二苯并噻吩单加氧酶催化特性及其催化吲哚合成靛类化合物的研究
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摘要
在生物脱硫的“4S”途径中,有4个酶参与,其中二苯并噻吩单加氧酶(Dibenzothiophene monooxygenase,DszC)是该途径第一个起作用的酶,连续两步氧化二苯并噻吩(Dibenzothiophene,DBT)生成二苯并噻吩砜(Dibenzothiophene sulfone,DBTO_2)。DszC活力的发挥需要NADH∶黄素氧化还原酶(NADH∶flavin oxidoreductase,DszD)为其提供还原黄素。DszC和DszD构成黄素依赖型双组分单加氧酶体系。DszC是该体系的单加氧酶组分,DszD是该体系的还原酶组分。黄素依赖型双组分单加氧酶体系的单加氧酶组分一般是黄素专一性的,一些酶专一性利用FMNH_2,另外一些酶则是专一性利用FADH_2。既能够利用FMNH_2又利用FADH_2的单加氧酶组分是非常稀少的,目前只有两个单加氧酶组分报道有此能力。一个是Acinetobacter baumannii的p-羟基苯乙酸羟化酶(p-Hydroxyphenylacetate hydroxylase)的C_2组分,另一个是Streptomycesviridifacienshave MG456-hF10的异丁胺N-羟基化酶(IsobutylamineN-hydroxylase)。DszC利用黄素的专一性尚未明确,该酶是否能利用FADH_2尚无确切结论。此外,黄素依赖型双组分单加氧酶体系也是体内活性氧的重要来源,过量活性氧的存在会对菌体造成毒害,因而抗氧化蛋白的表达对保持细胞内活性氧的平衡和细胞的正常活力有重要意义。目前尚无与“4S”途径脱硫相关的抗氧化蛋白的报道。生物降解和生物催化互相关联,DszC的底物谱不仅限于DBT类化合物,该酶在生物催化领域的应用潜力尚需挖掘。
本实验室长期从事生物脱有机硫的研究,筛选到多株有价值的脱硫菌株。本文以来自一株嗜热脱硫菌Mycobacterium goodii X7B的DszC为研究对象,详细研究了该单加氧酶对还原黄素利用的特点及黄素对酶活力的影响。考察了多株菌株中脱硫基因的表达与菌体的过氧化氢酶、超氧化物歧化酶表达的关系。探讨了DszC在生物催化吲哚羟基化从而合成靛类化合物中应用的潜力。
从一株嗜热脱硫菌M.goodii X7B克隆了dszC基因,并在大肠杆菌中大量表达后进行纯化,获得了在SDS-PAGE电泳上表现为单一条带(45 kDa)的纯酶。全波长扫描表明该酶在280 nm处有明显光吸收,在300-700 nm没有光吸收,说明该酶不结合血红素或黄素。通过使用既能催化FMN还原又能催化FAD还原的DszD,证明DszC既能够使用FMNH_2,也能使用FADH_2作为底物催化DBT生成DBTO_2。DszC的活力受到黄素种类、浓度、DszC与DszD之比的影响。使用FMNH_2时的DszC比活力比使用FADH_2时的比活力高,无论向DszCD的耦联体系中添加FMN或FAD,低浓度的黄素都会增强酶活力,但是高浓度的黄素强烈抑制DszC的活力。在DszCD耦联体系发挥作用时,还原态的黄素需要从DszD传递到DszC,在这个传递过程中,还原态黄素容易被自氧化,自氧化的发生削弱了流向DszC的还原态黄素,同时还原态黄素自氧化产生的大量过氧化氢会使DszC和DszD失活,造成活力下降。提高黄素的浓度会加强这种自氧化作用,提高DszC的比例在一定程度上可以缓解这种自氧化造成的活力下降,通过向DszCD体系中添加过氧化氢酶及时移走过氧化氢,可以将DszC的活力提高一倍。
研究了原始脱硫菌株和基因工程菌株中脱硫基因表达和超氧化物歧化酶(Superoxide dismutase,SOD)、过氧化氢酶(Catalase)表达之间的关系。分别使用Na_2SO_4、二甲基亚砜(Dimethyl sulfoxide,DMSO)和DBT作为唯一硫源,考察原始脱硫菌中SOD表达图谱的变化。在红平红球菌Rhodococcus erythropolis1awq中,发现了一个和脱硫基因共同表达的超氧化物歧化酶,该诱导型酶在其他几株原始脱硫菌株中不存在。目前,还没有关于和脱硫基因共表达的SOD的报道。测定原始脱硫菌株和基因工程菌中过氧化氢酶活力的变化,发现脱硫基因的异源表达会造成宿主菌自身的过氧化氢酶活力严重下降,但是在原始脱硫菌株中没有这种情况发生。
最近十年,研究人员已经发现了大量的黄素依赖型单加氧酶。这些单加氧酶可以高效而专一性氧化有机化合物,而且这类氧化反应通常难以用化学方法催化。然而,到目前为止,科研人员只研究了少数几种单加氧酶在催化合成中的应用。造成这种状况的主要原因有两个:单加氧酶难以表达和分离;另一个原因就是这些单加氧酶需要昂贵的NAD(P)H,不能以化学计量添加。为此,从枯草芽孢杆菌168菌株克隆葡萄糖脱氢酶(Glucose dehydrogenase,GDH)基因转入大肠杆菌大量表达。利用从大肠杆菌纯化得到的GDH构建了NADH的再生体系,经证明该体系可以代替NADH的直接加入,为DszCD耦联体系提供源源不断的NADH来催化DBT的氧化。该项结果为DszC用于生物催化奠定了基础。
