CAJ | 학술논문

对降解多环芳烃的芽孢杆菌 Bacillus sp. NAPZ 进行了萘诱导蛋白的研究,以揭示此类细菌参与萘降解的关键蛋白组分及可能的降解机制.将NAPZ活化、培养、镜检,测量其在600 nm处的吸光值绘制生长曲线,选择指数生长期对NAPZ进行萘诱导,在特定时间收集菌体,浓缩后进行超声破碎,使蛋白释放,样品进行 SDS-PAGE 电泳和考马斯亮蓝染色,之后对电泳结果进行分析.形态学观察表明,NAPZ 为杆状细菌,革兰氏染色结果呈阳性,产芽孢.生长曲线显示培养8h 时细菌进入指数生长期,12 h时生长速率最大.对菌体破碎研究表明,超声破碎5 min(55 w,超声5 s,间隔10 s)效果较好.SDS-PAGE 电泳结果显示,与对照相比,诱导培养12 h 的 NAPZ 蛋白样品中12-1(Mr约53000)、12-2(Mr约51000)、12-4(Mr约36000)有明显表达量升高,另外出现一新增条带12-3(Mr约38000),其相对分子质量大小与诱导型龙胆酸双加氧酶一致,推测该菌可能通过龙胆酸途径降解萘,而不同于一般革兰氏阴性细菌的邻苯二酚途径.本研究对降萘芽孢杆菌 NAPZ 进行了生长曲线的测定和超声破壁条件的研究,并发现了诱导及非诱导条件下 NAPZ 蛋白表达量差异,初步推测高表达蛋白及新增蛋白与菌株对萘的降解利用有关.
대강해다배방경적아포간균 Bacillus sp. NAPZ 진행료내유도단백적연구,이게시차류세균삼여내강해적관건단백조분급가능적강해궤제.장NAPZ활화、배양、경검,측량기재600 nm처적흡광치회제생장곡선,선택지수생장기대NAPZ진행내유도,재특정시간수집균체,농축후진행초성파쇄,사단백석방,양품진행 SDS-PAGE 전영화고마사량람염색,지후대전영결과진행분석.형태학관찰표명,NAPZ 위간상세균,혁란씨염색결과정양성,산아포.생장곡선현시배양8h 시세균진입지수생장기,12 h시생장속솔최대.대균체파쇄연구표명,초성파쇄5 min(55 w,초성5 s,간격10 s)효과교호.SDS-PAGE 전영결과현시,여대조상비,유도배양12 h 적 NAPZ 단백양품중12-1(Mr약53000)、12-2(Mr약51000)、12-4(Mr약36000)유명현표체량승고,령외출현일신증조대12-3(Mr약38000),기상대분자질량대소여유도형룡담산쌍가양매일치,추측해균가능통과룡담산도경강해내,이불동우일반혁란씨음성세균적린분이분도경.본연구대강내아포간균 NAPZ 진행료생장곡선적측정화초성파벽조건적연구,병발현료유도급비유도조건하 NAPZ 단백표체량차이,초보추측고표체단백급신증단백여균주대내적강해이용유관.
In order to investigate the naphthalene (a polycyclic aromatic hydrocarbon, PAH) degrading proteins as well as the degra-dation mechanism of Bacillus which showed potency in biodegradation, a PAH degrading Bacillus sp. NAPZ was studied on the naphthalene induction related proteins. NAPZ were activated, cultivated and microscopic examined to monitor the growth dynamic curve with the A600nm of the culture. Then NAPZ in exponential growth period was induced with naphthalene. NAPZ cells in different cultivation time were sampled, concentrated and ultrasonicated to release proteins, respectively. After that, the soluble proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and shown by Coomassie brilliant blue stain-ing. The electrophoretogram was used for analysis. Morphological observation showed that strain NAPZ was rod shaped, Gram-positive, and spore forming. Bacterium growth curve showed strain NAPZ reached exponential growth period at 8 h, and the strain reached the max growth rate at 12 h. Cell disruption study showed that the optimum condition of cell disruption was ultrasonication for 5 min (55 w, ultrasound for 5 s, interval 10 s). The results of SDS-PAGE showed that soluble proteins of NAPZ induced by naph-thalene for 12h harbored 3 strips with higher expression than the controlled. The molecular weights of these proteins were 12-1 (Mr 53 000), 12-2 (Mr 51 000) and 12-4 (Mr 36 000) respectively. Besides, a new protein strip appeared in naphthalene induced NAPZ, 12-3 (Mr 38 000), with the same molecular weight to the induced-type gentisate dioxygenase, which implied strain NAPZ utilized naphthalene via gentisate pathway, not catechol pathway. In this research, the growth curve and cell disruption conditions of NAPZ were studied. The protein expression differences were investigated between the induced NAPZ and the controlled. It can be deduced that the higher or newly expressed proteins might be responsible for the utilization of naphthalene.