April - June 2007 - Kasetsart University

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352 The enzymes were also increased at stationary growth phase to show the non-growth associated enzyme production. However, enzyme was quite stable in fermenter culture. Oxygen might confirm its important role during declining growth phase in promoting enzyme stability. Further investigation will be conducted on optimizing an effect of oxygen for the production of chitosanases. The properties of crude chitosanases The crude chitosanases after cell removal, prepared from the 2-l fermenter mentioned earlier were used without any further treatment for studying the pH and temperature optimum and stability of enzyme. Effect of pH The optimal pH of crude chitosanases was at pH 6.5 (Figure 3). At lower pH 3.0 and higher pH 9.0, the relative enzyme activities were 47.18 and 56.64%, respectively. This optimal pH was comparable to Bacillus cereus S1 chitosanases (pH 6.0) (Kurakake et al., 2000) and similar to Relative activity (%) Kasetsart J. (Nat. Sci.) 41(2) chitosanases from Bacillus circulans MH-K1 (Yabuki et al., 1988) and Bacillus sp. No. 7-M (Uchida and Ohtakara, 1988). This, however, differed totally from those produced by Bacillus subtilis IMR-NK1 (Chiang et al., 2003) and Bacillus megaterium P1 (Pelletier and Sygusch, 1990) which were optimized at pH 4.0 and 4.5- 6.5, respectively. As previously reported, the optimal pH’s for various chitosanases were in a broad range of 4.0-8.0 (Somasheka and Joseph, 1996) depending on the bacterial strains. Bacillus cereus TP12.24 chitosanases were found stable at a wide pH range of 3.0-8.0 retaining more than 70% activity after preincubation at 40°C for 60 min. However, at pH 9.0 and 11.0, the relative activities were decreased to 47.14 and 34.29%, respectively. Different chitosanases showed different pH stability, such as pH 6.0-11.0 for Bacillus cereus S1 chitosanases (Kurakake et al., 2000) and pH 5.0-9.0 for Bacillus subtilis IMR-NK1 chitosanases after preincubation at 25°C for 1 h (Chiang et al., 2003). 100 pH optimum pH stability 80 60 40 20 3 4 5 6 7 8 9 10 11 Figure 3 Optimal pH and pH stability of Bacillus cereus TP12.24 chitosanases. pH
Relative activity (%) 100 80 60 40 Effect of temperatures The activity of crude chitosanases from Bacillus cereus TP12.24 was found optimal at 55°C (Figure 4). At lower or higher temperatures, the relative activities were reduced to 86.36 and 85.86% at 30 and 70°C, respectively. This optimal temperature was slightly lower than that of Bacillus cereus S1 (60°C) (Kurakake et al., 2000), but higher than those of Bacillus subtilis IMR-NK1 (45°C) (Chiang et al., 2003) and Bacillus megaterium P1 (50°C) (Pelletier and Sygusch, 1990). Bacillus cereus TP12.24 chitosanases were stable at temperature of 30-50°C showing 74.26-81.19% activity. However the enzyme activity was decreased at temperature higher than 50°C. Chitosanases from Bacillus cereus S1 were ever reported to be stable at temperature higher than 60°C at pH 5.0 for 30 min (Kurakake et al., 2000). Therefore, Bacillus cereus TP12.24 chitosanases were not the thermostable enzyme. The enzyme could be used at moderate temperature and neutral pH under the wide pH range of stability. Kasetsart J. (Nat. Sci.) 41(2) 353 Temperature optimal Temperature stability 20 30 40 50 60 70 80 Temperature (°C) Figure 4 Optimal temperature and temperature stability of Bacillus cereus TP12.24 chitosanases. ACKNOWLEDGEMENTS The present study was financially supported by the National Center for Genetic Engineering and Biotechnology (Biotec), Thailand and partially supported by DNA Technology Laboratory, Kasetsart University Kamphaeng Saen Campus in association with the Commission on Higher Education, Thailand. The laboratory at the department of Biotechnology, Kasetsart University Bangkhen Campus was greatly acknowledged for providing the fermentation facilities. LITERATURE CITED Boucher, I., A. Dupuy, P. Vidal, W. A. Neugebuer and R. Brzezinski. 1992. Purification and characterization of a chitosanase from Streptomyces N174. Appl. Microbiol. Biotechnol. 38: 188-193. Cheng, C. Y. and Y. K. Li. 2000. An Aspergillus chitosanase with potential for large-scale preparation of chitosan oligosaccharides. Biotechnol. Appl. Biochem. 32: 197-203.
