Wang W K, Kruus K, Wu J H
Department of Chemical Engineering, University of Rochester, New York 14627-0166.
J Bacteriol. 1993 Mar;175(5):1293-302. doi: 10.1128/jb.175.5.1293-1302.1993.
Clostridium thermocellum ATCC 27405 produces an extracellular cellulase system capable of hydrolyzing crystalline cellulose. The enzyme system involves a multicomponent protein aggregate (the cellulosome) with a total molecular weight in the millions, impeding mechanistic studies. However, two major components of the aggregate, SS (M(r) = 82,000) and SL (M(r) = 250,000), which act synergistically to hydrolyze crystalline cellulose, have been identified (J. H. D. Wu, W. H. Orme-Johnson, and A. L. Demain, Biochemistry 27:1703-1709, 1988). To further study this synergism, we cloned and sequenced the gene (celS) coding for the SS (CelS) protein by using a degenerate, inosine-containing oligonucleotide probe whose sequence was derived from the N-terminal amino acid sequence of the CelS protein. The open reading frame of celS consisted of 2,241 bp encoding 741 amino acid residues. It encoded the N-terminal amino acid sequence and two internal peptide sequences determined for the native CelS protein. A putative ribosome binding site was identified at the 5' end of the gene. A putative signal peptide of 27 amino acid residues was adjacent to the N terminus of the CelS protein. The predicted molecular weight of the secreted protein was 80,670. The celS gene contained a conserved reiterated sequence encoding 24 amino acid residues found in proteins encoded by many other clostridial cel or xyn genes. A palindromic structure was found downstream from the open reading frame. The celS gene is unique among the known cel genes of C. thermocellum. However, it is highly homologous to the partial open reading frame found in C. cellulolyticum and in Caldocellum saccharolyticum, indicating that these genes belong to a new family of cel genes.
嗜热栖热菌（Clostridium thermocellum）ATCC 27405可产生一种能够水解结晶纤维素的胞外纤维素酶系统。该酶系统包含一个总分子量达数百万的多组分蛋白质聚集体（纤维小体），这阻碍了对其作用机制的研究。不过，已鉴定出该聚集体的两个主要组分，即SS（分子量为82,000）和SL（分子量为250,000），它们协同作用水解结晶纤维素（J. H. D. 吴、W. H. 奥姆 - 约翰逊和A. L. 德梅因，《生物化学》27:1703 - 1709，1988年）。为进一步研究这种协同作用，我们利用一种简并的、含次黄苷的寡核苷酸探针克隆并测序了编码SS（CelS）蛋白的基因（celS），该探针的序列源自CelS蛋白的N端氨基酸序列。celS的开放阅读框由2241个碱基对组成，编码741个氨基酸残基。它编码了天然CelS蛋白的N端氨基酸序列以及两个内部肽段序列。在基因的5'端鉴定出一个假定的核糖体结合位点。一个由27个氨基酸残基组成的假定信号肽毗邻CelS蛋白的N端。预测分泌蛋白的分子量为80,670。celS基因包含一个保守的重复序列，该序列编码在许多其他梭菌属cel或xyn基因所编码的蛋白质中发现的24个氨基酸残基。在开放阅读框下游发现了一个回文结构。celS基因在嗜热栖热菌已知的cel基因中是独特的。然而，它与解纤维素梭菌（C. cellulolyticum）和嗜糖栖热放线菌（Caldocellum saccharolyticum）中发现的部分开放阅读框高度同源，表明这些基因属于一个新的cel基因家族。
