Expression and characterization of a novel halohydrin dehalogenase from Rhodospirillaceae bacterium / 生物工程学报

As a class of multifunctional biocatalysts, halohydrin dehalogenases are of great interest for the synthesis of chiral β-substituted alcohols and epoxides. There are less than 40 halohydrin dehalogenases with relatively clear catalytic functions, and most of them do not meet the requirements of scientific research and practical applications. Therefore, it is of great significance to excavate and identify more halohydrin dehalogenases. In the present study, a putative halohydrin dehalogenase (HHDH-Ra) from Rhodospirillaceae bacterium was expressed and its enzymatic properties were investigated. The HHDH-Ra gene was cloned into the expression host Escherichia coli BL21(DE3) and the target protein was shown to be soluble. Substrate specificity studies showed that HHDH-Ra possesses excellent specificity for 1,3-dichloro-2-propanol (1,3-DCP) and ethyl-4-chloro-3-hydroxybutyrate (CHBE). The optimum pH and temperature for HHDH-Ra with 1,3-DCP as the reaction substrate were 8.0 and 30 °C, respectively. HHDH-Ra was stable at pH 6.0-8.0 and maintained about 70% of its original activity after 100 h of treatment. The thermal stability results revealed that HHDH-Ra has a half-life of 60 h at 30 °C and 40 °C. When the temperature is increased to 50 °C, the enzyme still has a half-life of 20 h, which is much higher than that of the reported enzymes. To sum up, the novel halohydrin dehalogenase from Rhodospirillaceae bacterium possesses good temperature and pH stability as well as catalytic activity, and shows the potential to be used in the synthesis of chemical and pharmaceutical intermediates.

Customization of enzyme molecular machine and cell factory, leading the future of biomanufacturing industry / 生物工程学报

The development and application of industrial enzymes have penetrated major industrial fields. China faces a major challenge as a large country in applying enzyme but a small one in producing enzyme. Biocatalysis has become an important technology and strategy of industrial development in the world since chemical catalysis encounters the crises from resource, energy and environment. The application of efficient and clean biocatalysis is one of the important ways to realize the sustainable development of chemical industry and to modernize the fermentation industry. From perspective of the industry-university-research cooperation, we reviewed the current status and the future development of enzyme engineering from the aspects of enzyme resources, customization of enzyme molecular machine and cell factory.

Asymmetric synthesis of atorvastatin intermediate by Pichia pastoris X-33 / 生物工程学报

Ethyl (R)-3-hydroxy-5-(1,3-dioxoisoindolin-2-yl)-pentanoate is a potential intermediate for the synthesis of HMG-CoA reductase inhibitor (atorvastatin) that can lower the cholesterol level in human blood. In this study, in order to synthesize ethyl (R)-3-hydroxy-5-(1,3-dioxoisoindolin-2-yl)-pentanoate by bioreduction, the yeast strains in our lab were screened. Ethyl (R)-3-hydroxy-5-(1,3-dioxoisoindolin-2-yl)-pentanoate was found to be produced efficiently from ethyl 5-(1,3-dioxoisoindolin-2-yl)-3-oxopentanoate by Pichia pastoris X-33. The effects of initial substrate concentration, reaction time, co-substrate, amount of yeast cells, pH, as well as the temperature on the yield and enantiomeric excesses (e.e. value) of product were examined in mono-phase system. The optimal reaction conditions are as fallows: substrate concentration 7 g/L, cell concentration 120 g/L, glucose concentration 120 g/L, pH 6.5, temperature 35 degrees C, reaction time 12 h, and the yield 93.12% with the high e.e. value of 98.55%.

Cloning, heterologous expression and purification of a 3-ketosteroid-9alpha-hydroxylase (KSH) from Mycobacterium sp. NwIB-01 / 生物工程学报

3-ketosteroid-9alpha-hydroxylase (KSH), a key enzyme in the microbial steroid degradation, is highly valuable for the production of some steroid drugs. Degenerate primers were designed by comparing the ksh from Rhodococcus erythropolis SQ1 and its homologous sequences in the reported genome of Mycobacteria. Subsequently, a gene fragment of KSH was cloned from Mycobacterium sp. NwIB-01, a sterol-transforming bacterium isolated from soil in our lab. According to the conservative sequence, the full-length 1188 bp gene encoding ksh (designated as M.S.-ksh) was obtained by chromosome walking, which showed 85% identity with the ksh of M. smegmatis mc(2)155. The heterologous expression of KSH was achieved in Escherichia coli BL21(DE3) using the pET-32a-c(+) vector system. The expressed KSH protein was mostly in soluble form after IPTG induction at 30 degreesC and accounted for more than 30% of total bacterial proteins according to SDS-PAGE electrophoresis. The molecular mass of KSH was about 45 kD, which was exactly the size predicted. After Ni2+ affinity chromatography, the purity of the target protein was more than 90%. Our work will definitely contribute to the industrial production of some steroid drugs by developing KSH genetically engineered bacteria.

