Fibre Degrading Enzymes from Butyrivibrio proteoclasticus

Abstract

The rumen harbours a large and diverse microbial population that is responsible for the breakdown of plant material into smaller compounds, which can then be utilised by the animal. Butyrivibrio proteoclasticus is an anaerobic, Gram-positive bacterium originally isolated from the rumen of New Zealand cows. The entire genome of B. proteoclasticus has been sequenced. This revealed that a large proportion of the genome is devoted to polysaccharide degradation and reassembly1. Prior to the start of the research described in this thesis, 44 of the genes from the B. proteoclasticus genome annotated as being involved in fibre degradation had been cloned and expression of many has been tested. Two of these enzymes were expressed, purified and had had their 3D structures determined. Further characterisation of these two enzymes is presented here, with site directed mutagenesis used to probe the proposed mechanism for each. The results support the proposed catalytic mechanisms of both enzymes. Kinetic parameters were measured with model substrates for both enzymes and this allowed comparison with similar enzymes produced by other organisms. A further nine fibre degrading enzymes from B. proteoclasticus were chosen for structural and/or functional investigation. Preliminary structural investigation gave crystallisation conditions for one enzyme, Xsa43E. The structure of Xsa43E is presented along with functional analysis of the enzyme. Four residues were found to be important for catalysis, three previously identified catalytic acidic residues common to all GH43 enzymes and a fourth residue, a histidine that is important for pKa modulation of the catalytic acid. The large central ion present in the structure of Xsa43E is identified as calcium and shown to be important both for activity and structural stability.