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Involvement of phosphatidate phosphatase in the biosynthesis of triacylglycerols in Chlamydomonas reinhardtii

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Journal of Zhejiang University-Science B(Biomedicine & biotechnology) 2013年第12期14卷 1121-1131页

作者： Xiao-dong DENG Jia-jia CAI Xiao-wen FEI Key Laboratory of Tropical Crop biotechnology Ministry of Agriculture Institute of Tropical Bioscience and Biotechnology Chinese Academy of Tropical Agricultural Sciences School of Science Hainan Medical College

Lipid biosynthesis is essential for eukaryotic cells, but the mechanisms of the process in microalgae remain poorly understood. Phosphatidic acid phosphohydrolase or 3-sn-phosphatidate phosphohydrolase(PAP) catalyzes the dephosphorylation of phosphatidic acid to form diacylglycerols and inorganic orthophosphates. This reaction is integral in the synthesis of triacylglycerols. In this study, the mRNA level of the PAP isoform CrPAP2 in a species of Chlamydomonas was found to increase in nitrogen-free conditions. Silencing of the CrPAP2 gene using RNA interference resulted in the decline of lipid content by 2.4%–17.4%. By contrast, over-expression of the CrPAP2 gene resulted in an increase in lipid content by 7.5%–21.8%. These observations indicate that regulation of the CrPAP2 gene can control the lipid content of the algal cells. In vitro CrPAP2 enzyme activity assay indicated that the cloned CrPAP2 gene exhibited biological activities.

关键词： Phosphatidate phosphohydrolase 2 Triacylglycerol biosynthesis RNAi Chlamydomonas reinhardtii Nitrogen deprivation Over-expression

Development of surface displaying system for heterologous protein expression in Candida tropicalis

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Systems Microbiology and Biomanufacturing 2023年第2期3卷 298-311页

作者： Tingting Wang Haibing Zhang Mengxuan Li Jiaxi Zheng Yuanyuan Xia Haiquan Yang Wei Shen Xianzhong Chen Key Laboratory of Industrial biotechnology Ministry of EducationJiangnan UniversityWuxi 214122China School of biotechnology Jiangnan UniversityWuxiChina

The purpose of this study was to assess the potential application of cell surface display in Candida *** display gene cassettes were constructed using five anchoring proteins from Saccharomyces cerevisiae,three of which[(suppression of exponential defect protein,SED1),(cell wall protein 2,CWP2)and(delayed anaerobic protein 4,DAN4)]were reported to show higher activity of heterologous proteins thanα-agglutinin(AGα1).The performance of yeast-enhanced green fluorescent protein(yeGFP)was evaluated using laser scanning confocal microscopy and flow *** results showed that the three anchoring regions(SED1,CWP2 and AGα1)successfully displayed yeGFP on the cell *** investigate the effect of the three anchoring proteins on the surface display of Rhizopus oryzaeα-amylase(ROA1)and Aspergillus aculeatusβ-glucosidase(BGL1)in ***,we constructed surface display gene cassettes for ROA1 and BGL1,*** strains containing the anchoring proteins SED1 and CWP2 showed higher activity of ROA1 and BGL1 than the strains containing the anchoring protein AGα*** highest ROA1 and BGL1 activities of strains with SED1 were 6.37 U/g CDW and 7.93 U/g CDW,respectively,which were sixfold and eightfold higher than those of strain with AGα*** addition,we also optimized signal *** results indicated that signal peptides have an impact on enzyme activity.

关键词： Yeast surface display Anchoring protein Glycosylphosphatidylinositol(GPI) α-Amylase β-Glucosidase Whole cell catalysis

A monoclonal antibody against Lates calcarifer vitellogenin and a competitive ELISA to evaluate vitellogenin induction after exposure to xenoestrogen

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Journal of Environmental Sciences 2019年第1期31卷 325-333页

作者： Witchuda Prasatkaew Phochit Nanthanawat Nanthika Khongchareonporn Sutin Kingtong Environmental Science program Faculty of Science Burapha University Department of biotechnology Faculty of Science Burapha University The Institute of biotechnology and Genetic Engineering Chulalongkorn University Department of Biology Faculty of Science Burapha University