DszC可以催化吲哚生成被广泛应用的蓝色染料——靛蓝,该化合物具有重要的工业应用价值。以吲哚作为底物,研究了GDH的加入和DszC的催化活力之间的关系。GDH加入量的提高可以为DszCD体系提供在单位时间内提供更多的NADH,增强DszCD的表观催化能力,但是GDH的加入量仍然需要和DszD的活力保持一个平衡。GDH提供的NADH可以增强DszD的催化能力,体系中产生了更多的FMNH_2,FMNH_2固然可以增加DszC的活力,然而FMNH_2的增加也会导致其自氧化的增强,并最终导致DszCD催化体系活力的下降。该辅因子再生体系的建立为利用黄素依赖型双组分单加氧酶进行体外催化奠定了基础。
全细胞催化体系可以减少还原黄素自氧化对催化活力的影响,提高催化效率,因此研究了重组大肠杆菌全细胞催化吲哚合成靛蓝的应用潜力。将DszD和DszC共表达可以大大提高细胞催化吲哚羟基化生成靛蓝的能力,但是在此基础上再共表达GDH则会削弱DszCD的表达,从而使细胞的催化能力下降。研究表明,当菌体生长至指数生长中期,加入1 mM IPTG,转入27℃继续培养8 h后收集细胞,可以得到催化能力最高的菌体。转化反应适宜在pH 6-7之间进行,增高pH会降低靛蓝产量,可能是影响了中间体向靛蓝的转化。细胞浓度对于终产物的浓度影响较大,控制装液量为反应器体积的20%时,理想的细胞浓度为OD_(600)在20左右。在1-4 mM范围内增加吲哚的浓度可以提高靛蓝的浓度,但是相对于底物的转化率则在降低,更高浓度的吲哚导致靛蓝浓度降低大幅降低。这可能是吲哚破坏细胞膜造成的,可以考虑使用有机溶剂耐受菌株作为宿主来进行该催化反应。
在重组大肠杆菌全细胞催化吲哚过程中检测到一个紫红色物质,初步鉴定为靛玉红。靛玉红已被证明是有效的癌症治疗药物,多项研究表明该化合物在诸多疾病的治疗中都能起到作用,因而靛玉红的规模生产具有重要意义,通过取代基的变化,可以制作更适合药用的靛玉红类化合物。实验证明,表达DszC的全细胞也能够以多种5-位取代吲哚作为底物,生成蓝色和红色化合物,推测为相应的靛蓝和靛玉红取代物,说明DszC和表达DszC的全细胞在催化合成靛玉红类化合物中具有很大的应用潜力。
Four enzymes are involved in the biocatalytic desulfurization via the "4S" pathway.Dibenzothiophene monooxygenase initiates the desulfurization by catalyzing dibenzothiophene(DBT) to dibenzothiophene sulfone(DBTO_2) through two consecutive oxidations.DszC requires an independent NADH:flavin oxidoreductase(DszD) to provide reduced flavins to sustain its activity.DszC and DszD consist of a flavin-dependent two component monooxygenase system.DszC is the monooxygenase unit,and DszD is the reductase one.Usually the monooxygenase unit is strictly dependent on FMNH_2 or FADH_2.Until now only two enzymes have been reported to have this unusual ability.One is the C_2 component of p-hydroxyphenylacetate hydroxylase from Acinetobacter baumannii, and the other is the isobutylamine N-hydroxylase from Streptomyces viridifacienshave MG456-hF10.However,utilization of FADH_2 by DszC has never been elucidated clearly.Flavin dependent monooxygenase is one of the sources of reactive oxygen species(ROS).Overproduction of ROS would cause damage to the cell.Therefor, antioxidant proteins play an important role in keeping the ROS at proper levels and the cells work well.Any antioxidant protein related to "4S" pathway has never been reported.Biodegradtion and biocatalysis is always related.DszC has broader substrate than DBTs,and it is a potential biocatalyst in synthesis of valuble products.
