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Biomedical subjects

G J Shen

Publications and source records attributed to G J Shen.

At least 19 recordsLinked to original sources
PubMed2001

Microbial glycosyltransferases for carbohydrate synthesis: alpha-2,3-sialyltransferase from Neisseria gonorrheae.

The alpha-2,3-sialyltransferase from Neisseria gonorrheae was overproduced in E. coli for exploitation of its substrate specificity and synthetic utility. Several potential acceptor substrates were synthesized in this study, including mono- and oligosaccharides, glycolipids, and glycopeptides and their sulfate derivatives. Some CMP-sialic acid derivatives with modification at the C-5 position were also prepared for evaluation as donor substrates. It was found that the enzyme exhibits a broader acceptor substrate specificity when compared to other sialyltransferases, though the donor specificity is quite limited. Application of the enzyme to the preparative synthesis of representative sialyl glycoconjugates has been demonstrated. On the basis of this work and the work of others, this enzyme is the most versatile and synthetically useful among all sialyltransferases known to date, especially for the synthesis of sulfate-containing glycoconjugates.

Carbohydrates↗
PubMed2001

[Simultaneous separation of organic acid and organic salts by electrostatic ion chromatography].

The electrostatic ion chromatographic column was prepared by coating conjugated acid salt micelles on the surface of octadecyl silica stationary phase. Pure water was used as mobile phase, and the conductance detector was connected on-line to electrostatic ion chromatograph. The conditions under which organic acid and organic salts were detected were studied. The mechanism for the above separation is discussed. Sodium benzoate and citric acid in Lichee drink were separated and determined. This method is rapid, simple with little interference and good reproducibility without any pollution since the mobile phase is water. This is an environmental friendly analytical method.

Beverages↗
PubMed2000

[Detection of specific antibodies in saliva of patients with chronic schistosomiasis japonica].

OBJECTIVE: To investigate the feasibility of detecting specific antibodies in the saliva of schistosomiasis patients. METHODS: Specific antibodies in saliva samples of 32 schistosomiasis patients and 140 normal individuals were detected by using ELISA and the results were compared with those detected for specific antibodies in serum. RESULTS: The sensitivity and specificity of the ELISA were 90.6% and 94.4%, respectively, being slightly lower than that of the 100% sensitivity and 96.2% specificity for the serum. No significant difference between them was found (P > 0.05). CONCLUSION: Detecting specific antibodies in saliva can be used for immunodiagnosis of schistosomiasis japonica as a noninvasive method in field surveys.

Adolescent↗
PubMed1999

Expression of alpha2,8/2,9-polysialyltransferase from Escherichia coli K92. Characterization of the enzyme and its reaction products.

The capsular polysaccharide of Escherichia coli K92 contains alternating -8-NeuAcalpha2- and -9-NeuAcalpha2- linkages. The enzyme catalyzing this polymerizing reaction has been cloned from the genomic DNA of E. coli K92. The 1.2-kilobase polymerase chain reaction fragment was subcloned in pRSET vector and the protein was expressed in the BL21(DE3) strain of E. coli with a hexameric histidine at its N-terminal end. The enzyme was isolated in the supernatant after lysis of the cells and fractionated by ultracentrifugation. Western blotting using anti-histidine antibody showed the presence of a band that migrated at about 47.5 kDa on both reducing and nonreducing SDS-polyacrylamide gel electrophoresis, indicating a monomeric enzyme. Among the carbohydrate acceptors tested, N-acetylneuraminic acid and the gangliosides G(D3) and G(Q1b) were preferred substrates. The cell-free enzyme reaction products obtained were characterized by NMR and mass spectrometry, which indicated the presence of both alpha2,9- and alpha2,8-linked polysialyl structure. The K92 neuS gene was used to transform the K1 strain of E. coli, the capsule of which contains only -8-NeuAcalpha2- linkages. Analysis of the polysaccharides isolated from these transformed cells is consistent with the presence of both -8-NeuAcalpha2- and -9-NeuAcalpha2- linkages. Our results suggest that the neuS gene product of E. coli K92 catalyzes the synthesis of polysialic acid with alpha2,9- and alpha2,8-linkages in vitro and in vivo.

