深渊沉积物中盐单胞菌(Halomonas sp.)NyZ771菌株的分离培养及其降解芳香酸的研究

【背景】海洋是地球上最大的碳库,也是地球生物最大的栖息地。在这个庞大的生态系统中拥有多种多样的微生物,它们在全球碳循环中扮演了重要的角色。海斗深渊(海平面6 000 m以下的海域)由于高静水压和表层沉积汇集了大量有机质,形成了包含丰富生物资源的特殊生境。【目的】从马里亚纳海沟海斗深渊沉积物样品中分离培养能够以芳香酸为唯一碳源和能源生长的微生物,并研究其降解特性。【方法】通过模拟原位高压环境富集培养和常压条件下芳香酸选择性分离培养获得深渊来源的纯培养细菌,并根据形态学观察和16S rRNA基因序列系统发育分析进行种属鉴定,利用不同芳香酸进行培养和生物转化,通过HPLC和LC/MS鉴定芳香酸代谢中间产物。【结果】从马里亚纳海沟6 300 m沉积物样本中分离获得了一株盐单胞菌(Halomonas sp.)NyZ771。该菌株能够利用苯甲酸和4-羟基苯甲酸作为唯一碳源生长。其代谢4-羟基苯甲酸的中间产物鉴定为原儿茶酸。【结论】从深渊沉积物样本分离得到一株能降解苯甲酸和4-羟基苯甲酸的盐单胞菌NyZ771,丰富了深渊来源的微生物资源,为今后研究深渊中微生物的芳香酸降解及海洋微生物驱动的碳循环提供了一定的理论基础。     

三氯卡班降解菌株Sphingomonas sp. YL-JM2C中多个邻苯二酚双加氧酶的功能

【目的】研究Sphingomonas sp.YL-JM2C菌株的生长特性,确定以三氯卡班作为碳源的生长情况。挖掘菌株YL-JM2C潜在的邻苯二酚1,2-双加氧酶及邻苯二酚2,3-双加氧酶基因,在大肠杆菌(Escherichia coli)中异源表达邻苯二酚双加氧酶基因并研究其酶学性质。【方法】优化S.sp.YL-JM2C菌株以三氯卡班作为碳源时的培养条件,并利用全自动生长曲线测定仪测定菌株生长情况,绘制生长曲线。通过生物信息学方法挖掘潜在的邻苯二酚双加氧酶基因,并分别在Escherichia coli BL21(DE3)中进行异源表达,通过AKTA快速纯化系统纯化蛋白,分别以邻苯二酚、3-和4-氯邻苯二酚为底物检测重组蛋白的酶学特性。【结果】菌株在pH为7.0-7.5时生长最优。在以浓度为4-8 mg/L的三氯卡班做为底物时,菌株适宜生长。当R2A培养基仅含有0.01%酵母提取物和无机盐时,加入终浓度为4 mg/L的三氯卡班可促进菌株生长。挖掘到6个潜在的邻苯二酚双加氧酶基因stcA1、stcA2、stcA3、stcE1、stcE2和stcE3,表达并通过粗酶液分析证明其中5个基因stcA1、stcA2、stcA3、stcE1和stcE2编码的酶均具有邻苯二酚双加氧酶和氯邻苯二酚双加氧酶的活性;纯化酶的底物范围研究揭示了StcA1、StcA2和StcA3均属于Ⅱ型邻苯二酚1,2-双加氧酶,StcE1和StcE2为两个新型邻苯二酚2,3-双加氧酶;它们酶动力学分析研究证明了5个酶对邻苯二酚的亲和力和催化效率最高,4-氯邻苯二酚次之。【结论】在同一菌株中发现了5个具有功能的邻苯二酚双加氧酶基因,stcA1、stcA2和stcA3编码的酶均属于Ⅱ型邻苯二酚1,2-双加氧酶,stcE1和stcE2为两个新型邻苯二酚2,3-双加氧酶编码基因。5个酶均具有催化邻苯二酚和氯邻苯二酚开环反应的功能,这为更好地理解微生物基因组内代谢邻苯二酚及其衍生物氯代邻苯二酚基因的多样性奠定了基础。     

