Chapter 16 – Direct Cellulase Gene Amplification From Hot Spring Using the Guidance of 16S rRNA Amplicon Metagenomics

Abstract Thermostable cellulase plays an important role in the process of converting lignocellulosic waste to biofuel. A culture-dependent approach may not effectively discover new thermostable cellulase, hemicellulase, or lignin-acting enzymes, since many thermophiles are not culturable in general laboratory setups. Due to this limitation, culture-independent techniques—for instance, the metagenomic approach—may be a good technique for finding novel genes. This current chapter demonstrates a method to amplify complete genes from a hot spring complex metagenome without the need to isolate bacterium or perform a clonal library. To achieve this, one needs to perform sampling (water, sediment, or biofilm from hot spring), extract the environmental DNA material, and perform 16S rRNA amplicon sequencing using a next-generation sequencer. From the microbial population data, we can then identify the genera of interest and their cellulase genes, followed by the designing of specific or degenerate primers, and finally amplification of the genes of interest using conventional PCR. This methodology will likely be an economic and powerful approach to discover novel genes that are present in complex environments. This chapter also describes the challenges of using such an approach. Using this method, an interesting heat-tolerant enzyme for biomass degradation was cloned, expressed, and purified. The enzyme exhibited optimum activity at a pH of 5.5 and at 90°C, a much higher temperature than other known enzyme counterparts. This enzyme is highly active on complex polysaccharide carboxymethyl cellulose. This technique will be useful for any gene amplification from hot springs using the guidance of 16S rRNA amplicon metagenomics.