Blotting, Western↗
PubMed1996

Overexpression, one-step purification and characterization of UDP-glucose dehydrogenase and UDP-N-acetylglucosamine pyrophosphorylase.

Two enzymes of the Leloir pathway, UDP-GlcNAc pyrophosphorylase and UDP-Glc dehydrogenase, which are involved in the synthesis of activated sugar nucleotides have been cloned, overexpressed in Escherichia coli, and purified to homogeneity in only one step by chelation-affinity chromatography. The gene KfaC of E. coli K5 was thus demonstrated to encode UDP-Glc DH. Some properties of the cloned enzymes, such as stability, pH dependence, and substrate kinetics, were studied in order to facilitate the use of these enzymes in carbohydrate synthesis, especially in the synthesis of hyaluronic acid.

Amino Acid Sequence↗
PubMed1996

[Etiological investigation on diarrhoeal diseases in the rural area of Suixian county, Yu Dong plain in China].

To study the etiology of diarrhoeal diseases in the rural area, we carried out a study on diarrhoeal diseases in two natural villages. 15 kinds of diarrhoeal pathogens including 172 strains were isolated from 318 patients with diarrhoea making the isolation rate 49.37%; 12 kinds of diarrhoeal pathogens and 69 strains were isolated from 310 healthy controls and the isolation rate was 22.26%; 6 kinds of diarrhoeal pathogens and 44 strains were isolated in 405 samples obtained from the envronmenfal samples. The major pathogens isolated from various samples were ETEC (LT) and proteus, followed by Shigella, Rotavirus. Analysis on the pathogenetic agents and preventive measurements were also carried out.

China↗
PubMed1995

CMP-KDO synthetase: overproduction and application to the synthesis of CMP-KDO and analogs.

CTP:CMP-3-deoxy-manno-octulosonate cytidylyltransferase (CMP-KDO synthetase, EC 2.7.7.38) has been cloned and overexpressed in Escherichia coli. The structure gene was amplified from the total DNA of E. coli K-235 through the primer-directed polymerase chain reaction. The gene was then cloned into lambda ZAP vector at the EcoRI and XbaI restriction sites and overexpressed in E. coli Sure strain at a level approximately 400 times as much as that produced in the host strain. Application of the enzyme to the synthesis of cytidine 5'-monophospho-3-deoxy-D-manno-2-octulosonic acid (CMP-KDO) and analogs was studied. Of several KDO analogs tested, 5-fluoro-2-keto-3,5-dideoxyoctulosonic acid (5-FKDO) was found to be a good substrate of the enzyme, and the product (CMP-5-FKDO) was prepared and characterized, representing the first stable CMP-KDO analog prepared enzymatically to date. The natural enzyme product, CMP-KDO, was however quite unstable (t1/2 = 19 min, in 50 mM MgCl2, 0.2 M Tris buffer, pH 9.0). A mechanism for the decomposition of CMP-KDO involving the hydrogen bonding interactions between the OH groups of C-5 and C-7 (and/or C-8) and the phosphate oxygens was proposed.

Base Sequence↗
PubMed1995

A new strategy for the cloning, overexpression and one step purification of three DHAP-dependent aldolases: rhamnulose-1-phosphate aldolase, fuculose-1-phosphate aldolase and tagatose-1,6-diphosphate aldolase.

Three DHAP-dependent aldolases, rhamnulose-1-phosphate aldolase (Rham-1PA), fuculose-1-phosphate aldolase (Fuc-1PA) and tagatose-1,6-diphosphate aldolase (TDPA) have been cloned and overexpressed in Escherichia coli using two different expression vectors: pTrcHis for the expression of Rham-1PA and Fuc-1PA and pRSET for the expression of TDPA. In each case the recombinant enzyme is synthesized as a fusion protein with a hexahistidine tag on the N-terminus. The three enzymes have been purified in only one step by chelation affinity chromatography. The effects of cultivation temperature and concentration of inducer have been studied in order to optimize the expression of the recombinant proteins and to avoid the formation of inclusion bodies.

Aldehyde-Lyases↗
PubMed1995

Cloning and overexpression of rhamnose isomerase and fucose isomerase.

