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Kasetsart J. (Nat. Sci.) 40 : 121 - 135 (2006) Physiological Study and Alcohol Oxidase Gene(s) of Thermotolerant Methylotrophic Yeasts Isolated in Thailand Nantana Srisuk 1 *, Savitree Limtong 1 , Hiroya Yurimoto 2 , Yasuyoshi Sakai 2 and Nobuo Kato 2 ABSTRACT Two thermotolerant methylotrophic yeasts, Candida sithepensis sp. nov. and Pichia siamensis sp. nov. isolated in Thailand were studied for their physiology and alcohol oxidase gene(s). Optimum and maximum temperatures for growth on methanol of C. sithepensis sp. nov. were 34°C and 37°C whereas those of P. siamensis sp. nov. were 37°C and 40°C, respectively. Effect of methanol on the methylotrophic growth of the two yeast strains was investigated within the ranges of 0.25% to 2% (v/v) methanol. At 34°C, C. sithepensis sp. nov. exhibited a decline in µ max from 0.16 h -1 to 0.13 h -1 with respect to increase in methanol concentration. When cultivated at 37°C, the µ max of C. sithepensis sp. nov. was found to be drastically decreased from 0.16 h -1 to 0.10 h -1 within the same ranges of methanol concentration studied. P. siamensis sp. nov. showed an insignificant decrease of µ max with respect to an increase in methanol supplement when cultivated at 37°C. Key enzymes involved in methanol dissimilatory pathway, i.e., alcohol oxidase (AOD), glutathione-dependent formaldehyde dehydrogenase (FLD), formate dehydrogenase (FDH) and catalase (CAT) of the two yeasts were assessed. As for the favorable growth of C. sithepensis sp. nov. at its optimum temperature, fairly high level of specific AOD activity was observed but lower level of specific AOD activity was exhibited at maximum temperature. In contrast, specific FLD and FDH activity profiles were found to be higher when the yeast C. sithepensis sp. nov. was grown at its maximum temperature compared to the methylotrophic growth at its optimum temperature. Catalase production appeared to be slightly disturbed under all conditions studied. In case of P. siamensis sp. nov., stationary phase growth under low methanol supplement at its optimum temperature appeared soon after 24h cultivation. Nucleotide sequences of AOD gene(s) of the two yeasts were investigated. C. sithepensis sp. nov. showed two AOD genes designated as AOD1 and AOD2 whereas P. siamensis sp. nov. revealed only one AOD gene within the genome. Key words: methylotrophic yeasts, alcohol oxidase gene, thermotolerant, physiological study 1 Department of Microbiology, Faculty of Science, Kasetsart University, Bangkok 10900, Thailand. 2 Laboratory of Microbial Biotechnology, Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan. * Corresponding author, e-mail: fscints@ku.ac.th Received date : 25/07/05 Accepted date : 06/02/06
122 INTRODUCTION Methylotrophic yeasts are widely used for heterologous protein production (Cereghino and Cregg, 2000; Gellissen, 2000; Faber et al., 1995). Two yeast genera, i.e., Pichia spp. and Candida spp. have now been recognized as methylotrophic (Houard et al., 2002). Several species: P. pastoris, P. methanolica, P. angusta, C. boidinii and C. methanolovescens are known as representatives of these two genera (Demain et al., 1998). Methanol metabolism starts with the oxidation of methanol to formaldehyde and hydrogen peroxide. The reaction is catalyzed by alcohol oxidase and the hydrogen peroxide produced is subsequently removed by the reaction catalyzed by catalase. This beginning part of methanol metabolism is localized in peroxisomes whereas the subsequent reactions in methanol assimilation and dissimilation are localized in the cytosol ( Fukui et al., 1975; Douma et al., 1985; Goodman, 1985; Veenhuis and Harder, 1987). Dissimilation of formaldehyde proceeds to formate and further to CO 2, and is catalyzed by formaldehyde dehydrogenase and formate dehydrogenase, respectively, resulting in the generation of two molecules of NADH (Veenhuis and Harder, 1987). The merits of methylotrophic yeasts applied to various processes are due to the capability to utilize methanol as the sole carbon and energy source. However, industrial applications have mostly been limited to the inability of these yeast strains to grow at high temperature. This affects not only the yeast growth but also the production of yeast proteins or metabolites. It is, therefore of advantage to search for thermotolerant methylotrophic yeast strains to overcome this detrimental effect of high temperature. Thailand is one of the tropical countries having reasonably high ambient temperature throughout the year. As a result of such climate, the thermotolerant microbial resources Kasetsart J. (Nat. Sci.) 40(1) have therefore been the target of interest. Limtong et al. (2004) described three new thermotolerant methylotrophic yeasts isolated in Thailand, i.e., Candida krabiensis sp. nov., C. sithepensis sp. nov. and Pichia siamensis sp. nov. In this study it was aimed to determine the optimum and maximum temperatures for growth of the two new yeast isolates described by Limtong et al. (2004). In addition, the effect of methanol on the