嗜盐古菌Haloferax sp. D1227中超氧化物歧化酶功能鉴定及其增强细菌耐盐性的研究

【背景】细菌、酵母或植物来源的超氧化物歧化酶(Superoxide dismutase,SOD)编码基因在异源宿主中表达并提高宿主耐盐性的研究已有一些报道,其异源宿主也多为植物,而古菌来源的超氧化物歧化酶编码基因在细菌中成功表达并提高其耐盐性的研究尚无报道。【目的】寻找嗜盐古菌Haloferax sp.D1227中的超氧化物歧化酶编码基因并鉴定其功能,将其在4-硝基苯酚降解细菌Burkholderia sp.SJ98中表达,研究该古菌的超氧化物歧化酶对菌株SJ98耐盐性和降解4-硝基苯酚功能的影响。【方法】通过生物信息学方法寻找嗜盐古菌D1227中潜在的超氧化物歧化酶编码基因,利用表达载体p ET-28a和广泛宿主载体p BBR1MCS-2将其分别在E.coli BL21(DE3)和4-硝基苯酚的降解菌株SJ98中异源表达,检测细胞抽提液和纯化蛋白的超氧化物歧化酶比活力。分别以葡萄糖和4-硝基苯酚为碳源,在M9培养基和添加500 mmol/L Na Cl(Na Cl含量约3%)的M9培养基中分别培养细菌SJ98的重组菌株和空载体重组菌株,利用全自动生长曲线分析仪和高效液相色谱等方法检测重组菌株的生长能力和对4-硝基苯酚的降解能力。【结果】通过生物信息学分析,在嗜盐古菌D1227基因组中发现了潜在的超氧化物歧化酶编码基因sod A,其在E.coli BL21(DE3)和菌株SJ98中分别异源表达均具有超氧化物歧化酶活力[细胞抽提液的比活力分别为21.07±0.02 U/mg和84.56±0.16 U/mg,从BL21(DE3)菌株纯化的蛋白Sod AD1227比活力为179.46±3.43 U/mg]。在添加500 mmol/L Na Cl的M9培养基中培养时,以葡萄糖为碳源,重组菌株SJ98[p BBR-sod A]仍可正常生长,而空载体对照菌株SJ98[p BBR1MCS-2]几乎丧失了生长能力;以4-硝基苯酚为碳源,菌株SJ98[p BBR-sod A]保持了利用底物生长和降解底物的能力,而菌株SJ98[p BBR1MCS-2]的生长和降解能力几乎丧失。用软件Phyre2模拟分析Sod AD1227的单体结构,该蛋白拥有Fe/Mn-SOD家族的典型结构特征,推测其属于Fe/Mn-SOD家族。【结论】本研究为利用古菌SOD对细菌进行改造以适应高盐环境中降解有机污染物的应用提供了潜在的可行性。     

