Document Detail
Hemicellulases from extremely thermophilic microorganisms as wood pulp bleaching aids
Abstract/OtherAbstract :
Restricted Item. Print thesis available in the University of Auckland Library or available through Inter-Library Loan. The Kraft pulping process is the predominant method used for the production of paper. This process involves the high temperature cooking of wood fibres in alkali, followed by the extraction of the coloured lignin components of wood using chlorine. The treatment of wood pulp with hemicellulases has been shown to be a feasible way to enhance the extraction of lignin in the quest for environmentally benign methods of paper manufacture. This enzymatic pre-treatment method leads either to higher final paper brightness or to reductions in the consumption of bleaching chemicals. The resultant minimisation of the consumption of chlorine-based bleaching agents which give rise to the formation of toxic chlorinated organic compounds is particularly important. The enzymes should function optimally in the high temperature and alkali conditions of pulp after cooking in order for enzymes to replace chlorine with minimal changes to the process conditions. This thesis describes the isolation, characterisation and expression of three hemicellulase genes from extremely thermophilic organisms. These genes code for enzymes which may be especially suited for use as Kraft pulp bleaching aids. The hemicellulase gene manA from the thermophilic anaerobe Caldicellulosiruptor saccharolyticus was previously characterised by a member of this research group, and shown to code for a 346 amino acid mannanase. However, evidence suggested that the sequence of manA had been misinterpreted, and that the gene coded for a larger multidomain enzyme. I describe here the correction of the sequence of manA, and show that the gene codes for an enzyme of 1332 amino acids in length which has an amino-terminal mannanase domain, a carboxy-terminal domain which can hydrolyse both cellulose and xylan, and two central cellulose binding domains (CBDs). Each of these four domains is separated from the next by long stretches of proline and threonine residues (called PT-linkers). This multidomain structure has been found to be a common feature for other cellulolytic enzymes from C. saccharolyticus, and the general structure of: catalytic domain – PT linker - CBD - PT-linker - CBD - PT-linker - catalytic domain, is unique to enzymes from this organism. A homolog of the mannanase domain of ManA, was trialed for its ability to release reducing sugars from Pinus radiata Kraft pulp. A gene from an organism related to C. saccharolyticus isolated from an alkaline pool was also studied. A genomic library was constructed for the thermophilic anaerobe Caldicellulosiruptor sp. Rt8B.4. It was expected that the secreted hemicellulases from this organism would function optimally in alkaline conditions. The Rt8B.4 genomic library was screened for the expression of thermophilic hemicellulase genes, and a mannanase gene, manA, was isolated and sequenced. The manA gene was found to code for a putative 911 amino acid multidomain enzyme with a carboxy-terminal mannanase domain and two amino-terminal domains of unknown function. The DNA coding for the mannanase domain of ManA was isolated and expressed in a controlled expression vector. However, the enzyme was found to be active across a very narrow pH range, and was not active at alkaline pH. The temperature optimum of the recombinant mannanase domain was only 65°C, which is lower than the 70°C temperature at which Rt8B.4 grows. Although not proven, evidence is provided which suggests that the mannanase domain of ManA requires the amino-terminal domains for activity at higher temperatures and pH. A genomic library was constructed for the extremely thermophilic anaerobe Dictyoglomus sp. Rt46B.1. This organism was isolated from a New Zealand geothermal pool. A recombinant phage expressing xylanase activity was recovered, and a portion of the insert was sequenced to show that it contained a gene encoding a xylanase, XynA, with a putative length of 352 amino acids. Homology comparisons showed that XynA is related to the family F group of xylanases. The temperature and pH optima of the recombinant enzyme were determined to be 85°C and pH 6.5 respectively. However, the enzyme was active across a broad pH range, with over 50% activity between pH5.5 and 9.5. In addition, it was shown to be a true endo-acting xylanase, capable of hydrolysing xylan to xylotriose and xylobiose, but it could not hydrolyse xylobiose to xylose. XynA was shown to hydrolyse the xylan component of Kraft pulp, and to improve the brightness of elemental chlorine free (ECF) bleached Kraft pulps from softwoods and hardwoods. The equivalent xylanase gene was isolated from the related bacterium Dictyoglomus thermophilum and DNA sequencing showed the genes are identical, and other evidence is given which supports the proposition of Patel et al. (1987)who suggested that Rt46B.1 was a strain of D. thermophilum. A second xylanase gene was also isolated from Rt46B.1 using consensus PCR techniques. This gene, xynB, codes for a xylanase which is related to the family G group of xylanases. During the course of this work, a PCR-based method was developed which uses consensus oligonucleotide primers to facilitate the isolation of family F and family G xylanase genes. Consensus primers were designed to bind to DNA coding for groups of amino acids which are highly conserved in xylanases from a broad range of bacterial species. Preliminary trials have shown that this method of gene isolation may prove to be useful for the rapid identification and isolation of xylanase genes from culturable and unculturable thermophilic bacteria which produce enzymes suited to application in Kraft pulp bleaching.
Authors :
Gibbs, Moreland David
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Publication Detail :
Publisher :  ResearchSpace@Auckland     Type :  Thesis     Format :  -    
Date Detail :
2007-07-13, 2007-07-13, 1995
Subject :
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Coverage :
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Relation :
UoA609451
Source :
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Copyright Information :
Restricted Item. Print thesis available in the University of Auckland Library or available through Inter-Library Loan.
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Languages :  en, en    
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