Biocatalytic desulfurization has been studied for a long time at our laboratory, and several valuable strains were isolated.In this study,dszC from a thermophilic biodesulfurizing strain Mycobacterium goodii X7B was cloned and expressed in Escherichia coli,and DszC was purified.Preference to reduced flavins by this DszC and factors that affected the DszC activity was discussed.Relationship between desulfurizing enzymes expression and antioxidant proteins expression was studied. Research was also conducted to estimate the potential application of DszC (microorganism containing of) in the biocatalytic synthesis of indigoids.
Purified DszC showed a single band of about 45 kDa on the SDS-PAGE. Wavescan of the enzyme showed that DszC had strong absorbance at 280 nm,but no specific absorbtion in the range of 300-700 nm,indicating that no heine or flavin was bounded.As a monooxygenase unit of the flavin-dependent two-component monooxygenase,DszC activity is significantly dependent on the reduced flavins provided by DszD.Using a DszD that reduced either FMN or FAD,DszC was proved to be able to utilize FADH_2 as well as FMNH_2 to catalyze DBT to DBTO_2. DszC activity is dependent on the type and concentration of the flavin,and the ratio of DszC to DszD.DszC was much more active with FMNH_2 than that with FADH_2. Either flavin at low concentration stimulates the DszC activity but at high concentrations inhibits the activity of DszC due to the autocatalytic oxidation of reduced flavins.Autooxidation depressed the flow of reduced flavin to DszC,and hydrogen peroxide formed due to the autooxidation,which caused the inactivation of DszC and DszD and led to the decrease in DszC activity.The autooxidation was enhanced when flavins concentration increased,and released by the increase of DszC amount in the DszCD system.Addition of catalase destructed H_2O_2 as soon as it formed and thus increased the activity of DszC.
Relationships between expressions of desulfurizing enzymes and the antioxidant proteins were investigated in both wild types of desulfurizing strains and engineering strains.Using undenaturing gel electrophoresis and active staining,a superoxide dismutase(SOD) induced by DBT or dimethyl sulfoxide was for the first time discovered in Rhodococcus erythropolis 1 awq.Expression of desulfurizing genes in heterogeneous engineering strains would cause a serious decrease in catalase activity; such phenomenon was not observed in the wild strains.
Although more and more flavin dependent monooxygenase have been discovered in the past decade,seldom of them was applied in the biocatalytic synthesis of valuable compounds that were hard to obtain with traditionally chemical catalysts.Two obstacles exist on the way to explore scaled-up production by the flavin dependent monooxygenase.First,monooxygenase is usually difficult to express and purify,and second,expensive cofactors of NAD(P)H is required to drive the reaction,and stoichemsitroic addition of these cofactors are not economical. Glucose dehydrogenase(GDH) gene was cloned from Bacillus subtilis subsp,subtilis str.168 and transferred to E.coli for overexpression.Using GDH purified from the engineered E.coli,a system for regeneration of NADH was constructed.It could provide NADH continuously to the DszCD coupling reaction to catalyze DBT to DBTO_2.Effect of GDH on the DszC activity was investigated with indole as the substrate of DszC.Increase of DszC provided more NADH to DszCD,hence DszCD activity was enhanced.However,GDH should be kept at a proper ratio to DszD.DszD activity can be elevated by NADH supply,and thus more FMNH_2 is formed.As described previously,excessive FMNH_2 would inevitably lead to decrease of DszCD activity.