深渊来源Citricoccus sp.strain NyZ702的分离培养及其降解4-羟基苯甲酸的特性

【背景】深渊沉积物中存在丰富的微生物细胞和活跃的微生物碳周转,因此,分离培养微生物资源对于认识深渊中的物质循环、能量代谢具有重要意义。芳香化合物在环境中广泛存在,基于组学分析揭示了深渊中具有潜在的芳香化合物代谢菌株,然而深渊来源的芳香化合物降解微生物纯培养和相关的代谢机理研究仍然缺乏。【目的】从马里亚纳海沟沉积物样本中分离培养具有降解芳香化合物能力的微生物,对其代谢途径、中间产物和降解酶活力进行初步鉴定。【方法】以4-羟基苯甲酸为唯一碳源对马里亚纳海沟沉积物样本中的降解菌株进行分离培养,结合形态观察、16S rRNA基因扩增与序列分析对菌株进行鉴定,通过底物生长实验验证其降解能力,通过高效液相色谱和超高效液相色谱-飞行时间质谱联用仪初步鉴定全细胞生物转化中间产物,利用紫外分光光度计测定其粗酶液催化4-羟基苯甲酸的活力,进而推测菌株降解4-羟基苯甲酸的代谢途径。【结果】从深渊沉积物中分离培养获得一株好氧细菌,16SrRNA基因序列分析显示该菌株隶属于柠檬球菌属(Citricoccus),命名为Citricoccus sp. strain NyZ702。该菌株在LB固体培养基上经30°C培养4 d后呈柠檬黄色、不透明、表面光滑、边缘整齐、凸出于培养基表面、直径约为1-2 mm的圆形菌落。扫描电镜表明菌体呈球形,直径为0.4-0.6μm,无鞭毛结构。该菌株为耐盐菌,最适生长盐浓度范围为2%-8%(质量体积分数)。该菌株可利用4-羟基苯甲酸为唯一碳源进行生长,可转化4-羟基苯甲酸至中间产物原儿茶酸,推测该菌株通过原儿茶酸途径降解4-羟基苯甲酸。菌株NyZ702的粗酶液具有4-羟基苯甲酸单加氧酶活力,对4-羟基苯甲酸的催化反应需要还原型烟酰胺腺嘌呤二核苷酸磷酸(NADPH)作为辅因子。【结论】从深渊沉积物样本分离得到一株4-羟基苯甲酸降解菌Citricoccus sp. strain NyZ702,该菌株以原儿茶酸为中间代谢产物降解4-羟基苯甲酸,丰富了深渊来源的微生物菌种资源,为深渊中的芳香化合物降解研究提供了一定的理论基础。     

恶臭假单胞菌ND6菌株catA基因的克隆和表达及其儿茶酚裂解途径探讨

恶臭假单胞菌ND6菌株的萘降解质粒pND6-1中编码儿茶酚1,2-双加氧酶的catA基因在大肠杆菌中进行了克隆和表达,并研究表达产物的酶学性质。结果表明:酶的Km为0.019μmol/L,Vmax为1.434μmol/(min.mg);具有很好的耐热性,在50℃保温45min后仍能够保留酶活力的93.7%;Fe2 对酶活性有显著的促进作用,其比活力是对照反应的292%;酶对4-氯儿茶酚的催化活性非常低,属于Ⅰ型儿茶酚1,2-双加氧酶。以萘为底物生长时,ND6菌株的细胞提取液中既存在催化邻位裂解途径的儿茶酚1,2-双加氧酶活性,也存在催化间位裂解途径的儿茶酚2,3-双加氧酶活性。以苯甲酸、对羟基苯甲酸和苯乙酸为唯一碳源生长时,ND6菌株细胞提取液的儿茶酚1,2-双加氧酶活性远远大于儿茶酚2,3-双加氧酶活性。表明ND6菌株既能通过儿茶酚间位裂解途径降解萘,也能通过儿茶酚邻位裂解途径降解萘,而以苯甲酸、对羟基苯甲酸和苯乙酸为诱导物时只利用儿茶酚邻位裂解途径。     

