Molecular cloning of xyloglucanase in suspension-cultured poplar cells

从白杨培养基中分离纯化出一种内切-1，4-β-葡聚糖酶(EC 3.2.1.4)。细胞通过顺序阴离子交换、疏水和凝胶过滤层析。胞外β-葡聚糖酶的制备在SDS-PAGE和天然PAGE上均一，SDS-PAGE测得的相对分子质量为50000，凝胶过滤测得的相对分子质量为40000。等电点为5.5。该酶催化羧甲基纤维素酶的最适pH为6.0，Km值为1.0 mg·m l~(-1)·g~(-1)。该酶特异地切割羧甲基纤维素、膨胀纤维素、地衣多糖和木葡聚糖的1，4-β-葡萄糖基，尽管最后一种酶的水解速度比其他受试底物慢。内切-1，4-β-葡聚糖酶的活性在对数生长期中期早期升高，然后迅速下降，表明β-葡聚糖酶在细胞发育之前就被诱导。为了阐明内切-1，4-葡聚糖酶的分子生物学和生理生化变化，我们克隆了编码内切-1，4-葡聚糖酶的基因。对胞外内切-1，4-β-葡聚糖酶的氨基末端进行了测序。与蛋白质序列相对应的寡核苷酸用于从生长中期对数生长期早期的杨树细胞cDNA文库中鉴定克隆。该基因编码一个前体多肽，随后被加工成一个含有467个氨基酸的成熟蛋白。前体含有N-末端信号序列(包含27个氨基酸)，它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.