Elsevier

Biochimie

Volume 90, Issue 9, September 2008, Pages 1291-1305
Biochimie

Research paper
Biochemical and molecular characterization of a detergent-stable serine alkaline protease from Bacillus pumilus CBS with high catalytic efficiency

https://doi.org/10.1016/j.biochi.2008.03.004Get rights and content

Abstract

We have described previously the potential use of an alkaline protease from Bacillus pumilus CBS as an effective additive in laundry detergent formulations [B. Jaouadi, S. Ellouz-Chaabouni, M. Ben Ali, E. Ben Messaoud, B. Naili, A. Dhouib, S. Bejar, A novel alkaline protease from Bacillus pumilus CBS having a high compatibility with laundry detergent and a high feather-degrading activity, Process Biochem, submitted for publication]. Here, we purified this enzyme (named SAPB) and we cloned, sequenced and over-expressed the corresponding gene. The enzyme was purified to homogeneity using salt precipitation and gel filtration HPLC. The pure protease was found to be monomeric protein with a molecular mass of 34598.19 Da as determined by MALDI-TOF mass spectrometry. The NH2-terminal sequence of first 21 amino acids (aa) of the purified SAPB was AQTVPYGIPQIKAPAVHAQGY and was completely identical to proteases from other Bacillus pumilus species. This protease is strongly inhibited by PMSF and DFP, showing that it belongs to the serine proteases superfamily. Interestingly, the optimum pH is 10.6 while the optimum temperature was determined to be 65 °C. The enzyme was completely stable within a wide range of pH (7.0–10.6) and temperature (30–55 °C). One of the distinguishing properties is its catalytic efficiency (kcat/Km) calculated to be 45,265 min−1 mM−1 and 147,000 min−1 mM−1 using casein and AAPF as substrates, respectively, which is higher than that of Subtilisin Carlsberg, Subtilisin BPN′ and Subtilisin 309 determined under the same conditions. In addition, SAPB showed remarkable stability, for 24 h at 40 °C, in the presence of 5% Tween-80, 1% SDS, 15% urea and 10% H2O2, which comprise the common bleach-based detergent formulation. The sapB gene encoding SAPB was cloned, sequenced and over-expressed in Escherichia coli. The purified recombinant enzyme (rSAPB) has the same physicochemical and kinetic properties as the native one. SapB gene had an ORF of 1149 bp encoding a protein of 383 aa organized into a signal peptide (29 aa), a pro-protein (79 aa) and a mature enzyme (275 aa). The deduced amino acid sequence inspection displays an important homology with other bacterial proteases. The highest homology of 98.1% was found with BPP-A protease from Bacillus pumilus MS-1, with only 8 aa of difference.

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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