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Articles by K. Khucharoenphaisan
Total Records ( 10 ) for K. Khucharoenphaisan
  K. Khucharoenphaisan , N. Sripairoj and K. Sinma
  Actinomycetes are a group of prokaryotic organisms belonging to Gram-positive bacteria and play an important ecological role in recycling substances in the nature. The objective of this study was to isolate and identify actinomycetes from termite’s gut against human pathogen. Total isolates were examined for antimicrobial activity and selected isolate was identified using morphological characters and molecular technique. The results showed that eighty-three strains of actinomycetes were isolated from guts of Termes sp. Among these, 66, 67, 7, 9 and 3 isolated strains were active against the tested pathogenic microorganism of Bacillus cereus, Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli and Candida utilis, respectively. Furthermore, some isolate of actinomycetes was able to inhibit both Gram-positive and Gram-negative pathogen. The isolated actinomycetes strain FSPNRU 102 having broad spectrum of inhibition was selected. The morphological character of this strain showed aerial mycelium with longitudinal spirales-type spore chain and light black soluble pigment. The aerial spore color varied from white to gray. Moreover, this strain contained LL-diaminopimelic acid of the peptidoglycan in the whole-cell hydrolysate of chemotaxonomical characteristic. These results assigned strain FSPNRU 102 to genus Streptomyces. Based on its 16S rDNA sequence and phylogenetic tree analysis, this new isolate belong to the Streptomyces niveoruber.
  K. Khucharoenphaisan and K. Sinma
  Xylanase is useful enzyme in various industrial such as conversion of lignocellulose to fermentable sugars for the production of chemicals and biofuels. Thermomyces lanuginosus is a potent thermophilic strain which produces high level of cellulose-free xylanase. The aim of this study was to investigate the effects of signal sequence on T. lanuginosus xylanase expression in Escherichia coli. The xylanase gene from those was amplified with and without signal sequence and expressed in E. coli. The result showed that the transformant (pUC/Xynsig) containing original signal sequence from T. lanuginosus SKR produced xylanase with 56% higher than that of another transformant (pUC/Xyn) lacking of original signal sequence. The recombinant enzymes were partially purified using ammonium sulfate precipitation and DEAE cellulose column and then determined some properties. The optimal pH and optimal temperature of xylanase from pUC/Xynsig was pH 6.0 and 50°C, respectively. In case of pUC/Xyn without signal sequence, the values of xylanase showed higher than those having pH 7.0 and temperature of 70°C in which was similar to original host. In conclusion, original signal sequence from T. lanuginosus SKR could increase xylanase production when expressed in E. coli but some properties of expressed enzyme were changed. However, this finding could apply to other expression system of various hosts in order to stimulate the level of the protein production.
  K. Khucharoenphaisan , K. Sinma and C. Lorrungruang
  Phytopathogenic fungus as Colletotricum gloeosporioides is a cause of disease on chilli and wide varieties of agricultural crops resulting in yield loss. The aim of this study was to screened actinomycetes according to its ability to produce various secondary metabolites with inhibition activity against chilli anthracnose. Firstly, actinomycetes from previously study were tested for antagonistic activity toward the fungus by the dual culture technique. Finally, extracellular antifungal metabolites produced by selected isolates were evaluated for antifungal potential toward the fungus with agar core technique. Eighty three strains of actinomycetes were screened for their antifungal as well as phytopathogenic activity. Among these, 26 isolates were shown the inhibition activities against Colletotricum gloeosporioides chi in which was isolated from infected chilli. The culture supernatants obtained from 21 actinomycetes strains were affective against the fungus. More interestingly, 7 isolates produced affective thermostable compound that having activity after treated with temperature of 121°C for 20 min. In total, the isolate R58 was most promising on the basis of its interesting antimicrobial activity and it could reduce anthracnose disease of chilli comparing to the absence of biocontrol agent. Based on morphological character, its 16S rDNA sequence and phylogenetic tree analysis, isolate R58 belong to the Streptomyces malaysiensis. These findings have increased the scope of agriculturally important actinomycetes.
