|Budget Amount *help
¥6,500,000 (Direct Cost : ¥6,500,000)
Fiscal Year 1993 : ¥1,000,000 (Direct Cost : ¥1,000,000)
Fiscal Year 1992 : ¥5,500,000 (Direct Cost : ¥5,500,000)
An endo-1, 4-beta-glucanase (EC 220.127.116.11) was purified to apparent homogeneity from the culture medium of poplar (Populus alba L.) cells by sequential anion-exchange, hydrophobic, and gel-filtration chromatography. The preparation of extracellular beta-glucanase was homogeneous on SDS-polyacrylamide gel electrophoresis (PAGE) and native PAGE.The molecular weight, as determined by SDS-PAGE was 50, 000, whereas that determined by gel filtration was 40, 000. The isoelectric point (pI) was 5.5. The purified enzyme catalyzed the endohydrolysis of carboxymethylcellulose with a pH optimum of 6.0 and a Km of 1.0 mg ml^<-1>. The enzyme specifically cleaved the 1, 4-beta-glucosyl linkages of carboxymethylcellulose, swollen cellulose, lichenan and xyloglucan, although the last was hydrolyzed more slowly than the other tested substrates. The activity of the endo-1, 4-beta-glucanase increased up to the early stage of the mid-logarithmic phase of growth and then decreased rapidly, suggesting that the b-glucanase is induced before cell development.
To elucidate the molecular biology of poplar endo-1, 4-beta-glucanase with biochemical and physiological changes, we have isolated a cDNA clone encoding the endo-1, 4-beta-glucanase. The amino termini of extracellular endo-1, 4-beta-glucanase were sequenced. Oligonucleotides corresponding to the protein sequences were used to identify a clone from a poplar cell cDNA library at the early stage of mid-logarithmic phase of growth. The cDNA encodes a precursor polypeptide which is subsequently processed to a mature protein containing 467 amino acids. The precursor incorporates the N-terminal signal sequence (containing 27 amino acids) which probably contains information relevant to the targeting of the enzyme to the cell wall.