Isolation and Expression of Genes for Hemicellulases from Extremely Thermophilic Culturable and Unculturable Bacteria

Abstract

Our current research has focused on the isolation of genes coding for thermostable hemicellulolytic enzymes that function under alkaline conditions, and we have isolated xylanase and mannanase genes from both culturable bacteria isolated after enrichment and non-culturable organisms from consortia obtained after enhanced growth. Genomic or biomass DNA is subjected to the polymerase chain reaction (PCR) using consensus primers for xylanases or mannanases that we have designed. The full length sequences are obtained either by genomic walking PCR or by using consensus amino- and carboxy-terminal PCR primers that allow the amplification of the entire gene in a form that allows direct cloning into an expression vector. A number of novel genes have been isolated and cloned. They have been expressed in mesophilic bacteria and fungi (such as yeast and Trichoderma), since the mesophilic proteins can be removed by a simple heating step, giving a facile 20-fold purification. Selected candidate enzymes have been tested for their ability to enhance the bleaching of conventional and oxygen-bleached Pinus radiata and Eucalyptus kraft pulp.