We produced (S)-4-cyano-3-(4-chlorophenyl)-butyrate by highly stereoselective biocatalyst in this study. A nitrilase-producing strain, named Gibberella intermedia WX12, was isolated by 3-(4-chlorophenyl)-glutaronitrile as substrate in the screening with phenol-sodium hypochlorite method. The fermentation conditions and catalytic properties of this strain were investigated. The preferred carbon and nitrogen sources for nitrilase production were lactose (30 g/L) and peptone (20 g/L). After being cultivated for 96 h, the cells were collected for use in biotransformation. The hydrolysis of 3-(4-chlorophenyl)-glutaronitrile was performed at 30 degrees C in phosphate buffer (pH 8.0, 50 mmol/L) for 24 h to give (S)-4-cyano-3-(4-chlorophenyl)-butyric acid with 90% yield and > 99% of ee, which can be used for the synthesis of (R)- and (S)-baclofen. The configuration of product was determined by chemically converting it to baclofen and comparison with the authentic sample by chiral HPLC analysis.
Aminohydrolases ; metabolism ; Baclofen ; chemical synthesis ; chemistry ; Biocatalysis ; Chlorophenols ; chemistry ; Gibberella ; enzymology ; Hydrolysis ; Nitriles ; chemistry ; Prodrugs ; chemical synthesis ; chemistry

Nitriles are an important type of synthetic intermediates in the production of fine chemicals because of their easy preparations and versatile transformations. The traditional chemical conversion of nitriles to carboxylic acids and amides is feasible but it requires relatively harsh conditions of heat, acid or alkali. Nitrile converting enzymes (nitrilase, nitrile hydratase and amidase) which are used as biocatalyst for the production of fine chemicals have attracted substantial interest because of their ability to convert readily available nitriles into the corresponding higher value amides or acids under mild conditions with excellent chemo-, regio- and stereo-selectivities. Many nitrile converting enzymes have been explored and widely used for the production of fine chemicals. In this paper, various examples of biocatalytic synthesis of pharmaceuticals and their intermediates, agrochemicals and their intermediates, food and feed additives, and other fine chemicals are presented. In the near future, an increasing number of novel nitrile converting enzymes will be screened and their potential in the production of useful fine chemicals will be further exploited.
Amides ; metabolism ; Amidohydrolases ; metabolism ; Aminohydrolases ; metabolism ; Carboxylic Acids ; metabolism ; Chemical Industry ; methods ; Hydro-Lyases ; metabolism ; Nitriles ; chemistry

The expression of the vgb gene in vivo could improve the fermentation density and then contribute the extracellular secretion of the product of bxn gene. Constructed the recombination plasmid pPIC9K-vgbbxn and transformed into Pichia pastoris GS115. The results of PCR and SDS-PAGE indicate that the vgb gene and bxn gene had integrated into the genome of Pichia pastoris GS115 and expressed in efficient level. Also, the protein activity of their products had been verified respectively. Shake flask fermentation experiments showed that the presence of VHb in yeast Pichia pastoris efficiently enhanced cell growth and secretive expression of bxn gene under hypoxic habitats.
Aminohydrolases ; genetics ; metabolism ; Bacterial Proteins ; genetics ; physiology ; Electrophoresis, Polyacrylamide Gel ; Pichia ; genetics ; Plasmids ; Polymerase Chain Reaction ; Recombinant Proteins ; biosynthesis ; Truncated Hemoglobins ; genetics ; physiology

Microbial nitrilases have attracted increasing attention in nitrile hydrolysis for carboxylic acid production in recent years. A bacterium with nitrilase activity was isolated and identified as Pseudomonas putida CGMCC3830 based on its morphology, physiological and biochemical characteristics, as well as 16S rRNA gene sequence. The nitrilase production was optimized by varying culture conditions using the one-factor-at-a-time method and response surface methodology. Glycerol 13.54 g/L, tryptone 11.59 g/L, yeast extract 5.21 g/L, KH2PO4 1 g/L, NaCl 1 g/L, urea 1 g/L, initial pH 6.0 and culture temperature 30 degrees C were proved to be the optimal culture conditions. It resulted in the maximal nitrilase production of 36.12 U/mL from 2.02 U/mL. Investigations on substrate specificity demonstrate P. putida nitrilase preferentially hydrolyze aromatic nitriles. When applied in nicotinic acid synthesis, 2 mg/mL P. putida cells completely hydrolyzed 20.8 g/L 3-cyanopyridine into nicotinic acid in 90 min. The results indicated P. putida CGMCC3830 displayed potential for industrial production of nicotinic acid.
Aminohydrolases ; biosynthesis ; Culture Media ; Hydrolysis ; Niacin ; biosynthesis ; Nitriles ; metabolism ; Pseudomonas putida ; enzymology ; Pyridines ; metabolism ; RNA, Ribosomal, 16S ; genetics ; Substrate Specificity ; Temperature

