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Journal of Environmental Sciences 2012年第6期24卷 1091-1098页

作者： Rajakumar Govindasamy Abdul Abdul Rahuman Unit of Nanotechnology and bioactive natural products Post Graduate and Research Department of ZoologyC.Abdul Hakeem CollegeMelvisharam-632509Vellore DistrictTamil NaduIndia

To evaluate the potential environmental effects of engineered nano metals,it is important to determine the adverse effects of various nanomaterials on aquatic *** tilapia （Oreochromis mossambicus） were maintained in 10 L glass aquaria,and exposed to a graded series of synthesized silver nanoparticles （Ag-NPs） at 25,50 and 75 mg/L for eight *** LC 50 value was 12.6 mg/*** activities of antioxidant enzymes and the contents of antioxidants were lowered in the gills and liver of fishes treated with AgNPs,which resulted in heavy accumulation of free *** results imply that the balance between the oxidative and antioxidant system in the fish was broken down during Ag-NPs *** principal concern related with the release of nanomaterials and their smaller particle may change the materials transport and potential toxicity to aquatic organisms compared to larger particles.

关键词： silver nanoparticles Oreochromis mossambicus oxidative stress analysis histopathological studies toxic effect

Combinatorial mutation on the β-glycosidase specific to 7-β-xylosyltaxanes and increasing the mutated enzyme production by engineering the recombinant yeast

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Acta Pharmaceutica Sinica B 2019年第3期9卷 626-638页

作者： Jing-Jing Chen Xiao Liang Fen Wang Yan-Hua Wen Tian-Jiao Chen Wan-Cang Liu Ting Gong Jin-Ling Yang Ping Zhu State Key Laboratory of bioactive Substance and Function of natural Medicines & NHC Key Laboratory of Biosynthesis of natural products Institute of Materia Medica Chinese Academy of Medical Sciences & Peking Union Medical College

Taxol is a 'blockbuster' antitumor drug produced by Taxus species with extremely low amount, while its analogue 7-β-xylosyl-10-deacetyltaxol is generally much higher in the plants. Both the fungal enzymes LXYL-P1à1 and LXYL-P1à2 can convert 7-β-xylosyl-10-deacetyltaxol into10-deacetyltaxol for Taxol semi-synthesis. Of them, LXYL-P1à2 is twice more active than LXYLP1à1, but there are only 11 significantly different amino acids in terms of the polarity and acidic-basic properties between them. In this study, single and multiple site-directed mutations at the 11 sites from LXYL-P1à1 to LXYL-P1à2 were performed to define the amino acids with upward bias in activities and to acquire variants with improved catalytic properties. Among all the 17 mutants, E12(A72 T/V91 S) was the most active and even displayed 2.8-and 3-fold higher than LXYL-P1à2 on β-xylosidase andβ-glucosidase activities. The possible mechanism for such improvement was proposed by homology modeling and molecular docking between E12 and 7-β-xylosyl-10-deacetyltaxol. The recombinant yeast GS115-P1 E12-7 was constructed by introducing variant E12, the molecular chaperone gene pdi and the bacterial hemoglobin gene vhb. This engineered yeast rendered 4 times higher biomass enzyme activity than GS115-3.5 K-P1à2 that had been used for demo-scale fermentation. Thus, GS115-P1 E12-7 becomes a promising candidate to replace GS115-3.5 K-P1à2 for industrial purpose.

关键词： β-Glycosidases Combinatorial mutation Improved catalytic property Molecular docking Engineered yeast Taxol

Interactions among SARS-CoV accessory proteins revealed by bimolecular fluorescence complementation assay

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Acta Pharmaceutica Sinica B 2015年第5期5卷 487-492页

作者： Jianqiang Kong Yanwei Shi Zhifang Wang Yiting Pan State Key Laboratory of bioactive Substance and Function of natural Medicines & Ministry of Health Key Laboratory of Biosynthesis of natural products Institute of Materia Medica Chinese Academy of Medical Sciences & Peking Union Medical College aState Key Laboratory of bioactive Substance and Function of natural Medicines & Ministry of Health Key Laboratory of Biosynthesis of natural products Institute of Materia Medica Chinese Academy of Medical Sciences & Peking Union Medical College School of Chemical Engineering Beijing Institute of Petrochemical Technology