  C. Lorrungruang , K. Sinma , P. Pantagrud , S. Wannasirisuk , K. Mahabandha and K. Khucharoenphaisan
  Cheese is a dairy product with high nutrition and usually made from cows, sheep and goat milk. In this study, cheeses production from soymilk by using Lactobacillus casei (L), Monascus purpureus (M) and combination of L. casei and M. purpureus (LM) were investigated. The result found that protein coagulation of soymilk could be performed by direct inoculated with Lactobacillus casei and combination of L. casei and M. purpureus which L. casei produced lactic acid to decrease pH to pI of protein in soymilk. While the curd was not occur in the soymilk inoculated with only M. purpureus. Red cheese was produced by adding LM to the cultured soymilk at 8 weeks of cultivation time compared with using M for protein coagulation. The growth of M. purpureus changed chemical compositions of the red cheese from both M and LM especially fat and protein contents. Fat content dramatically increased from 15.84±0.18-18.97±0.58% during ripening contrary to cheese using L. casei fermentation for protein coagulation without M. purpureus adjunction. Adhesiveness of red cheese M and LM increased from 12.58±0.26-0.17±0.70% and 19.36±0.75-6.99 ±0.63%, respectively. In contrast, protein content was decreased from 44.56±0.55-0.09±0.47% during ripening of cheese L. Red cheese M and LM decreased from 45.24±0.44-5.34±0.57% and 43.97±0.64-1.20±0.52%, respectively. In the sensory evaluation, the red cheese from soymilk had accepted more than that from cow’s milk and blue cheese in aspect of odour.
  K. Sinma and K. Khucharoenphaisan
  Micromonospora is one of actinomycetes belonging to Gram-positive bacteria. It was found both inside plant tissues and outside tissues as free-living bacteria. The objectives of this study were determined the distribution of Micromonospora in sugar cane tissues; root, leave and stem which obtained from 5 provinces in Thailand. The results showed that 147 endophytic actinomycetes were isolated. Most endophytic actinomycetes were found in plant root due to high root exudates in rhizosphere whereas the localization of most Micromonospora were found in plant stem. Among these, thirty-four isolates has identified as Micromonospora by using morphological character and meso-diaminopimelic acid. They were grouped into 5 different groups based on color of substrate mycelium. In this way, most of them belong to Gr. 1 with 10 members. The representative isolates of each group were analyzed 16S rDNA sequence and construct phylogenetic tree. The phylogenetic tree showed Gr. 1 to Gr. 5 were closed relationship to Micromonospora coriariae, M. brigensis, M. humi, M. rifamycinica and M. saelicesensis, respectively. This is the first descriptions of the presence of Micromonospora inside the sugar cane tissue.
  K. Khucharoenphaisan , U. Puangpetch , K. Puttaraksa and K. Sinma
  Actinomycetes are a group of prokaryotic organisms belonging to Gram-positive bacteria and play an important ecological role in recycling substances in the nature. To determine possible established correlation between isolated actinomycetes and its biochemical degradation (xylan, soluble starch, cassava, protein, lipid, uric acid, carboxymethyl cellulose and avicel degradation) and oxidation property (guaiacol), the actinomycetes were isolated from Termes sp. The dendogram was generated from UPGMA analysis with FreeTree software. In this work, 45 strains of actinomycetes were isolated from guts of Termes sp. Among these, 44 isolated strains could degrade protein, soluble starch and cassava starch. Twenty-three strains degraded uric acid and xylan. The isolated strains that able to degrade avicel and oxidize lignin were rare. The morphological character showed the variety of aerial hyphae and spore forming in each strain. The dendogram was constructed based on biodegradation activity of tested strains. The tested strains were classified into 5 clusters. Cluster 1, cluster 2, cluster 3, cluster 4 and cluster 5 contained 14, 9, 9, 9 and 4 isolated strains, respectively. The actinomycetes strains showed a similar biodegration activity within a same cluster. This result indicated the relation between biodegradation activity and actinomycetes strains. Dendogram based on the biodegradation activity was found to be an efficiencient tool for grouping purpose.
  K. Khucharoenphaisan , K. Rodbangpong , P. Saengpaen and K. Sinma
  Actinomycetes have been promised as biocontrol and stimulating agents for use in agriculture without detrimental effects to the environmental due to their antifungal with secondary metabolites produced. The aim of this study was to screen soil actinomycetes according to its ability to produce various secondary metabolites against Phytophthora sp. that causing root rot disease of cassava and stimulating agent has also determined. Firstly, soil actinomycetes were isolated and tested for antagonistic activity toward the fungus by the dual culture technique. After that the selected isolate was determined on the stimulating agent as IAA production. Finally, extracellular anti-fungal metabolites produced by selected isolates were evaluated for anti-fungal potential toward the fungus with agar core technique. The result showed that 98 isolates from soil samples were screened on their anti-fungal activity. Among these, 38 isolates showed the inhibition activities against Phytophthora sp. in which was isolated from infected cassava. The culture supernatants without cell obtained from 16 isolates were affective against the fungus whereas 10 isolates produced affective thermostable compound. In total, the isolate LB35 was most promising on the basis of its interesting antimicrobial activity and could produce IAA with 50 μg mL–1. Based on its 16S rDNA sequence and phylogenetic tree analysis, isolate LB35 belong to the Streptomyces malaysiensis. The addition of isolate LB35 as fresh inoculums to the cassava field, the cassava showed more height comparing to control experiment. Moreover, S. malaysiensis has not to be phytopathogenic microorganism of cassava. This finding has been increased scope of agriculturally important actinomycetes applications.