Rhamnose isomerase and fucose isomerase were overexpressed in E. coli, purified and characterized. The rhamnose isomerase gene was ligated to the restriction sites of PstI and Hind III of vector pTrcHis and the fucose isomerase gene was ligated to the EcoRI and PstI sites of vector pKK223-3 for overexpression of the enzymes in E. coli XL1-Blue MRF. Approximately 16,500 U of active fucose isomerase and 2400 of rhamnose isomerase can be obtained per liter of culture from these expression systems.

Aldose-Ketose Isomerases↗
PubMed1994

Cloning, overexpression and isolation of the type II FDP aldolase from E. coli for specificity study and synthetic application.

A stable overexpression E. coli strain containing the plasmid pKEN 2 for the production of the Zn(2+)-dependent FDP aldolase from E. coli has been developed. Approximately 14,000 U of the enzyme (specific activity = 23.3 U/mg) can be obtained from 4-L of growth medium. The enzyme was isolated, purified to homogeneity and used for the studies of stability, substrate specificity and metal ion replacement and dissociation. Crystals of the enzyme have been obtained for structural analysis. This E. coli strain was deposited with the American Type Culture Collection (ATCC #77472).

Base Sequence↗
PubMed1989

Ferredoxin and rubredoxin from Butyribacterium methylotrophicum: complete primary structures and construction of phylogenetic trees.

Complete amino acid sequences of ferredoxin and rubredoxin from Butyribacterium methylotrophicum, a methylotrophic hetero-acetogen, were determined by combination of protease digestion, Edman degradation, carboxypeptidase digestion, and/or partial acid hydrolysis. The ferredoxin was composed of 55 amino acids with a molecular weight of 5,732 excluding iron and sulfur atoms and showed a typical 2[4Fe-4S]-type ferredoxin sequence with an internal repeat at the 14-23 and 42-51 positions. The rubredoxin was composed of 53 amino acids with a molecular weight of 5,672 excluding iron atom and showed a sequence similar to those of other anaerobic rubredoxins. The sequences were compared to those of corresponding proteins from six different bacteria to construct phylogenetic trees, which showed essentially the same topology. The relationships between the ferredoxin sequences from this bacterium and those of Clostridium thermoaceticum and Methanosarcina barkeri, both of which possess a carbonyl-dependent acetyl-CoA metabolic system, are also discussed.

Amino Acid Sequence↗
PubMed1989

Continuous Production of Thermostable beta-Amylase with Clostridium thermosulfurogenes: Effect of Culture Conditions and Metabolite Levels on Enzyme Synthesis and Activity.

A beta-amylase-overproducing mutant of Clostridium thermosulfurogenes was grown in continuous culture on soluble starch to produce thermostable beta-amylase. Enzyme productivity was reasonably stable over periods of weeks to months. The pH and temperature optima for beta-amylase production were pH 6.0 and 60 degrees C, respectively. Enzyme concentration was maximized by increasing biomass concentration by using high substrate concentrations and by maintaining a low growth rate. beta-Amylase concentration reached 90 U ml at a dilution rate of 0.07 h in a 3% starch medium. A further increase in enzyme activity levels was limited by acetic acid inhibition of growth and low beta-amylase productivity at low growth rates.

Journal Article↗
PubMed1989

Purification and properties of ferredoxin and rubredoxin from Butyribacterium methylotrophicum.

A ferredoxin and a rubredoxin from Butyribacterium methylotrophicum, which displays a carbonyl-dependent acetyl-coenzyme A synthesis, were purified to electrophoretic homogeneity. The two electron carriers showed absorption spectra similar to those in Clostridium species. The ferredoxin displayed absorption peaks at 280 and 391 nm, while rubredoxin displayed absorption peaks at 279, 382, and 482 nm. Minimum molecular weights calculated from the respective amino acid compositions were 5,727 for ferredoxin and 5,488 for rubredoxin, excluding iron and inorganic sulfur atoms. Both electron carriers were isolated as monomers, according to gel-filtration data. Electron spin resonance analysis revealed that the ferredoxin was a 2[4Fe-4S]-type and that both clusters had a midpoint redox potential value of -410 mV, whereas rubredoxin contained one acid-stable iron and had a redox value of -40 mV. The coupling of these electron carriers to hydrogenase and carbon monoxide dehydrogenase activities was investigated. Rubredoxin showed higher activity towards carbon monoxide dehydrogenase, whereas ferredoxin showed higher activity towards hydrogenase.

