重组大肠杆菌全细胞催化高效合成胆绿素北大核心CSCD

胆绿素作为一种重要的保护细胞的抗氧化剂,其传统生产方法主要由胆红素的化学氧化产生,但过程复杂、纯度不高。本研究提出了一种高效、绿色、安全的生产胆绿素的方法。通过比较,筛选得到了破伤风梭状芽孢杆菌(Clostridium tetani)来源的血红素加氧酶(heme oxygenase,HO)基因,并成功构建具备转化血红素合成胆绿素能力的重组大肠杆菌(Escherichiacoli)BL21/pETDuet-hoCt。在pH 7.0、35℃、100 mg/L底物浓度条件下胆绿素产量为32.9 mg/L。为提高还原力,构建了基于谷氨酸脱氢酶(glutamate dehydrogenase,GdhA)的NADPH辅酶再生系统,获得重组菌E.coli BL21/pETDuet-gdhAEc-hoCt,胆绿素产量为71.5 mg/L。此外,通过引入膜表面展示系统,构建重组菌E.coli BL21/pETDuet-gdhAEc-blc/hoCt,缩短转化时间的同时,胆绿素产量进一步得到提高,达到76.3 mg/L,是目前生物法合成胆绿素的最高研究报道。本研究为胆绿素的绿色生产奠定了良好的基础。     

Accelerating whole-cell biocatalysis by reducing outer membrane permeability barrier

Whole-cell biocatalysts are preferred in many biocatalysis applications. However, due to permeability barriers imposed by cell envelopes, whole-cell catalyzed reactions are reportedly 10-100-fold slower than reactions catalyzed by free enzymes. In this study, we accelerated whole-cell biocatalysis by reducing the membrane permeability barrier using molecular engineering approaches. Escherichia coli cells with genetically altered outer membrane structures were used. Specifically, a lipopolysaccarides mutant SM101 and a Braun's lipoprotein mutant E609L were used along with two model substrates that differ substantially in size and hydrophobicity, nitrocefin, and a tetrapeptide N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide. The reduction of the outer membrane permeability by genetic methods led to significant increases (up to 380%) in reaction rates of whole-cell catalyzed reactions. The magnitude of increase in biocatalysis rates was dependent on the substrates and on the nature of mutations introduced in the outer membrane structure. Notably, mutations in outer membrane can render the outer membrane completely permeable to one substrate, a barrierless condition that maximizes the reaction rate. The impact of the mutations introduced on the permeability barrier of the membranes was compared to the impact of polymixin B nonapeptide, a known potent permeabilizer acting on lipopolysaccharides. Our results suggest that genetic modifications to enhance the permeability of hydrophilic molecules should target the Lipid A region. However, strategies other than reduction of Lipid A synthesis should be considered. As we have demonstrated with tetrapeptide, membrane engineering can be much more effective in reducing a permeability barrier than are exogenous permeabilizers. This work, to our knowledge, is the first use of a molecular membrane engineering approach to address substrate permeability limitations encountered in biocatalysis applications.     

Efficient synthesis of simvastatin by use of whole-cell biocatalysis

Simvastatin is a semisynthetic derivative of the fungal polyketide lovastatin and is an important drug for lowering cholesterol levels in adults. We have developed a one-step, whole-cell biocatalytic process for the synthesis of simvastatin from monacolin J. By using an Escherichia coli strain overexpressing the previously discovered acyltransferase LovD (X. Xie, K. Watanabe, W. A. Wojcicki, C. C. Wang, and Y. Tang, Chem. Biol. 13:1161-1169, 2006), we were able to achieve >99% conversion of monacolin J to simvastatin without the use of any chemical protection steps. The key finding was a membrane-permeable substrate, alpha-dimethylbutyryl-S-methyl-mercaptopropionate, that was efficiently utilized by LovD as the acyl donor. The process was scaled up for gram-scale synthesis of simvastatin. We also demonstrated that simvastatin synthesized via this method can be readily purified from the fermentation broth with >90% recovery and >98% purity as determined by high-performance liquid chromatography. Bioconversion using high-cell-density, fed-batch fermentation was also examined. The whole-cell biocatalysis can therefore be an attractive alternative to currently used multistep semisynthetic transformations.     

