红平红球菌LSSE8–1的培养及其对二苯并噻吩中硫元素脱除的影响

红平红球菌(Rhodococcus erythropolis)LSSE8-1是一株新分离的专一性脱硫菌，HPLC分析表明该菌能选择性地脱除二苯并噻吩(DBT)中的硫，最终代谢产物是2-羟基联苯(2-HBP). 适宜的初始pH为6.5~9；能利用多种碳源，其中以甘油为最佳，适宜的甘油浓度为8 g/L；2 g/L的乙酸铵是菌体生长和脱硫的合适氮源；二甲基亚砜是提高该菌细胞收率和脱硫比活性的有效硫源.     

质粒平均分配基因parDE对甲酸脱氢酶NADH再生系统稳定性的影响

将质粒平均分配基因parDE引入重组质粒pDK7-fdh(携带甲酸脱氢酶基因fdh)中,得到基因重组菌F6.考察了F6与未引入parDE基因的基因重组菌FY(含重组质粒pDK6-fdh)和F-1(含重组质粒pMAL-p2X-fdh)的质粒遗传稳定性及甲酸脱氢酶活性的差异.结果显示,F6传代160代后,仅有3%的菌质粒丢失,而基因重组菌F-1和FY(均只携带fdh基因)传代160代后,丢失质粒的菌分别占93%和78%.传代160代后,F6的甲酸脱氢酶比酶活为2.77 U/mg,比传代前降低1.77%,FY为2.02 U/mg,F-1与野生型菌株M5al相近,为1.33 U/mg.结果表明,引入parDE基因的基因重组菌质粒稳定性和甲酸脱氢酶活性均远高于未引入parDE基因的基因重组菌.     

表面活性剂对微生物脱除柴油中有机硫的影响

采用假单胞菌(Pseudomonas delafieldii)菌株R-8和红色红球菌(Rhodococcus erythropolis)菌株N1-36研究了加氢精制柴油脱硫工艺，两株菌脱除柴油中有机硫的活性相近. 添加表面活性剂能提高菌株对柴油的脱硫率；当Tween80存在、搅拌转速为250 r/min时，菌株R-8最高可脱除硫含量<300 mg/L的柴油中72%的有机硫；但当硫含量超过1000 mg/L时，微生物脱硫率极低.     

红球菌H-412生长细胞脱除正十六烷中的有机硫

利用实验室筛选的能够通过“4S”途径进行脱硫代谢的红平红球菌H-412(Rhodococcus sp.H-412)作为生物脱硫催化剂,二苯并噻吩(DBT)为含硫模型化合物,考察了红球菌H-412生长细胞脱除正十六烷中有机硫的脱硫性能.在油相体积分数20％的反应体系中,相同浓度的生长细胞比休止细胞的脱硫效率高.利用红球菌H-412生长细胞在油相分率20％的情况下,初始发酵液菌体浓度为0.048 g/L,初始DBT浓度为0.25 mmol/L的条件下分批培养的脱硫效率较高,达到了57％.红球菌H-412生长细胞在油相体积分率分别为0,5％,10％和20％条件下进行脱硫,DBT的消耗量及二羟基联苯(2-HBP)的生成量随油相体积增加而增大,20％条件下均达最大值,脱硫效率最高.细胞生长量随油相体积增大而减少,无油存在的条件下,细胞生长量最大.     

重组苏云金芽孢杆菌丙酮酸脱氢酶克隆及表达条件的优化

根据Bc14579丙酮酸脱氢酶基因序列设计引物,从苏云金芽孢杆菌BMB171菌株总DNA中扩增得到相应的丙酮酸脱氢酶基因DNA片段.将DNA片段装载到大肠杆菌构建pET-E1表达系统,再通过优化重组菌的表达条件获得有生物活性的丙酮酸脱氢酶.结果表明, pET-E1表达系统构建获得成功;优化的表达条件如下:培养基为TB+M9(体积比1∶1)、起始菌体密度OD600为4～5.5、诱导剂IPTG浓度为0.04 mmol·L-1.     