OBJECTIVETo determine the efficacy of adenovirus mediated suicide gene transduction combined with prodrug 5-fluorocytosine (5FC) as a therapeutic protocol for pancreatic cancer.

METHODSCytosine Deaminase(CD) gene was cloned into pAdTrack-CMV-CD, pAdTrack-CMV-CD and pAdEasy-1 were recombined in bacteria. The newly recombined adenovirus (Ad)-CD containing green fluorescent protein (GFP) were packaged and propagated in 293 cells and purified by cesium chloride gradient centrifugation. Human pancreatic carcinoma cell line-Patu8988 was infected with this virus, then 5FC was added. XTT assay was used to estimate relative numbers of viable cells. In vivo model of pancreatic cancer was established by injecting 1.0 x 10(7) Patu8988 cells subcutaneously in Balb/c nude mice. When tumors were palpable, Ad-CD was injected into each tumor and 5FC was administered.

RESULTSPositive clones were selected using endonuclease to digest the recombinants and the concentration of viral liquids containing the CD gene was 2 x 10(11) pfu /ml. Significant cytotoxic activity as shown for 5FC in the CD gene transduced 8988 cell line, while little effect was found in the nontransduced pancreatic carcinoma cells. Antitumor effect was observed in Patu8988 xenograft nude mice with in situ CD gene transduction.

CONCLUSIONSCD gene mediated by adenovirus has high infectivity and may be useful for gene therapy in pancreatic carcinoma. These data demonstrate the use of an enzyme prodrug strategy in experimental pancreatic cancer.

Adenoviridae ; genetics ; Animals ; Cytosine Deaminase ; Gene Transfer Techniques ; Genetic Therapy ; Genetic Vectors ; Humans ; Mice ; Mice, Inbred BALB C ; Nucleoside Deaminases ; genetics ; Pancreatic Neoplasms ; therapy

BACKGROUND: Scrub typhus may cause complications such as pneumonia, meningoencephalitis, liver failure, and renal failure, although most of patients with scrub typhus are treated with appropriate antibiotics. Scrub typhus has varying degree of disease severity, from mild to fatal, therefore, early recognition of the clinical parameters representing the severity of the disease is a very important factor for effective treatment. MATERIALS AND METHODS: We investigated clinical, laboratory results and simple chest X-ray images retrospectively by reviewing the medical records of 212 scrub typhus patients, admitted to Catholic University St. Vincent's Hospital from September 2000 to November 2004. RESULTS: Patients demographics revealed a mean age of 59.7, (age: 18 to 90), and 145 of the 212 patients were female (68.4%). Cure was achieved in 211 (99.5%) of the 212 patients with the 1-week course of daily 200 mg doses of doxycycline. Fifty-nine (27.8%) of the patients revealed the abnormal findings, such as interstitial, pneumonic infiltrations, or pleural effusions, in the results of simple chest X-ray images, and cure was achieved in 58 (98.3%) of 59 patients. The median time to defervescence was 27.1 h for the group showing abnormal simple chest X-ray findings, 23.3 h for the group showing normal chest X-ray findings. The titers of serum C-reactive protein and adenine deaminase were significantly higher, and initial hemoglobin and serum albumin levels were lower, in the group with abnormal simple chest X-ray findings, compared to the group with normal simple chest X-ray findings. CONCLUSION: The 1-week course of daily 200-mg doses of doxycycline was effective for the treatment of scrub typhus. Initial simple chest X-ray findings may be useful in the selection of patients, who might show more favorable laboratory findings.
Adenine ; Aminohydrolases ; Anti-Bacterial Agents ; C-Reactive Protein ; Demography ; Doxycycline ; Female ; Hemoglobins ; Humans ; Liver Failure ; Medical Records ; Meningoencephalitis ; Pleural Effusion ; Pneumonia ; Renal Insufficiency ; Retrospective Studies ; Scrub Typhus ; Serum Albumin ; Thorax

