【作者】 李华；

【导师】 邵宗泽；

【作者基本信息】 厦门大学 ， 细胞生物学， 2007， 硕士

【摘要】 “21世纪是手性化学发展的世纪”, 2001年诺贝尔化学奖就授予分子手性催化的主要贡献者。很多药物及生物活性物质是具有手性特征的,手性制药是目前医药行业的前沿领域和高速增长的领域,是各大制药企业竞争的热点之一,手性技术也是21世纪势必快速发展的领域。不仅仅是医药领域,食品,化工,微电子,材料,等众多领域都会因为手性技术的发展得到巨大改变。而目前所有手性催化剂中,将酮催化为具有手性的酯的最佳催化剂是在不动杆菌中发现的环己酮单加氧酶。本文从太平洋深海获得了一株可利用环己酮为唯一碳源进行生长的微球菌CN1,该菌不仅表现出除转化环己酮外的诸多性能。如不具有致病性,生长速度快,而且可耐受高浓度环己酮(>44%),并且在16%(V/V)的环己酮中生长最好。CN1可在12m M的重铬酸钾中生长,可利用高浓度氰化钾(250mM),还能产生植物生长素——吲哚乙酸。而CN1表现的最重要的功能是,它可以高转化率,高手性率的将极为廉价的非手性药物中间体双环[3,2,0] -2-双键-6-酮加氧转化为极为昂贵的手性酯,生成物与反应物的市场价格相差近1000倍。不同于已有报道,环戊酮却不能被CN1转化,表明CN1中的环己酮加氧酶有着较为特别的底物利用范围。环己酮可被CN1降解,环己醇可转化为环己酮后降解,环己醇又阻碍自身通过环己酮途径降解。环己酮在氧气不充足或环己酮较多的情况下,转化为环己醇。通过分析环己酮降解的中间代谢产物发现,环己酮有多种途径进行代谢,不仅可通过β-氧化途径降解,通过转化为环己醇后合成其他物质,还有可能直接水解。通过Southern杂交,结合基因组文库构建,获得了环己酮降解的完整基因簇。整个基因簇与具有高GC含量的红球菌,节杆菌的基因簇比较接近,而与目前研究的较深入的不动杆菌的基因簇无论从单个基因还是基因排布还是调控基因的类型上都差别很大。通过反转录结合PCR发现CN1的这个基因簇与阿拉伯糖操纵子有很高的相似性,其两个启动子紧挨着位于基因簇中部,并在环己酮诱导的情况下向相反的方向转录。利用GC-MS验证环己醇脱氢酶和环己酮加氧酶的转化功能,并且还发现在环己醇脱氢酶与环己酮加氧酶共表达的条件下,前者的活性远远高于后者。在大肠杆菌中异源表达了CN1的环己酮单加氧酶,发现大都形成了包涵体,通过稀释复性从而获得了有活性的酶。通过构建了CN1环己酮单加氧酶及所有BVMO家族蛋白的三维结构模型,结合系统发育分析,发现BVMO家族蛋白中心相当保守,发现并推测一条保守的多肽对于酶的性质影响很大,不认同一些学者对BVMO催化中心的见解,提出了BVMO蛋白有额外的底物结合区,和新的催化机理模型。此外,通过组合碳源,富集培养基,筛选培养基等要素,从印度洋热液口的一个泥样中分离到30个属的116株细菌,其中16株菌的16SRNA序列与网上比对的最高相似度低于或等于97%。通过GC-MS检测发现其中有19株可转化环己酮,随机选9株菌进行southern杂交验证,发现这些菌中确实含有环己酮加氧酶。与已报道的筛菌方法相比不仅开创了新的环己酮降解菌的高效筛选方法,而且在一个泥样中获得的环己酮降解菌的数目为全世界已发表的环己酮降解菌的总和的两倍。另外还研究了包括CN1的20株菌的降解范围和降解效率。通过扩增烷烃羟化酶alkB和细胞色素氧化酶P450,发现这两个基因与环己烷的降解存在正相关性。更多还原

