Characterization of novel extracellular protease produced by marine bacterial isolate from the Indian Ocean

Abstract

Out of the vast pool of enzymes, proteolytic enzymes from microorganisms are the most widely used in different industries such as detergent, food, peptide production etc. Several marine microorganisms are known to produce proteases with commercially desirable characteristics. We have isolated nine different cultures from marine samples of the Indian Ocean. All of them were i) motile ii) rod shaped iii) non spore forming iv) catalase and amylase positive v) able to grow in presence of 10 % NaCl. They produced acid from glucose, fructose and maltose and grew optimally at 30 °C temperature and pH 7.0-8.0. None of them could grow above 45 °C and below 15 °C. Only one of them (MBRI 7) exhibited extracellular protease activity on skim milk agar plates. Based on 16S rDNA sequencing, it belonged to the genus Marinobacter (98% sequence similarity, 1201 bp). The cell free extract was used to study effects of temperature and pH on protease activity. The optimum temperature and pH for activity were found to be 40 °C and 7.0 respectively. The crude enzyme was stable at temperature range of 30-80 °C and pH 5.0-9.0. It retained 60 % activity at 80 °C after 4 h and more than 70 % activity at 70 °C after 1 h. D value was found to be 342 minutes and 78 minutes for 40 °C and 80 °C respectively. Interestingly the enzyme remained 50 % active at pH 9.0 after 1 h. Comparison with other proteases from different microbial sources indicated that the neutral protease from the halotolerant marine isolate MBRI 7 is a novel enzyme with high thermostability.

Keywords: Halotolerant; mesophilic; neutral protease; thermostable.

Figure 1

Growth curve of an isolate MBRI 7 (present work). The cells were inoculated in skim milk broth, incubated at 30 °C, 120 rpm and growth was determined by measuring absorbance at 660 nm. Protease activity was estimated at different time intervals and maximum activity was observed after 16 h (♦ optical density, ■ % residual activity).

Figure 2

a) Effect of temperature on protease activity. The assay was carried out using crude culture supernatant at different temperatures ranging from 30 to 70 °C. b) Effect of pH on protease activity. The assay was carried out using crude culture supernatant at different pH in the range of 5.0–10.0.

Figure 2

a) Effect of temperature on protease activity. The assay was carried out using crude culture supernatant at different temperatures ranging from 30 to 70 °C. b) Effect of pH on protease activity. The assay was carried out using crude culture supernatant at different pH in the range of 5.0–10.0.

Figure 3

a) Kinetics of thermal inactivation of crude enzyme. Culture supernatant was exposed to different temperatures ranging from 30–80 °C. Enzyme activity was measured at time intervals of 30 minutes, 1 h, 2 h, and 4 h. b) Stability of crude enzyme at various pH. Culture supernatant was exposed to various pH ranging from 5.0–9.0. Enzyme activity was measured at time intervals of 30 minutes and 60 minutes.(100 % activity corresponds to 52 U mL^-1) (▓ - 0 minutes, □ - 30 minutes, ░ - 60 minutes).

Figure 3

a) Kinetics of thermal inactivation of crude enzyme. Culture supernatant was exposed to different temperatures ranging from 30–80 °C. Enzyme activity was measured at time intervals of 30 minutes, 1 h, 2 h, and 4 h. b) Stability of crude enzyme at various pH. Culture supernatant was exposed to various pH ranging from 5.0–9.0. Enzyme activity was measured at time intervals of 30 minutes and 60 minutes.(100 % activity corresponds to 52 U mL^-1) (▓ - 0 minutes, □ - 30 minutes, ░ - 60 minutes).