Soong, Chee Leong (1996) Purification and Characterization of Endoxylanases Cloned from Fibrobacter Succinogenes S85 and Expressed in Escherichia Coli HB101. Masters thesis, Universiti Putra Malaysia.
The xylanase enzyme from Escherichia coli HB 101 containing the xylanolytic recombinant plasmid pBX6 was purified to homogeneity using ultrafiltration, DEAESepharose, CM-Sepharose and Sephadex 0-200 chromatography. Three xylanases, namely, Xyn A, Xyn BI and Xyn BII were obtained and were found to have the same molecular weight and optimum pH which were estimated to be 60.3 kDa and pH 7.0 respectively. The optimum assay temperature for both Xyn A and Xyn BI was 50°C, while for Xyn BII, it was 40°C. The xylanases were stable up to 45°C at pH 7.2 for 30 min. Approximately 80% of the enzyme activity was retained at the pH range of 5.0 to 8.0. The isoelectric point for Fraction A, Fraction BI and Fraction BII was 8.2, 8.5 and 5.5, respectively. The respective apparent K", and Vmax value on oat-spelt xylan was 12.2 mg/ml and 47.9 µmol xylose/min/mg protein for Xyn A; 10.8 mglml and 52.1 µmol xylose/min/mg protein for Xyn BI; 8.7 mg/ml and 54.2 µmol xylose/min/mg protein for Xyn BII. From the hydrolysis products of oat-spelt xylan analysed on thinlayer chromatography, the xylanases hydrolysed xylan through an endo-acting mechanism as no xylose, xylobiose or arabinose was detected. Thus, the xylanases were classified as an endoxylanase. The xylanases showed no activity toward carboxymethyl cellulose (CMC), crystalline cellulose (Avicel) and cellulose filter paper. The xylanases were not affected by potassium chloride, EDT A and EGTA at concentrations of 10 mM. Calcium chloride and magnesium chloride at the same concentrations enhanced the xylanase activities by 50%. Mercury chloride at 1.0 mM concentration completely inhibited the activities of all the purified xylanases. From zymogram analysis and characteristics of the xylanases investigated, multiplicity of xylanases in E. coli HB 101 (pBX6) was probably due to post-translational modification of a single gene product.
|Item Type:||Thesis (Masters)|
|Chairman Supervisor:||Associate Professor Abdullah Sipat, PhD|
|Call Number:||FSAS 1996 10|
|Faculty or Institute:||Faculty of Environmental Studies|
|Deposited By:||Nurul Hayatie Hashim|
|Deposited On:||03 Dec 2010 04:03|
|Last Modified:||11 Jun 2012 07:38|
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