April - June 2007 - Kasetsart University

More documents

Recommendations

Info

Kasetsart J. (Nat. Sci.) 41 : 346 - 355 (2007) Production and Partial Characterization of Chitosanases from a Newly Isolated Bacillus cereus Sutee Wangtueai 1 , Wanchai Worawattanamateekul 1 *, Mathana Sangjindavong 1 , Nuanphan Naranong 2 and Sarote Sirisansaneeyakul 3 ABSTRACT The production of chitosanases by a newly isolated Bacillus cereus TP12.24 was studied both in shake flask and fermenter cultures. The M9-chitosan medium was found most suitable with 0.5% chitosan as a sole carbon source optimized under aerobic growth conditions at pH 6.0 and 30°C. The specific rates of growth, substrate consumption, and enzyme production were improved using controlled completely aerobic conditions in 2-l fermenter. While the yield of biomass was considerably increased, the enzyme yield was on the contrary decreased. As a result, the volumetric chitosanases productivity was 43.55 U/l h, which was 1.2 times that obtained from shake flask culture due to higher specific rates of chitosan consumption and chitosanases production. In this work, the crude chitosanases from Bacillus cereus TP12.24 showed their optimal pH and temperature at 6.5 and 55°C, while the stabilities to pH and temperature were found at 3.0-8.0 and 30-50°C, respectively. The Bacillus cereus chitosanases could be used for preparing the chitosano-oligosaccharides under mild temperature. Key words: chitosanases, chitosan, Bacillus cereus, optimization, batch culture INTRODUCTION Chitosan (poly-β-(1→4)-2-amino-2deoxy-D-glucose) is a long chain polymer derived from chitin by deacetylation (Kumar et al., 2000). Mostly, the sources of chitin in Thailand are solid wastes derived from the shrimp processing industries. Chitosan has been utilized as multipurpose products in food, semi-food and non-food industries. Whereas the production of chitosanderived oligosaccharides shows its potential as high value added food product, the enzymatic hydrolysis rather than chemical degradation that provides an attractive process is obviously limited. Chitosanase (EC 3.2.1.132) is exploited for the production of chitosano-oligosaccharides. Various sources of enzyme could be obtained from soil fungi and bacteria, such as Bacillus circulans MH- K1 (Yabuki et al., 1988), Bacillus sp. No.7-M (Uchida and Ohtakara, 1988), Bacillus licheniformis UTK (Uchida et al., 1992), Bacillus cereus S1 (Kurakake et al., 2000), Streptomyces N-174 (Boucher et al., 1992), Streptomyces sp. No.6 (Price and Storck, 1975), Amycolatopsis sp. CsO-2 (Okajima et al., 1994), and Burkholderia gladioli strain CHB101 (Shimosaka et al., 2000). 1 Department of Fishery Products, Faculty of Fisheries, Kasetsart University, Bangkok 10900, Thailand. 2 Department of Applied Biology, Faculty of Science, King Mongkut’s Institute of Technology Ladkrabang, Bangkok 10520, Thailand. 3 Department of Biotechnology, Faculty of Agro-Industry, Kasetsart University, Bangkok 10900, Thailand. * Corresponding author, e-mail: ffiswcw@ku.ac.th Received date : 21/04/06 Accepted date : 14/12/06
The chitosano-oligosaccharides are water-soluble and possess versatile bioactivities such as immunopotentiating, bacteriostatic activities which have their advantages in food materials, agricultural and medical, and antitumor activity (Tominaga and Tsujisaka, 1975; Price and Storck, 1975; Suzuki et al., 1984; Papineau et al., 1991; Somashekar and Joseph, 1996; Jeon and Kim, 1998, 2000). The purpose of the present work was to optimize the production of chitosanases from the newly isolated Bacillus cereus TP12.24 (Wangtueai et al., 2006). The crude chitosanases were also partly characterized for their optimal and stability based on pH and temperature. MATERIALS AND METHODS Microorganism Bacillus cereus TP12.24, a newly isolated soil bacterium (Wangtueai et al., 2006) was used throughout the experiments. The stock culture was maintained on the chitosanasedetection agar medium (CDA) (Cheng and Li, 2000) and freshly transferred every 2 weeks. Factors affecting enzyme production in shake flask culture All experiments were carried out in shake flask cultures using 500-ml Erlenmeyer flask containing 250-ml M9-chitosan medium at 250rpm for 72 h. Samples were taken every 6 h for determining total viable cells, dry cell weight, residual chitosan and enzyme activity. The culture conditions and all analyses have been described previously (Wangtueai et al., 2006). Effect of pH The study on pH optimum for enzyme production was carried