Molecular cloning of xyloglucanase in suspension-cultured poplar cells

从杨树（Populus alba L.）细胞培养基中通过阴离子交换、疏水和凝胶过滤层析纯化出一种内切- 1,4 - β -葡聚糖酶（EC 3.2.1.4）。细胞外β -葡聚糖酶的制备在sds -聚丙烯酰胺凝胶电泳（PAGE）和天然PAGE上均呈均匀性。SDS-PAGE测定的分子量为50000，而凝胶过滤测定的分子量为40000。等电点pI为5.5。纯化后的酶催化羧甲基纤维素的内水解，最适pH为6.0，Km为1.0 mg ml^<-1>。该酶特异地裂解羧甲基纤维素、膨胀纤维素、地衣和木葡聚糖的1,4 - β -葡萄糖键，尽管后者的水解速度比其他测试底物慢。内切- 1,4 - β -葡聚糖酶活性在细胞生长中期呈上升趋势，随后迅速下降，表明b-葡聚糖酶在细胞发育前就已被诱导。为了阐明杨树内- 1,4 - β -葡聚糖酶的分子生物学及其生化生理变化，我们分离了一个编码内- 1,4 - β -葡聚糖酶的cDNA克隆。测定了胞外内切- 1,4 - β -葡聚糖酶的氨基端序列。利用该蛋白序列对应的寡核苷酸，从杨树细胞cDNA文库中鉴定了生长中期早期的一个克隆。cDNA编码一个前体多肽，该前体多肽随后被加工成含有467个氨基酸的成熟蛋白质。前体包含n端信号序列（包含27个氨基酸），其可能包含与酶靶向细胞壁相关的信息。

An endo-1, 4-beta-glucanase (EC 3.2.1.4) 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.