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April - June 2007 Volume 41 Number
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KASETSART JOURNAL NATURAL SCIENCE T
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Kasetsart J. (Nat. Sci.) 41 : 205 -
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harvesting time which started from
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y Chen and Paull (2000). Figure 1 a
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pineapple fruit allowed the accumul
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Kasetsart J. (Nat. Sci.) 41(2) 215
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Cluster analysis Cluster analysis u
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Table 3 (Continued) 1 2 3 4 5 6 7 8
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CONCLUSION AFLP markers classified
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difference of soil texture used in
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under wet soil condition planting.
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tropics. Following the expansion, w
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sealed in a plastic box with 1 cm w
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moisture content increment after so
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Frequency Frequency 30 20 10 0 20.0
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School, Kasetsart University. The a
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for combining ability of lines (Lam
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selective mass sibbing within indiv
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Although most of S 2-interfamily hy
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composite sets as used in this stud
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days. The numbers of calli producin
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the report of Ching (1982) showing
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clearly amplified bands generated b
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caused by either the recombination
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Kuhlmann, V. and B. Foroughi-Wehr.
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Xanthomonas fuscans subsp. aurantif
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was collected and washed with 70% e
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expected size was amplified with 35
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eaction technique has been used for
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Schaad, N.W., D. Opgenorth and P. G
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MATERIALS AND METHODS Model descrip
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calculated TF value calculated TF v
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sincere gratitude and deep apprecia
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1989). In monogastrics, the inclusi
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of all LLS samples in this study we
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Melbourne, Parkville, Victoria. Goe
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considered responsible for low prod
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mineral supplementation suggested b
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Body weight chane (kg/head) 32 30 2
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CONCLUSION AND RECOMMENDATION With
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SAS. (Statistical Analysis System).
Page 101 and 102: genes covering a variety of desirab
Page 103 and 104: mg/l NAA and 0.5 mg/l zeatin) incub
Page 105 and 106: 5. Purification by various sucrose
Page 107 and 108: as the culture period increased. So
Page 109 and 110: culture, pp. 1-20. In L.C. Fowke an
Page 111 and 112: Kasetsart J. (Nat. Sci.) 41 : 311 -
Page 113 and 114: GC. Analytical methods Total carboh
Page 115 and 116: ash. The crude hot water polysaccha
Page 117 and 118: spirulan (H-SP), and a desulfated c
Page 121 and 122: cells often displayed an increased
Page 123 and 124: LITERATURE CITED Bell, T.A. and D.V
Page 125 and 126: for alternative sources of supply.
Page 127 and 128: BR2.1.2 formed the same clade with
Page 129 and 130: supplement with glutamate (Iida et
Page 131 and 132: optimal for S. limacinum BR2.1.2 fo
Page 133 and 134: fluorescent lamp at 1.5 kLux develo
Page 137 and 138: Wisconsin, USA). Total extracted RN
Page 139 and 140: RESULTS Cloning and sequencing of m
Page 141 and 142: Expression of rmIL-2 and purificati
Page 143 and 144: other strains of mice have differen
Page 145 and 146: dose interleukin-2 therapy promotes
Page 147 and 148: The chitosano-oligosaccharides are
Page 149 and 150: enzyme and cells were maximized by
Page 151: of crude chitosanases. Fortunately,
Page 155 and 156: Uchida, K. Tateishi, O. Shida and K
Page 157 and 158: MATERIALS AND METHODS 1. Materials
Page 159 and 160: without coating and coated with pec
Page 161 and 162: in Table 5-7. It was found that the
Page 165 and 166: the cultures at 10,200 × g for 15
Page 167 and 168: incubation and reached the final pH
Page 169 and 170: y lysine- and tyrosine-decarboxylas
Page 171 and 172: during the stages of ripening as su
Page 175 and 176: As the 25 companies were divided in
Page 177 and 178: All the data comes up with informat
Page 179 and 180: as piece “A” or straight as pie
Page 181 and 182: et al. (1998); Zeng (2000); and Tal
Page 183 and 184: p 1, i * Kasetsart J. (Nat. Sci.) 4
Page 185 and 186: c ≤ c2 jq2 j n q p k p * 2, j , ,
Page 187 and 188: algorithm would consider RM 5, prio
Page 189 and 190: Kasetsart J. (Nat. Sci.) 41(2) 389
Page 191 and 192: the maximum deviation of about 10%.
Page 193 and 194: Stock Quantities Using Heuristic Op
Page 195 and 196: which dynamically and automatically
Page 197 and 198: 2. Framework architecture and compo
Page 199 and 200: Risk (VaR) calculation which was im
Page 201 and 202: Throughput (job/sec) 7 6 5 4 3 2 1
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Speed up 40 35 30 25 20 15 10 5 0 F
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and discovery, resource selection a
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April - June 2007 - Kasetsart University

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