BACKGROUND: Scrub typhus may cause complications such as pneumonia, meningoencephalitis, liver failure, and renal failure, although most of patients with scrub typhus are treated with appropriate antibiotics. Scrub typhus has varying degree of disease severity, from mild to fatal, therefore, early recognition of the clinical parameters representing the severity of the disease is a very important factor for effective treatment. MATERIALS AND METHODS: We investigated clinical, laboratory results and simple chest X-ray images retrospectively by reviewing the medical records of 212 scrub typhus patients, admitted to Catholic University St. Vincent's Hospital from September 2000 to November 2004. RESULTS: Patients demographics revealed a mean age of 59.7, (age: 18 to 90), and 145 of the 212 patients were female (68.4%). Cure was achieved in 211 (99.5%) of the 212 patients with the 1-week course of daily 200 mg doses of doxycycline. Fifty-nine (27.8%) of the patients revealed the abnormal findings, such as interstitial, pneumonic infiltrations, or pleural effusions, in the results of simple chest X-ray images, and cure was achieved in 58 (98.3%) of 59 patients. The median time to defervescence was 27.1 h for the group showing abnormal simple chest X-ray findings, 23.3 h for the group showing normal chest X-ray findings. The titers of serum C-reactive protein and adenine deaminase were significantly higher, and initial hemoglobin and serum albumin levels were lower, in the group with abnormal simple chest X-ray findings, compared to the group with normal simple chest X-ray findings. CONCLUSION: The 1-week course of daily 200-mg doses of doxycycline was effective for the treatment of scrub typhus. Initial simple chest X-ray findings may be useful in the selection of patients, who might show more favorable laboratory findings.
Adenine ; Aminohydrolases ; Anti-Bacterial Agents ; C-Reactive Protein ; Demography ; Doxycycline ; Female ; Hemoglobins ; Humans ; Liver Failure ; Medical Records ; Meningoencephalitis ; Pleural Effusion ; Pneumonia ; Renal Insufficiency ; Retrospective Studies ; Scrub Typhus ; Serum Albumin ; Thorax

OBJECTIVES: To study the association of maternal methylenetetrahydrofolate dehydrogenase 1 (MTHFD1) and methylenetetrahydrofolate dehydrogenase 2 (MTHFD2) gene polymorphisms with congenital heart disease (CHD) in offspring. METHODS: A hospital-based case-control study was conducted. The mothers of 683 children with CHD alone who attended Hunan Children's Hospital, from November 2017 to March 2020 were enrolled as the case group, and the mothers of 740 healthy children who attended the same hospital during the same period and did not have any deformity were enrolled as the control group. A questionnaire survey was performed to collect related exposure data, and then venous blood samples (5 mL) were collected from the mothers to detect MTHFD1 and MTHFD2 gene polymorphisms. A multivariate logistic regression analysis was used to evaluate the association of MTHFD1 and MTHFD2 gene polymorphisms with CHD. The four-gamete test in Haploview 4.2 software was used to construct haplotypes and evaluate the association between haplotypes and CHD. The generalized multifactor dimensionality reduction method and logistic regression analysis were used to examine gene-gene interaction and its association with CHD. RESULTS: The multivariate logistic regression analysis showed that maternal MTHFD1 gene polymorphisms at rs11849530 (GA vs AA: OR=1.49; GG vs AA: OR=2.04) andat rs1256142 (GA vs GG: OR=2.34; AA vs GG: OR=3.25) significantly increased the risk of CHD in offspring (P<0.05), while maternal MTHFD1 gene polymorphisms at rs1950902 (AA vs GG: OR=0.57) and MTHFD2 gene polymorphisms at rs1095966 (CA vs CC: OR=0.68) significantly reduced the risk of CHD in offspring (P<0.05). The haplotypes of G-G-G (OR=1.86) and G-A-G (OR=1.35) in mothers significantly increased the risk of CHD in offspring (P<0.05). The gene-gene interaction analyses showed that the first-order interaction between MTHFD1 rs1950902 and MTHFD1 rs2236222 and the second-order interaction involving MTHFD1 rs1950902, MTHFD1 rs1256142, and MTHFD2 rs1095966 might be associated with risk of CHD (P<0.05). CONCLUSIONS Maternal MTHFD1 and MTHFD2 gene polymorphisms and their haplotypes, as well as the interaction between MTHFD1 rs1950902 and MTHFD1 rs2236222 and between MTHFD1 rs1950902, MTHFD1 rs1256142, and MTHFD2 rs1095966, are associated with the risk of CHD in offspring.
Aminohydrolases/genetics* ; Case-Control Studies ; Child ; Female ; Genetic Predisposition to Disease ; Heart Defects, Congenital/genetics* ; Humans ; Methylenetetrahydrofolate Dehydrogenase (NADP)/genetics* ; Minor Histocompatibility Antigens/genetics* ; Mothers ; Multifunctional Enzymes/genetics* ; Polymorphism, Single Nucleotide ; Risk Factors