The accessory proteins(3a, 3b, 6, 7a, 7b, 8a, 8b, 9b and ORF14), predicted unknown proteins(PUPs) encoded by the genes, are considered to be unique to the severe acute respiratory syndrome coronavirus(SARS-Co V) genome. These proteins play important roles in various biological processes mediated by interactions with their partners. However, very little is known about the interactions among these accessory proteins. Here, a EYFP(enhanced yellow fluorescent protein) bimolecular fluorescence complementation(BiFC) assay was used to detect the interactions among accessory proteins. 33 out of 81 interactions were identified by BiFC, much more than that identified by the yeast two-hybrid(Y2H)system. This is the first report describing direct visualization of interactions among accessory proteins of SARS-CoV. These findings attest to the general applicability of the BiFC system for the verification of protein-protein interactions.

关键词： SARS-CoV Accessory proteins Y2H Bimolecular fluorescence complementation assay

The enzymatic biosynthesis of acylated steroidal glycosides and their cytotoxic activity

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Acta Pharmaceutica Sinica B 2018年第6期8卷 981-994页

作者： Ming Liu Jian-Qiang Kong State Key Laboratory of bioactive Substance and Function of natural Medicines & Ministry of Health Key Laboratory of Biosynthesis of natural products Institute of Materia Medica Chinese Academy of Medical Sciences & Peking Union Medical College

Herein we describe the discovery and functional characterization of a steroidal glycosyltransferase(SGT) from Ornithogalum saundersiae and a steroidal glycoside acyltransferase(SGA) from Escherichia coli and their application in the biosynthesis of acylated steroidal glycosides(ASGs). Initially,an SGT gene, designated as OsSGT1, was isolated from O. saundersiae. OsSGT1-containing cell free extract was then used as the biocatalyst to react with 49 structurally diverse drug-like compounds. The recombinant OsSGT1 was shown to be active against both 3β-and 17β-hydroxyl steroids. Unexpectedly,in an effort to identify OsSGT1, we found the bacteria lacA gene in lac operon actually encoded an SGA,specifically catalyzing the acetylations of sugar moieties of steroid 17β-glucosides. Finally, a novel enzymatic two-step synthesis of two ASGs, acetylated testosterone-17-O-β-glucosides(AT-17β-Gs) and acetylated estradiol-17-O-β-glucosides(AE-17β-Gs), from the abundantly available free steroids using OsSGT1 and EcSGA1 as the biocatalysts was developed. The two-step process is characterized by EcSGA1-catalyzed regioselective acylations of all hydroxyl groups on the sugar unit of unprotected steroidal glycosides(SGs) in the late stage, thereby significantly streamlining the synthetic route towards ASGs and thus forming four monoacylates. The improved cytotoxic activities of 30-acetylated testosterone17-O-β-glucoside towards seven human tumor cell lines were thus observable.

关键词： Steroidal glycosyltransferase Steroidal glycoside acyltransferase Ornithogalum saundersiae Acylated steroidal glycosides LacA

Maytenus erythroxylon Reissek(Celastraceae) ethanol extract presents antidiarrheal activity via antimotility and antisecretory mechanisms

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World Journal of Gastroenterology 2017年第24期23卷 4381-4389页

作者： Rodrigo de Oliveira Formiga Zelma Glebya Maciel Quirino Margareth de Fátima Formiga Melo Diniz Alexsandro Fernandes Marinho Josean Fechine Tavares Leonia Maria Batista Postgraduate Program in natural products and bioactive synthetics Health Sciences CenterFederal University of Paraiba Applied Science and Education Center Federal University of Paraiba