化学-酶法合成他汀类手性内酯结构单元

以红球菌Rhodococcus erythropolis AJ270催化的3-苄氧基-5-烯-己腈立体选择性水解反应产物(R)-3-苄氧基-5-烯-己酰胺为原料,通过简单的水解反应和碘内酯化反应,方便地获得了他汀类降脂药美伐他汀和洛伐他汀的手性内酯结构单元。产物的结构经过IR、MS、1HNMR、13C NMR及元素分析确证。     

红球菌YZ用于加氢柴油脱硫的影响因素研究

筛选了一株红球菌(Rhodococcus sp.)YZ.利用菌株YZ制备生物催化剂,研究了其脱硫率与脱硫活力及相关因素对红球菌YZ催化加氢柴油脱硫的影响.结果表明,菌株YZ能够在20 mmol·L-1 Na2SO4中保留94.6%的脱硫活性,而2-HBP的存在对菌株YZ的脱硫具有强烈的抑制作用,10 mmol·L-1 2-HBP能使其脱硫活性降低98.7%.菌株YZ在处理柴油15 h后还保留了90.6%的脱硫活性.利用YZ制备的生物催化剂平均每脱除1 mg S所消耗的葡萄糖质量变化不大,约0.36 g.     

重组甲酸脱氢酶对合成1,3-丙二醇的促进作用

在甘油厌氧发酵生产1,3-丙二醇的过程中,需要消耗还原当量NADH,NADH的有效供给决定了1,3-丙二醇的产量。本文从Candida boidinii基因组DNA中克隆了甲酸脱氢酶基因fdh,利用表达质粒pMAL TM-p2X-fdh转化到1,3-丙二醇生产菌 Klebsiella pneumoniae YMU2中,构建了具有NADH再生系统的重组菌Klebsiella pneumoniae F-1。在5 L发酵罐培养中,F-1合成1,3-丙二醇浓度和产率分别达到了78. 6 g·L-1 和1. 33 g·L-1·h-1,分别比YMU2提高了12. 5% 和41. 2%。根据F-1和YMU2菌株的主要代谢产物的生成情况比较了二者的代谢流分布。     

重组大肠杆菌不对称还原2-羟基苯乙酮合成(R)-苯基乙二醇

通过对近平滑假丝酵母(R)-专一性羰基还原酶（rCR）进行氨基酸序列分析并根据其序列的保守性设计PCR引物,以近平滑假丝酵母基因组为模板利用PCR技术得到目的基因rcr。该基因全长1011bp,共编码336个氨基酸。将该基因与表达载体pET21c连接后构建重组质粒pETRCR,并转化入Escherichia coli BL21（DE3）中进行表达。重组大肠杆菌具有(R)-专一性羰基还原酶活性,可催化不对称还原2-羟基苯乙酮合成(R)-苯基乙二醇。研究发现,在重组菌培养过程中,同添加IPTG诱导培养的情况相比,不添加IPTG诱导培养的酶活及不对称转化(R)-苯基乙二醇的效果较好。在反应过程中,转化48 h及在pH值 8～9的条件下更有利于不对称反应的进行。另外,较高的底物浓度会对反应产生抑制作用,通过提高重组菌细胞浓度可显著提高转化效果。在5 g/L的底物浓度条件下,利用0.3 g/mL重组菌细胞可还原2-羟基苯乙酮得到(R)-苯基乙二醇,产物光学纯度为95.5% e.e,产率为92.6%。     

无乳链球菌GapC蛋白和鼠伤寒沙门菌鞭毛蛋白的融合表达及纯化

目的构建无乳链球菌(Streptococcus agalactiae,S.agalactiae)gapC基因和鼠伤寒沙门菌(Salmonella typhimu-rium,S.typhimurium)fliC基因的融合基因gapC-fliC,并在大肠杆菌中表达融合蛋白。方法应用PCR技术分别扩增鼠伤寒沙门菌鞭毛蛋白fliC基因和无乳链球菌gapC基因片段,通过柔性肽(Gly4Ser)2编码序列将二者串联并克隆至质粒pQE-30上,构建重组原核表达质粒fliC-gapC-pQE30,转化E.coli XL1-Blue,IPTG诱导表达,并进行SDS-PAGE分析。表达产物经镍离子亲和层析柱纯化后,进行Western blot分析及融合蛋白活性检测。结果重组表达质粒fliC-gapC-pQE-30经双酶切和测序证明构建正确;表达的融合蛋白FliC-GapC相对分子质量约95 000,表达量占菌体总蛋白的58%,主要以包涵体形式存在,且可与小鼠抗鼠伤寒沙门菌和抗无乳链球菌多抗血清发生特异性反应,具有较好的甘油醛-3-磷酸脱氢酶(GAPDH)活性。结论成功构建了重组表达质粒fliC-gapC-pQE-30,并在E.coli XL1-Blue中表达了融合蛋白,为下一步动物免疫保护性试验的研究奠定了基础。     