A monoclonal antibody specific to sea bass(Lates calcarifer) vitellogenin(VTG) was developed,for use as a tool for monitoring endocrine disrupting chemicals(EDCs). VTG was induced in sea bass by intramuscular injection of 17β-estradiol(E_2: 2 mg/kg) every three days. Blood was collected three days after the last injection. Plasma VTG was then purified by chromatography in hydroxyapatite and a sephacryl-S300 column. Characterizations of purified VTG were done by phospholipoglycoprotein staining on a native-PAGE with confirmation by mass spectrometry(LC-MS/MS). Antibody was raised in mice by injection of purified VTG. After monoclonal antibody production, the hybridoma clone No. 41(MAb-sea bass VTG 41)was selected and developed for quantification of VTG by competitive enzyme-linked immunosorbent assay(ELISA). The ELISA method was sensitive with a detection limit of VTG 40 ng/mL. MAb-sea bass VTG 41 was specific to VTG from E_2-treated sea bass and others EDCs(Nonylphenol, Benzo[a]pyrene and CdCl_2). Moreover, cross-reactivity was also found in E_2-treated coral grouper(Epinephelus corallicola). The ELISA method obtained from this work can be further applied for the assessment of EDCs in Thailand and Southeast Asia's aquatic environment.

关键词： Monoclonal antibody Vitellogenin Biomarker Endocrine disrupting chemicals(EDCs) ELISA

Whole‑cell catalytic synthesis of trehalose by Corynebacterium glutamicum displaying trehalose synthase on its cell surface

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Systems Microbiology and Biomanufacturing 2023年第3期3卷 489-497页

作者： Ming Fang Qiang Wang Zhi Wang Ruiqi Lu Rufan Xing Xian Zhang Zhiming Rao Key Laboratory of Industrial biotechnology of Ministry of Education School of BiotechnologyJiangnan UniversityWuxi 214122Jiangsu ProvincePeople’s Republic of China

Trehalose is a disaccharide with many applications in cosmetics,refrigeration,and *** synthase is a significant enzyme in trehalose *** coli is usually used to express this enzyme *** *** is a pathogenic strain,we chose Corynebacterium glutamicum ATCC13032 as an engineering strain in this study for food safety *** of its poor permeability,we constructed two recombinant *** strains using two anchor proteins,PorH,and short-length NCgl1337,to anchor trehalose synthase from Streptomyces coelicolor on the cell surface and synthesize trehalose directly from *** on enzymatic properties indicated that NCgl1337S–ScTreSK246A had better enzyme activity and thermal stability than the free *** optimizing the whole-cell transformation,the optimal transformation condition was 35°C,pH 7.0,and OD600 of *** this condition,the conversion rate of 300 g/L maltose reached 69.5%in a 5 L *** relative conversion rate was still above 75%after repeated five times.

关键词： Trehalose synthase Corynebacterium glutamicum Surface display Whole-cell catalyses

Enhanced catalytic efficiency and substrate specificity of Streptomyces griseus trypsin by evolution‑guided mutagenesis

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Systems Microbiology and Biomanufacturing 2023年第2期3卷 287-297页

作者： Jingcheng Shi Chaofan Duan Bo Pang Yang Wang Guocheng Du Zhen Kang The Science Center for Future Foods Jiangnan UniversityWuxi 214122China The Key Laboratory of Carbohydrate Chemistry and biotechnology Ministry of EducationSchool of BiotechnologyJiangnan University1800 Lihu RoadWuxi 214122JiangsuChina The Key Laboratory of Industrial biotechnology Ministry of EducationSchool of BiotechnologyJiangnan UniversityWuxi 214122China

Streptomyces griseus trypsin(SGT)is a bacteria-sourced trypsin that could be potentially applied to industrial insulin ***,SGT produced by microbial hosts displayed low catalytic efficiency and undesired preference to lysine *** this study,by engineering theαsignal peptide in Pichia pastoris,we increased the SGT amidase activity to 67.91 U mL^(−1)in shake flask ***,we engineered SGT by evolution-guided mutagenesis and obtained three variants A45S,V177I and E180M with increased catalytic *** this basis,we performed iterative combinatorial mutagenesis and constructed a mutant A45S/V177I/E180M which the amidase activity reached 98 U mL^(−1)in shake flasks and 2506 U mL^(−1)in 3-L fed-batch ***,single mutation T190 to S190 increased the substrate catalytic preference of R to K and the R/K value was improved to 7.5,which was 2 times better than the animal-sourced trypsin.

关键词： Trypsin enzyme engineering Pichia pastoris Streptomyces griseus

Isolation and mechanism analysis of a catalytic efciency improved L‑aspartateβ‑decarboxylase toward 3‑methylaspartic acid

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Systems Microbiology and Biomanufacturing 2022年第1期2卷 157-164页

作者： Yufeng Liu Mingzhu Hao Zhemin Zhou Zhongmei Liu School of biotechnology Jiangnan UniversityWuxiJiangsuChina

L-Aspartateβ-decarboxylase from Acinetobacter radioresistens(ArASD)has been modifed to convert 3-methylaspartic acid into 2-aminobutyric acid,which activated a novel process for biosynthesis of 2-aminobutyric ***,the process is limited by the low activity of the ***,the activity of ArASD was signifcantly improved by modifcation based on sequence alignment and structural *** 38th residue of ArASD is speculated to be the key residue for regulating the conformation of the internal aldimine,and site-directed mutagenesis on R38 residue was carried out.A variant,K18A/R38K/V287I,with 2.2 times higher specifc activity was *** dynamics simulation indicated that the torsion angle of the imine bond of the variant decreased,which was benefcial to the protonation of the internal aldimine and the increase in the initial energy of the ***,the energy barrier of the transition state was reduced,resulting in improved catalytic activity toward 3-methylaspartic *** results provide a reference and a new point of view for enzyme modifcation by increasing the energy of the initial state.

