Isolation of Xylanase Genes from Extremely Thermophilic Bacteria as Potential Kraft Pulp Bleaching Aids

Abstract

Xylanases can be used in the pulp and paper industry to address the environmental concerns associated with the bleaching of kraft pulps with chlorine compounds. When used prior to chemical bleaching, xylanases can enhance the bleachability of kraft pulps, resulting in either a reduction in the consumption of chlorine-based bleaching chemicals (and consequently, a reduction in the environmental loading of chloro-organic compounds), or an increase in brightness of pulps bleached using oxygen-based bleaching chemicals. Commercial xylanase preparations are available to the pulp and paper industry, however, the optimal characteristics of the constituent enzymes in these preparations are poorly suited to the extreme conditions of temperature (60-80°C) and pH (>pHl0.0) encountered during kraft pulp bleaching. Consequently there is a need for xylanases which are both thermostable and alkalitolerant. The research described in this thesis involves the isolation of xylanase genes from extremely thermophilic bacterial strains encoding enzymes for potential uses in kraft pulp bleacing applications. The xylanase gene repertoires of six thermoanaerobic bacterial strains (TG453, TG456, TG457, TG479, TG53 and TG631) isolated from thermal springs around the central volcanic plateaux (New Zealand) were analysed by PCR with degenerate oligonucleotide primers. These strains, which were isolated using identical in situ enrichment techniques, were found to be very closely related bacteria of the genus Caldicellulosiruptor. Three family F xylanase genes (xynA, xynB and xynC) and a single family G xylanase gene (xynD) were identified from the priority strain. TG456. The remaining strains also contained these four xylanase genes, with the exception of TG453, which only possessed the three family F xylanase gene. Primers designed previously to amplify the xynA gene from TG453 could be used to amplify the respective xynA gene-fragments from the five remaining strains. The complete nucleotide sequences of the TG456 xynB, xynC and xynD genes were obtained by genomic-walking PCR. The XynB and XynC xylanases were both highly modular enzymes, incorporating at least l0 and 8 discrete domains each, and represented the largest xylanases reported to date. The XynD family G xylanase was a low molecular weight, two-domain enzyme. The TG456 XynA-D xylanases showed optimal activity at between 65-70°C, at pH6.0 to 6.5. The family G xylanase gene (xynB) from Dictyoglomus thermophilum strain Rt46B.l (isolated from thermal spring in Rotorua) was also identified and sequenced using consensus PCR and GWPCR techniques. The native Rt46B.l XynB xylanase consisted of a family G xylanase domain joined by a short linker to a non-catalytic domain, and was homologous in sequence and architecture to the XynD family G xylanase from Caldicellulosiruptor spp. TG456. However, Rt46B.l XynB was optimally active at 85°C, at pH6.5. Seven different XynB mutants (XynB1-7) were prepared in order to examine the contributions of the XynB N-terminal region and C-terminal domain on the characteristics of XynB. The XynBl-7 xylanases ranged in temperature optima from 70°C to 85°C, and in overall expression level from 0.05 to 25 XU/ml culture. Minor deletions of mature N-terminal peptide sequence had an additive effect on the reduction in thermostability of XynB. Full-length XynB enzymes were also less thermostable as compared to the isolated xylanase catalytic domains, at least in the absence of complex lignocellulosic substrates. Preliminary bleaching trials of XynB were performed at FRI, NZ Ltd. (Rotorua) and CSIRO, Forestry and Forest Products Division (Clayton, Victoria). These trials demonstrated that XynB could enhance the bleachability of Pinus radiata kraft-oxygen pulp to elemental chlorine-free (ECF) bleaching, and eucalypt kraft-oxygen pulp to both ECF and total chlorine-free (TCF) bleaching. In contrast, previous bleaching trials with the family F xylanase from Rt46B.l (XynA) suggested that this enzyme had a negligible effect on the ECF bleachability of pine and eucalypt kraft pulps. A comparative study on the characteristics of Rt46B.l XynA and XynB demonstrated that these enzymes differed with respect to their specific activities, hydrolysis products from kraft pulps (in terms of the oligomeric composition of the xylose-based hydrolysis products, and the amounts chromophoric materials solubilised from the pulp), and their enzyme-pulp interactions. These latter two differences may be associated with the enhanced bleachboosting effect of XynB over XynA.