【Abstract】 “The 21th century is the century of chiral chemistry”, the Nobel Price was donated to the main contributor of the asymmetric catalysis in 2001. Many medicament and biological active substance has the chiral character. Chiral-drug making is hot spot in which economic soars nowadays, as well as a hot spot that allures the pharmaceutical company to compete vehemently, The chiral technique certainly will also develop rapidly in the new century. Not only pharmaceutical industry will benefit from chiral technique ,kinds of fields like food, chemistry , microelectronic et.al will all change amazingly by this tequnique. While, maybe to anybody`s surprise ,the best asymmetric catalyst that transform cyclic ketone to chiral lactone is cyclohexanone monooxygenase in Acinetobacter.A cyclohexanone-degrading bacterium, Micrococcu letus CN1, was obtained by screening with cyclohexanone as the sole carbon source from the strains originally isolated from Pacific Ocean sediment. Except for cyclohexanone degradation ,it shew many other functional merit,for example ,it can grow rapidly at room temperature ,and the OD600 can reach about 15 after 48h`s culture, the optimal growth conditions were determined as 25℃-37℃,pH 6-9,salinity 6%. It can tolerate high concentration of cyclohexanone (44%), and grew most vigorously in medium containing 16.7% cyclohexanone (V/V), it can grow well in medium with 12mM potassium dichromate and 250mM potassium cyanide, respectively. Additionally, It can generate auxin (IAA).CN1 can transform cyclohexanol to cyclohexanone, and then degraded and mineralized cyclohexanone quickly. But different with previously reported cyclohexanone degraders, this strain can not degrade cyclopentanone, indicating that the cyclohexanone monooxygenase was responsible for the difference. Additionally, cyclohexanol can inhibit cyclohexanone degradation to some degree. On anaerobic conditions, cyclohexanone can also be transformed to cyclohexanol. All of these indicate that there is complex mechanism and subtle regulation involved in cyclohexanone degradation by CN1.The whole gene cluster for cyclohexanone degradation was obtained by Southern blotting combined with genomic library construction. The cluster is similar to the counterpart in Phodococcus and Arthrobacter ,who also have high content of GC. But, the cluster is very different from the counterpart in well studied Acinetobacter in gene sequence, gene pattern ,type of regulator .The cluster is similar to arabinose operator in ORF pattern, location and transcriptional direction of predicted promoter .The transformational functions of cyclohexanol dehydrogenase and cyclohexanone monooxygenase was tested by GC-MS. The activity of cyclohexanol dehydrogenase is much higher than that of cyclohexanone monooxygenase as co-expression . The cyclohexanone monooxygenase was expressed in E.coli alone via various expression vectors. All the protein expressed formed inclusion body , and the activity was obtained by dilution renature.The predicted three-dimensional models of CN1 cyclohexanone monooxygenase and all other monooxygenases of BVMO family were constructed. Combined with analysis of their homology, the center of BVMO was found very conservative ,and a conservative polypeptide was supposed to affect the activity of enzyme deeply, opinions of many scholars was denied, the extra locus for substrate combination was proposed, and a new model explaining catalysis mechanism emerged. Additionally,116 isolates of 30 genera were isolated from sediment of Indian Ocean by recombination of factors like carbon souce, culture medium for enrichment , medium plate for isolation et,al. 55 isolates from them were selected to transform cyclopentanone ,cyclohexanone,cyclohe- xanol and phenylacetone,ten bacteria of them can degrade cyclohexanone completely, six bacteria of them can degrade phenylacetone, five bacteria can transform cyclohexanone to cyclohexanol with no further degradation, five bacteria can transform cyclohexanol to cyclohexanone with no further degradation. The alkane hydroxylase alkB and cytochrome P450 were shown to positively correlate to cyclohexane degradation.更多还原