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.

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.

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.

Antileptospiral activity of xanthones from Garcinia mangostana and synergy of gamma-mangostin with penicillin G.

BACKGROUND: Leptospirosis, one of the most widespread zoonotic infectious diseases worldwide, is caused by spirochetes bacteria of the genus Leptospira. The present study examined inhibitory activity of purified xanthones and crude extracts from Garcinia mangostana against both non-pathogenic and pathogenic leptospira. Synergy between γ-mangostin and penicillin G against leptospires was also determined. METHODS: Minimal inhibitory concentrations (MIC) of crude extracts and purified xanthones from G. mangostana and penicillin G for a non-pathogenic (L. biflexa serovar Patoc) and pathogenic (L. interrogans serovar Bataviae, Autumnalis, Javanica and Saigon) leptospires were determined by using broth microdilution method and alamar blue. The synergy was evaluated by calculating the fractional inhibitory concentration (FIC) index. RESULTS: The results of broth microdilution test demonstrated that the crude extract and purified xanthones from mangosteen possessed antileptospiral activities. The crude extracts were active against all five serovars of test leptospira with MICs ranging from 200 to ≥ 800 µg/ml. Among the crude extracts and purified xanthones, garcinone C was the most active compound against both of pathogenic (MIC =100 µg/ml) and non-pathogenic leptospira (MIC = 200 µg/ml). However, these MIC values were higher than those of traditional antibiotics. Combinations of γ-mangostin with penicillin G generated synergistic effect against L. interrogans serovars Bataviae, Autumnalis and Javanica (FIC = 0.52, 0.50, and 0.04, respectively) and no interaction against L. biflexa serovar Patoc (FIC =0.75). However, antagonistic activity (FIC = 4.03) was observed in L. interrogans serovar Saigon. CONCLUSIONS: Crude extracts and purified xanthones from fruit pericarp of G. mangostana with significant antibacterial activity may be used to control leptospirosis. The combination of xanthone with antibiotic enhances the antileptospiral efficacy.