恶臭假单胞菌ND6菌株catA基因的克隆和表达及其儿茶酚裂解途径探讨

恶臭假单胞菌ND6菌株的萘降解质粒pND6-1中编码儿茶酚1,2-双加氧酶的catA基因在大肠杆菌中进行了克隆和表达,并研究表达产物的酶学性质。结果表明:酶的K_m为0.019μmol/L,V_max为1.434μmol/(min.mg);具有很好的耐热性,在50℃保温45min后仍能够保留酶活力的93.7%;Fe²⁺对酶活性有显著的促进作用,其比活力是对照反应的292%;酶对4-氯儿茶酚的催化活性非常低,属于Ⅰ型儿茶酚1,2-双加氧酶。以萘为底物生长时,ND6菌株的细胞提取液中既存在催化邻位裂解途径的儿茶酚1,2-双加氧酶活性,也存在催化间位裂解途径的儿茶酚2,3-双加氧酶活性。以苯甲酸、对羟基苯甲酸和苯乙酸为唯一碳源生长时,ND6菌株细胞提取液的儿茶酚1,2-双加氧酶活性远远大于儿茶酚2,3-双加氧酶活性。表明ND6菌株既能通过儿茶酚间位裂解途径降解萘,也能通过儿茶酚邻位裂解途径降解萘,而以苯甲酸、对羟基苯甲酸和苯乙酸为诱导物时只利用儿茶酚邻位裂解途径。     

嗜麦芽寡养单胞菌PX1的降解芘特性及定殖效能

为丰富多环芳烃降解菌菌种库、降低农作物的污染风险,本研究对一株可高效降解多环芳烃(PAHs)的植物内生菌进行筛选鉴定,并初步探究其降解途径以及定殖效能。结果表明: 菌株PX1为嗜麦芽寡养单胞菌。该菌株对多环芳烃的降解具有广谱性,7 d几乎可彻底降解PAH无机盐培养基中的萘,在分别含有50.0 mg·L^-1菲、20.0 mg·L^-1芘、20.0 mg·L^-1荧蒽和10.0 mg·L^-1苯并[a]芘的培养体系中,对菲、芘、荧蒽、苯并[a]芘的降解率分别为72.6%、50.7%、31.9%和12.9%。选取芘作为PAHs模型研究菌株PX1的降解特性。酶活性试验表明,芘可诱导菌株PX1体内邻苯二甲酸双加氧酶、邻苯二酚-1,2-双加氧酶和邻苯二酚-2,3-双加氧酶的活性。在芘降解过程中检测到4,5-环氧化芘、4,5-二羟基芘、龙胆酸/原茶儿酸、水杨酸、顺-己二烯二酸/2-羟粘糠酸半醛、顺-2′-羧基苯丙酮酸、1-羟基-2-萘甲酸、水杨醛等中间产物。浸种定殖试验表明,菌株PX1可高效定殖到空心菜和小麦体内,显著促进空心菜和小麦生长,并能够将空心菜、小麦体内及其生长基质中的芘浓度分别降低29.8%～50.7%、52.4%～67.1%和8.0%～15.3%。表明菌株PX1主要通过“水杨酸途径”和“邻苯二甲酸途径”降解芘,且可以定殖到植物体内,促进植物生长。     

柴油食烷菌B-5及其卤代烷烃脱卤酶DadA对卤代化合物的降解

【目的】柴油食烷菌(Alcanivorax dieselolei)B-5是重要的石油降解菌。为研究其对卤代化合物的降解范围和降解机制,【方法】以不同的卤代化合物作为唯一碳源,观察菌株B-5在其中的生长情况;通过多重序列比对、系统发育分析和三维结构同源建模,分析该菌株基因组内一个假定的卤代烷烃脱卤酶(Haloalkane dehalogenase,HLD)DadA;利用大肠杆菌异源表达、纯化DadA,并测定了其对46个卤代底物的酶活。【结果】菌株B-5能够利用C3-C18链长范围的多种卤代化合物为唯一碳源生长;在系统进化树中,DadA相对独立于其他HLD-II亚家族成员,但具有典型的HLD-II亚家族的催化五联体残基;DadA确实具有脱卤活性,但该酶特异性高,底物范围明显小于其他已鉴定的HLDs,仅对1,2,3-三溴丙烷、1,2-二溴-3-氯丙烷和2,3-二氯-1-丙烯有脱卤酶活。【结论】因为DadA对很多B-5菌株可以利用做碳源的卤代底物没有脱卤酶活,所以推测B-5菌中可能还有其他脱卤酶参与了卤代烷烃的降解。菌株B-5及其卤代烷烃脱卤酶DadA在卤代烷烃污染物的生物降解方面具有应用潜力。     

