Research paperBiochemical and molecular characterization of a detergent-stable serine alkaline protease from Bacillus pumilus CBS with high catalytic efficiency
Introduction
Microbial proteases make approximately 40% of the total worldwide production of enzymes [1]. Subtilisins (EC 3.4.21.14) are some of the largest selling proteases. Interestingly, bacteria, belonging to the genus Bacillus, produced the most commercial proteases used today [2], [3]. Bacillus licheniformis, Bacillus subtilis and Bacillus pumilus are the most known species used in industry for alkaline protease production [4]. The performance of alkaline protease in detergent is influenced by several factors such as pH and temperature of washing solution as well as detergent composition. Ideally, proteases used in detergent formulations should have high activity and stability within a broad range of pH and temperatures, and should also be compatible with various detergent components along with oxidizing and sequestering agents [5].
The most available commercial detergent proteases are Subtilisin Carlsberg (SC), Subtilisin Novo (Bacterial Protease Nagase BPN′), Alcalase™, Esperase™ and Savinase™ (SB 309), where the last four are from Novozymes A/S (Denmark) [6]. These enzymes were reported to be stable at conditions of elevated temperatures and pH. However, the most of these enzymes are relatively unstable in the presence of non-ionic surfactants (Tween-80), anionic surfactants (SDS) and peroxide agents (H2O2), which are the common ingredients in modern bleach-based detergent formulations [6], [7]. Therefore, various attempts have been made to enhance stability of alkaline proteases by site-directed mutagenesis [8] and protein engineering [9]. For example, Subtilisin Carlsberg has been engineered in order to obtain a bleach-stable alkaline protease [9].
Isolation and screening of micro-organisms from naturally occurring alkaline habitats or from alkaline wastewater are expected to provide new strains producing active and stable enzymes in highly alkaline conditions and resisting chemical denaturant agents present in detergents. In addition, studies of this kind of biocatalyst at molecular level will allow valuable information for further understanding their properties. Few published reports are available on the compatibility of the alkaline proteases from high yielding Bacillus genus, with detergent ingredients [10], [11]. Those already reported are from Bacillus pumilus species concerned with some of the following applications: alkaline protease used in the inactivation of RNase during RNA purification from cell homogenates [12], the coagulation of soybean milk [13], [14], the cleaning of ultrafiltration membranes [15], the dehairing of leather [16] and recently the production of zein hydrolysates [17]. Patent literature showed a number of Subtilisins from Bacillus pumilus species to be appropriate for detergents. In fact, Damodaran and Han [18] described an interesting detergent-stable alkaline protease (termed Protease Q) from Bacillus pumilus strain ATCC 202073. Vetter et al. [19] demonstrated a suitable use of two alkaline proteases, named Protease P46 and Protease P415, from Bacillus pumilus DSM 5777, in composition for cleaning and washing purposes. Recently, Merkel et al. [20] reported a novel Subtilisin-type alkaline protease from Bacillus pumilus, with potential for use in washing and cleaning agents. Even though a relatively high number of Bacillus pumilus proteases have already been reported for detergent, few of these have a high optimum pH (>10) and thermoactivity/thermostability (>65 °C) as well as an important compatibility and stability against oxidants, bleaches and denaturing agents.
During a screening process on protease-producing strains, Bacillus pumilus CBS strain producing a thermostable serine alkaline protease (termed SAPB) was previously reported as a good candidate for biotechnological and industrial applications [64]. In this paper we report the purification and the biochemical characterization of a thermoactive serine alkaline protease with high catalytic efficiency from Bacillus pumilus CBS. We also report gene cloning, amino acid sequence inspection, and heterologous expression of SAPB in Escherichia coli DH5α.
Section snippets
Bacterial strains, media and plasmids
The alkaline protease from Bacillus pumilus CBS strain was produced at pH 10.0 using the medium M [composed of (g/L): gelatin, 10; yeast extract, 5; CaCl2, 1; K2HPO4, 1; KH2PO4, 1; and oligoelements 1‰ (v/v) which was composed of (g/L): ZnCl2, 0.4; FeSO4 · 7H2O, 2; H3BO3, 0.065; and MoNa2O4 · 2H2O, 0.135]. The culture conditions were as described previously (Jaouadi et al., submitted for publication). Escherichia coli DH5α (F− supE44 Φ80 δlacZ ΔM15 Δ(lacZYA-argF) U169 endA1 recA1 hsdR17 (rk−, mk+)
Protease purification
Supernatant obtained by centrifugation of the Bacillus pumilus CBS culture broth (500 ml) was used as the crude enzyme solution. The SAPB was precipitated between 40% and 60% ammonium sulphate saturation. The precipitate formed was collected by centrifugation (15,000 × g, 20 min at 4 °C), dissolved in a minimum amount of buffer A and then dialyzed overnight against repeated changes of the same buffer. Purification to homogeneity was achieved by HPLC using a Shodex Protein WK 802-5 column. The
Conclusion
It is very important to obtain enzymes with high stability and activity in the presence of oxidants, surfactants, bleaching and denaturant agents, even at high washing temperatures and pH values for their application in industrial purposes. Considering this fact, we report here the biochemical characterization of a detergent-stable serine alkaline protease named SAPB, which has a very important catalytic efficiency. The purification of SAPB was achieved to homogeneity after two-steps: ammonium
Acknowledgements
The authors are very thankful for the financial support provided by the Tunisian Government “Contrat Programme CBS-LEMP/Code: RL02CBS01” (Centre de Biotechnologie de Sfax-Laboratoire d'Enzymes et de Métabolites des Procaryotes). This work is part of a Doctoral Thesis by Bassem Jaouadi. We are very grateful to Dr E. Ben Messaoud, Dr M.A. Borgi and Mr B. Naili (LEMP/CBS) and Dr A. Aloulou (Gelb Research Group, USA) for their invaluable help and constructive discussions during the preparation of
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2021, Process BiochemistryCitation Excerpt :The proteins were visualized by Coomassie brilliant blue R-250 staining. Alternatively, a zymography technique was performed by incorporating 1 mg/mL azo-casein into the separating gel [22]. The specific rSAPNs bands were sliced and transferred to a polyvinylidene difluoride blotting membrane, and their N-terminal residues were identified by Edman degradation with an ABI Automated Protein Sequencer (Model 473A).