methylotrophic growth at optimum and maximum temperatures was evaluated. Methanol utilizing enzymes of these yeast isolates were investigated under different growth conditions. Furthermore, alcohol oxidase gene(s) of the isolates of interest were also studied. MATERIALS AND METHODS Yeast strains All yeast isolates, except Pichia angusta, were isolated in Thailand from <strong>natural</strong> samples (flowers, fruits, leaves, soils and water) by the selective and enrichment isolation technique. Pichia angusta, on the other hand, was obtained from Laboratory of Microbial Biotechnology, Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Japan. Cultivation and media Yeast isolates were maintained in YPD slant culture. Methanol synthetic medium (Sakai et al., 1995) was used for the shake flask cultivation. Methanol concentration varied as 1% (v/v) for determination of optimum and maximum temperatures for growth, 0.25-2% (v/v) to study the effect of methanol on methylotrophic yeast growth and 0.75-2% (v/v) for the production of methanol utilizing enzymes. Yeast cultivation (100 ml) to determine temperatures and methanol concentrations critical to yeast growth was conducted aerobically at various temperatures, i.e.,
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January - March 2006 Volume 40 Numb
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KASETSART JOURNAL NATURAL SCIENCE T
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In sacco Degradation Characteristic
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2 and management practices a sorghu
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4 the greater plant height was obta
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6 at Wolenchity (Figure 3) that led
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8 Stover and aboveground biomass Co
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10 Radford, B.J., A.J. Dry, L.N. Ro
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12 At present, new cultivars with h
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14 2000), grapevine (Lavee, 2000),
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16 repeatability of FW, FLT, FLW, S
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18 (Table 5) indicating that select
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Kasetsart J. (Nat. Sci.) 40 : 20 -
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22 measured to define fruit shape i
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24 for higher fruit number and yiel
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28 (Table 2). The numbers of flower
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30 to the color of fruit and seed c
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32 1999. Carrots and related vegeta
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34 MATERIALS AND METHODS Laboratory
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36 DISCUSSION Extensive studies hav
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38 from Gossypium hirsutum. J. Inse
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40 The natural resistance of plants
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42 4. Data collection and statistic
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44 was not significantly different
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46 showed the highest yield compare
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48 Kessmann, H., T. Staub, C. Hofma
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50 There are approximately 60 comme
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52 in PBST containing 2% ovalbumin
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54 Kasetsart J. (Nat. Sci.) 40(1) T
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56 Virus-free orchid plants show fa
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60 feed source was based on purchas
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62 intake of DM, CP, ether extract
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64 relatively slow and short. The p
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66 week 4. Milk composition was not
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68 Agricultural Experiment Station.
Page 75 and 76: 70 et al., 2002). In contrast to ma
Page 77 and 78: 72 level and showed marked polymorp
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Page 81 and 82: 76 loam (Eutric Fluvisol) soil and
Page 83 and 84: 78 Eugenia caryophyllata, Echinops
Page 85 and 86: 80 CONCLUSION In conclusion, out of
Page 87 and 88: 82 of chromic acid titration method
Page 89 and 90: 84 initials at the time of pinning
Page 91 and 92: 86 vessels a point where the number
Page 93 and 94: 88 Kasetsart J. (Nat. Sci.) 40(1) T
Page 95 and 96: 90 CONCLUSIONS In the investigation
Page 97 and 98: 92 1994; Strand et al., 1997). Now
Page 99 and 100: 94 The clones were grown overnight
Page 101 and 102: 96 isozymes. The copy number for nu
Page 103 and 104: 98 Helianthus annuus. Theor. Appl.
