Wang W K, Kruus K, Wu J H
University of Rochester, Department of Chemical Engineering, NY 14627-0166.
Appl Microbiol Biotechnol. 1994 Nov;42(2-3):346-52. doi: 10.1007/BF00902740.
Clostridium thermocellum ATCC 27405 produces an extremely complicated multi-component cellulase aggregate (cellulosome) highly active on crystalline cellulose. From the cellulosome, two subunits, CelS (or Ss; M(r) = 82,000) and CelL (or SL, CipA; M(r) = 250,000), have been identified as essential for crystalline cellulose degradation [Wu et al. (1988) Biochemistry 27:1703]. We have determined the DNA sequence of the celS gene from four cloned DNA fragments encompassing this gene [Wang et al. (1993) J Bacteriol 175:1293]. To express the entire celS gene in Escherichia coli, the celS structural gene was amplified by the polymerase chain reaction (PCR) employing the PCR primers corresponding to sequences flanking the desired gene. This PCR product (2.1 x 10(3) bases; 2.1 kb) was cloned into an E. coli expression vector pRSET B. Subsequent expression of the cloned gene resulted in a fusion protein (rCelS; M(r) = 86,000) as inclusion bodies. The rCelS protein was recognized specifically by an anti-CelS antiserum in a Western blot analysis. The inclusion bodies were purified and solubilized in 5 M urea. The refolded rCelS produced very little reducing sugar from carboxymethylcellulose. However, it showed a higher activity on the crystalline cellulose (Avicel) and an even higher activity on phosphoric-acid-swollen Avicel. These results indicate that the CelS is an exoglucanase.
嗜热栖热菌ATCC 27405产生一种极其复杂的多组分纤维素酶聚集体（纤维小体），对结晶纤维素具有高活性。从纤维小体中，已鉴定出两个亚基，即CelS（或Ss；分子量 = 82,000）和CelL（或SL、CipA；分子量 = 250,000），它们对结晶纤维素的降解至关重要[Wu等人（1988年）《生物化学》27:1703]。我们从包含该基因的四个克隆DNA片段中确定了celS基因的DNA序列[Wang等人（1993年）《细菌学杂志》175:1293]。为了在大肠杆菌中表达整个celS基因，使用与所需基因侧翼序列相对应的PCR引物，通过聚合酶链反应（PCR）扩增celS结构基因。该PCR产物（2.1×10³个碱基；2.1 kb）被克隆到大肠杆菌表达载体pRSET B中。随后克隆基因的表达产生了一种融合蛋白（rCelS；分子量 = 86,000），以包涵体形式存在。在蛋白质免疫印迹分析中，rCelS蛋白能被抗CelS抗血清特异性识别。包涵体被纯化并溶解于5 M尿素中。复性后的rCelS从羧甲基纤维素产生的还原糖很少。然而，它对结晶纤维素（微晶纤维素）显示出较高活性，对磷酸膨胀微晶纤维素的活性甚至更高。这些结果表明CelS是一种外切葡聚糖酶。
