EXPRESSION AND CHARACTERIZATION OF XYLANASES FROM TRICHODERMA HARZIANUM AND AN ANAEROBIC BACTERIUM

Sibtain, Ahmed (2007) EXPRESSION AND CHARACTERIZATION OF XYLANASES FROM TRICHODERMA HARZIANUM AND AN ANAEROBIC BACTERIUM. Doctoral thesis, University of Agriculture Faisalabad.

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Abstract

Xylan is the major component of hemicellulose and xylan should be fully utilized to improve the efficiencies of a biobased economy. Interest in xylanases from different sources has increased markedly in the past decade because of applications of these enzymes in the pulp and paper industry. Xylanases have received great attention in the development of environment friendly technologies in the paper and pulp industry. The use of xylanases could greatly improve the overall lignocellulosic materials for the generation of liquid fuels and chemicals. Trichoderma harzianum strain E58 produced extracellular xylanases, when grown in liquid medium cultures containing oat spelt xylan as inducer. Trichoderma harzianum E-58 was grown in Vogel's medium with different carbon sources such as glucose, maltose, carboxymethy1cellulose (CM C), wheat bran and xylan at 28°C with shaking at 150 rpm to check the induction of xylanase. Expression of xylanase was induced maximally by xylan and repressed by glucose. The pH and temperature optima of the crude xylanase were 5 and 60°C, respectively. The apparent Km and Vmax values of the crude xylanase using birchwood xylan as a substrate were 2 mg mL-1 and 3.35 µmol min-1 mg-1, respectively. Successful amplification of the xylanase gene was achieved by PCR and RT-PCR. The PCR product (750 bp) was inserted into pUC18 and pFLAG-ATS vector and transformed in E. coli for expression. Congo red staining showed the presence of clear zones of hydrolysis around colonies suggesting that xylan hydrolysis was obtained. pUC18 expression plasmid resulted in lower xylanase activity as compared to xylanase activity of T. harzianum. High level expression of re comb in ant xylanase (133.78 IU/mL) was achieved by using pFLAG-ATS vector. An anerobic, thermophilic xylanase-producing bacterium designated as JW/SA-NVT was isolated. DNA was extracted from the bacterium by using ultra clean Microbiol DNA Kit. 16S rDNA gene was amplified and sequenced. According to initial phylogenetic analysis by BLASTN search the closet relative of the strain JW/SA-NVT was Thermovenabulum ferriphilus Z9801T (A Y033493) showing 97% similarity. Thermosediminibacter oceani JW/IW-1228PT (A Y703478) and Thermosediminibacter litoriperuensis JW/ YJL-123-0-7/2T (A Y703479) showed 95% similarity respectively whereas Thermoanaerobacter ethanolics had shown 90% similarity. Several phylogenetic trees were constructed by changing he positions of reference organisms. In a phylogenetic tree constructed by neighbour joining method it was found that JW/SA-NVT had closest relative Thermosediminibacter oceani JW/IW-1228PT (A Y703478) and Thermovenabulum ferriphilus Z9801T (A Y033493). 16S rDNA sequence analysis clearly show strain JW/SA-NVT falls in genus Thermosediminibacter. JW/SA-NVT represents a novel group with Thermovenabu/um and Caldanaerobacter. JW/SA-NVT is the first species belonging to the genus Thermosediminibacter isolated from hot spring. The cells were rod shaped, 0.3- 0.5µm in diameter and 2.0-10.0 µm in length. The temperature range for growth of JW/SA-NVT at pH25C 8.5 was 54-74°C with an optimum around 69 to 72°C. The pH25C range for growth was from 6.5 to 8.75 with an optimum at 8.5. JW/SA-NVT is strictly anaerobic. Substrates utilized include xylose, glucose, mannnose, xylan, pyruvate, fructose, ribose, sucrose, casamino acids, manitol, tryptone, peptone, cellobiose, yeast extract. No indication of sulfate or Fe (III) reduction could be found. Yeast extract enhanced the growth but was not necessary for growth. The G + C mol % contents of JW/SA-NVT were 39.39 %. The type strain is JW/SA-NVT (DSM 18802; ATCC BAA-1454). On the basis of results described above, we propose that the strain JW/SA-NVT represents a novel species of genus Thermosediminibacter sp. Novo

Item Type: Thesis (Doctoral)
Uncontrolled Keywords: xylanases, trichoderma harzianum, anaerobic bacterium, hemicellulose, anacerboic xylanolytic bacterial species, thermosediminibacter terrestris
Subjects: Q Science > QD Chemistry
Depositing User: Muhammad Khan Khan
Date Deposited: 07 Sep 2016 07:26
Last Modified: 07 Sep 2016 07:26
URI: http://eprintstest.hec.gov.pk/id/eprint/1039

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