Purification and Characterization of a Novel Collagenase from Bacillus pumilus Col-J

Abstract

The collagenase, produced extracellular by Bacillus pumilus Col-J, was purified by ammonium sulfate precipitation followed by two gel filtrations, involving Sephadex G-100 column and Sepharose Fast Flow column. Purified collagenase has a 31.53-fold increase in specific activity of 87.33 U/mg and 7.00% recovery. The collagenase has a relative molecular weight of 58.64 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The optimal temperature for the enzyme reaction was 45 degrees C. More than 50% of the original activity still remained after 5 min of incubation at 70 degrees C or 10 min at 60 degrees C. The maximal enzyme activity of collagenase was obtained at pH 7.5, and it was stable over a pH range of 6.5-8.0. The collagenase activity was strongly inhibited by Mn(2+), Pb(2+), ethylenediamine tetraacetic acid, ethylene glycol tetraacetic acid, and beta-mercaptoethanol. However, Ca(2+) and Mg(2+) greatly increased its activity. The collagenase from B. pumilus Col-J showed highly specific activity towards the native collagen from calf skin. The K(m) and V(max) of the enzyme for collagen were 0.79 mg/mL and 129.5 U, respectively.

Production and Characterization of Collagenase by Penicillium sp. UCP 1286 Isolated From Caatinga Soil

“…The enzyme was stable between pH 6.0 and 10.0 during the first 8 h, but after 28 h its stability was restricted to a pH range from 7.0 (50.7%) to 9.0 (75.1%). In accordance with Wu et al [6], collagenase from B. pumilus is stable between pH 6.5 and 8, the enzyme retained above 84% of full activity after 30 min. By retaining more than 80% of its stability at pH 8 and 9, the enzyme described in the present work proved to be very stable in comparison with the collagenase produced by other microorganisms.…”

“…Comparing results of temperature assays, Wu et al [6] found for B. pumilus collagenase an optimum temperature of 45 °C, and with heating, the collagenase retained above 50% activity at 70 °C. Baehaki et al [35] assayed the collagenase activity between 30 and 90 °C, being observed an increase in enzymatic activity between 30 and 50 °C, with a strong reduction occurring above 60 °C.…”

“…The collagenase from Klebsiella pneumoniae had an optimum pH range from 6.0 to 8.0, with maximal activity at 7.0 in Tris-HCl buffer while Bacillus cereus collagenase activity was recorded as the highest in the pH range of 5.4-8.2 [2]. The collagenase from Bacillus pumilus [6] was evaluated in a range from 3 to 10 being the highest activity at pH 7.5. Baehaki et al [35] used a pH range between 2 and 12 and observed the maximum activity at pH 9.0.…”

“…Collagenases are specific enzymes that can hydrolyze both . native and denatured collagens [5,6]. These enzymes can degrade native triple helix of collagen to small fragments and play an important role in connective tissue metabolism [7,8].…”

“…These enzymes can degrade native triple helix of collagen to small fragments and play an important role in connective tissue metabolism [7,8]. Collagenolytic enzymes are a kind of proteases that are related to various physiological and pathological processes and have several applications in industry, medicine and biotechnology [6,[9][10][11]. With biotechnology accelerated growth, applications of proteases have expanded to new areas such as clinical, medicinal and analytical chemistry [12].…”