环境有机碳源抑制食弧菌嗜盐噬菌弧菌(Halobacteriovorax vibrionivorans)的捕食生长

【背景】蛭弧菌类群(Bdellovibrio-And-Like Organisms,BALOs)的生长所需碳源主要来源于宿主菌,而环境中各类碳源对其生长的影响还有待探究。【目的】探究不同环境有机碳源对食弧菌嗜盐噬菌弧菌(Halobacteriovorax vibrionivorans)捕食生长的影响,为其后续捕食机制和微生物菌剂的应用研究提供理论基础。【方法】以溶藻弧菌(Vibrio alginolyticus)为宿主,采用96孔板测定细胞吸光度法和双层平板法测定不同糖类化合物、酵母提取物和胰蛋白胨对食弧菌嗜盐噬菌弧菌Y22捕食生长的影响,设置热致死宿主和活宿主、人工海水和Tris-HCl (25 g/L NaCl)培养体系的对比试验,结合菌株基因组信息分析,探索其可能的生长影响机制。【结果】食弧菌嗜盐噬菌弧菌Y22不具备转运外界糖类的相关基因,不能利用环境中的糖类物质作为碳源;宿主溶藻弧菌可以利用蔗糖、麦芽糖、甘露醇生长且产酸,降低人工海水混合培养体系的pH值,从而抑制菌株Y22的捕食;葡萄糖不仅可以改变人工海水混合培养体系的pH值,而且可影响宿主的细胞特性,抑制菌株Y22的捕食识别过程;宿主无法利用淀粉、α-乳糖生长,该类碳源不影响菌株Y22捕食生长。菌株Y22具有蛋白和多肽膜转运蛋白基因,可以通过分解并摄取宿主细胞蛋白质类物质以获取碳源和氮源。外源添加质量浓度为1-5 g/L酵母提取物和胰蛋白胨都会抑制菌株Y22捕食生长,抑制效果随浓度增加而加强,酵母提取物浓度超过4g/L、胰蛋白胨浓度超过5g/L,菌株Y22捕食现象几乎不可见,抑制效果最强。【结论】环境中糖类可通过影响宿主菌代谢产酸或细胞特性,从而影响食弧菌嗜盐噬菌弧菌的捕食生长。食弧菌嗜盐噬菌弧菌可以吸收利用环境中的蛋白或多肽,并因此抑制其捕食生长。该研究结果将为嗜盐噬菌弧菌捕食机制的进一步探究和嗜盐噬菌弧菌微生物菌剂的开发应用提供重要的理论基础。     

放射污染区古菌分离及多样性分析

【目的】研究放射污染区古菌多样性。【方法】放射污染区采集土样,采用甘油-精氨酸培养基(GJ)、甘油-天冬氨酸培养基(C1)、海藻糖-肌酸培养基(B7)、甘露醇-丙氨酸培养基(Z5)、干酪素-甘露醇培养基(CMKA)、壳聚糖-天冬酰胺培养基(F6)、甘露醇-酸水解酪蛋白培养基(GW1)、CM培养基、HP培养基和KC培养基10种分离培养基,采用梯度稀释法对古菌进行分离,将分离获得的菌株经形态特征,16S rRNA基因片段扩增及限制性内切酶酶切,选取酶切图谱中存在差异性的条带进行测序,最终通过序列比对,聚类分析,获得不同种类的古菌资源。【结果】从该土样中共获得了256株古菌,最终筛选出71株不同类型的古菌,这71株古菌均属于广古菌门,盐杆菌纲,盐杆菌目,盐杆菌科,分布于盐陆生菌属(Haloterrigena)、纳白菌属(Natrialba)、盐球菌属(Halococcus)、盐红菌属(Halorubrum)、盐长寿菌属(Halovivax)、纳线菌属(Natrinema)、盐碱球菌属(Natronococcus)、盐二型菌属(Halobiforma)、盐惰菌属(Halopiger)、盐池栖菌属(Halostagnicola)、富盐菌属(Haloferax)11个属,26个种,其中31株菌的16S rRNA基因序列与已有效发表菌株的序列相似性小于98%,Haloterrigena为该土样的优势菌属。对于分离效果较好的F6培养基采用了梯度营养成分的稀释,最终获得了19株古菌,这些菌株相互之间存在一定的差异性。【结论】本次分离获得了大量的古菌,表明放射污染区存在着较为丰富的古菌资源,其中蕴藏着多种新的物种类型,具有较大的研究价值。     