脱硫菌Rhodococcus sp.LY822专一性脱硫活性及相关基因的研究

以专一性脱硫菌Rhodococcus sp. LY822质粒DNA为模板,利用已知的脱硫基因序列设计引物,PCR扩增得到了3个脱硫基因片段dszA, dszB和dszC. 构建了相应的表达质粒pETA, pETB和pETC,在转化大肠杆菌BL21(DE3)中表达,得到了dszA, dszB和dszC基因的融合表达产物. SDS-PAGE分析结果显示,蛋白带分子量分别为50, 40和45 kDa. 3种重组菌BL21(DE3)(pETA), BL21(DE3)(pETB)和BL21(DE3)(pETC)的无细胞粗提液(2 mL)的活性检测结果表明,DszC酶的粗提液反应30 min能够将0.02 mmol/L二苯并噻吩完全转化为二苯并噻吩砜,DszA酶的粗提液能够将0.01 mmol/L二苯并噻吩砜完全转化为羟基联苯亚磺酸盐,DszA和DszB酶的粗提液联合作用能够将0.01 mmol/L二苯并噻吩砜完全转化为2-羟基联苯,证明LY822对二苯并噻吩的降解符合专一性脱硫的"4S途径".     

红串红球菌脱硫酶的活性调节

研究了分别以葡萄糖或乙醇为唯一碳源对红串红球菌脱硫活性的效应．结果表明乙醇作为碳源可以促进红串红球菌3种脱硫基因的表达和脱硫酸的生产,并可促进辅酶FMNH2的生物合成,进而提高红串红球菌脱硫酸（dszC,A）的活性．     

手性醇脱氢酶与甲酸脱氢酶的融合蛋白体系的构建

为了在手性催化的同时实现辅酶的再生,利用源于红平红球菌Rhodococcus erythropolis的手性醇脱氢酶chiral alcohol dehydrogenase(READH)与来源于博伊丁假丝酵母Candida boidinii的甲酸脱氢酶formate dehydrogenase(CbFDH)构建了一系列具有双酶活性的融合蛋白体系。分别为：1）READH的N端与麦芽糖结合蛋白maltose binding protein(MBP)的C端融合;2）CbFDH的N端与MBP的C端融合;3）READH的N端与CbFDH的C端融合;4）READH的C端与CbFDH的N端融合。结果表明：在所有的融合策略中,READH的活性都受到了较大的影响。READH在N端融合在CbFDH的C端后活性最高,但是此时CbFDH不具有活性。相反,READH的C端与CbFDH的N端融合后,CbFDH具有最高的活性。在READH的C端与CbFDH的N端融合的策略下,两者都具有活性。表明通过合理设计具有双重功能的融合蛋白,有望能提高其催化效率,为新型辅酶再生系统的建立提供基础。     

An integrated biodesulfurization process,including inoculum preparation,desulfurization and sulfate removal in a single step,for removing sulfur from oils

BACKGROUND: A single‐stage reactor, in which the growth of bacterial culture, induction of desulfurizing enzymes, and desulfurization reaction are carried out in a single step, was adopted to investigate desulfurization of dibenzothiophene (DBT) at high cell densities. Rhodococcus erythropolis, IGTS8 was used as the biocatalyst. Optimal conditions for bacterial growth and DBT desulfurization were investigated. RESULTS: Optimization of fermentation conditions was necessary to obtain high cell densities including controlling accumulation of acetate. Under optimal operating conditions, the maximum optical density at 600 nm (OD₆₀₀) was measured to be 26.6 at 118 h of cultivation. When biodesulfurization of DBT in model oil with a high cell density culture of IGTS8 was investigated, accumulation of sulfate was found to limit the extent of desulfurization. A sulfate removal step was added to obtain a single‐stage integrated biodesulfurization process. Sulfate removal was achieved via an aqueous bleed stream and use of a separation unit to recycle the organic phase. CONCLUSION: A proof of principle of a complete system capable of biocatalyst growth, induction, desulfurization and by‐product separation was demonstrated. This system enables simplification of the biodesulfurization process and has potential to lower the operating cost of the bioprocess. Copyright © 2008 Society of Chemical Industry     

n-Hexadecane and pyrene biodegradation and metabolization by Rhodococcus sp. T1 isolated from oil contaminated soil

