Isolation and Characterisation of a Bacteria with Cellulase Activity from Local Hot Springs.
Zvidzai, Cuthbert Johnson
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A sporulating, aerobic Bacillus subtilis sp. was isolated from the Chimanimani hot spring in Zimbabwe. The microorganism was characterised using classical identification tools and molecular techniques. The microbial strain was designated Bacillus subtilis CHZ1. It produced an endoglucanase enzyme when cultured on M162 medium with an initial value of pH within the range 5.0 – 9.0 and a temperature range between 30ºC and 50ºC. The optimal conditions for the production of the endoglucanase enzyme were a pH value of 6.0 and temperature of 50ºC. The enzyme was produced constitutively when the microorganism was cultured in M162 medium supplemented with either starch, cellobiose, carboxymethylcellulose, sucrose, glucose, galactose, Avicel, lactose, mannose or maltose as carbon sources. The Bacillus subtilis CHZ1 was found to produce hydrolytic extracellular enzymes when cultured on a medium extracted from opaque beer brewery wastewater supplemented with defatted soya, spent yeast and malt flour. The medium from the food waste materials was named CWW medium. Elimination of one raw material component during CWW medium preparation revealed that all the components were essential for maximum enzyme production. The absence of spent yeast during CWW medium preparation greatly reduced the levels of enzyme production. In addition, the microorganism produced endoglucanase, amylase, polygalacturonase, xylanase and protease when cultured on CWW medium. All assayed enzymes reached high levels within 5 – 10 h during fermentation at 40ºC and pH maintained at 6.0 in fermentor experiments. A cultivation temperature of 50ºC resulted in a rapid drop in all the enzyme levels after 12 h of cultivation. High biomass and enzyme levels were obtained in fermentor cultivations compared to shake flask experiments. This showed that food wastes could be used to produce hydrolytic enzymes from B. subtilis CHZ1. A proteolytic enzyme produced by a B. subtilis CHZ1 was purified using ammonium sulphate precipitation, gel filtration and cationic exchange on S-Sepharose fast flow column chromatography. Production of the protease enzyme was higher when the Bacillus strain was cultured in a synthetic medium, M162, supplemented with 0.3 % (w/v) organic compared to inorganic nitrogen sources. Enzyme production was growth associated and its production was highest when tryptone was used as the nitrogen source. When run on SDS-PAGE gel, the purified enzyme gave a 35 kDa band, suggesting that it consisted of a single polypeptide chain. High protease enzyme activity was observed in the pH range of 6 – 10 with a maximum value at pH 8.0 when 0.5 % (w/v) azocasein was used as the substrate for activity assay. Maximum temperature for protease activity was found to be 50ºC, and the enzyme had considerable thermal stability for 5½ h retaining about 90 % residual activity at this temperature. At 2.5 mM concentration, PMSF, Ag+ and Hg+ caused marked reduction in activity on the protease enzyme. Natural biological metal cofactors that include Mn2+, Mg2+ and Fe2+ increased the enzyme activity, while Zn2+, Cu2+ and Ca2+ did not affect the enzyme's activity. The protease pH and thermal stability as well as high activity expressed by small amounts of the enzyme can be exploited for industrial applications. The endoglucanase gene (celG) from B. subtilis CHZ1 was amplified by the polymerase chain reaction, cloned and expressed in Escherichia coli DH5a. The amplified fragment with the full celG structural gene was 2 589 bp and its nucleotide sequence was determined and analysed. The amplified fragment had an open reading frame of 1 524 bp encoding a predicted protein of 56 472 daltons in size containing 508 amino acids. The most likely ribosomal binding site resembling that of B. subtilis s43 RNA polymerase was identified upstream of the highly likely initiation start codon. The gene contains a secretory signal sequence encoding a peptide of 38 amino acid residues. A BLASTN search using the 2 589 bp sequence showed 98 % identity to endo-b-1,4-glucanase genes in the databases. Its predicted protein structure was that of an endoglucanase comprising a catalytic domain (CD) of 278 amino acids linked to a cellulose-binding domain (CBD-3) with 83 residues. The enzyme was identified as belonging to the family-5A glycosyl hydrolases. Amino acid alignment of the predicted protein with the aid of SWISSPro computer program gave high homology. The regular seven conserved regions of the endoglucanase enzymes were identified on Cel5G. The celG gene was overexpressed in E. coli DH5a and could be induced 10 fold with 1.0 mM IPTG. The cloned endoglucanase enzyme optimal activity conditions were the same as those of the wild type strain. It was however observed that about 70 % of the endoglucanase activity from the clone was cell bound. Site directed mutagenesis at position 1168 of the celG endoglucanase enzyme resulted in the production of an inactive enzyme.