一株太平洋深海环己酮降解菌的筛选与功能研究

Screening and function analysis of a cyclohexanone-degrading bacterium CN1 from deep sea sediment

从太平洋深海菌株中筛选到一株能以环己酮为唯一碳源生长的微球菌（CN1）,其最适生长温度为25℃~37℃,最适生长pH8,最适生长盐度6%。该菌可耐受高浓度环己酮（〉44% V/V）,并且在16.7%（V/V）的环己酮中生长最好。CN1可转化环己醇成环己酮,环己酮又可被快速降解、矿化。这表明该菌含有环己醇脱氢酶并且很可能还含有环己酮单加氧酶。通过兼并PCR克隆到450bp环己酮单加氧酶基因片段,其编码的氨基酸序列不仅具有Baeyer-Villiger单加氧酶家族的保守序列,而且与节杆菌（Arthrobacter BP2）的环己酮单加氧酶同源性最高（80%）,而与研究较深入的不动杆菌（Acinetobacter sp.NCIMB 9871）单加氧酶的同源性仅为53%。由于目前报道的环己醇和环己酮的降解都是通过环己酮单加氧酶进行的,所以CN1的环己酮单加氧酶应该负责环己酮的降解。目前报道的环己酮降解菌都可以降解环戊酮,而CN1不可降解环戊酮,暗示了CN1的环己酮单加氧酶比较特别。另外,我们还首次发现在CN1中环己醇对环己酮的降解有一定的抑制作用。

Micrococcu luteus CN1 was found to be able to utilize cyclohexanone well from the strains originally isolated from Pacific Ocean sediment. The optimum conditions for its growth were determined as 25℃-37℃,pH 8,salinity 6%. It could survive in the medium with high concentration of cyclohexanone （〉44% V/V）, and grew most vigorously in medium with 16.7%（V/V） cyclohexanone. CN1 could transform cyclohexanol to cyclohexanone which could be further degraded and mineralized quickly. This indicated the presence of cyclohexanol dehydrogenase and probable presence of cyclohexanone monooxygenase. With degenerate PCR for cloning part of cyclohexanone monooxygenase gene,the DNA fragment of 450bp was gotten. Amino acid sequence analysis showed that it owned the conserved sequence of the Baeyer-Villiger monooxygenase family and had the highest homology of 80% with cyclohexanone monooxygenase from Arthrobacter sp.BP2, only 53% with that from Acinetobacter sp. NCIMB 9871 which had been the most deeply investigated. So far as we know, both cyclohexanol and cyclohexanone degradation resorted to cyclohexanone monooxygenase. So this gene should be responsible for cyclohexanone degradation in CN1. All the cyclohexanone-degraders previously reported could degrade cyclopentanone, but, CN1 did not degrade cyclopentanone. This indicated that cyclohexanone monooxygenase in CN1 was special. Additionally, it was found for the first time that cyclohexanol could inhibit cyclohexanone degradation to certain degree in CN1.