Recent advances in enzyme assays using fluoremetry / 生物工程学报

Enzymes play such a pivotal role in cellular metabolism that enzyme assays are important for bio-engineering, disease diagnoses and drug discovery. Among the reported methods, fluoremetry has attracted more and more attention due to its high sensitivity and possibility of continuous dynamic monitoring. The recent progresses and applications in enzyme assays using fluorescent probes were reviewed. Different methods were classified into direct fluorescence detection and indirect fluorescence detection according to their labeled substrates and detection mechanisms. Our writing purpose is to provide the readers with a flavor of the kinds of tools and strategies available in enzyme assays with fluorescent probes. Also, the research situation and prospects were disucssed

Citrus fiavonoids simultaneous HPLC analysis / 中成药

AIM: To develop a rapid HPLC method for quality control of traditional Chinese medicinal ingredients consisted of citrus flavonoids, naringin, hesperidin, neohesperidin, sinensetin and nobiletin. METHODS:Gradient elution with non-salt mobile phase ( methanol and water only) HPLC method on a Kromasil column ( 100-5C18-250A, 4.6 mm ×250 mm, 5 μm, C18 reverse phase) with peaks identification through DAD full UV wavelength scan. UV 284 nm and 332 nm profiles were observed. RESULTS: Satisfactory resolution, linearity, 95%～ 102% of recovery and 1.88 ～ 2.93% of repeatability were obtained for those five citrus flavonoids. Content of 6 Citrus aurantium L. based TCM ingredients were analyzed and identified. CONCLUSION: Rapid HPLC test method on citrus flavonoids was developed and can be in LC-MS identification.

A modified simple RFLP-PCR method for single nucleotide polymorphism (SNP) typing

We describe a modified single nucleotide polymorphism (SNP) typing method based on the restriction fragment length polymorphism polymerase chain reaction (RFLP-PCR). This is a simple, economical method without the need for special equipment. For most SNP loci, a common restriction endonuclease (Hind III, EcoR I or BamH I) recognizing site (RER) can be introduced into one allelic form, but not the other by two rounds of mismatched PCR. The flanking regions can be changed by as many as five bases after PCR amplification with specially designed mismatching primers so the genotypes can be distinguished after digestion of the PCR products with corresponding endonucleases.

Effects of cleistocalyx operculatus on oxygenic injury of nerve cells / 中国组织工程研究

BACKGROUND: Cleistocalyx operculatus is a dried alsbastrum of myrtle. It is reported that cleistocalyx operculatus extracts can improve cardiac contraction through inhibiting the activity of Na+/K+-ATPase, and decrease rate of contraction. Do cleistocalyx operculatus extracts have the biological activity of antioxidation?OBJECTIVE: To observe the effects of cleistocalyx operculatus on oxidative injury of PC12 nerve cells induced by H2O2.DESIGN: Non-randomized controlled study.SETTING: State Key Laboratory of Bioreactor Engineering, New World Institute of Biotechnology, East China University of Science and Technology.MATERIALS: The experiment was conducted at New World Institute of Biotechnology, State Key Laboratory of Bioreactor Engineering of East China University of Science and Technology, from May to November 2002.Eight adult male Kunming mice were selected. PC12 nerve cells were supplied by Shanghai Cell Institute of the Chinese Academy of Sciences.METHODS: Model of oxygenic injury of PC12 nerve cells was estabPC12 cells were cultured in 96-well plates. Cleistocalyx operculatus was diluted with RPMI1640 culture medium into five concentrations of 0.001,0.01, 0.1, 0.5 and 1 g/L with 3 wells in each concentration; each well had 2×103 cells. Blank control group, or non-drug culture medium group, was set. Under the standard condition, cells were cultured for 48 hours and ascells were inoculated in 96-well plate with the density of 2×103 for 24-hour wall adhering, and then divided into normal control group (normal cell without H2O2 or cleistocalyx operculatus extracts), 0, 0.01, 0.1, 0.5 and 1 g/L cleistocalyx operculatus. Cells in all groups except normal control group were treated with 200 μmol/L H2O2 at 37℃ for 3 hours, then cleistocalyx operculatus of various concentrations was added and survival rate was asfree radicals: PC12 cells with oxygen-derived free radicals were treated in the same way as done for cell survival rate assay and measured with CDCFH staining method.fect of cleistocalyx operculatus extracts on intracellular and extracellular oxygen-derived free radicals in PC12 nerve cells induced by oxidative injury.operculatus could protect nerve cells; however, at 0.055-1.00 g/L the effect on cell growth did not significantly differ from that of blank control extracts had no protective effect on the injury of PC12 nerve cells induced by H2O2. At 1.00 g/L, it had strong plerosis for oxidative injury of PC12 and extracellular oxygen-derived free radicals in PC12 nerve cells was increased; however, at 0.01 g/L concentration of cleistocalyx operculatus extracts, the level was lower than that in model group.dation of membrane lipid of hepatic microsome, but also protect against oxcleistocalyx operculatus extracts is related to its concentration. At 1.00 g/L,it has great capacity of oxidation plerosis, and at 0.01 g/L it can decrease the level of oxygen-derived free radicals inside and outside cells.