OBJECTIVETo elucidate the killing activity of yeast cytosine deaminase/5-fluorocytosine (YCD/5-FC) gene therapy system on gene-transferred tumorigenic cell line K562B in vivo.

METHODK562B cell was infected with high titer virus and a gene transferred cell clone, YCD-K562B, was selected. Twelve male SCID mice of 4 week old were divided into 2 groups at random and both YCD-K562B and K562B cells were implanted to each mice. 5-FC or saline was given i. p for 10 days after tumor developed, and relative tumor volume was measured every 3 days. At the end of experiment, animals were sacrificed and the specimens were processed for histopathological examination.

RESULTSAt the end of experiment (21 days after tumor cell implantation), the relative tumor volume of the 4 groups were: YCD-K562B + 5-FC 2.922 +/- 0.581, YCD-K562B + saline 24.434 +/- 4.790, K562B + 5-FC 22.701 +/- 2.350 and K562B + saline 24.460 +/- 1.670; t-test analysis showed that 5-FC could kill cells (YCD-K562B) in vivo (P = 0.0001), but had no effect on the growth of gene-untransferred cells (K562B) (P = 0.096). In YCD-K562B + 5-FC group, relative tumor volume reduced in 3 approximately 6 days after treatment (the minimum was 0.681). Necrosis around artery could be found in the tumor of YCD-K562B + 5-FC group.

CONCLUSIONYCD/5-FC suicide gene therapy system has a significant in vivo killing activity to gene-transferred tumorigenic YCD-K562B cell.

Animals ; Cytosine Deaminase ; Flucytosine ; metabolism ; pharmacology ; Genetic Therapy ; methods ; Humans ; K562 Cells ; Male ; Mice ; Mice, SCID ; Neoplasm Transplantation ; Neoplasms, Experimental ; genetics ; therapy ; Nucleoside Deaminases ; genetics ; metabolism ; Saccharomyces cerevisiae ; enzymology ; Transfection ; Treatment Outcome ; Xenograft Model Antitumor Assays

OBJECTIVETo investigate the anticancer activity of three-pronged approach: liposome transfer cytosine deaminase (CD) gene, ionizing radiation, and dendritic cells (DCs).

METHODSAfter rectal cancer cells were killed by CD and 5-FC and radiotherapy with improved liposome transfection and radiosensitization, the killed cancer cells, DCs and T lymphocytes were incubated together. The uptake of (3)H-TdR weighed the immune activity of DCs. The effect of anticancer was monitored by MTT.

RESULTSDCs acquired the apoptosis body and other antigen from the killed cancer cells and presented them to T-lymphocytes. The induced CTL killed the cancer cells powerfully and selectively. The mortality of rectal cancer cells was 98.1%, 76.2%, 37.8% at the ratio of efficacy to target of 30:1, 15:1, 1:1, respectively.

CONCLUSIONThe three-pronged approach may represent a powerful and safe means to selectively destroy cancer cells.

Combined Modality Therapy ; Cytosine Deaminase ; Dendritic Cells ; Gamma Rays ; therapeutic use ; Genetic Therapy ; Humans ; In Vitro Techniques ; Liposomes ; Nucleoside Deaminases ; genetics ; therapeutic use ; Radiation, Ionizing ; Rectal Neoplasms ; mortality ; radiotherapy ; therapy