Purification, characterization, and overexpression of an endo-1,4-ß-mannanase from thermotolerant Bacillus sp. SWU60.

Endo-ß-1,4-mannanases are important catalytic agents in several industries. The enzymes randomly cleave the ß-1,4-linkage in the mannan backbone and release short ß-1,4-mannooligosaccharides and mannose. In the present study, mannanase (ManS2) from thermotolerant Bacillus sp. SWU60 was purified, characterized, and its gene was cloned and overexpressed in Escherichia coli. ManS2 was purified from culture filtrate (300 ml) by using hydrophobic, ion-exchange, and size-exclusive liquid chromatography. The apparent molecular mass was 38 kDa. Optimal pH and temperature for enzyme activity were 6.0 and 60 °C, respectively. The enzyme was stable up to 60 °C for 1 h and at pH 5-9 at 4 °C for 16 h. Its enzyme activity was inhibited by Hg2+. The full-length mans2 gene was 1,008 bp, encoding a protein of 336 amino acids. Amino acid sequence analysis revealed that it belonged to glycoside hydrolase family 26. Konjac glucomannan was a favorable substrate for recombinant ManS2 (rManS2). rManS2 also degraded galactomannan from locust bean gum, indicating its potential for production of glucomanno- and galactomanno-oligosaccharides. Both native and recombinant ManS2 from Bacillus sp. SWU60 can be applied in several industries especially food and feed.

Association of Single Nucleotide Polymorphisms of Endothelin, Orexin and Vascular Endothelial Growth Factor Receptor Genes with Obstructive Sleep Apnea among Thai Ethnic.

Background: Obstructive sleep apnea (OSA) is a complex disorder characterized by repetitive collapse of upper airway during sleep which strongly influenced by genetic factors, especially those affect regulation of the sleep-wake cycle and endothelial function. Objective: This study investigated the association between single nucleotide polymorphisms (SNPs) in endothelin (EDNRA), orexin (OX1R, OX2R) and vascular endothelial growth factor (VEGFR1) receptor genes with risk of OSA in Thai population. Material and Method: All subjects were diagnosed by overnight polysomnography (PSG) before divided into OSA (59) and NOSA (60) groups based on their apnea-hypopnea index (AHI). Serum lipid levels were examined by using enzymatic colorimetric and homogeneous methods. DNAs were extracted and genotyped the SNPs by polymerase chain reaction (PCR) and high-resolution melting (HRM) analysis. Genotype distribution were analyzed using Chi-square test of SPSS program version 15.0. Results: The triglycerides level of OSA patients was significantly higher than NOSA (p-value = 0.002). The SNPs in EDNRA (rs5335), OX1R (rs2271933), OX2R (rs2292040, rs10456182) and VEGFR1 (rs11149523) genes showed no association with OSA. However, the SNP (rs17675063) in EDNRA gene showed significant differences in genotype distribution in the subjects with and without OSA (p-value = 0.002, odds ratio = 3.29 and 95% CI = 1.86-5.82). Conclusion: Obstructive sleep apnea, Single nucleotide polymorphisms, Endothelin receptor type A, Orexin receptor 1, Orexin receptor 2, Vascular endothelial growth factor receptor type 1.

Association of Cholinergic Muscarinic 2 Receptor Gene Polymorphisms with Learning Aptitude among Medical and Fine Arts Students.

Background: Cholinergic muscarinic 2 receptor (CHRM2) is believed to be involved in neuronal excitability, synaptic plasticity and feedback regulation of acetylcholine release. Polymorphism in the CHRM2 gene had been shown a significant association with intelligence. Objective: To investigate single nucleotide polymorphisms (SNPs) in CHRM2 gene with the learning aptitude among medical and fine arts students at Srinakharinwirot university, Thailand. Material and Method: A total of two hundred blood samples were withdrawn from medical (100) and fine arts (100) students. DNAs were extracted using DNA extraction kit. SNP primers were designed and screened. Genotyping was performed by real-time PCR and high-resolution melting (HRM) analysis and confirmed by sequencing. The difference in genotypic distribution was analyzed using Pearson's Chi-square test implemented in SPSS program version 11.5. Significant level was set at p<0.05. Results: Two SNPs in CHRM2 gene, rs2061174 and rs6948054, showed significant difference in genotype distribution between medical and fine arts students (p<0.05). The rs2061174 showed significant at p = 0.001, OR and 95% CI were 3.78 (2.00-7.14), whereas the rs6948054 was significant at p = 0.012, OR and 95% CI were 2.50 (1.32-4.77). Conclusion: Two SNPs in CHRM2 gene, rs2061174 and rs6948054, may be used as biomarker to distinguish the learning aptitude among Thai individual.

Detection of Pathogenic Leptospires by Loop-Mediated Isothermal Amplification Targeting LipL32 Gene.

BACKGROUND: Leptospirosis is a worldwide re-emerging infectious disease caused by pathogenic leptospires including Leptospira interrogans. OBJECTIVE: In the present study, a loop-mediated isothermal amplification (LAMP) was developed to detect L. interrogans using lipL32 as a gene target. MATERIAL AND METHOD: Four specific primers were designed based on the conserved region of lipL32 gene of various serovars ofpathogenic leptospires. LAMP reaction was performed at 65 °C for 1 hour The LAMP products were detected by agarose gel electrophoresis andfluorescence dye. RESULTS: The lipL32 LAMP assay showed highly specificity to the reference stains of L. interrogans serovar Autumnalis, Bataviae, Javanica, Pyrogenes, Icterohaemorrhagiae, and Saigon. No product was produced from non-pathogenic leptospire (L. biflexa), human, or Escherichia coli. The lower limit of detection analyzed by agarose gel electrophoresis andfluorescence dye visualization was 0.02 pg/µl which equivalent to 4 genomic equivalents/reaction. Moreover the clinical strain of leptospires including pathogenic and intermediate group of L. interrogans were detected by lipL32 LAMP CONCLUSION: The developed lipL32 LAMP is high specificity and sensitivity that can be applied to detect pathogenic leptospires in clinical samples.

Apolipoprotein E receptor 2 Gene Polymorphisms Associated with Dyslipidemia among Thai Population.

BACKGROUND: Dyslipidemia is an abnormal amount of lipids and/or lipoproteins in the blood. It is a major risk factor of coronary heart disease and atherosclerosis. OBJECTIVE: This study investigated two single nucleotide polymorphisms (SNPs) in the apolipoprotein E receptor 2 (ApoER2) gene in association with risk of dyslipidemia in the Thai patients. MATERIAL AND METHOD: Four hundred blood samples including dyslipidemia patient (200) and unrelated normal control (200) were included in this study. Serum lipids were examined. DNAs were extracted and genotyped by using polymerase chain reaction (PCR) followed by high-resolution melting (HRM) analysis. The differences in genotype distribution between patient and normal control were assessed by Chi-square test of the SPSS software version 11.5. RESULTS: The data analysis revealed that two SNPs (rs3737984 and rs2297660) in ApoER2 gene had significant association with dyslipidemia. The rs3 737984 showed significant association at p-value = 0.001, in which A alleles informed the decreased risk of dyslipidemia [odds ratio and 95% CI of A allele, 0.42 (0.28-0.65)]. In contrast, the rs2297660 exhibited strongest association with an increase risk ofdyslipidemia [p-value = 0.001, odds ratio and 95% CI for theA allele was 2.38 (1.49-3.80)]. CONCLUSION: The rs2297660 may be used as biomarker for the risk of dyslipidemia in Thai ethnic.