The high-molecular weight polycyclic aromatic hydrocarbons(PAHs) pyrene and typical long chain alkane nhexadecane are both difficult to degrade. In this study, n-hexadecane and pyrene degrading strain Rhodococcus sp. T1 was isolated from oil contaminated soil. Strain T1 could remove 90.81% n-hexadecane(2 vol%) and 42.79% pyrene(200 mg·L~(-1)) as a single carbon within 5 days, respectively. Comparatively, the degradation of pyrene increased to 60.63%, but the degradation of n-hexadecane decreased to 87.55% when these compounds were mixed. Additionally, identification and analysis of degradation metabolites of Rhodococcus sp. T1 in the above experiments showed that there were significant changes in alanine, methylamine, citric acid and heptadecanoic acid between sole and dual substrate degradation. The optimal conditions for degradation were then determined based on analysis of the pH, salinity, additional nutrient sources and liquid surface activity.Under the optimal conditions of pH 7.0, 35 °C, 0.5% NaCl, 5 mg·L~(-1) of yeast extract and 90 mg·L~(-1) of surfactant,the degradation increased in single or dual carbon sources. To our knowledge, this is the first study to discuss metabolite changes in Rhodococcus sp. T1 using sole substrate and dual substrate to enhance the long-chain alkanes and PAHs degradation potential.     

Biodesulfurization of dibenzothiophene using recombinant Pseudomonas strain

BACKGROUND: The sulfur content in crude oil available from various sources ranges from 0.03 to values as high as 8.0 wt%. These high quantities of sulfur must be removed before the crude oil is processed because combustion of this oil would result in severe environmental pollution, such as acid rain. Due to high utility and operating costs, the conventional hydrodesulfurization process (HDS) is considered to be uneconomic. The biotechnological option, biodesulfurization (BDS) seems an attractive low cost, environmentally benign technology. RESULTS: This paper reports the development of a recombinant strain of bacteria designed by introducing desulfurizing, dsz genes containing plasmid pSAD 225‐32, which was isolated from Rhodococcus erythropolis IGTS8 into a gram negative solvent‐tolerant bacterium, Pseudomonas putida (MTCC 1194). This recombinant bacterium can desulfurize the dibenzothiophene (DBT) in the sulfur selective 4S‐pathway. It has been observed that for the same concentration of DBT, the recombinant strain's growth rate is greater than that of the parent strain. Increasing the concentration of DBT resulted in an increase of lag phase as well as decreased growth rate, which shows that the bacteria is following substrate inhibition type kinetics. This genetically modified bacterium can desulfurize 73.1% of 1.2 mmol L^?1 DBT (dissolved in ethanol) in 67 h of cultivation time using growing cells. CONCLUSIONS: It is concluded that further research in this area of biodesulfurization using genetically modified organisms may remove the bottlenecks presently in the way of commercialization of the BDS process. Copyright © 2007 Society of Chemical Industry     

海藻酸钠-聚乙烯醇共固定化细胞脱除燃料油中的有机硫

从孤岛油田油浸土壤中筛选得到能降解二苯并噻吩(DBT)的红平红球菌DS-3, 对其进行了固定化研究.选取海藻酸钠(SA〖DK〗)-聚乙烯醇(PVA)为包埋固定化载体, 以1 mmol·L^-1二甲基亚砜培养菌体, 固定化最佳操作条件为4℃交联, 8%PVA和2%SA混合, 细胞在胶液中的浓度为0.1 g·ml^-1, 氯化钙含量为2%,此时固定化细胞不但具有良好的脱硫性能, 而且脱硫重复性好, 其寿命可以达到250 h以上.固定化细胞在3次循环后能使模拟柴油中的DBT含量从0.5 mmol·L^-1降至0.011 mmol·L^-1, 总脱硫率达到93％, 平均脱硫效率约为0.225 mg DBT· (g DCW)^-1·h^-1.同样条件下能将精制柴油的硫含量由340 mg·L^-1降到42 mg·L^-1, 总脱硫率为87.64%.DS-3能利用醇类、饱和的C₈～C₁₅脂肪烃, 但不能利用环烷烃、芳香烃、小于C₈或大于C₁₅的脂肪烃, DS-3优先利用碳源的顺序是乙醇、葡萄糖和烷烃.脱硫前后的精制柴油经GC-FID分析证实, 固定化细胞对C—S键具特异性, 不降低柴油的热值.