Paisarn Khawsak

Purification and Characterization of Organic Solvent and Detergent Tolerant Lipase from Thermotolerant Bacillus sp. RN2

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.

Multiplex RT-PCR assay for simultaneous detection of six viruses of penaeid shrimp

In the present study, multiplex reverse transcription-polymerase chain reaction (mRT-PCR) was developed for simultaneously detection of six major shrimp viruses including yellow-head virus (YHV), white spot syndrome virus (WSSV), Taura syndrome virus (TSV), hepatopancreatic parvovirus (HPV), infectious hypodermal and hematopoietic necrosis virus (IHHNV) and monodon baculovirus (MBV). The six primer sets could amplify viral nucleic acids resulting in PCR products with different sizes. They were highly specific and did not cross-hybridize with other viral or shrimp nucleic acids. The sensitivity of the multiplex RT-PCR was 0.15pg for IHHNV, 0.15pg for TSV, 1.00pg for HPV, 1.5pg for MBV, 5.00pg for WSSV and 10.00pg for YHV. In the field application, 42 samples including whole tissue of post-larvae and hepatopancreas of Penaeus monodon collected from ponds in the central and southern parts of Thailand during 2002-2005 were examined by multiplex RT-PCR. The results revealed that a single infection was dominant and WSSV was the highest prevalence at that time. Dual infection was found in one sample. This developed multiplex RT-PCR will be useful for simultaneous detection of six major viruses of penaeid shrimp and benefit to shrimp cultured industry.

Multiplex real-time PCR and high-resolution melting analysis for detection of white spot syndrome virus, yellow-head virus, and Penaeus monodon densovirus in penaeid shrimp

A multiplex real-time PCR and high-resolution melting (HRM) analysis was developed to detect simultaneously three of the major viruses of penaeid shrimp including white spot syndrome virus (WSSV), yellow-head virus (YHV), and Penaeus monodon densovirus (PmDNV). Plasmids containing DNA/cDNA fragments of WSSV and YHV, and genomic DNAs of PmDNV and normal shrimp were used to test sensitivity of the procedure. Without the need of any probe, the products were identified by HRM analysis after real-time PCR amplification using three sets of viral specific primers. The results showed DNA melting curves that were specific for individual virus. No positive result was detected with nucleic acids from shrimp, Penaeus monodon nucleopolyhedrovirus (PemoNPV), Penaeus stylirostris densovirus (PstDNV), or Taura syndrome virus (TSV). The detection limit for PmDNV, YHV and WSSV DNAs were 40fg, 50fg, and 500fg, respectively, which was 10 times more sensitive than multiplex real-time PCR analyzed by agarose gel electrophoresis. In viral nucleic acid mixtures, HRM analysis clearly identified each virus in dual and triple infection. To test the capability to use this method in field, forty-one of field samples were examined by HRM analysis in comparison with agarose gel electrophoresis. For HRM analysis, 11 (26.83%), 9 (21.95%), and 4 (9.76%) were infected with WSSV, PmDNV, and YHV, respectively. Agarose gel electrophoresis detected lesser number of PmDNV infection which may due to the limit of sensitivity. No multiple infection was found in these samples. This method provides a rapid, sensitive, specific, and simultaneous detection of three major viruses making it as a useful tool for diagnosis and epidemiological studies of these viruses in shrimp and carriers.

Cloning, Expression, and Characterization of Thermotolerant Manganese Superoxide Dismutase from Bacillus sp. MHS47

A superoxide dismutase gene from thermotolerant Bacillus sp. MHS47 (MnSOD47) was cloned, sequenced, and expressed. The gene has an open reading frame of 612 bp, corresponding to 203 deduced amino acids, with high homology to the amino acid sequences of B. thuringiensis (accession no. EEN01322), B. anthracis (accession no. NP_846724), B. cereus (accession no. ZP_04187911), B. weihenstephanensis (accession no. YP_001646918), and B. pseudomycoides. The conserved manganese-binding sites (H28, H83, D165, and H169) show that MnSOD47 has the specific characteristics of the manganese superoxide dismutase (MnSOD) enzymes. MnSOD47 expressed an enzyme with a molecular weight of approximately 22.65 kDa and a specific activity of 3537.75 U/mg. The enzyme is active in the pH range 7–8.5, with an optimum pH of 7.5, and at temperatures in the range 30–45 °C, with an optimum temperature of 37 °C. Tests of inhibitors and metal ions indicated that the enzyme activity is inhibited by sodium azide, but not by hydrogen peroxide or potassium cyanide. These data should benefit future studies of MnSODs in other microorganisms and the biotechnological production of MnSOD47, and could also be used to develop a biosensor for the detection of antioxidants and free radical activity. In the future, this basic knowledge could be applicable to the detection of cancer risks in humans and therapeutic treatments.

Isolation, cloning and molecular characterization of a thermotolerant xylanase from Streptomyces sp. THW31

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-β -xylanase activity using xylan as a substrate and bound to insoluble xylan. Hydrolysis of xylan by the xylanase yielded xylobiose as the principal product. Keywords : Gene expression, hemicellulase, Streptomyces , xylan, xylanase African Journal of Biotechnology Vol. 12(4), pp. 427-437