全细胞催化生产苯乙酰-7-氨基-3-脱乙酰氧基头孢烷酸的条件优化

头孢类抗生素由于广谱性和低毒性被广泛用于细菌感染的治疗。7-氨基-3-脱乙酰氧基头孢烷酸(7-aminodeacetoxycephalosporanic acid,7-ADCA)作为半合成头孢类抗生素的重要中间体,需求量逐渐增加,而7-ADCA主要由G-7-ADCA(苯乙酰-7ADCA)脱酰基得到。工业上多以化学法合成G-7-ADCA,成本高,污染严重。迫切需要对环境友好且经济高效的合成方法。在前期研究中,构建了一株可以将青霉素G转化为G-7-ADCA的代谢工程菌(E.coli H7/PG15)。本研究通过单因素试验对E.coli H7/PG15的G-7-ADCA合成过程进行优化,包括底物组成及其最适浓度,转化条件(菌体浓度、p H、青霉素浓度、MOPS浓度、葡萄糖浓度,铁离子浓度和时间等)。优化后,建立了全细胞催化法生产G-7-ADCA的工艺流程,使G-7-ADCA的产量稳定在15 mmol/L左右,转化率达到30%,具有操作简便、高效和经济的优势。     

Characterization of detergent purified recombinant rat liver monoamine oxidase B expressed in Pichia pastoris

The high level expression and purification of rat monoamine oxidase B (rMAOB) in the methylotrophic yeast Pichia pastoris is reported. Nearly 100 mg of purified rMAOB is obtained from 130 g (wet weight) of cells (0.5 L of culture). The MALDI-TOF mass spectrum of the purified protein shows a single species with a molecular mass of 59.228 ± 0.064 kDa, which agrees with the calculated molecular weight of 59.172 kDa for the rMAOB protein sequence assuming one mole of covalent FAD per mole of the enzyme. Consistent with the MALDI-MS data, purified rMAOB shows a single band near 60 kDa in Coomassie-stained SDS–PAGE gel as well as on Western blot analyses performed using antisera raised against human MAOA and BSA-conjugated FAD. A partial amino acid sequence of the purified protein is confirmed to be that of the wild type rMAOB by in-gel trypsin digestion and MALDI-TOF-MS analyses of the liberated peptide fragments. Steady state kinetic data show that purified rMAOB exhibits a K_m(amine) of 176 ± 15 μM and a k_cat of 497 ± 83 min⁻¹ for benzylamine oxidation, and a K_m(O₂) of 170 ± 10 μM. Kinetic parameters obtained for purified rMAOB are compared with those reported earlier for recombinant human liver MAOB expressed in P. pastoris.     

Human recombinant monoamine oxidase B as reliable and efficient enzyme source for inhibitor screening

Interest in inhibitors of monoamine oxidase type B (MAO B) has grown in recent years, due to their therapeutic potential in aging-related neurodegenerative diseases, such as Parkinson's disease and Alzheimer's disease. This study is devoted to the use of human recombinant MAO B obtained from a Baculovirus expression system (Supersomes MAO B, BD Gentest, MA, USA) as reliable and efficient enzyme source for MAO B inhibitor screening. Comparison of inhibition potencies (pIC50 values) determined with human cloned and human platelet MAO B for the two series of MAO B inhibitors, coumarin and 5H-indeno[1,2-c]pyridazin-5-one derivatives, showed that the difference between pIC50 values obtained with the two enzyme sources was not significant (P>0.05, Student's t-test). Hence, recombinant enzyme is validated as convenient enzyme source for MAO B inhibitor screening.     

Enzyme-based glucose delivery as a high content screening tool in yeast-based whole-cell biocatalysis

The influence of glucose release on growth and biotransformation of yeasts was examined by using the medium EnBase^® Flo in shake flasks. The medium contains a polysaccharide acting as substrate, which is degraded to glucose by the addition of an enzyme. In the present paper, this medium was adapted for the cultivation of yeasts by increasing the complex components (booster) and the enzyme concentrations to guarantee a higher glucose release rate. Important changes were an increase of the complex component booster to 10–15% and an increased glucose release by increasing the enzyme content to 15 U L^?1. The 20 yeasts investigated in the present work showed an improvement of growth and biomass production when cultivated with the EnBase medium in comparison to yeast extract dextrose (YED) medium. Values of optical densities (OD₆₀₀) of approximately 40 AU (corresponding to over 60 g L^?1 wet cell weight) were achieved for all 20 yeast strains tested. During the following screening of the yeasts in whole-cell biotransformation, an improvement of the conversion for 19 out of the 20 yeasts cultivated with the EnBase Flo medium could be observed. The biomass from the EnBase Flo cultivation showed a higher conversion activity in the reduction of 2-butanone to (R/S)-2-butanol. The enantioselectivity (ee) of 15 yeast strains showed an improvement by using the EnBase medium. The number of yeasts with an ee >97% increased from zero with YED to six with EnBase medium. Thus, the use of a glucose release cultivation strategy in the screening process for transformation approaches provides significant benefits compared to standard batch approaches.     

