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Title of Thesis

Sibtain Ahmed
Institute/University/Department Details
Department of Chemistry/ University of Agriculture Faisalabad
Number of Pages
Keywords (Extracted from title, table of contents and abstract of thesis)
xylanases, trichoderma harzianum, anaerobic bacterium, hemicellulose, anacerboic xylanolytic bacterial species, thermosediminibacter terrestris

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

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S. No. Chapter Title of the Chapters Page Size (KB)
1 0 Contents
419.44 KB
2 1 Introduction 1
216.67 KB
  1.1 Objectives Of The Study 6
3 2 Review Of Literature 7
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  2.1 Trichoderma Harzianum , A Mesophilic Xylanase Producing Species Of The Genus Trichoderma 7
  2.2 Components Of The Cell Wall 8
  2.3 Hemicellulases 10
  2.4 An Overview Of Xylanases 16
  2.5 Microbial Production 21
  2.6 Multiplicity Of Xylanases 24
  2.7 Regulation Of Xylanase Gene Expression 26
  2.8 Cloning Of Bacterial Xylanase Genes 30
  2.9 Cloning Of Fungal Xylanase Genes 30
  2.10 Application Of ’-Xylanases 49
  2.11 Isolation Of A Novel Anacerboic Xylanolytic Bacterial Species 52
4 3 Materials And Methods 57
701.79 KB
  3.1 Section A : Xylanase : Induction, Gene Isolation, Cloning And Expression From T. Harzianum (3.1-3.9) 57
  3.2 Section B : Isolation Of A Novel Anaerobic Xylanolytic Bacterium(3.10-3.23) 77
5 4 Results And Discussion : Part-A: Xylanase From Trichoderma Harzianum : Enzyme Characterization, Gene Isolation , Cloning And Expression 83
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  4.1 Growth of Trichoderma harzianum 83
  4.2 Optimum pH for growth and xylanase production from Trichoderma harzianum 84
  4.3 Temperature and shaking 84
  4.4 Optimization of xylanase assay 85
  4.5 Effect of different carbon sources on xylanase induction 86
  4.6 Activity of cellulase from T. harzianum 93
  4.7 Effect of pH on crude xylanase activity 95
  4.8 Effect of temperature on crude xylanase activity 95
  4.9 Kinetic Parameters K m and V max 96
  4.10 Isolation of xylanase gene from T. harzianum 97
  4.11 Cloning in pUC18 expression vector 109
  4.12 Expression of xylanase gene in Flag expression system 113
  4.13 Conclusions and Future Prospective 117
6 5 Results And Discussion: Part-B Isolation Of A New Anaerobic Xylanolytic Thermophilic Bacterium From Trego Hot Spring, Nevada, Usa 119
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  5.1 Isolation 119
  5.2 Habitat 120
  5.3 Maintenance 120
  5.4 Morphology 120
  5.5 Physiological and Biochemical characterization 122
  5.6 pH range 122
  5.7 Temperature range 124
  5.8 Substrate utilization 127
  5.9 Utilization of yeast extract 129
  5.10 Doubling time and Fe (III) Reduction 130
  5.11 G+Cmol % 131
  5.12 Genomic DNA extraction 132
  5.13 16S rDNA Amplification 133
  5.14 Cloning and sequencing of 16S rDNA gene of JW/SA-NVT 134
  5.15 Phylogenetic analysis 135
  5.16 Deposition of JW/SA-NV T in Gennan culture collection (DSMZ) and American type culture collection (ATCC ) 137
  5.17 Description of Thermosediminibacter terrestris sp. nov. , 137
7 6 Summary 138
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8 7 Literature Cited 140
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