Page 105 and 106: 100 MATERIALS AND METHODS Plant mat
Page 107 and 108: 102 The amount of Na + (%) 6 5 4 3
Page 109 and 110: 104 NGE, 7-16 folds). Finally, all
Page 111 and 112: 106 Anal. Biochem. 162: 156-159. Cr
Page 113 and 114: 108 grown for academic purpose at N
Page 115 and 116: 110 Cluster A was further separated
Page 117 and 118: 112 Table 2 Similarity index of 19
Page 119 and 120: 114 subjected to UPGMA and resultin
Page 121 and 122: 116 Tabel 3 The similarity index of
Page 123 and 124: 118 formation as seen from phylogen
Page 125: 120 Genet. 101: 860-864. Lerceteau,
Page 129 and 130: 124 RESULTS AND DISCUSSION Screenin
Page 131 and 132: 126 optimum temperature, insignific
Page 133 and 134: 128 medium of C. boidinii (Volfova
Page 135 and 136: 130 was observed soon after 24h cul
Page 137 and 138: 132 Kasetsart J. (Nat. Sci.) 40(1)
Page 139 and 140: 134 ACKNOWLEDGEMENTS This work was
Page 143 and 144: 138 Determination of enzyme activit
Page 145 and 146: 140 Table 1 Optimum and stability o
Page 147 and 148: 142 for example recombinant E. coli
Page 149 and 150: 144 were studied for β-1,3-1,4-glu
Page 151 and 152: 146 ACKNOWLEDGEMENT This work was s
Page 155 and 156: 150 vulcanization times were calcul
Page 157 and 158: 152 RESULTS AND DISCUSSION Mechanic
Page 159 and 160: 154 Kasetsart J. (Nat. Sci.) 40(1)
Page 161 and 162: 156 blend with 0.5 phr Irganox. How
Page 165 and 166: 160 (AA-680 ShiMADZU, Atomic Absorp
Page 167 and 168: 162 warm, relatively shallow and hi
Page 169 and 170: 164 cycle in crustacean (Chen et al
Page 171 and 172: 166 Salaenoi, J. 2004. Changes of e
Page 173 and 174: 168 MATERIALS AND METHODS This rese
Page 175 and 176: 170 DISCUSSION The average total am
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174 repacked and scanned three time
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176 found the most in the shrimp fe
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178 PLS model The comparison betwee
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180 brix value and dry matter of ma
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182 approach to increase immunity t
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184 higher (P
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186 levels were significantly eleva
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190 currents. Zone 4: River outlet
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192 standing waters, thus the occur
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194 and moving to 59%TL in juvenile
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198 thin membrane (Figure 1A, B). T
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200 were clearly seen. The presence
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202 could change to female at 6-12
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206 water until the water became cl
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208 reported by Thu and Preston (19
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210 cavalcade hay. The crude protei
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212 CONCLUSIONS Ruminal disappeared
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214 ∅rskov, E.R. and I. McDonald.
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216 is constrained mainly due to fa
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218 SNF and fat composition in raw
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220 in terms of both manual samplin
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222 Harding, F. 1995. Compositional
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224 proteins described by Barbut an
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226 starch/flour, shaken vigorously
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228 % Stress relaxation 54 49 44 39
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230 Kasetsart J. (Nat. Sci.) 40(1)
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234 categories including puffed sna
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236 Cohesiveness of mass Crispness
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238 Cohesiveness of mass Sweetness
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242 3. Physicochemical properties 3
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246 ACKNOWLEDGEMENTS This research
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248 are now becoming more and more
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250 downstream control. Lam Chae re
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254 Inflow (mcm.) Inflow (mcm.) Inf
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256 were above 0.70. The testing re
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258 1999-2000 could be used for tra
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262 we assume that r ≥ 2 and d r
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266 Figure 1 Prototype domains. num
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268 DISCUSSION Consequences indicat
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272 George, B. K. 1997. The GIS Boo
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discussed; and appropriateness of t
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LITERATURE CITED FORMAT Manuscripts
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