Expression and characterization of recombinant GH11 xylanase from thermotolerant Streptomyces sp. SWU10.

Xylans are major hemicellulose components of plant cell wall which can be hydrolyzed by xylanolytic enzymes. Three forms of endo-ß-1,4-xylanases (XynSW1, XynSW2A, and XynSW2B) produced by thermotolerant Streptomyces sp. SWU10 have been reported. In the present study, we described the expression and characterization of the fourth xylanase enzyme from this bacteria, termed XynSW3. The gene containing 726 bp was cloned and expressed in Escherichia coli. The recombinant enzyme (rXynSW3) was purified from cell-free extract to homogeneity using Ni-affinity column chromatography. The apparent molecular mass of rXynSW3 was 48 kDa. Amino acid sequence analysis revealed that it belonged to a xylanase of glycoside hydrolase family 11. The optimum pH and temperature for enzyme activity were 5.5-6.5 and 50 °C, respectively. The enzyme was stable up to 40 °C and in wide pH ranges (pH 0.6-10.3). Xylan without arabinosyl side chain is the most preferable substrate for the enzyme. By using birch wood xylan as substrate, rXynSW3 produced several oligosaccharides in the initial stage of hydrolysis, and their levels increased with time, demonstrating that the enzyme is an endo-acting enzyme. The major products were xylobiose, triose, and tetraose. The rXynSW3 can be applied in several industries such as food, textile, and biofuel industries, and waste treatment.

Purification, characterization of GH11 endo-ß-1,4-xylanase from thermotolerant Streptomyces sp. SWU10 and overexpression in Pichia pastoris KM71H.

We have previously described two forms of an endo-ß-1,4-xylanase (XynSW2A and XynSW2B) synthesized by thermotolerant Streptomyces sp. SWU10. Here, we describe another xylanolytic enzyme, designated XynSW1. The enzyme was purified to homogeneity from 2 L of culture filtrate. Its apparent molecular mass was 24 kDa. The optimal pH and temperature were pH 5.0 and 40 °C, respectively. The enzyme was stable in a wide pH ranges (pH 1-11), more than 80 % of initial activity remained at pH 2-11 after 16 h of incubation at 4 °C and stable up to 50 °C for 1 h. Xylobiose and xylotriose were the major xylooligosaccharides released from oat spelt xylan by the action of XynSW1, indicating of endo-type xylanase. The complete xynSW1 gene contains 1,011 bp in length and encode a polypeptide of 336 with 41 amino acids of signal peptide. The amino acid sequence analysis revealed that it belongs to glycoside hydrolase family 11 (GH11). The mature xynSW1 gene without signal peptide sequence was overexpressed in Pichia pastoris KM71H. The recombinant XynSW1 protein showed higher molecular mass due to the differences in glycosylation levels at the six N-glycosylation sites in the amino acid sequence and exhibited better physicochemical properties than those of the native enzyme including higher optimal temperature (60 °C), and specific activity, but lower optimal pH (4.0). Because of their stability in a wide pH ranges, both of native and recombinant enzymes of XynSW1, may have potential application in several industries including food, textile, biofuel, and also waste treatment.

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.

Purification, characterization, and overexpression of thermophilic pectate lyase of Bacillus sp. RN1 isolated from a hot spring in Thailand.

A thermophilic pectate lyase, Pel SWU, was isolated from a culture filtrate of Bacillus sp. RN1 isolated from a hot spring in Ranong Province, Thailand. The enzyme was purified to homogeneity using cation-exchange and hydrophobic column chromatographies. The molecular mass of Pel SWU was estimated to be 33 kDa. The specific substrate was demethylated galacturonic acid. The enzyme was stable at pH 4.0-10.0 and at temperatures up to 70 degrees C in the presence of calcium and polygalacturonic acid (PGA). The optimum pH and temperature were 10.0 and 90 degrees C. The pel gene encoding Pel SWU was 1,023 bp, which corresponds to 341 amino acids. The properties of the recombinant enzyme was similar to those of Bacillus Pel SWU. Unsaturated di- and trigalacturonic acids were formed mainly as the final products of degradation by Pel SWU, as revealed by high-performance anion-exchange chromatography (HPAEC) and electrospray ionization mass spectrometry (ESI-MS) analyses. This thermophilic pectate lyase should be useful in the degradation of pectin networks at high temperature.

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.