Aerobic metabolism of 4-hydroxybenzoic acid in Archaea via an unusual pathway involving an intramolecular migration (NIH shift)

A novel haloarchaeal strain, Haloarcula sp. strain D1, grew aerobically on 4-hydroxybenzoic acid (4HBA) as a sole carbon and energy source and is the first member of the domain Archaea reported to do so. Unusually, D1 metabolized 4HBA via gentisic acid rather than via protocatechuic acid, hydroquinone, or catechol. Gentisate was detected in 4HBA-grown cultures, and gentisate 1,2-dioxygenase activity was induced in 4HBA-grown cells. Stoichiometric accumulation of gentisate from 4HBA was demonstrated in 4HBA-grown cell suspensions containing 2,2'-dipyridyl (which strongly inhibits gentisate 1,2-dioxygenase). To establish whether initial 1-hydroxylation of 4HBA with concomitant 1,2-carboxyl group migration to yield gentisate occurred, 2,6-dideutero-4HBA was synthesized and used as a substrate. Deuterated gentisate was recovered from cell suspensions and identified as 3-deutero-gentisate, using gas chromatography-mass spectrometry and proton nuclear magnetic resonance spectroscopy. This structural isomer would be expected only if a 1,2-carboxyl group migration had taken place, and it provides compelling evidence that the 4HBA pathway in Haloarcula sp. strain D1 involves a hydroxylation-induced intramolecular migration. To our knowledge, this is the first report of a pathway which involves such a transformation (called an NIH shift) in the domain ARCHAEA:     

NADP-dependent isocitrate dehydrogenase from the halophilic archaeon Haloferax volcanii: cloning,sequence determination and overexpression in Escherichia coli

A gene encoding NADP-dependent Ds-threo-isocitrate dehydrogenase was isolated from Haloferax volcanii genomic DNA by using a combination of polymerase chain reaction and screening of a lambda EMBL3 library. Analysis of the nucleotide sequence revealed an open reading frame of 1260 bp encoding a protein of 419 amino acids with 45837 Da molecular mass. This sequence is highly similar to previously sequenced isocitrate dehydrogenases. In the alignment of the amino acid sequences with those from several archaeal and mesophilic NADP-dependent isocitrate dehydrogenases, the residues involved in dinucleotide binding and isocitrate binding are well conserved. We have developed methods for the expression in Escherichia coli and purification of the enzyme from H. volcanii. This expression was carried out in E. coli as inclusion bodies using the cytoplasmic expression vector pET3a. The enzyme was refolded by solubilisation in 8 M urea followed by dilution into a buffer containing EDTA, MgCl(2) and 3 M NaCl. Maximal activity was obtained after several hours incubation at room temperature.     