To investigate the acute toxicity, phytochemical profile, antidiarrheal activity and mechanisms of action of Maytenus erythroxylon (M. erythroxylon) ethanol extract. METHODSA castor oil-induced diarrhea model was used to evaluate antidiarrheal activity. Intestinal transit and gastric emptying protocols were used to evaluate a possible antimotility effect. KATP channels, nitric oxide, presynaptic α2-adrenergic and tissue adrenergic receptors were investigated to uncover antimotility mechanisms of action and castor oil-induced enteropooling to elucidate antisecretory mechanisms. RESULTSAll tested doses of the extract (62.5, 125, 250 and 500 mg/kg) possessed antidiarrheal activity, with a significant decrease of the evacuation index. This activity is possibly related to a reduced gastric emptying (125, 250 and 500 mg/kg) and to a decreased percentage of intestinal transit for all tested doses. That last effect seems to be modulated by nitric oxide, KATP channels and tissue adrenergic receptors. Besides, the extract also presented antisecretory effect due to a decrease of intestinal fluid accumulation. CONCLUSIONThe antidiarrheal effect of M. erythroxylon found in this study involves antimotility and antisecretory mechanisms that may be attributed to the chemical compounds found in this species: saponins, flavonoids, tannins, triterpenes and steroids.

关键词： Medicinal plants Celastraceae Maytenus erythroxylon Diarrhea Antidiarrheal activity

Structural diversification of bioactive bibenzyls through modular co-culture leading to the discovery of a novel neuroprotective agent

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Acta Pharmaceutica Sinica B 2023年第4期13卷 1771-1785页

作者： Yuyu Liu Xinnan Li Songyang Sui Jingshu Tang Dawei Chen Yuying Kang Kebo Xie Jimei Liu Jiaqi Lan Lei Wu Ridao Chen Ying Peng Jungui Dai State Key Laboratory of bioactive Substance and Function of natural Medicines CAMS Key Laboratory of Enzyme and Biocatalysis of Natural Drugsand NHC Key Laboratory of Biosynthesis of Natural ProductsInstitute of Materia MedicaChinese Academy of Medical Sciences and Peking Union Medical CollegeBeijing 100050China

Bibenzyls,a kind of important plant polyphenols,have attracted growing attention for their broad and remarkable pharmacological ***,due to the low abundance in nature,uncontrollable and environmentally unfriendly chemical synthesis processes,these compounds are not readily ***,one high-yield bibenzyl backbone-producing Escherichia coli strain was constructed by using a highly active and substrate-promiscuous bibenzyl synthase identified from Dendrobium officinale in combination with starter and extender biosynthetic *** types of efficiently postmodifying modular strains were engineered by employing methyltransferases,prenyltransferase,and glycosyltransferase with high activity and substrate tolerance together with their corresponding donor biosynthetic *** different bibenzyl derivatives were tandemly and/or divergently synthesized by co-culture engineering in various combination ***,a prenylated bibenzyl derivative(12)was found to be an antioxidant that exhibited potent neuroprotective activity in the cellular and rat models of ischemia ***-seq,quantitative RT-PCR,and Western-blot analysis demonstrated that 12 could up-regulate the expression level of an apoptosis-inducing factor,mitochondria associated 3(Aifm3),suggesting that Aifm3 might be a new target in ischemic stroke *** study provides a flexible plug-and-play strategy for the easy-to-implement synthesis of structurally diverse bibenzyls through a modular co-culture engineering pipeline for drug discovery.

关键词： Bibenzyls Modular co-culture engineering Promiscuous enzymes Neuroprotective agent Aifm3 Dendrobium officinale

Functional characterization of a cycloartenol synthase and four glycosyltransferases in the biosynthesis of cycloastragenol-type astragalosides from Astragalus membranaceus

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Acta Pharmaceutica Sinica B 2023年第1期13卷 271-283页

作者： Yangyang Duan Wenyu Du Zhijun Song Ridao Chen Kebo Xie Jimei Liu Dawei Chen Jungui Dai State Key Laboratory of bioactive Substance and Function of natural Medicines CAMS Key Laboratory of Enzyme and Biocatalysis of Natural DrugsNHC Key Laboratory of Biosynthesis of Natural ProductsInstitute of Materia MedicaChinese Academy of Medical Sciences and Peking Union Medical CollegeBeijing 100050China