关键词： L-Aspartateβ-decarboxylase 3-Methylasparic acid The internal aldimine enzyme engineering Molecular dynamics simulation

Purification and studies on characteristics of cholinesterases from Daphnia magna

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Journal of Zhejiang University-Science B(Biomedicine & biotechnology) 2013年第4期14卷 325-335页

作者： Yan-xia YANG Li-zhi NIU Shao-nan LI Institute of Pesticide and Environmental Toxicology College of Agriculture and BiotechnologyZhejiang University

Due to their significant value in both economy and ecology,Daphnia had long been employed to investigate in vivo response of cholinesterase(ChE) in anticholinesterase exposures,whereas the type constitution and property of the enzyme remained unclear.A type of ChE was purified from Daphnia magna using a three-step procedure,i.e.,Triton X-100 extraction,ammonium sulfate precipitation,and diethylaminoethyl(DEAE)-Sepharose Fast-Flow *** to sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE),molecular mass of the purified ChE was estimated to be 84 *** on substrate studies,the purified enzyme preferred butyrylthiocholine iodide(BTCh) [with maximum velocity(Vmax)/Michaelis constant(Km)=8.428 L/(min·mg protein)] to acetylthiocholine iodide(ATCh) [with V max /Km =5.346 L/(min·mg protein)] as its *** of the purified enzyme was suppressed by high concentrations of either ATCh or *** studies showed that the purified enzyme was more sensitive towards inhibition by tetraisopropylpyrophosphoramide(iso-OMPA) than by 1,5-bis(4-allyldimethylammoniumphenyl) pentan-3-one dibromide(BW284C51).Result of the study suggested that the purified ChE was more like a type of pseudocholinesterase,and it also suggested that Daphnia magna contained multiple types of ChE in their bodies.

关键词： Crustacea Pseudocholinesterase Cholinesterase Substrate preference Selective inhibitors

Cell fusion in the pathogenesis of COVID-19

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Military Medical Research 2022年第3期9卷 387-388页

作者： You Zheng Lu-Lin Zhou Yan Su Qiang Sun Laboratory of Cell Engineering Institute of BiotechnologyResearch Unit of Cell Death MechanismChinese Academy of Medical Science2021RU00820 Dongda StreetBeijing 100071China

Dear Editor,Infectionw ithsevereacuterespirator ysy ndrome coronavirus2(SARS-CoV-2),whichcausescoronavirus disease 2019 (COVID-19),is initiated by the virus spike (S)glycoprotein binding to its cellular receptor such as angiotensin converting enzyme 2 (ACE2)[1,2].Subsequently,the virus may take two routes for its entry into the host cells,including an endocytic route that ends up in endosomal compartment,and a plasma membrane route on the cell *** both routes,fusion between the viral and plasma membrane was required to introduce the viral genome into the cytoplasm[3].Similarly,SARS-CoV-2 infection results in the expression of fusogenic S glycoprotein on the host cell membrane.

关键词： ACE2 glycoprotein pathogenesis

A Review on General Nutritional Compounds and Pharmacological Properties of the Lentinula edodes Mushroom

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Food and Nutrition Sciences 2014年第12期5卷 1095-1105页

作者： Tiane Cristine Finimundy Aldo José Pinheiro Dillon Joao Antonio Pêgas Henriques Mariana Roesch Ely Laboratory of Genomics Proteomics and DNA RepairInstitute of BiotechnologyUniversity of Caxias do SulCaxias do SulBrazil Laboratory of enzyme and Biomass Institute of BiotechnologyUniversity of Caxias do SulCaxias do SulBrazil

Lentinula edodes?is a macrofungus with great potential for therapeutic applications and serves as a model for investigating functional fungi properties and isolating pure compounds for pharmaceutical use. Mushrooms have a great nutritional value and present medicinal molecules including polysaccharides, terpenoids, sterols and lipids, that participate actively in several human disorders and modulate mechanisms involved in the immune system regulation. This review will focus on general nutritional compounds and pharmacological properties of L. edodes.

关键词： Lentinula edodes β-Glucans Therapeutic Applications Review