The aim of this study was to characterize the organic solvent and detergent tolerant properties of recombinant lipase isolated from thermotolerant Bacillus sp. RN2 (Lip-SBRN2). The isolation of the lipase-coding gene was achieved by the use of inverse and direct PCR. The complete DNA sequencing of the gene revealed that the lip-SBRN2 gene contains 576 nucleotides which corresponded to 192 deduced amino acids. The purified enzyme was homogeneous with the estimated molecular mass of 19 kDa as determined by SDS-PAGE and gel filtration. The Lip-SBRN2 was stable in a pH range of 9–11 and temperature range of 45–60 °C. The enzyme was a non metallo-monomeric protein and was active against pNP-caprylate (C8) and pNP-laurate (C12) and coconut oil. The Lip-SBRN2 exhibited a high level of activity in the presence of 108% benzene, 102.4% diethylether and 112% SDS. It is anticipated that the organic solvent and detergent tolerant enzyme secreted by Bacillus sp. RN2 will be applicable as catalysts for reaction in the presence of organic solvents and detergents.

ABSTRACT:Gene encoding for endo-1,4-β-mannanase (EC 3.2.1.78) from Bacillus licheniformis THCM 3.1 was cloned and over-expressed in pET 100/D TOPO vector. The molecular weight of the purified enzyme was about 40 kDa. This enzyme had an optimum pH of 9 and an optimum temperature of 45°C and retained up to 77% of its activity after incubation for 48 h. The activity of the enzyme was inhibited by 10 mM of Pb 2+ , Ag + , Fe 3+ , Sn 2+ , Cu 2+ , and EDTA. Although partially inhibited, the enzyme retained much of its activity when the reaction solution was mixed with 15% (v/v) of the organic solvents acetone, toluene, benzene, dimethyl sulphoxide, 2-propanol, acetonitrile, or cyclohexane.

Thermotolerant bacteria isolated from soil and water samples taken from 76 hot springs in Thailand were investigated for their biosurfactant, biocatalytic, and antimicrobial properties. DNA samples purified from 148 pure isolates were PCR amplified using primers specific for the 16S rDNA hypervariable region of the genus Bacillus.

A xylan-degrading Streptomyces sp. THW31 was isolated from rubbish compost in Thailand. Analysis of a genomic library of nucleotide sequences from Streptomyces sp. THW31 revealed that the complete open reading frame (ORF) of xylanase (xlnW31) was 999 bp, and this gene encoded a member of the glycosyl hydrolase family 11. Sequence homology of the predicted amino acid sequence encoded by xlnW31 demonstrated that the enzyme consists of a signal peptide, catalytic and substrate-binding domains. The XlnW31 enzyme shared the highest identity (90%) to a xylanase family 11 member from Streptomyces lividans TK24. Cloning and expression of xlnW31 in Escherichia coli resulted in the production and purification of a 31.0 kDa enzyme. Purified XlnW31 show the highest activity at pH 6.0 and at a temperature 65 to 70°C. Enzyme stability tests indicated that XlnW31 retained its activity over a broad pH range (5.0 to 11.0) and at temperatures reaching 60°C for 2 h. Purified XlnW31 also exhibited endo-1,4--xylanase activity using xylan as a substrate and bound to insoluble xylan. Hydrolysis of xylan by the xylanase yielded xylobiose as the principal product.