Fontes C M G A, Gilbert H J, Hazlewood G P, Clarke J H, Prates J A M, McKie V A, Nagy T, Fernandes T H, Ferreira L M A
CIISA-Faculdade de Medicina Veterinária, Pólo Universitário do Alto da Ajuda, Rua Professor Cid dos Santos, 1300-477 Lisboa, Portugal1.
Department of Biological and Nutritional Sciences, University of Newcastle upon Tyne, Newcastle upon Tyne NE1 7RU, UK2.
Microbiology (Reading). 2000 Aug;146 ( Pt 8):1959-1967. doi: 10.1099/00221287-146-8-1959.
Hydrolysis of the plant cell wall polysaccharides cellulose and xylan requires the synergistic interaction of a repertoire of extracellular enzymes. Recently, evidence has emerged that anaerobic bacteria can synthesize high levels of periplasmic xylanases which may be involved in the hydrolysis of small xylo-oligosaccharides absorbed by the micro-organism. Cellvibrio mixtus, a saprophytic aerobic soil bacterium that is highly active against plant cell wall polysaccharides, was shown to express internal xylanase activity when cultured on media containing xylan or glucose as sole carbon source. A genomic library of C. mixtus DNA, constructed in lambdaZAPII, was screened for xylanase activity. The nucleotide sequence of the genomic insert from a xylanase-positive clone that expressed intracellular xylanase activity in Escherichia coli revealed an ORF of 1137 bp (xynC), encoding a polypeptide with a deduced M(r) of 43413, defined as xylanase C (XylC). Probing a gene library of Pseudomonas fluorescens subsp. cellulosa with C. mixtus xynC identified a xynC homologue (designated xynG) encoding XylG; XylG and xynG were 67% and 63% identical to the corresponding C. mixtus sequences, respectively. Both XylC and XylG exhibit extensive sequence identity with family 10 xylanases, particularly with non-modular enzymes, and gene deletion studies on xynC supported the suggestion that they are single-domain xylanases. Purified recombinant XylC had an M(r) of 41000, and displayed biochemical properties typical of family 10 polysaccharidases. However, unlike previously characterized xylanases, XylC was particularly sensitive to proteolytic inactivation by pancreatic proteinases and was thermolabile. C. mixtus was grown to late-exponential phase in the presence of glucose or xylan and the cytoplasmic, periplasmic and cell envelope fractions were probed with anti-XylC antibodies. The results showed that XylC was absent from the culture media but was predominantly present in the periplasm of C. mixtus cells grown on glucose, xylan, CM-cellulose or Avicel. These data suggest that C. mixtus can express non-modular internal xylanases whose potential roles in the hydrolysis of plant cell wall components are discussed.
植物细胞壁多糖纤维素和木聚糖的水解需要多种胞外酶的协同作用。最近，有证据表明厌氧细菌能够合成高水平的周质木聚糖酶，这些酶可能参与微生物吸收的小的木寡糖的水解。混合纤维弧菌是一种对植物细胞壁多糖具有高活性的腐生需氧土壤细菌，当在以木聚糖或葡萄糖作为唯一碳源的培养基上培养时，它表现出内部木聚糖酶活性。构建于λZAPII中的混合纤维弧菌DNA基因组文库被筛选木聚糖酶活性。来自一个在大肠杆菌中表达细胞内木聚糖酶活性的木聚糖酶阳性克隆的基因组插入片段的核苷酸序列揭示了一个1137 bp的开放阅读框（xynC），编码一个推导分子量为43413的多肽，定义为木聚糖酶C（XylC）。用混合纤维弧菌的xynC探测荧光假单胞菌纤维素亚种的基因文库，鉴定出一个编码XylG的xynC同源物（命名为xynG）；XylG和xynG与相应的混合纤维弧菌序列分别有67%和63%的同一性。XylC和XylG与第10家族木聚糖酶具有广泛的序列同一性，特别是与非模块化酶，并且对xynC的基因缺失研究支持了它们是单结构域木聚糖酶的观点。纯化的重组XylC的分子量为41000，并表现出第10家族多糖酶的典型生化特性。然而，与先前表征的木聚糖酶不同，XylC对胰蛋白酶的蛋白水解失活特别敏感且不耐热。混合纤维弧菌在葡萄糖或木聚糖存在下生长至指数后期，并用抗XylC抗体探测细胞质、周质和细胞包膜部分。结果表明，XylC不存在于培养基中，但主要存在于在葡萄糖、木聚糖、羧甲基纤维素或微晶纤维素上生长的混合纤维弧菌细胞的周质中。这些数据表明混合纤维弧菌能够表达非模块化的内部木聚糖酶，并讨论了它们在植物细胞壁成分水解中的潜在作用。