Astragalosides are the main active constituents of traditional Chinese medicine Huang-Qi,of which cycloastragenol-type glycosides are the most typical and major bioactive *** kind of compounds exhibit various biological functions including cardiovascular protective,neuroprotective,*** to the limitations of natural sources and the difficulties encountered in chemical synthesis,re-engineering of biosynthetic machinery will offer an alternative and promising approach to producing ***,the biosynthetic pathway for astragalosides remains elusive due to their complex structures and numerous reaction types and ***,guided by transcriptome and phylogenetic analyses,a cycloartenol synthase and four glycosyltransferases catalyzing the committed steps in the biosynthesis of such bioactive astragalosides were functionally characterized from Astragalus ***1,the first reported cycloartenol synthase from Astragalus genus,is capable of catalyzing the formation of cycloartenol;AmUGT15,AmUGT14,AmUGT13,and AmUGT7 are four glycosyltransferases biochemically characterized to catalyze 3-O-xylosylation,3-O-glucosylation,25-O-glucosylation/O-xylosylation and 2’-O-glucosylation of cycloastragenol glycosides,*** findings not only clarified the crucial enzymes for the biosynthesis and the molecular basis for the structural diversity of astragalosides in Astragalus plants,also paved the way for further completely deciphering the biosynthetic pathway and constructing an artificial pathway for their efficient production.

关键词： Cycloastragenol-type astragalosides Cycloartenol synthase Glycosyltransferase Biosynthesis Astragalus membranaceus

Steroids hydroxylation catalyzed by the monooxygenase mutant 139-3 from Bacillus megaterium BM3

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Acta Pharmaceutica Sinica B 2017年第4期7卷 510-516页

作者： Xing Liu Jian-qiang Kong State Key Laboratory of bioactive Substance and Function of natural Medicines&Ministry of Health Key Laboratory of Biosynthesis of natural products Institute of Materia MedicaChinese Academy of Medical Sciences and Peking Union Medical CollegeBeijing 100050China

The search of new substrates with pharmaceutical and industrial potential for biocatalysts including cytochrome P450 enzymes is always challenging. Cytochrome P450 BM3 mutant 139-3, a versatile biocatalyst, exhibited hydroxylation activities towards fatty acids and alkanes. However, there were limited reports about its hydroxylation activity towards steroids. Herein, an Escherichia coli-based whole-cell extract containing the recombinant 139-3 protein was used as the biocatalyst to screen 13 steroids. Results revealed that 139-3 was able to specifically hydroxylate androstenedione (1) at 1α-position, generating a hydroxylated steroid 1α-OH-androstenedione (1a). To investigate whether C-1α hydroxylation catalyzed by BM3 mutant 139-3 could be industrially used, an optimization of catalyzing conditions was performed. Accordingly, the BM3 mutant 139-3 enzyme was observed to display maximum activity at 37 °C, under pH 7.0 for 4 h, with 37% transformation rate. Moreover, four 139-3 variants were generated by random mutagenesis with the aim of improving its activity and expanding substrate scope. Surprisingly, these mutants, sharing a common mutated site R379S, lost their activities towards androstenedione (1). These data clearly indicated that arginine residue located at site 379 played key role in the hydroxylation activities of 139-3. Overall, these new findings broadened the substrate scope of 139-3 enzyme, thereby expanding its potential applications as a biocatalyst on steroids hydroxylation in pharmaceutical industry.

