Metagenomic cellulases from Bacteria linking IL-tolerance, halotolerance and thermostability

Cellulose is a ubiquitous biopolymer consisting of β-1,4-linked D-glucose that can be used as a source for glucose and subsequent molecules, e.g. ethanol. The enzymatic hydrolysis has one major drawback: Crystalline cellulose is not soluble in water and the enzymes need an aquatic medium for efficient hydrolysis. Some ionic liquids (ILs) have been described to dissolve cellulose and would therefore provide an alternative reaction medium, if cellulases were functional in the presence of ILs. The aim of this work was to discover novel metagenomic cellulases that are active and stable in the presence of ILs. Therefore hydrolytic habitats were evaluated on activity and biodiversity level. Metagenomic libraries derived from the most auspicious habitats, elephant faeces (20,000 clones), a biogas plant sample (9,600 clones) and an enrichment culture inoculated with the shipworm Teredo navalis (3,600 clones), were screened with a function based approach. Altogether 14 cellulolytic clones were identified. The most active clones were sequenced via pyrosequencing and were evaluated for their performance in the presence of 30% (vol/vol) IL and after overnight incubation in 60% (vol/vol) IL. Though many of the clones lost almost their complete activity in the presence of the majority of ILs used in this study, two clones from the library constructed with DNA derived from the biogas plant (pFosCelA2 and pFosCelA3) and several clones from the library constructed with DNA derived from elephant faeces (among others pFosCelA84) were active and stable in the presence of ILs. Therefore the respective enzymes were purified and furthermore investigated. CelA2 showed highest identity (41%) to a glycoside hydrolase (GH) family 9 protein from Clostridium cellulovorans and comprised an additional Ig-like domain. CelA3 was closest related (60% identity) to a GH5 cellulase from Clostridium cellulolyticum and included a carbohydrate binding module while CelA84 did not comprise any additional domain and was 41% identical to a GH5 enzyme from Fibrobacter succinogenes. In addition to their IL-tolerance, all three enzymes shared the features of moderate to high thermostability and high halotolerance. For this reason, the enzymes were compared with two further thermostable cellulases; CelA was a GH12 endoglucanase from Thermotoga maritima and Cel5K was a metagenomic cellulase derived from the Avachinsky Crater in Kamchatka with highest identity (61%) to a GH5 enzyme from Thermus caldophilus. Both enzymes did not comprise any additional domain. CelA3 showed comparable activity in the presence of 30% (vol/vol) of all tested ILs (about 1 U/mg at 37°C and 1-2 U/mg at 70°C). Cel5K also showed activity in the presence of all tested ILs. The highest activity was observed in the presence of 30% (vol/vol) [EMIM][OTF] at 70°C (7.1 U/mg). CelA2, CelA84 and CelA were almost inactive in the chloride containing ILs [BMIM]Cl and [BMMIM]Cl though they were active in the presence of the other ILs. CelA84 showed about 0.5 U/mg in the other tested ILs at 37°C while CelA2 exhibited 6.4 U/mg in [EMIM][OTF] at 55°C. CelA exhibited with 11.4 U/mg in 30% (vol/vol) [EMIM][OTF] at 83°C the highest overall activity. Some of the cellulases also exhibited very high long term stability in the presence of ILs that has never been described before. At room temperature, CelA3 was stable over 4 days in the presence of 60% (vol/vol) of several ILs ([BMIM]Cl, [BMIM][OTF] and [BMMIM]Cl). Cel5K did not lose any activity after 4 days in 60% (vol/vol) [BMIM][OTF] and [EMIM][OTF]. In contrast, the three enzymes that were inactive in the presence of 30% (vol/vol) [BMIM]Cl and [BMMIM]Cl exhibited considerably lower long term stability in the presence of ILs. At the respective optimum temperatures, none of the enzymes was stable in the presence of any of the tested ILs after a pre-incubation period of 1 to 3 days. With the characterisation of the described cellulases it was shown that (A) metagenomics is an appropriate tool for the discovery of novel cellulases which are (B) very active and (C) long term stable in the presence of high IL concentrations, that (D) IL-tolerance depends on the reaction temperature, (E) halotolerance is linked to IL-tolerance, (F) thermostability is linked to IL-tolerance, (G) the phylogenetic derivation of cellulases does not correlate with IL-tolerance unless it is coherent with other properties related to IL-tolerance, (H) additional domains like carbohydrate binding modules and Ig-like domains are not necessary for IL-tolerance and that (I) each enzyme has to be investigated individually, as each enzyme has individual requirements for solvent properties.