Degradation of 3-phenylpropionic acid by Haloferax sp. D1227

Haloferax sp. D1227, isolated from soil contaminated with highly saline oil brine, is the first halophilic archaeon to demonstrate the utilization of aromatic compounds (i.e., benzoic acid, cinnamic acid, and 3-phenylpropionic acid) as sole carbon and energy sources for growth. The degradation of 3-phenylpropionic acid in this strain was studied to examine the strategies utilized by Archaea to metabolize aromatic compounds. Based on our findings of (1) the extracellular accumulation of cinnamic acid, benzoic acid, 3-hydroxybenzoic acid, and gentisic acid in cultures of Haloferax D1227 grown on 3-phenylpropionic acid, (2) the presence of an 3-phenylpropionylCoA dehydrogenase, (3) the ATP, CoA, and NAD-dependent conversion of cinnamic acid to benzoylCoA, and (4) the presence of gentisate 1,2-dioxygenase, we propose that Haloferax D1227 metabolizes 3-phenylpropionic acid by initial 2-carbon shortening of the side chain to benzoylCoA via a mechanism similar to fatty acid β-oxidation, fol-lowed by aromatic degradation using a gentisate pathway. The upper aliphatic pathway from 3-phenylpropionic acid to benzoic acid is regulated separately from the lower gentisate pathway. Received: January 7, 1998 / Accepted: July 22, 1998     

Biochemical and molecular characterization of a ring fission dioxygenase with the ability to oxidize (substituted) salicylate(s) from Pseudaminobacter salicylatoxidans

The gene coding for a dioxygenase with the ability to cleave salicylate by a direct ring fission mechanism to 2-oxohepta-3,5-dienedioic acid was cloned from Pseudaminobacter salicylatoxidans strain BN12. The deduced amino acid sequence encoded a protein with a molecular mass of 41,176 Da, which showed 28 and 31% sequence identity, respectively, to a gentisate 1,2-dioxygenase from Pseudomonas alcaligenes NCIMB 9867 and a 1-hydroxy-2-naphthoate 1,2-dioxygenase from Nocardioides sp. KP7. The highest degree of sequence identity (58%) was found to a presumed gentisate 1,2-dioxygenase from Corynebacterium glutamicum. The enzyme from P. salicylatoxidans BN12 was heterologously expressed in Escherichia coli and purified as a His-tagged enzyme variant. The purified enzyme oxidized in addition to salicylate, gentisate, 5-aminosalicylate, and 1-hydroxy-2-naphthoate also 3-amino- and 3- and 4-hydroxysalicylate, 5-fluorosalicylate, 3-, 4-, and 5-chlorosalicylate, 3-, 4-, and 5-bromosalicylate, 3-, 4-, and 5-methylsalicylate, and 3,5-dichlorosalicylate. The reactions were analyzed by high pressure liquid chromatography/mass spectrometry, and the reaction products were tentatively identified. For comparison, the putative gentisate 1,2-dioxygenase from C. glutamicum was functionally expressed in E. coli and shown to convert gentisate but not salicylate or 1-hydroxy-2-naphthoate.     

Identification of a novel gentisate 1,2-dioxygenase from <Emphasis Type="Italic">Silicibacter pomeroyi</Emphasis>

A 1,125-bp long ORF encoding a novel gentisate 1,2-dioxygenase with two-domain bicupins was cloned from Silicibacter pomeroyi DSS-3 and expressed in Escherichia coli. The resulting product was purified to homogeneity and partially characterized. Non-reductive SDS-PAGE and gel filtration showed that the active recombinant gentisate 1,2-dioxygenase had an estimated molecular mass of 132 kDa, and reductive SDS-PAGE indicated an approximate size of 45 kDa. The enzyme thus appears to be a homotrimeric protein. This is in contrast to the homotetrameric or dimeric protein of the gentisate 1,2-dioxygenases that have been characterized thus far. The K (m) and K (cat)/K (m) for gentisate were 12 muM and 653 x 10(4) M(-1 )s(-1); the pI was 4.6-4.8. It was optimally active at 40 degrees C and pH 8.0.     

