Kälin M, Neujahr H Y, Weissmahr R N, Sejlitz T, Jöhl R, Fiechter A, Reiser J
Institute for Biotechnology, Swiss Federal Institute of Technology, ETH-Hönggerberg, Zürich.
J Bacteriol. 1992 Nov;174(22):7112-20. doi: 10.1128/jb.174.22.7112-7120.1992.
A cDNA clone encoding phenol hydroxylase from the soil yeast Trichosporon cutaneum was isolated and characterized. The clone was identified by hybridization screening of a bacteriophage lambda ZAP-based cDNA library with an oligonucleotide probe which corresponded to the N-terminal amino acid sequence of the purified enzyme. The cDNA encodes a protein consisting of 664 amino acids. Amino acid sequences of a number of peptides obtained by Edman degradation of various cleavage products of the purified enzyme were identified in the cDNA-derived sequence. The phenol hydroxylase cDNA was expressed in Escherichia coli to yield high levels of active enzyme. The E. coli-derived phenol hydroxylase is very similar to the T. cutaneum enzyme with respect to the range of substrates acted upon, inhibition by excess phenol, and the order of magnitude of kinetic parameters in the overall reaction. Southern blot analysis revealed the presence of phenol hydroxylase gene-related sequences in a number of T. cutaneum and Trichosporon beigelii strains and in Cryptococcus elinovii but not in Trichosporon pullulans, Trichosporon penicillatum, or Candida tropicalis.
从土壤酵母皮状丝孢酵母中分离并鉴定了一个编码苯酚羟化酶的cDNA克隆。通过用与纯化酶的N端氨基酸序列相对应的寡核苷酸探针杂交筛选基于λ噬菌体ZAP的cDNA文库来鉴定该克隆。该cDNA编码一个由664个氨基酸组成的蛋白质。在cDNA衍生序列中鉴定出了通过对纯化酶的各种裂解产物进行埃德曼降解获得的多个肽段的氨基酸序列。苯酚羟化酶cDNA在大肠杆菌中表达，产生了高水平的活性酶。就作用底物范围、过量苯酚的抑制作用以及整个反应中动力学参数的数量级而言，大肠杆菌衍生的苯酚羟化酶与皮状丝孢酵母的酶非常相似。Southern印迹分析显示，在一些皮状丝孢酵母和贝氏丝孢酵母菌株以及埃利诺夫隐球菌中存在苯酚羟化酶基因相关序列，但在普鲁兰丝孢酵母、青霉状丝孢酵母或热带假丝酵母中不存在。