PROPERTIES OF 3-GALACTOSIDASE FROM BACILLUS STEAROTHERMOPHILUS / 微生物学通报

A themostable intracellular β-galactosidase from a thermophilic Bacillus stearothermophilus was purified by a combination of (NH4)2SO4 fractionation, ion-exchange (DEAE-22)and gel filtration (Sephades G-75). The optimum temperature and pH of the enzyme acivity were 60Cand pH6.4 respectively. The β-galatosidase activity exhibited thermosttability at 50 C. The enzyme was significaantly activated by alkali and alkali-earth metal ions. The activity was inhibited by Zn2+ 、 Fe3+ 、 Cu2+Reducing agents enhanced β- galactosidase activity. Thiol-binding agents drastically decreased the enzyme activity. The enzyme was specific for β-D glycosidic linkages,and the identity of the aglycone moiety also influenced enzyme activity. At 55Cthe Km for O-nitrophenyl-β-D-galactosidase (ONPG)and lactose were 2. 63mmol/L and 4.39mmol/L, respectively,and Vmax for both substrates were 1.93 × 10-5mmol. min-1 mg-1protein6.54 ×105 mmol. min-1. mg-1protein,respectively. The enzyme was inhibited by glucose (the products of lactose hydrolysis,ki 2.47mmol/L),but not by galactose. In addition,the enzyme possessed transgalactosylation activity. Galacto-oligosaccharides,both tri- and tetrasaccharide,were involved in the products during lactose hydrolysi

Determination of procyanidin B2 in grape seed extract by RP-HPLC / 中成药

AIM: To establish the method of determining procyanidin B2 content in grape seed extract by HPLC. METHODS: The determination was carried out with ZORBAX SB-C_ 18 column. The mobile phase consisted of acetonitrile-2% acetate buffer under the gradient elution condition, detection wavelength was at 280nm. RESULTS: There was a good linear relationship between the peak area and concentration in the range of 1~30 ?g/mL for procyanidin B2. The average of procyanidin B2 (n=5) was 99.29% and RSD=1.64%, respectively. CONCLUSION: The method is simple, accurate, reproducible and can be used for assay of procyanidin B2.

PROPERTIES OF ?-GALACTOSIDASE FROM BACILLUS STEAROTHERMOPHILUS / 微生物学通报

A themostable intracellular ? galactosidase from a thermophilic Bacillus stearothermophilus was purified by a combination of (NH 4) 2SO 4 fractionation,ion exchange (DEAE 22)and gel filtration (Sephades G 75).The optimum temperature and pH of the enzyme acivity were 60℃and pH6.4 respectively.The ? galatosidase activity exhibited thermosttability at 50 ℃.The enzyme was significaantly activated by alkali and alkali earth metal ions.The activity was inhibited by Zn 2+ 、 Fe 3+ 、 Cu 2+ Reducing agents enhanced ? galactosidase activity.Thiol binding agents drastically decreased the enzyme activity.The enzyme was specific for ? D glycosidic linkages,and the identity of the aglycone moiety also influenced enzyme activity.At 55℃the Km for O nitrophenyl ? D galactosidase(ONPG)and lactose were 2.63mmol/L and 4.39mmol/L, respectively,and Vmax for both substrates were 1.93?10 5 mmol.min 1 .mg 1 protein6.54?10 5 mmol.min 1 .mg 1 protein,respectively.The enzyme was inhibited by glucose (the products of lactose hydrolysis,ki 2.47mmol/L),but not by galactose.In addition,the enzyme possessed transgalactosylation activity.Galacto oligosaccharides,both tri and tetrasaccharide,were involved in the products during lactose hydrolysis.