Yield of indigo from indole was improved when whole cells of recombinant E. coli were used instead of isolated enzymes,in which case the autooxdiation of reduced flavins was depressed greatly.Co-expression of DszD and DszC significantly increased the activity of the biocatalysts,but further co-expression of GDH led to a decrease in the yield of indigo.It could be attributed to that the over expression of GDH made a sharp decrease in the expression of DszC and DszD. When the cells grew to the exponential phase,1 mM IPTG was added and the cells were further induced at 27℃for 8 h.The biotransformtion was carried out at pH 6-7;higher pH would reduce the indigo production.Cell density had an obvious effect on the production;an optimized cell density was OD_(600) at about 20 when the reaction volume was one fifth of the total volume.In the range of 1-4 mM,increase of indole concentration led to indigo production increase,but the yield of product to substrate decreased.Higher indole concentration than 4 mM decreased the final concentration of indigo,which was possibly due to the cell toxicity of indole.
A pink compound was primarily identified as inirubin,which was proved as an effective anti cancer drug,and might be applied in the therapeutics of several diseases. Whole cells expressing DszC also could catalyze various substituted indole at the C-5 site to form the blue and pink compounds,assumed to be derivatives of indigo and indirbin.It suggested that DszC(whole microorganism containing of) had great potential in the production of various valuable indigoids.
本实验室长期从事生物脱有机硫的研究,筛选到多株有价值的脱硫菌株。本文以来自一株嗜热脱硫菌Mycobacterium goodii X7B的DszC为研究对象,详细研究了该单加氧酶对还原黄素利用的特点及黄素对酶活力的影响。考察了多株菌株中脱硫基因的表达与菌体的过氧化氢酶、超氧化物歧化酶表达的关系。探讨了DszC在生物催化吲哚羟基化从而合成靛类化合物中应用的潜力。
从一株嗜热脱硫菌M.goodii X7B克隆了dszC基因,并在大肠杆菌中大量表达后进行纯化,获得了在SDS-PAGE电泳上表现为单一条带(45 kDa)的纯酶。全波长扫描表明该酶在280 nm处有明显光吸收,在300-700 nm没有光吸收,说明该酶不结合血红素或黄素。通过使用既能催化FMN还原又能催化FAD还原的DszD,证明DszC既能够使用FMNH_2,也能使用FADH_2作为底物催化DBT生成DBTO_2。DszC的活力受到黄素种类、浓度、DszC与DszD之比的影响。使用FMNH_2时的DszC比活力比使用FADH_2时的比活力高,无论向DszCD的耦联体系中添加FMN或FAD,低浓度的黄素都会增强酶活力,但是高浓度的黄素强烈抑制DszC的活力。在DszCD耦联体系发挥作用时,还原态的黄素需要从DszD传递到DszC,在这个传递过程中,还原态黄素容易被自氧化,自氧化的发生削弱了流向DszC的还原态黄素,同时还原态黄素自氧化产生的大量过氧化氢会使DszC和DszD失活,造成活力下降。提高黄素的浓度会加强这种自氧化作用,提高DszC的比例在一定程度上可以缓解这种自氧化造成的活力下降,通过向DszCD体系中添加过氧化氢酶及时移走过氧化氢,可以将DszC的活力提高一倍。
研究了原始脱硫菌株和基因工程菌株中脱硫基因表达和超氧化物歧化酶(Superoxide dismutase,SOD)、过氧化氢酶(Catalase)表达之间的关系。分别使用Na_2SO_4、二甲基亚砜(Dimethyl sulfoxide,DMSO)和DBT作为唯一硫源,考察原始脱硫菌中SOD表达图谱的变化。在红平红球菌Rhodococcus erythropolis1awq中,发现了一个和脱硫基因共同表达的超氧化物歧化酶,该诱导型酶在其他几株原始脱硫菌株中不存在。目前,还没有关于和脱硫基因共表达的SOD的报道。测定原始脱硫菌株和基因工程菌中过氧化氢酶活力的变化,发现脱硫基因的异源表达会造成宿主菌自身的过氧化氢酶活力严重下降,但是在原始脱硫菌株中没有这种情况发生。
最近十年,研究人员已经发现了大量的黄素依赖型单加氧酶。这些单加氧酶可以高效而专一性氧化有机化合物,而且这类氧化反应通常难以用化学方法催化。然而,到目前为止,科研人员只研究了少数几种单加氧酶在催化合成中的应用。造成这种状况的主要原因有两个:单加氧酶难以表达和分离;另一个原因就是这些单加氧酶需要昂贵的NAD(P)H,不能以化学计量添加。为此,从枯草芽孢杆菌168菌株克隆葡萄糖脱氢酶(Glucose dehydrogenase,GDH)基因转入大肠杆菌大量表达。利用从大肠杆菌纯化得到的GDH构建了NADH的再生体系,经证明该体系可以代替NADH的直接加入,为DszCD耦联体系提供源源不断的NADH来催化DBT的氧化。该项结果为DszC用于生物催化奠定了基础。