Brain monoamine oxidase in schizophrenia

The content of SH-groups and substrate specificity have been studied in purified preparations of monoamine oxidase (MAO) from human brain. It has been shown that both in schizophrenic and mentally normal persons MAO occurs in a partially oxidized state. The enzyme contains 2 SH-groups per 10(5) daltons of protein and deaminates MAO substrates (serotonin, beta-phenylethylamine) along with histamine, diamine oxidase substrate. Reduction of the partially oxidized SH-groups of MAO in schizophrenics up to 15 SH-groups per 10(5) daltons of protein (the normal value for human brain MAO) does not eliminate the histamine deaminase activity as is the case in experiments with MAO from the normal brain but, on the contrary, considerably potentiates it. The data suggest certain structural alteration of MAO in schizophrenia.     

Structure-activity relations in the oxidation of phenethylamine analogues by recombinant human liver monoamine oxidase A

The interaction of recombinant human liver monoamine oxidase A (MAO A) with a series of phenethylamine substrate analogues has been investigated by steady-state and stopped-flow kinetic techniques. Substrate analogues with para substituents exhibit large deuterium kinetic isotope effect on k(cat), on k(cat)/K(m), and on the limiting rate of enzyme reduction in reductive half-reaction experiments. These kinetic isotope effect values range from 5 to 10 with the exception of tyramine, which exhibited smaller steady-state isotope effects (2.3-3.5) than that observed on the rate of flavin reduction (6.9). The stopped-flow data show that imine release from the reduced enzyme is slower than the rate of catalytic turnover. Phenethylamine oxidation by MAO A can be described as the C-H bond cleavage step being rate limiting in catalysis and with oxygen reacting with the reduced enzyme-imine complex. In the case of tyramine, the product release from the oxidized enzyme-imine complex contributes to the rate limitation in catalysis. The binding affinities of a series of para-substituted phenethylamine analogues to MAO A show an increase in affinity of the deprotonated amine with increasing van der Waals volume of the substituent. The limiting rate of enzyme reduction decreases with increasing van der Waals volume of the substituent in a linear manner with no observable electronic contribution as observed previously with benzylamine reduction of MAO A [Miller, J. R., and Edmondson, D. E. (1999) Biochemistry 38, 13670-13683]. Examination of side chain analogues of phenethylamine show 3-phenylpropylamine to be oxidized 2.5-fold more slowly and bound 75-fold more tightly than phenethylamine. 4-Phenylbutylamine is not a substrate for MAO A but is a good competitive inhibitor with a K(i) value of 31 +/- 5 microM. Analysis of the effect of alkyl side chain alterations on binding affinities of a series of arylalkylamine analogues taken from this study and from the literature show a linear correlation with the Taft steric value (E(s)) of the side chain. These results suggest that the binding site for the aryl ring is identical for phenethylamine and for benzylamine analogues and that steric interactions of the alkyl side chain with the enzyme strongly contribute to the binding affinities of a series of reversible inhibitors of MAO A.     

Metabolism of acetylpolyamines by monoamine oxidase,diamine oxidase and polyamine oxidase

N¹-Monoacetylspermine, N¹,N¹²-diacetylspermine and N¹-monoacetylspermidine were found to be good substrates for rat liver polyamine oxidase, but not for rat liver mitochondrial monoamine oxidase. N⁸-Monoacetylspermidine, monoacetylcadaverine, monoacetylputrescine and monoacetyl-1,3-diaminopropane were oxidized by the monoamine oxidase when the substrate concentration was 10.0 mM, but not by the polyamine oxidase. All the acetylpolyamines except N¹,N¹²-diacetylspermine were also oxidized by hog kidney diamine oxidase although their affinities for the oxidase appeared low. The present data suggest that acetylpolyamines are not easily metabolized in vivo by either monoamine oxidase or diamine oxidase in mammalian tissues although N¹-monoacetylspermine, N¹,N¹²-diacetylspermine and N¹-monoacetylspermidine are attacked by polyamine oxidase.