The aim of this work was the identification and characterization of novel microbial enzymatic activities involved in lignin degradation and the isolation of their corresponding genes. The rumen is one of the environments where lignin seems to be degraded, owing to the presence of bacteria and fungi described as potential utilizers of lignin or its constituents. For these reasons this "biofermentor" was chosen as a model for our investigation. Rumen microorganisms showed ability to modify preferentially transferulic and p-coumaric acid. These phenolic acids are very important in the structure and stability of lignocellulose since they form a bridge between the lignin aromatic biopolymer and cell wall polysaccharides. They are the main aromatic compounds released from the plant cell wall during lignocellulose mineralization and they could represent an interesting renewable source for the industrial production of value-added aromatic substances. The enzyme which seems to be responsible for the modification of ferulic and pcoumaric acid in a wide range of rumen bacteria, and particularly in Bacillus species, was identified and purified in our laboratory from a Bacillus pumilus strain isolated from cow rumen fluid. This activity has been demonstrated to be a decarboxylase which is able to modify ferulic acid to 4-vinylguaiacol and p-coumaric acid to 4-vinylphenol, is inducible by the substrates and is specific for these two cinnamic compounds. The B. pumilus ferulate decarboxylase gene (fdc) was identified and isolated by its ability to promote ferulic and p-coumaric acid decarboxylation in Escherichia coli DH5a. The DNA sequence of the gene was determined and the recombinant enzyme produced in E. coli was purified and characterized. The deduced aminoacid sequence did not show homology to any known protein. This gene was expressed in a Saccharomyces cerevisiae strain under the control of yeast regulatory sequences and its product was more active toward ferulic and p-coumaric acid than the endogenous phenylacrylic acid decarboxylase of the host. The regulation of the fdc gene as well as the metabolic role of this decarboxylase function, are interesting questions which warrant further investigation.
|Titolo:||Ligninolytic Enzymes in the Ruminal Ecosystem: Molecular Analysis of the Bacterial Ferulic Acid Decarboxylase Gene and its Expression in Yeast|
|Relatore/i esterni:||Bruschi, Carlo Vito|
|Data di pubblicazione:||22-gen-1996|
|Appare nelle tipologie:||8.1 PhD thesis|