Characterization of a cellulolytic enzyme from wood degrading bacteria, Bacillus circulans

This report describes the purification and characterization of an enzyme that exhibits cellulase activity produced by the wood degrading bacteria, Bacillus circulans. The enzyme was purified by ion-exchange chromatography using CM-Sepharose CL-6B, and shown to exhibit hydrolytic activity on carboxymethylcellulose. The molecular weight of the purified enzyme was determined to be 43 KDa by means of SDS-PAGE. The kinetic parameters, and the effects of pH and temperature on the purified enzyme were determined. The enzyme was 4.37 fold and showed a specific activity of 29.13 μg of glucose produced/min/mg protein. The apparent Km value for the hydrolysis of carboxymethylcellulose was 1.061 ± 1.17 mg/ml with a Vmax of 13.75 ± 1.51 μg of glucose produced/ml/min. The enzyme showed an optimum pH value of 9.0 and the optimum temperature was 50 °C. Alkalophilicity and moderate thermostability of this enzyme are some of its essential characteristics that may make it suitable for industrial and biotechnological applications.Keywords: Bacillus circulans, cellulase, decayed wood, optimum, bacteria, enzym

Isolation and kinetic properties of arginase in the gut of grasshopper (Zonocerus variegatus Linn)

Arginase (EC 3.5.3.1) catalyzes the hydrolysis of arginine to ornithine and urea. Arginase was purified and characterized from the gut of Zonocerus variegatus through DEAE-cellulose and biogel-P100 gel filtration chromatography. The specific activity of the enzyme was 3.7 μmoles/min  per mg of protein and a yield of 14.7%. An apparent molecular weight of 143,000 daltons was estimated by gel filtration on biogel P-100. The Michaelis constant (Km) of the enzyme was 40 mM with arginine as substrate. The optimum pH was 8.0 and the optimum temperature was 40 oC for Z. variegatus arginase. The enzyme was stable up to 40 oC for 20 min and lost all of its activity at 80 oC. The enzyme was specific for arginine as substrate. The enzyme was strongly enhanced in the presence of Mn2+, Na+, NH4 + and Hg2+ showed similar activation. Ni2+ and Zn2+ slightly inhibited Z. variegatus. Chelating (EDTA, citrate, ascorbic acid and urea) and thio (2-mercaptoethanol and cystein) compounds inhibited the activity of arginase in Z. variegatus. While amino acids (proline, lysine, aspartate and valine) showed no inhibition on arginase activity. The presence of arginase in the gut of Zonocerus variegatus could be for other functions rather than urea production in urea cycle. © 2013 International Formulae Group. All rights reserved. Keywords: Arginase; Zonocerus variegatus; Distribution; Physicochemical Properties

Properties of Arginase from the Hepatopancreas of Giant Freshwater Prawn (Macrobrachium rosenbergii, de Man).

We describe the hepatopancreas arginase activity of freshwater prawn (Macrobrachium rosenbergii). The enzyme was isolated using reactive blue 2- agarose affinity chromatography and gel filtration on Sephadex G-150. The enzyme had a specific activity of 5.70 μmol/min/mg of protein. The enzyme exhibited a maximal activity at pH 8.5 and Km of 12.5 mM. The enzyme was capable of hydrolysing L-arginine and to a lesser extent, L-arginine monohydrochlorate and L-arginine monohydrate. The optimum temperature of the enzyme was 35 0C. The molecular weight as determined by gel filtration was approximately 160,000 dalton and SDS-PAGE, was 22,000 dalton. The different amino acids (L-lysine, L-cysteine, Lvaline, L-proline, L-aspartic acid, L-glutamic acid and L-serine) and metal ions (Ni2+, Co2+, Zn2+, Mn2+ and Mg2+) did not show any inhibition on the enzyme activity. The enzyme was activated with Mn2+ and different concentration of Mn2+ had no effect on the enzyme activity. EDTA, citrate and urea showed considerable inhibition on the enzyme activity.Key words: Freshwater prawn; arginase; uricotelism; invertebrates; hepatopancrea

Inhibition and Kinetic Studies of Tortoise (Kinixys erosa) Liver arginase

The effect of amino acid on tortoise liver arginase showed that L-lysine, L-valine, L-serine, L-aspartic acid and L aspartic acid had significant inhibitory effect on the enzyme but proline and glutamic acid showed slight inhibition. Ethylenediaminetetraacetic acid (EDTA), citrate, ascorbic acid, boric acid and sodium borate completely inactivated the tortoise liver arginase. Inhibition studies on the enzyme with a number of cations showed decreased arginase activity. While Mn2+ satisfies the metal ion requirement of tortoise arginase, Sn2+, Hg2+, Ba2+ and Co2+, to a lesser extent inhibited the enzyme. The enzyme was markedly sensitive to inhibition by Zn2+, Ni2+ and Mg2+. The enzyme was also completely inactivated by thiol compounds: reduced glutathione (GSH), cysteine and 2-mercaptoethanol