DszC可以催化吲哚生成被广泛应用的蓝色染料——靛蓝,该化合物具有重要的工业应用价值。以吲哚作为底物,研究了GDH的加入和DszC的催化活力之间的关系。GDH加入量的提高可以为DszCD体系提供在单位时间内提供更多的NADH,增强DszCD的表观催化能力,但是GDH的加入量仍然需要和DszD的活力保持一个平衡。GDH提供的NADH可以增强DszD的催化能力,体系中产生了更多的FMNH_2,FMNH_2固然可以增加DszC的活力,然而FMNH_2的增加也会导致其自氧化的增强,并最终导致DszCD催化体系活力的下降。该辅因子再生体系的建立为利用黄素依赖型双组分单加氧酶进行体外催化奠定了基础。
全细胞催化体系可以减少还原黄素自氧化对催化活力的影响,提高催化效率,因此研究了重组大肠杆菌全细胞催化吲哚合成靛蓝的应用潜力。将DszD和DszC共表达可以大大提高细胞催化吲哚羟基化生成靛蓝的能力,但是在此基础上再共表达GDH则会削弱DszCD的表达,从而使细胞的催化能力下降。研究表明,当菌体生长至指数生长中期,加入1 mM IPTG,转入27℃继续培养8 h后收集细胞,可以得到催化能力最高的菌体。转化反应适宜在pH 6-7之间进行,增高pH会降低靛蓝产量,可能是影响了中间体向靛蓝的转化。细胞浓度对于终产物的浓度影响较大,控制装液量为反应器体积的20%时,理想的细胞浓度为OD_(600)在20左右。在1-4 mM范围内增加吲哚的浓度可以提高靛蓝的浓度,但是相对于底物的转化率则在降低,更高浓度的吲哚导致靛蓝浓度降低大幅降低。这可能是吲哚破坏细胞膜造成的,可以考虑使用有机溶剂耐受菌株作为宿主来进行该催化反应。
在重组大肠杆菌全细胞催化吲哚过程中检测到一个紫红色物质,初步鉴定为靛玉红。靛玉红已被证明是有效的癌症治疗药物,多项研究表明该化合物在诸多疾病的治疗中都能起到作用,因而靛玉红的规模生产具有重要意义,通过取代基的变化,可以制作更适合药用的靛玉红类化合物。实验证明,表达DszC的全细胞也能够以多种5-位取代吲哚作为底物,生成蓝色和红色化合物,推测为相应的靛蓝和靛玉红取代物,说明DszC和表达DszC的全细胞在催化合成靛玉红类化合物中具有很大的应用潜力。
Four enzymes are involved in the biocatalytic desulfurization via the "4S" pathway.Dibenzothiophene monooxygenase initiates the desulfurization by catalyzing dibenzothiophene(DBT) to dibenzothiophene sulfone(DBTO_2) through two consecutive oxidations.DszC requires an independent NADH:flavin oxidoreductase(DszD) to provide reduced flavins to sustain its activity.DszC and DszD consist of a flavin-dependent two component monooxygenase system.DszC is the monooxygenase unit,and DszD is the reductase one.Usually the monooxygenase unit is strictly dependent on FMNH_2 or FADH_2.Until now only two enzymes have been reported to have this unusual ability.One is the C_2 component of p-hydroxyphenylacetate hydroxylase from Acinetobacter baumannii, and the other is the isobutylamine N-hydroxylase from Streptomyces viridifacienshave MG456-hF10.However,utilization of FADH_2 by DszC has never been elucidated clearly.Flavin dependent monooxygenase is one of the sources of reactive oxygen species(ROS).Overproduction of ROS would cause damage to the cell.Therefor, antioxidant proteins play an important role in keeping the ROS at proper levels and the cells work well.Any antioxidant protein related to "4S" pathway has never been reported.Biodegradtion and biocatalysis is always related.DszC has broader substrate than DBTs,and it is a potential biocatalyst in synthesis of valuble products.
Biocatalytic desulfurization has been studied for a long time at our laboratory, and several valuable strains were isolated.In this study,dszC from a thermophilic biodesulfurizing strain Mycobacterium goodii X7B was cloned and expressed in Escherichia coli,and DszC was purified.Preference to reduced flavins by this DszC and factors that affected the DszC activity was discussed.Relationship between desulfurizing enzymes expression and antioxidant proteins expression was studied. Research was also conducted to estimate the potential application of DszC (microorganism containing of) in the biocatalytic synthesis of indigoids.
