Cellulases from extremely thermophilic anaerobic bacteria: a comparison of several new cellulolytic isolates and the partial purification and characterisation of components of the cellulase complex from one isolate

Abstract

Forty-seven thermal pool sites in the central volcanic plateau of the North Island of New Zealand were sampled and enrichment cultures on crystalline cellulose screened for growth and cellulase activity. Eight anaerobic cellulolytic cultures resulted. Cellulolytic isolates were obtained from five of these cultures. All were obligate anaerobic non-sporing rods, staining Gram-negative. Most grew well at 75°C but none grew at 80°C. CMCase activity from some isolates exhibited exceptional thermal stability, with half-lives at 85°C in excess of 10 hours. Isolates from two sources, designated TP8 and TP10, were capable of completely hydrolysing crystalline cellulose and accumulated reducing sugar and avicelase activity in the growth medium. Levels of CMCase and avicelase in our most active culture supernatants were between 10 and 35% of those of Clostridium thermocellum grown on the same medium (but at 60°C). All produced acetic acid and ethanol in varying proportions, with a consequent tendency to decrease the pH of the growth medium. Cellobiose at 0.2%(w/v) repressed production of CMCase activity by most of the isolates by about 50%, but led to a slightly increased CMCase production by two of them. One of the isolates capable of completely hydrolysing crystalline cellulose (TP8.T6.3.3.1) was subjected to repeated subculture, reselection and reisolation to ensure its purity. The cell-free cellulase complex of this organism was found to produce partial solubilisation of a range of natural cellulosics, and also contained β-glucosidase activity. Ammonium sulphate precipation and adsorption onto crystalline cellulose were the best of the methods tested for concentrating the low levels of cellulolytic activity in the culture supernatant of TP8.T6.3.3.1. Adsorption onto cellulose was the cheapest alternative and also doubled as a preliminary fractionation and purification step. Three fractions, separated on the basis of their affinities for crystalline cellulose, were found to interact synergistically in hydrolysing crystalline cellulose when recombined. Conventional ion exchange and gel permeation column chromatography served only to subdivide the TP8.T6.3.3.1 cellulase complex into subcomplexes. These were fractionated further using preparative SDS-PAGE and IEF. The cellulose-binding component of the cellulase complex was found to contain an unexpectedly large number of active components (20 being a conservative estimate). Several of these component proteins exhibited clear differences in relative reactivities towards a range of cellulosic substrates. When rerun on SDS-PAGE, almost all produced single protein bands with mobilities indicative of molecular weights covering the range 20,000 to 140,000. Isoelectric points were all in the range pH 4.5-5.0. However, narrow range isoelectric focussing (pH 4.5-5.0) produced further subdivision of these single bands. The bands produced by isoelectric focussing (7-10 per SDS-PAGE band) each bore a share of the endoglucanase activity. We were unable to individually characterize the component activities at this level, and it was not clear whether these multiple components were artefacts of the isoelectric focussing, distinct products of multiple genes, modified products of a relatively small number of genes or microheterogeneity due to ageing of the proteins.