  K. Khucharoenphaisan , S. Tokuyama , K. Ratanakhanokchai and V. Kitpreechavanich
  The effect of carbon sources on the production of β-xylanase by Thermomyces lanuginosus TISTR 3465 was investigated. Xylan showed the highest inductive effect on the enzyme formation whereas xylobiose and xylooligosaccharides resulted in lesser effect. β-Xylanase was also produced at low level with xylose as well as other sugars tested. Xylan concentration at 15 g L-1 gave the maximum inductive effect on β-xylanase formation, whereas xylooligosaccharides and xylose were effective at a lower concentration of 2.5 g L-1. High concentrations of these sugars significantly repressed the enzyme formation. Crude enzyme from the supernatants, without and with other sugars produced a single xylanase band on non-denaturing PAGE gels. However, an intense xylanase activity band was observed from the supernatant of media with xylan, xylobiose and xylooligosaccharides as the carbon sources. An intense protein band of 24.9 kDa from the culture filtrate of xylan medium was observed. Xylan increased β-xylanase production by the fungus 16-fold when it was added to the xylose medium after cultivation for 3 days. In contrast, addition of xylose to the xylan medium decreased β-xylanase production 3-fold. A distinct appearance and disappearance of a 24.9 kDa protein and the activity band coincided with an increase and decrease of xylanase activity, respectively. This indicated the synthesis of xylanase by this strain was most induced by xylan. Moreover, the level of xylanase induction has no related to amino acid sequence of the enzyme.
  K. Khucharoenphaisan and K. Sinma
  Thermomyces lanuginosus is thermophilic fungus in which was isolated from widespread material. A high number of this fungus was found in composts especially mushroom composts. This fungus has been reported to produce a high level xylanase when cultivated in the medium containing xylan and corn cob as a carbon source. Various strains of T. lanuginosus produced a single xylanase with molecular masses in range of 22.0 to 29.0 kDa. Pure β-xylanase obtained from various strains of this fungus exhibited highly stability at high temperature and wide pH range. The optimal temperature and optimal pH of pure β-xylanase from various strains of T. lanuginosus have been reported in range of 60-75°C and pH 6.0-7.0, respectively. The great thermal stability was resulting from the present of hydrophilic amino acid on beta sheet of the surface of xylanase structure. Moreover, the relatedness between high and low xylanase producing strains can be distinguish by random amplification of polymorphic DNA (RAPD). Based on nucleotide sequences and T. lanuginosus xylanase gene has been classified to be a member of family 11 (formerly known as cellulase family G) glycosyl hydrolases. This enzyme was endo-type xylanase having main product are xylose and xylobiose. The expression of xylanase gene from T. lanuginosus was achieved in Escherichia coli and methylotrophic yeast Pichia pastoris. The ability of T. lanuginosus in which produced large amount of high thermos stable xylanase has made this fungus to be a source of xylanase production for biobleaching in pulp and paper process.
  K. Khucharoenphaisan and K. Sinma
  The strain PNR11 was isolated from gut of termite during the screening for uric acid degrading actinomyces. This strain was able to produce an intracellular uricase when cultured in fermentation medium containing uric acid as nitrogen source. Base on its morphological characters and 16S rDNA sequence analysis, this strain belong to the genus Saccharopolyspora. This is the first report of uricase produced from the genus Saccharopolyspora. The aim of this study was to investigate the effects of different factors on uricase production by new source of Saccharopolyspora. Saccharopolyspora sp. PNR11 was cultured in production medium in order to determine the best cultivation period. The result showed that the time period required for maximum enzyme production was 24 h on a rotary shaker operating at 180 rpm. Optimized composition of the production medium consisted of 1% yeast extract, 1% maltose, 0.1% K2HPO4, 0.05% MgSO4 7H2O, 0.05% NaCl and 1% uric acid. The optimum pH and temperature for uricase production in the optimized medium were pH 7.0 and 30°C, respectively. When the strain was cultured at optimized condition, the uricase activity reached to 216 mU mL-1 in confidential level of 95%. The crude enzyme had an optimum temperature of uricase was 37°C and it was stable up to 30°C at pH 8.5. The optimum pH of uricase was 8.5 and was stable in range of pH 7.0-10.0 at 4°C. This strain might be considered as a candidate source for uricase production in the further studies. Present finding could be fulfill the information of uricase produce from actinomycetes.
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