Bacillus pumilus S124A carboxymethyl cellulase; a thermo stable enzyme with a wide substrate spectrum utility

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Abstract

Bacillus pumilus S124A was identified as carboxymethyl cellulase producing bacteria from Azorean Bacillus collection (Lab collection), which was isolated in local soils. The bacterium was identified by 16S rRNA sequence and designated as Bacillus pumilus S124A. NCBI-blast analysis showed B. pumilus S124A; 16S rRNA sequence has high identity to other B. pumilus strains. Phylogenetic analysis showed B. pumilus S124A close to B. pumilus LZBP14 strain. CMcellulase was purified from cells-free supernatants and post mano-Q purification; 5.39% protein folds, and 0.88% recoveries were obtained. SDS-PAGE analysis showed molecular weight of the purified CMcellulase was estimated ∼40 kDa and composed of a single subunit. NonoLC ESI-MS/MS analysis was yielded four peptides, and protein has identity to other cellulases. Purified CMcellulase showed high activity to cellobiose followed by CMcellulose. Kinetic analysis showed Km, and Vmax were determined as 2.12 mg/ml, 239 μmol/min/mg, respectively. Optimum temperature and pH for the purified CMcellulase activity were found at 50 °C and pH 6.0, respectively. Purified CMcellulase was maintained about 75% activity in a pH range of 4–8 and 70% activity in a temperature range of 40–70 °C. CMcellulase activity was highly reduced by HgCl2, followed by EDTA, PMSF whereas CoCl2 was activated CMcellulase activity.

Introduction

Carboxymethyl cellulose is a cellulose derivative with carboxymethyl groups (–CH2–COOH) bound to some of the hydroxyl groups of the glucopyranose monomers that make up the cellulose backbone. The carboxymethyl cellulose structure is based on the β-(1-4)-d-glucopyranose polymer of cellulose, and it is often used as its sodium salt, sodium carboxymethyl cellulose [1]. Cellulases are comprised of independently folding, structurally and functionally discrete units called domains or modules, making cellulases modular structure [2].

The conversion of cellulose biomass by microorganisms is a sustainable approach to develop novel bio-processes and products [3]. Cellulose biomass is great potential resources for the production of bio-fuels because it is largely abundant, inexpensive and production of such resources is environmentally sound [2]. Agricultural residues are a considerable source of cellulose biomass, which is a renewable, chiefly unexploited, economic and also cellulose waste from industrial and agricultural processes [4]. Use of cellulases in bio-ethanol production from various lignocellulosic substrates has been reported by many authors recently [5], [6]. Among the group of cellulase enzymes, carboxymethyl cellulase has been used predominantly in the process of ethanol production [7], [8]. The bio-degradation of cellulose has been studied for many years, and a number of cellulolytic enzymes, especially cellulases produced by fungi and bacteria, have been characterized [9]. Commercial cellulases are mainly produced by fungi (Aspergillus and Trichoderma sp.); however, bacteria have also been considered as robust and versatile enzyme producers because of their high growth rate, stability at extreme conditions, and presence of multi enzyme complexes. Among these, Bacillus sp. has been revealed to be the most potent extracellular enzyme producer [10], [11]. The proteases from Bacillus sp. are used in various industries such as food, chemical, detergent, and leather industries [12], also can be benefited from the proteases from Bacillus pumilus [3].

Over the years, culturable, cellulase producing bacteria have been isolated for industrial use from a wide variety of sources such as composting heaps, decaying plant material from forestry or agricultural waste, the feces of ruminants such as cows, soil and organic matter, and extreme environments like hot-springs [13]. Bacteria produce enzymes that are stable under extreme conditions, which may be present in the bio-conversion process and this may increase rates of enzymatic hydrolysis, fermentation, and product recovery [2]. Cellulases produced by bacteria are often more effective catalysts, among them Bacillus species can produce numbers of extracellular polysaccharide hydrolyzing enzymes [14]. Isolation and characterization of cellulose producing bacteria will continue to be an important aspect of bio-fuel and bio-product research.

Different bacterial cellulase proteins were reported in Bacillus amyoliquefaciens DL-3 [10], Bacillus sp. D04 [15], Bacillus brevis VS-1 [16], B. pumilus EB3 [17], Bacillus subtilis YJ1 [18], Bacillus strains CH43 and HR68 [19], B. pumilus [20], Bacillus sphaericus JS1 [21], Bacillus sp. [22], B. subtilis strain LFS3 [23], B. subtilis strain I15 [24] and B. subtilis [25]. In this present study, a bacterium hydrolyzing carboxymethyl cellulose was identified from Azorean Bacillus collection and designate as B. pumilus S124A. Characterization of purified carboxymethyl cellulase from B. pumilus not yet well described. In this study, we describe about 40 kDa protein purification and biochemical characterization of thermo stable with wide substrate utility, including agricultural waste degrading carboxymethyl cellulase produced by B. pumilus S124A for the utilization of abundant cellulose biomass.

Section snippets

Screening of carboxymethyl cellulase producing bacteria and strain conservation

Bacillus sp. was isolated from local soil (São Miguel) samples and established Azorean Bacillus collection (Lab collection). We screened 391 isolates from the Bacillus collection on carboxymethyl cellulose medium. The bacteria that showed cellulase activity were cryopreserved in 20% glycerol and stored at −80 °C. They were also preserved lyophilized in filter paper and conserved at room temperature on sealed glass vials [26].

Morphological identification

Bacteria isolate S124A was identified based on (i) Gram stains (ii)

Bacteria screening on carboxymethyl cellulose

Bacteria screening was performed on carboxymethyl cellulose agar using the Congo red assay for identifying cellulase producers. We screened 391 isolates, among them 78 isolates showed positive signs for carboxymethyl cellulase activity. The isolate S124A remarkably showed lysis on carboxymethyl cellulose and demonstrated more positive results than the other strain (Fig. 1a).

B. pumilus S124A morphology and 16S rRNA sequence analysis

Isolate S124A hydrolyzing carboxymethyl cellulose was assessed to be B. pumilus based on the 16S rRNA sequence. The B.

Discussion

Strain B. pumilus S124A has been shown cellulose hydrolyzing bacteria by Congo red screening from the Azorean Bacillus collection. Besides we identified few other bacteria that also can hydrolyze carboxymethyl cellulose, but the activity was less. Among the screened bacteria, isolate S124A showed strong signs of growth on carboxymethyl cellulose agar and exhibited more positive results in the Congo red test. Carboxymethyl cellulose was the sole carbon source in the medium; therefore, cellulase

Acknowledgements

This work was supported by Fundo Regional Science and Technology (FRCT), Azores, Portugal; N. Balasubramanian gratefully acknowledges for the post-doctoral grant (M3.1.7/F/009A/2009), and acknowledges IBBA, Azores far financial support (M2.1.2/I/025/2008-RTF/2). We also acknowledge to Dr. Carla Cabral, Mario Teixeira, Duarte Toubarro and Mafalda Raposa.

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