cl, 0.1% NaNO3 in brewer’s spent grain based medium with 50% moisture content, inoculated with 2.8×107 spores and incubated at 30oC for 6 days. Out of twenty seven factors, four variables including soybean meal, FeSO4, MnSO4 and NaNO3 were selected to study the interactive effects and optimum level of these variables in central composite design of response surface methodology. The final mannanase yield was 193 IU/g which was active at broader temperature and pH range and could result in 26.6% reduction in kappa number with 4.93% higher tear index and 1% increase in brightness when used to treat the wheat straw based kraft pulp. The hydrolytic potential of enzyme was demonstrated on both locust bean gum and guar gum.

Production and Biochemical Characterization of a High Maltotetraose (G4) Producing Amylase fromPseudomonas stutzeriAS22

Amylase production and biochemical characterization of the crude enzyme preparation fromPseudomonas stutzeriAS22 were evaluated. The highestα-amylase production was achieved after 24 hours of incubation in a culture medium containing 10 g/L potato starch and 5 g/L yeast extract, with initial pH 8.0 at 30°C under continuous agitation at 200 rpm. The optimum temperature and pH for the crudeα-amylase activity were 60°C and 8.0, respectively. The effect of different salts was evaluated and it was found that bothα-amylase production and activity were Ca2+-dependent. The amylolytic preparation was found to catalyze exceptionally the formation of very high levels of maltotetraose from starch (98%, w/w) in the complete absence of glucose since the initial stages of starch hydrolysis (15 min) and hence would have a potential application in the manufacturing of maltotetraose syrups.

Factor influencing the production and properties of extracellur protease from Streptomyces bikiniensis

The actinomycete Streptomyces bikiniensis collected from laboratory stock culture, Department of Microbiology, University of Chittagong was found to be a protease producer. Effect of various culture conditions for the production of protease from S. bikiniensis and its properties were determined in the present study. The organism showed maximum protease yield at temperature 37°C with an initial culture, pH 7.0 after 4 days of incubation period under stationary condition. The crude enzyme preparation exhibited highest activity at substrate (casein) concentration of 1.0% with a reaction time of 75 min, at 40°C and pH 6.0. EDTA was found to a potent inhibitor of protease of the isolate while, 1,2- epoxy-3-(P-nitrophenoxy) propane exhibited low inhibitory effect on the enzyme activity. The enzyme was partially purified by precipitation method using ammonium sulfate fractionation and the maximum amount of the enzyme was precipitated at 60% saturation. DOI: http://dx.doi.org/10.3329/cujbs.v4i1.13399 The Chittagong Univ. J. B. Sci.,Vol. 4(1&2):153-163, 2009

Characterization of a NewProvidenciasp. Strain X1 Producing Multiple Xylanases on Wheat Bran

Providenciasp. strain X1 showing the highest xylanase activity among six bacterial isolates was isolated from saw-dust decomposing site. Strain X1 produced cellulase-free extracellular xylanase, which was higher in wheat bran medium than in xylan medium, when cultivated at pH 8.0 and 35°C. Zymogram analysis of crude preparation of enzymes obtained while growing on wheat bran and birchwood xylan revealed the presence of seven and two distinct xylanases with estimated molecular weight of 33; 35; 40; 48; 60; 75; and 95 kDa and 33 and 44 kDa, respectively. The crude xylanases were produced on wheat bran medium and showed optimum activity at pH 9.0 and 60°C. The thermotolerance studies showed activity retention of 100% and 85% at 40°C and 60°C after 30 min preincubation at pH 9.0. It was tolerant to lignin, ferulic acid, syringic acid, and guaiacol and retained 90% activity after ethanol treatment. The enzyme preparation was also tolerant to methanol and acetone and showed good activity retention in the presence of metal ions such as Fe2+, Mg2+, Zn2+, and Ca2+. The crude enzyme preparation was classified as endoxylanase based on the product pattern of xylan hydrolysis. Pretreatment of kraft pulp with crude xylanases for 3 h at 60°C led to a decrease in kappa number by 28.5%. The properties of present xylanases make them potentially useful for industrial applications.