关键词： Cytochrome P450 BM3 139-3 Steroids hydroxylation 1α-OH-androstenedione Biocatalyst

Construction of acetyl-CoA and DBAT hybrid metabolic pathway for acetylation of 10-deacetylbaccatin Ⅲ to baccatin Ⅲ

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Acta Pharmaceutica Sinica B 2021年第10期11卷 3322-3334页

作者： Hao Wang Bo-Yong Zhang Ting Gong Tian-Jiao Chen Jing-Jing Chen Jin-Ling Yang Ping Zhu State Key Laboratory of bioactive Substance and Function of natural Medicines NHC Key Laboratory of Biosynthesis of Natural ProductsCAMS Key Laboratory of Enzyme and Biocatalysis of Natural DrugsInstitute of Materia MedicaChinese Academy of Medical Sciences&Peking Union Medical CollegeBeijing 100050China

10-DeacetylbaccatinⅢ(10-DAB)C10 acetylation is an indispensable procedure for Taxol semi-synthesis,which often requires harsh conditions.10-DeacetylbaccatinⅢ-10-β-O-acetyltransferase(DBAT)catalyzes the acetylation but acetyl-CoA supply remains a key limiting *** we refactored the innate biosynthetic pathway of acetyl-CoA in Escherichia coli and obtained a chassis with acetyl-CoA productivity over three times higher than that of the host ***,we constructed a microbial cell factory by introducing DBAT gene into this chassis for efficiently converting 10-DAB into baccatinⅢ.We found that baccatinⅢcould be efficiently deacetylated into 10-DAB by DBAT with CoASH and K+under alkaline ***,we fed acetic acid to the engineered strain both for serving as a substrate of acetyl-CoA biosynthesis and for alleviating the deacetylation of baccatinⅢ.The fermentation conditions were optimized and the baccatinⅢtiters reached 2,3 and 4.6 g/L,respectively,in a 3-L bioreactor culture when 2,3 and 6 g/L of 10-DAB were *** study provides an environmentfriendly approach for the large scale 10-DAB acetylation without addition of acetyl-CoA in the industrial Taxol *** finding of DBAT deacetylase activity may broaden its application in the structural modification of pharmaceutically important lead compounds.

关键词： Microbial cell factory Biosynthetic pathway of acetyl-CoA Bioconversion of 10-deacetylbaccatinⅢto baccatinⅢ 10-DeacetylbaccatinⅢ-10-β-Oacetyltransferase Deacetylation of baccatinⅢ High-cell-density fermentation Taxol semi-synthesis

Expression and characterization of a bifunctional alginate lyase named Al163 from the Antarctic bacterium Pseudoalteromonas sp. NJ-21

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Journal of Oceanology and Limnology 2018年第4期36卷 1304-1314页

作者： XIE Maisheng LI Jiang HE Peiqing LIN Xuezheng Key Laboratory of Marine bioactive Substances The First Institute of Oceanography State Oceanic Administration (SOA) Qingdao 266061 China Key Laboratory of Marine natural products of Qingdao Qingdao 266061 China

In this study, an endolytic alginate lyase, named Al163, was identified, cloned, and characterized from the Antarctic bacterium Pseudoalteromonas sp. NJ-21. Comparative sequence analysis showed that the predicted amino acid sequence encoded by al163 belongs to the polysaccharide lyase 6(PL-6) family and has a molecular mass of about 80 kDa. Recombinant enzyme was purified by Ni-Sepharose affinity chromatography. Recombinant Al163 exhibited maximum activity(258 U/mg) at pH 7.0 and 40℃, and thermal stability assays showed retention of almost 90% activity after incubation at 30℃ for 30 min. Al163 activity was stimulated by Cd^(2+), Ca^(2+), Fe^(3+), and Mn^(2+), but inhibited by Cu^(2+), Si^(2+), Fe^(2+), and Ni^(2+). Thin-layer chromatographic analysis indicated that Al163 degraded sodium alginate, poly M, and poly G, generating disaccharides and trisaccharides as the final products. Only a few bacterial strains that produce a bifunctional alginate lyase have been reported. Our results indicate that recombinant Al163 exhibits broad substrate specificity and its products exhibit low degrees of polymerization. Both properties imply high potential for use of the enzyme in several industrial fields, including cosmetics and pharmaceuticals, based on the high demand for biologically active oligosaccharides.

关键词： Pseudoalteromonas sp. alginate lyase PL-6 family