out by varying the pH values of M9-0.5% chitosan medium at 4.0 to 8.0 at 30°C. Kasetsart J. (Nat. Sci.) 41(2) 347 Effect of chitosan The M9-chitosan media containing 0.1, 0.5, 1.0 and 2.0 % chitosan were used for the production of chitosanases under optimized initial pH 6.0 at 30°C. Effect of temperature The temperatures of 30, 40 and 50°C were investigated for growth and enzyme production under optimized initial pH 6.0 and 0.5% chitosan concentration. Enzyme production in 2-l fermenter The 2-l fermenter (EYELA Mini jar fermenter, Model M-100, Tokyo Rikakikai Co., Ltd.) which contained 1.5-l M9-chitosan medium with 0.5% chitosan was used for the production of chitosanases from Bacillus cereus TP12.24. The fermentation conditions were controlled automatically at 30°C, pH 6.0, 1 vvm aeration rate and 400 rpm agitation rate for 58.5 h of cultivation time. The samples were taken every 6 h for determining the total viable cells, dry cell weight, residual chitosan and enzyme activity (Wangtueai et al., 2006). The fermentation kinetics of bacterial growth and chitosanases production were studied based on the experimental results. Characterization of crude chitosanases The crude chitosanases were prepared by growing cells in 2-l fermenter under the optimal conditions obtained in this work. The enzyme supernatant was collected from the culture broth after centrifugation at 8,000 rpm, 4°C for 20 min. This supernatant as crude chitosanases was used for the determination of enzyme optimal and stability on the basis of pH and temperature. Optimal pH The crude chitosanase activity was measured at various pH values, using 80% deacetylated chitosan as a substrate. The reaction mixtures consisting of 1.0 ml of 1% soluble
Page 1 and 2:
April - June 2007 Volume 41 Number
Page 3 and 4:
KASETSART JOURNAL NATURAL SCIENCE T
Page 5 and 6:
Kasetsart J. (Nat. Sci.) 41 : 205 -
Page 7 and 8:
harvesting time which started from
Page 9 and 10:
y Chen and Paull (2000). Figure 1 a
Page 11 and 12:
pineapple fruit allowed the accumul
Page 13 and 14:
Page 15 and 16:
Kasetsart J. (Nat. Sci.) 41(2) 215
Page 17 and 18:
Cluster analysis Cluster analysis u
Page 19 and 20:
Page 21 and 22:
Table 3 (Continued) 1 2 3 4 5 6 7 8
Page 23 and 24:
Page 25 and 26:
CONCLUSION AFLP markers classified
Page 27 and 28:
Page 29 and 30:
difference of soil texture used in
Page 31 and 32:
under wet soil condition planting.
Page 33 and 34:
tropics. Following the expansion, w
Page 35 and 36:
sealed in a plastic box with 1 cm w
Page 37 and 38:
moisture content increment after so
Page 39 and 40:
Frequency Frequency 30 20 10 0 20.0
Page 41 and 42:
School, Kasetsart University. The a
Page 43 and 44:
for combining ability of lines (Lam
Page 45 and 46:
selective mass sibbing within indiv
Page 47 and 48:
Although most of S 2-interfamily hy
Page 49 and 50:
composite sets as used in this stud
Page 51 and 52:
Page 53 and 54:
days. The numbers of calli producin
Page 55 and 56:
the report of Ching (1982) showing
Page 57 and 58:
clearly amplified bands generated b
Page 59 and 60:
caused by either the recombination
Page 61 and 62:
Kuhlmann, V. and B. Foroughi-Wehr.
Page 63 and 64:
Xanthomonas fuscans subsp. aurantif
Page 65 and 66:
Page 67 and 68:
was collected and washed with 70% e
Page 69 and 70:
expected size was amplified with 35
Page 71 and 72:
eaction technique has been used for
Page 73 and 74:
Schaad, N.W., D. Opgenorth and P. G
Page 75 and 76:
MATERIALS AND METHODS Model descrip
Page 77 and 78:
Page 79 and 80:
calculated TF value calculated TF v
Page 81 and 82:
sincere gratitude and deep apprecia
Page 83 and 84:
1989). In monogastrics, the inclusi
Page 85 and 86:
of all LLS samples in this study we
Page 87 and 88:
Melbourne, Parkville, Victoria. Goe
Page 89 and 90:
considered responsible for low prod
Page 91 and 92:
Page 93 and 94:
mineral supplementation suggested b
Page 95 and 96: Body weight chane (kg/head) 32 30 2
Page 97 and 98: CONCLUSION AND RECOMMENDATION With
Page 99 and 100: 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: dose interleukin-2 therapy promotes
Page 149 and 150: enzyme and cells were maximized by
Page 151 and 152: of crude chitosanases. Fortunately,
Page 153 and 154: Relative activity (%) 100 80 60 40
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
Page 203 and 204:
Speed up 40 35 30 25 20 15 10 5 0 F
Page 205 and 206:
and discovery, resource selection a
show all

April - June 2007 - Kasetsart University

You also want an ePaper? Increase the reach of your titles

Delete template?

Save as template?