Purified DszC showed a single band of about 45 kDa on the SDS-PAGE. Wavescan of the enzyme showed that DszC had strong absorbance at 280 nm,but no specific absorbtion in the range of 300-700 nm,indicating that no heine or flavin was bounded.As a monooxygenase unit of the flavin-dependent two-component monooxygenase,DszC activity is significantly dependent on the reduced flavins provided by DszD.Using a DszD that reduced either FMN or FAD,DszC was proved to be able to utilize FADH_2 as well as FMNH_2 to catalyze DBT to DBTO_2. DszC activity is dependent on the type and concentration of the flavin,and the ratio of DszC to DszD.DszC was much more active with FMNH_2 than that with FADH_2. Either flavin at low concentration stimulates the DszC activity but at high concentrations inhibits the activity of DszC due to the autocatalytic oxidation of reduced flavins.Autooxidation depressed the flow of reduced flavin to DszC,and hydrogen peroxide formed due to the autooxidation,which caused the inactivation of DszC and DszD and led to the decrease in DszC activity.The autooxidation was enhanced when flavins concentration increased,and released by the increase of DszC amount in the DszCD system.Addition of catalase destructed H_2O_2 as soon as it formed and thus increased the activity of DszC.
Relationships between expressions of desulfurizing enzymes and the antioxidant proteins were investigated in both wild types of desulfurizing strains and engineering strains.Using undenaturing gel electrophoresis and active staining,a superoxide dismutase(SOD) induced by DBT or dimethyl sulfoxide was for the first time discovered in Rhodococcus erythropolis 1 awq.Expression of desulfurizing genes in heterogeneous engineering strains would cause a serious decrease in catalase activity; such phenomenon was not observed in the wild strains.
Although more and more flavin dependent monooxygenase have been discovered in the past decade,seldom of them was applied in the biocatalytic synthesis of valuable compounds that were hard to obtain with traditionally chemical catalysts.Two obstacles exist on the way to explore scaled-up production by the flavin dependent monooxygenase.First,monooxygenase is usually difficult to express and purify,and second,expensive cofactors of NAD(P)H is required to drive the reaction,and stoichemsitroic addition of these cofactors are not economical. Glucose dehydrogenase(GDH) gene was cloned from Bacillus subtilis subsp,subtilis str.168 and transferred to E.coli for overexpression.Using GDH purified from the engineered E.coli,a system for regeneration of NADH was constructed.It could provide NADH continuously to the DszCD coupling reaction to catalyze DBT to DBTO_2.Effect of GDH on the DszC activity was investigated with indole as the substrate of DszC.Increase of DszC provided more NADH to DszCD,hence DszCD activity was enhanced.However,GDH should be kept at a proper ratio to DszD.DszD activity can be elevated by NADH supply,and thus more FMNH_2 is formed.As described previously,excessive FMNH_2 would inevitably lead to decrease of DszCD activity.
Yield of indigo from indole was improved when whole cells of recombinant E. coli were used instead of isolated enzymes,in which case the autooxdiation of reduced flavins was depressed greatly.Co-expression of DszD and DszC significantly increased the activity of the biocatalysts,but further co-expression of GDH led to a decrease in the yield of indigo.It could be attributed to that the over expression of GDH made a sharp decrease in the expression of DszC and DszD. When the cells grew to the exponential phase,1 mM IPTG was added and the cells were further induced at 27℃for 8 h.The biotransformtion was carried out at pH 6-7;higher pH would reduce the indigo production.Cell density had an obvious effect on the production;an optimized cell density was OD_(600) at about 20 when the reaction volume was one fifth of the total volume.In the range of 1-4 mM,increase of indole concentration led to indigo production increase,but the yield of product to substrate decreased.Higher indole concentration than 4 mM decreased the final concentration of indigo,which was possibly due to the cell toxicity of indole.
A pink compound was primarily identified as inirubin,which was proved as an effective anti cancer drug,and might be applied in the therapeutics of several diseases. Whole cells expressing DszC also could catalyze various substituted indole at the C-5 site to form the blue and pink compounds,assumed to be derivatives of indigo and indirbin.It suggested that DszC(whole microorganism containing of) had great potential in the production of various valuable indigoids.
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