Production, characterization, cloning and sequencing of yeast xylanase

Xylan is the second most abundant biopolymer, after cellulose. Two kinds of xylanolytic enzymes are generally involved in microbial hydrolysis of the main chain in xylan, endo-1,4- b -xylanase and b -xylosidase. Many different organisms, mostly bacteria and fungi produce large quantities of xylanases under variety of conditions. Although pentose-fermenting yeast species such as Pichia stipitis have been found that are capable of utilizing xylan, they are not strong xylanase producers. Commercial application of the yeasts in the processing of xylan is, therefore, hampered by low xylanase yield. In this study we have initiated a genetic and biochemical analysis of a xylanase from P. stipitis strain NRRL Y-11543. First, in order to improve the xylanolytic activity of P. stipitis , it was treated with a mutagenic agent (NTG). The most active mutant (NP54376) produced approximately twice as much xylanase as the parent strain. Some distinct differences were also recorded between the parental strain and the mutant strain NP54376, with regards to growth on various carbon sources and xylanase activity. The xylanase was purified about 45 fold with a 13% yield. The purified enzyme had a specific activity of 1075 U/mg of protein with optimal activity at about pH 4.8 and 45°C. It was a monomer with a molecular weight of 43 kDa.; We know little about mechanism of action of the P. stipitis xylanase. In view of this, genetic approaches and in particular investigation primary structure of xylanase was crucial. The xylanase (xynA ) gene of P. stipitis was cloned into pUC 19 in E. coli DH5 a F' and selected by growth on remazol brilliant blue-xylan as the sole carbon source. The nucleotide sequence of the P. stipitis xylanase gene had 1146 bp and encodes a protein of 381 amino acids with a molecular weight of 43,649 kDa. The sequence contains a 20-aa N-terminal signal sequence and 4 potential N-linked glycosylation sites. In the 5' noncoding region of the P. stipitis xynA gene a TATAAA element is found at nt --34, and two CCAAT potential recognition motifs were identified at nt --81 and nt --117. The E. coli clone secreted an inactive protein as determined by Western blotting using antibodies raised against P. stipitis xylanase. The cloned protein lacked natural glycosylation and this difference appeared to affect enzyme activity.