Catabolism of Substituted Benzoic Acids by Streptomyces Species

Four thermotolerant actinomycetes from soil, identified as Streptomyces albulus 321, Streptomyces sioyaensis P5, Streptomyces viridosporus T7A, and Streptomyces sp. V7, were grown at 45°C in media containing either benzoic acid or hydroxyl- and methoxyl-substituted benzoic acids as the principal carbon sources. Benzoic acid was converted to catechol; p-hydroxybenzoic, vanillic, and veratric acids were converted to protocatechuic acid; and m-hydroxybenzoic acid was converted to gentisic acid. Catechol, protocatechuic acid, and gentisic acid were cleaved by catechol 1,2-dioxygenase, protocatechuate 3,4-dioxygenase, and gentisate 1,2-dioxygenase, respectively. Dioxygenases appeared only in induced cultures. m-Hydroxybenzoic, m-anisic, and p-anisic acids were gratuitous inducers of dioxygenases in some strains. One strain converted vanillic acid to guaiacol.     

微生物降解4-羟基苯甲酸的研究进展

4-羟基苯甲酸(4HBA)是在自然界中广泛存在的芳香族化合物,也是很多天然产物和人工合成化合物的中间代谢产物。4HBA的代谢途径有原儿茶酸开环途径、脱碳酸途径和厌氧微生物的苯甲酰-CoA还原途径,以及尚未完全阐明的龙胆酸开环途径。从4HBA转化为龙胆酸的过程包含NIH重排反应步骤,本综述重点介绍NIH重排反应的研究进展并初步介绍了涉及4HBA降解过程中的酶。在本综述中,结合我们的研究工作介绍了一个嗜热Bacillus sp.B1菌株降解4HBA等芳香族化合物的代谢途径,最后对4HBA降解过程中的NIH重排反应研究进行了展望。     

芳香烃龙胆酸降解途径蛋白质组学的研究

芳香烃是一类重要的环境污染物,微生物降解是其主要的处理方法。研究显示降解过程中产生保守型和诱导型的各一组同工酶。目前,仅有保守型的龙胆酸加双氧酶（GDOI）及其下游片段被克隆。产碱假单胞菌NCIB9867（P25X）的突变株-SNZ28 GDOI被打断,在龙胆酸诱导的情况下,该突变株仍能检测到龙胆酸加双氧酶活性。采用二维蛋白电泳分析突变株SNZ28在有和没有龙胆酸诱导条件下的蛋白质表达差异。电泳结果显示了两者存在有15个蛋白点的差异。通过MALDI-TOF和Q—TOF分析,其中的12个蛋白质点与数据库中已知多肽片段有同源性。其中,P4点与青枯菌（Ralstonia species）龙胆酸1,2加双氧酶同源。该结果在蛋白质组学上证实了GDOII的存在。     

Gentisate pathway in Salmonella typhimurium: metabolism of m-hydroxybenzoate and gentisate

Abstract Salmonella typhimurium was shown to use the gentisate pathway to metabolize m -hydroxybenzoate and gentisate. m -Hydroxybenzoate hydroxylase and gentisate 1,2-dioxygenase were induced by growth on either gentisate or m -hydroxybenzoate. These enzymes were not detected when the bacteria were grown with glucose or glucose and either m -hydroxybenzoate or gentisate. However, both enzymes were induced when the bacteria were grown on succinate with either substrate. The maleylpyruvate isomerase required reduced glutathione and was irreversibly inhibited by N -ethylmaleimide.     

Gentisate 1,2-dioxygenase from Haloferax sp. D1227

Gentisate 1,2-dioxygenase from the extreme halophile Haloferax sp. D1227 (Hf. D1227) was purified using a three-step procedure. The enzyme was found to be a homotetramer of 42 000 ± 1000 Da subunits, with a native molecular weight of 174 000 ± 6000 Da. The optimal salt concentration, temperature, and pH for enzyme activity were 2 M KCl or NaCl, 45°C, and pH 7.2, respectively. The gene encoding Hf. D1227 gentisate 1,2-dioxygenase was cloned, sequenced, and expressed in Haloferax volcanii. The deduced amino acid sequence exhibited a 9.2% excess acidic over basic amino acids typical of halophilic enzymes. Four novel histidine clusters and a possible extradiol dioxygenase fingerprint region were identified. Received: November 19, 1997 / Accepted: May 12, 1998