Aldehyde Oxidoreductases↗
PubMed1988

Purification and characterization of a novel thermostable beta-amylase from Clostridium thermosulphurogenes.

An extracellular beta-amylase from Clostridium thermosulphurogenes was purified 811-fold to homogeneity, and its general molecular, physico-chemical and catalytic properties were determined. The native enzyme was a tetramer of 210 kDa composed of a single type subunit; its 20 amino acid N-terminus displayed 45% homology with Bacillus polymyxa beta-amylase. The beta-amylase was enriched in both acidic and hydrophobic amino acids. The pure enzyme displayed an isoelectric point of 5.1 and a pH activity optimum of 5.5. The optimum temperature for beta-amylase activity was 75 degrees C, and enzyme thermostability at 80 degrees C was enhanced by substrate and Ca2+ addition. The beta-amylase hydrolysed amylose to maltose and amylopectin and glycogen to maltose and limit dextrins, and it was inhibited by alpha- and beta-cyclodextrins. The enzyme displayed kcat. and Km values for boiled soluble starch of 400,000 min-1 per mol and 1.68 mg/ml, respectively. The enzyme was antigenically distinct from plant beta-amylases.

Amino Acid Sequence↗
PubMed1988

Ethanol production by thermophilic bacteria: biochemical basis for ethanol and hydrogen tolerance in Clostridium thermohydrosulfuricum.

The metabolic and enzymatic bases for growth tolerance to ethanol (4%) and H2 (2 atm [1 atm = 101.29 kPa]) fermentation products in Clostridium thermohydrosulfuricum were compared in a sensitive wild-type strain and an insensitive alcohol-adapted strain. In the wild-type strain, ethanol (4%) and H2 (2 atm) inhibited glucose but not pyruvate fermentation parameters (growth and end product formation). Inhibition of glucose fermentation by ethanol (4%) in the wild-type strain was reversed by addition of acetone (1%), which lowered H2 and ethanol production while increasing isopropanol and acetate production. Pulsing cells grown in continuous culture on glucose with 5% ethanol or 1 atm of H2 significantly raised the NADH/NAD ratio in the wild-type strain but not in the alcohol-adapted strain. Analysis of key oxidoreductases demonstrated that the alcohol-adapted strain lacked detectable levels of reduced ferredoxin-linked NAD reductase and NAD-linked alcohol dehydrogenase activities which were present in the wild-type strain. Differences in the glucose fermentation product ratios of the two strains were related to differences in lactate dehydrogenase and hydrogenase levels and sensitivity of glyceraldehyde 3-phosphate dehydrogenase activity to NADH inhibition. A biochemical model is proposed which describes a common enzymatic mechanism for growth tolerance of thermoanaerobes to moderate concentrations of both ethanol and hydrogen.

Clostridium↗
PubMed1986

Microbial oxidation of hydrocarbons and related compounds by whole-cell suspensions of the methane-oxidizing bacterium h-2.

Previously, a thermophilic obligate methane-oxidizing bacterium, H-2 (type I), was isolated in our laboratory. H-2 is a new type of methylotroph because of the G+C content of DNA; it uses both the ribulose monophosphate pathway and the serine pathway for carbon assimilation and possesses a new quinone. In addition, we found that resting cell suspensions of H-2 had the ability to oxidize a variety of compounds different from the other methane-oxidizing bacteria as follows. (i) C(1) to C(8)n-alkanes are hydroxylated and further oxidized, yielding mixtures of the corresponding alcohols, aldehydes, acids, and ketones. Liquid alkanes are transformed through a different oxidative pathway from that of gaseous ones. (ii) Both gaseous (C(2) to C(4)) and liquid (C(5), C(6)) n-alkenes are oxidized to their corresponding 1,2-epoxides. (iii) Liquid monochloro and dichloro n-alkanes (C(5), C(6)) are oxidized, yielding their corresponding acids or haloacids. (iv) Diethyl ether is oxidized to acetic acid; no ethanol and acetaldehyde are detected. (v) Cyclic and aromatic compounds are also oxidized. (vi) Secondary alcohols (C(3) to C(10)) are oxidized to their corresponding methyl ketones.

Journal Article↗