Hemicellulolytic microorganisms play a significant role in nature by recycling hemicellulose, one of the main components of plant polysaccharides. Xylanases (EC 3.2.1.8) catalyze the hydrolysis of xylan, the major constituent of hemicellulose. The use of these enzymes could greatly improve the overall economics of processing lignocellulosic materials for the generation of liquid fuels and chemicals. Recently cellulase-free xylanases have received great attention in the development of environmentally friendly technologies in the paper and pulp industry. In microorganisms that produce xylanases low molecular mass fragments of xylan and their positional isomers play a key role in regulating its biosynthesis. Xylanase and cellulase production appear to be regulated separately, although the pleiotropy of mutations, which causes the elimination of both genes, suggests some linkage in the synthesis of the two enzymes. Xylanases are found in a cornucopia of organisms and the genes encoding them have been cloned in homologous and heterologous hosts with the objectives of overproducing the enzyme and altering its properties to suit commercial applications. Sequence analyses of xylanases have revealed distinct catalytic and cellulose binding domains, with a separate non-catalytic domain that has been reported to confer enhanced thermostability in some xylanases. Analyses of three-dimensional structures and the properties of mutants have revealed the involvement of specific tyrosine and tryptophan residues in the substrate binding site and of glutamate and aspartate residues in the catalytic mechanism. Many lines of evidence suggest that xylanases operate via a double displacement mechanism in which the anomeric configuration is retained, although some of the enzymes catalyze single displacement reactions with inversion of configuration. Based on a dendrogram obtained from amino acid sequence similarities the evolutionary relationship between xylanases is assessed. In addition the properties of xylanases from extremophilic organisms have been evaluated in terms of biotechnological applications.
An extracellular endoglucanase (1,4-~-glucanohydrolas, EC 3.2.1.4) produced by Myceliophthora thermphila D-14 (ATCC 48104) has been purified to homogeneity by ammonium sulphate precipitation and two consecutive ion-exchange chromatographic steps on DEAE-Sephadex A-50 columns. The enzyme was purified 13.8-fold and was homogeneous by analytical PAGE and SDS-PAGE. It has a high apparent Mr, of about 100OOO. The pH and temperature optima for its activity were 4.8 and 65 "C respectively. The Km of the purified enzyme for CMC (sodium salt) was 3 mg ml-l. The enzyme displayed low activity toward salicin and p-nitrophenyl P-D-glucoside.The activity was enhanced in the presence of Na+, K+ and Ca2+ but effectively inhibited by Hg2+, Fe2+, Mg2+, Cu2+ and NHZ. Inhibition studies indicated that the enzyme may be a metalloprotein and/or that it requires metal ions for its optimum activity.
A recombinant plasmid construct, pLPX6.5, harbouring a 6.5 kb Hind III fragment of genomic DNA, from an alkalophilic, thermophilic Bacillus NCIM 59 and coding for xylanase activity, was electroporatically transformed into Bacillus subtilis MI 111. The expression of the recombinant xylanases was confirmed by cross-reactivity with antibodies raised against purified xylanase II (M r 15,800) from NCIM 59. However, as there were different xylan hydrolysis products from NCIM 59 and the host B. subtilis, the two xylanases appear to have different modes of action. Xylanase expression in the transformants was 6-fold higher than in the host. There was no significant enhancement in the expression of recombinant xylanases by adding xylan to the growth medium.
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