Spök A, Stubenrauch G, Schörgendorfer K, Schwab H
Institut für Biotechnologie, Technische Universität Graz, Austria.
J Gen Microbiol. 1991 Jan;137(1):131-40. doi: 10.1099/00221287-137-1-131.
Two pectinesterase-positive Escherichia coli clones, differing in expression levels, were isolated from a genomic library of Pseudomonas solanacearum. Both clones contained a common DNA fragment which included the pectinesterase-encoding region. The different expression levels found with the two clones could be ascribed to different positioning of the pectinesterase gene with respect to a vector promoter. Restriction analysis, subcloning, and further exonuclease deletion mapping revealed that the genetic information for pectinesterase was located within a 1.3 kb fragment. A protein of 41 to 42 kDa was expressed from this fragment. Nucleotide sequence analysis of the respective region disclosed an open reading frame of 1188 bp. The deduced polypeptide had a calculated molecular mass of 41,004 Da, which is consistent with the determined size of the pectinesterase protein. The predicted amino acid sequence showed significant homology to pectinesterases from Erwinia chrysanthemi and tomato. In cultures of E. coli clones up to 30% of total pectinesterase activity was transported into the medium. However, no significant pectinesterase activity could be detected in the periplasm.
从青枯假单胞菌的基因组文库中分离出两个果胶酯酶阳性的大肠杆菌克隆，它们的表达水平不同。两个克隆都包含一个共同的DNA片段，其中包括果胶酯酶编码区。两个克隆表达水平的差异可能归因于果胶酯酶基因相对于载体启动子的不同定位。限制性分析、亚克隆和进一步的外切核酸酶缺失图谱分析表明，果胶酯酶的遗传信息位于一个1.3 kb的片段内。从该片段表达出一种41至42 kDa的蛋白质。对相应区域的核苷酸序列分析揭示了一个1188 bp的开放阅读框。推导的多肽计算分子量为41,004 Da，这与所确定的果胶酯酶蛋白大小一致。预测的氨基酸序列与来自菊欧文氏菌和番茄的果胶酯酶具有显著同源性。在大肠杆菌克隆培养物中，高达总果胶酯酶活性30%的酶被转运到培养基中。然而，在周质中未检测到明显的果胶酯酶活性。
