RESUMO
Methyl parathion hydrolase (MPH) hydrolyses methyl parathion efficiently and specifically. Herein, we produced MPH from Plesiomonas sp. M6 using a Pichia pastoris multi-copy expression system. The original signal peptide sequence of the target gene was removed, and a modified coding sequence was synthesised. Multi-copy expression plasmids containing MPH were constructed using pHBM905BDM, and used to generate recombinant strains containing 1, 2, 3 or 4 copies of the MPH gene. The results showed that a higher target gene copy number increased the production of recombinant MPH (MPH-R), as anticipated. The expression level of the recombinant strain containing four copies of the MPH gene was increased to 1.9 U/ml using 500 ml shake flasks, and the specific activity was 15.8 U/mg. High-density fermentation further increased the target protein yield to 18.4 U/ml. Several metal ions were tested as additives, and Ni2+, Co2+ and Mg2+ at a concentration of 1 mM enhanced MPH-R activity by 196%, 201% and 154%, respectively. Enzyme immobilisation was then applied to overcome the difficulties in recovery, recycling and long-term stability associated with the free enzyme. Immobilised MPH-R exhibited significantly enhanced thermal and long-term stability, as well as broad pH adaptability. In the presence of inhibitors and chelating agents such as sodium dodecyl sulphate (SDS), immobilised MPH-R displayed 2-fold higher activity than free MPH-R, demonstrating its potential for industrial application.
Assuntos
Proteínas de Bactérias , Enzimas Imobilizadas , Expressão Gênica , Monoéster Fosfórico Hidrolases , Plesiomonas/genética , Saccharomycetales , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Enzimas Imobilizadas/biossíntese , Enzimas Imobilizadas/química , Enzimas Imobilizadas/genética , Enzimas Imobilizadas/isolamento & purificação , Monoéster Fosfórico Hidrolases/biossíntese , Monoéster Fosfórico Hidrolases/química , Monoéster Fosfórico Hidrolases/genética , Monoéster Fosfórico Hidrolases/isolamento & purificação , Plesiomonas/enzimologia , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Saccharomycetales/genética , Saccharomycetales/metabolismoRESUMO
Based on the research of plant taxonomy and botanical investigation, microscopic characteristics of the root, stem, leaf transverse section and powder of Atropa belladonna were studied for identification of the herb. The research detailed and made clear to the description identification and microscopic characteristics of officinal parts of the herbs. The work provided reference for the identification of A. belladonna herbs and pieces of work in the future, as well as a theoretical basis for the further research, development, medicinal use and the upgrading of quality standards.
Assuntos
Atropa belladonna/anatomia & histologia , Folhas de Planta/anatomia & histologia , Raízes de Plantas/anatomia & histologia , Caules de Planta/anatomia & histologia , Plantas Medicinais/anatomia & histologia , Atropa belladonna/citologia , Microscopia/métodos , Epiderme Vegetal/anatomia & histologia , Epiderme Vegetal/citologia , Folhas de Planta/citologia , Raízes de Plantas/citologia , Caules de Planta/citologia , Estômatos de Plantas/anatomia & histologia , Estômatos de Plantas/citologia , Plantas Medicinais/citologiaRESUMO
The RP-HPLC method was used to determinate the contents of forsythiaside B and poliumoside in different origin and parts from Callicarpa kwangtungensis. The linear ranges of forsythiaside B and poliumoside were 0. 106-3. 18 and 0. 105 2-3. 156 microg, respectively. The average recoveries of forythiaside B and poliumoside were 99. 01% ( RSD 1. 2%) and 100. 13% (RSD 0. 90% ), respectively. The contents of forsythiaside B and poliumoside were changed in different origin and parts from C. kwangtungensis. The sample from the area of Luxi, Pingxiang City, Jiangxi Province has the highest contents of forsythiaside B and poliumoside. The contents of forsythiaside B and poliumoside in different parts from C. kwangtungensis in Luxi are: leaf > stem > fruit. This result will provide a scientific basis for quality control and reasonable utilzation of C. kwangtungensis.