Khan A, Tewari R, Walia S
Department of Biological Sciences, Oakland University, Rochester, Michigan 48309-4401.
Appl Environ Microbiol. 1988 Nov;54(11):2664-71. doi: 10.1128/aem.54.11.2664-2671.1988.
Genes encoding 3-phenylcatechol dioxygenases were cloned from the chlorobiphenyl-degrading Pseudomonas putida strain OU83, using broad-host-range cosmid vector pCP13. Restriction enzyme analysis of DNA from 2,3-dioxygenase-positive chimeric cosmids showed DNA inserts ranging in size from 6.0 to 30 kilobases. The origin of the DNA insert in hybrid clones was established by using 32P-labeled hybrid clones (pOH101 and pOH810). A 2.3-kilobase HindIII fragment was common to two clones. The 2,3-dioxygenase from the parent P. putida strain, OU83, and the recombinant clones (pOH101 and pOH8101) showed similar characteristics as determined by isoelectric focusing and polyacrylamide gel electrophoresis. The 2,3-dioxygenase from the Escherichia coli recombinant cosmid showed a pI of 5.0, a Km of 14 microM, and broad substrate activity with catechol, 4-chlorocatechol, 4-methylcatechol, and 2,3-dihydroxybiphenyl.
利用广宿主范围黏粒载体pCP13，从降解氯代联苯的恶臭假单胞菌OU83菌株中克隆出编码3-苯基儿茶酚双加氧酶的基因。对来自2,3-双加氧酶阳性嵌合黏粒的DNA进行限制性酶切分析，结果显示DNA插入片段大小在6.0至30千碱基之间。通过使用32P标记的杂交克隆（pOH101和pOH810）确定了杂交克隆中DNA插入片段的来源。两个克隆共有一个2.3千碱基的HindIII片段。通过等电聚焦和聚丙烯酰胺凝胶电泳测定，亲本恶臭假单胞菌OU83菌株以及重组克隆（pOH101和pOH8101）中的2,3-双加氧酶表现出相似的特性。来自大肠杆菌重组黏粒的2,3-双加氧酶的等电点为5.0，米氏常数为14微摩尔，对儿茶酚、4-氯儿茶酚、4-甲基儿茶酚和2,3-二羟基联苯具有广泛的底物活性。
