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Author Report for: Wei D

Title: Alzheimer's Disease; Mechanism, Mutations, and Applications of Nano-Medicine
Maisam M, Khan MT, Lodhi MS, Mou K, Liu Z, Wei D
Ref: Front Biosci (Landmark Ed), 28:258, 2023 : PubMed
Abstract


ESTHER: Maisam_2023_Front.Biosci.(Landmark.Ed)_28_258
PubMedSearch: Maisam 2023 Front.Biosci.(Landmark.Ed) 28 258
PubMedID: 37919079

Abstract

BACKGROUND: In the past 10 years, significant progress has been made in understanding the pathogenic chain of events that causes Alzheimer's disease (AD). According to the most widely accepted concept, the production and aggregation of beta-amyloid (Abeta) peptides play a critical role in AD. As a result, therapeutic intervention with these processes is the focus of intense research. The Abeta peptide is cleaved by the alpha-secretase, beta-secretase, and gamma-secretase enzymes in a region near the pathogenic amyloid precursor protein (APP) and mutations occurring site. METHODS: In the current review, a complete picture of the risk factors behind AD has been investigated. Mutations involved in AD progression have also been screened in various studies. RESULTS: Most of the mutations in the amyloid precursor protein (APP) can lead to the accumulation of APP oligomers in the brain, leading to AD. Several point mutations in APP can cause familial AD (FAD), including the Swedish mutation (K>M670/671N>L) and the A673>V mutation. The pathogenic A673>V mutation and Swedish mutation (M670>K/N671>L) are present in the same region of amyloid precursor protein (APP). However, the A673>T mutation has been shown to confer protection against AD. CONCLUSION: More investigations are needed from geographically distinct regions on mutations associated with AD development and applications of nanomedicines for better management of the disease burden in the future. Nanotechnology-produced metal nanoparticles (NPs) have gotten much attention because of their wide range of uses in the medicinal and agricultural industries. Nanomedicine containing potential phytochemicals, including GX-50 and curcumin conjugated with NPs, maybe a potential candidate for treating AD.



        

Title: Identification, Characterization, and Computer-Aided Rational Design of a Novel Thermophilic Esterase from Geobacillus subterraneus, and Application in the Synthesis of Cinnamyl Acetate
Zhang J, Lin L, Wei W, Wei D
Ref: Appl Biochem Biotechnol, :, 2023 : PubMed
Abstract


ESTHER: Zhang_2023_Appl.Biochem.Biotechnol__
PubMedSearch: Zhang 2023 Appl.Biochem.Biotechnol
PubMedID: 37713064

Abstract

Investigation of a novel thermophilic esterase gene from Geobacillus subterraneus DSMZ 13552 indicated a high amino acid sequence similarity of 25.9% to a reported esterase from Geobacillus sp. A strategy that integrated computer-aided rational design tools was developed to select mutation sites. Six mutants were selected from four criteria based on the simulated saturation mutation (including 19 amino acid residues) results. Of these, the mutants Q78Y and G119A were found to retain 87% and 27% activity after incubation at 70 degreesC for 20 min, compared with the 19% activity for the wild type. Subsequently, a double-point mutant (Q78Y/G119A) was obtained and identified with optimal temperature increase from 65 to 70 degreesC and a 41.51% decrease in K(m). The obtained T(1/2) values of 42.2 min (70 degreesC) and 16.9 min (75 degreesC) for Q78Y/G119A showed increases of 340% and 412% compared with that in the wild type. Q78Y/G119A was then employed as a biocatalyst to synthesize cinnamyl acetate, for which the conversion rate reached 99.40% with 0.3 M cinnamyl alcohol at 60 degreesC. The results validated the enhanced enzymatic properties of the mutant and indicated better prospects for industrial application as compared to that in the wild type. This study reported a method by which an enzyme could evolve to achieve enhanced thermostability, thereby increasing its potential for industrial applications, which could also be expanded to other esterases.



        

Title: Pesticide Residues in Commonly Consumed Vegetables in Henan Province of China in 2020
Ma C, Wei D, Liu P, Fan K, Nie L, Song Y, Wang M, Wang L, Xu Q and Zeng X <10 more author(s)> Ma C, Wei D, Liu P, Fan K, Nie L, Song Y, Wang M, Wang L, Xu Q, Wang J, Shi J, Geng J, Zhao M, Jia Z, Huan C, Huo W, Wang C, Mao Z, Huang S, Zeng X (- 10)
Ref: Front Public Health, 10:901485, 2022 : PubMed
Abstract


ESTHER: Ma_2022_Front.Public.Health_10_901485
PubMedSearch: Ma 2022 Front.Public.Health 10 901485
PubMedID: 35757605

Abstract

BACKGROUND: Pesticides are widely used in agricultural production to control insect pests and regulate plant growth in China, which may result in the presence of some pesticide residues in the vegetables. However, few studies of monitoring pesticides have been conducted in Henan Province. The aim of this study was to evaluate the level of pesticide residues in commonly consumed vegetables in the regions of Henan Province. METHODS: In this study, we collected 5,576 samples of 15 different vegetables in 17 areas from Henan Province during 2020. Eight kinds of pesticides were analyzed by gas chromatography-mass spectrometry (GC-MS), including procymidone, lambda-cyhalothrin, cypermethrin, pendimethalin, isocarbophos, isazophos, fenthion and deltamethrin. The chi-square test was used to compare the detection rates of pesticide residues in different regions. RESULTS: Of all the pesticides above, procymidone, lambda-cyhalothrin, cypermethrin, pendimethalin and isocarbophos were detected in vegetables, the detection rates were 27.0%, 16.2%, 11.4%, 3.5%, and 1.9%, respectively. However, isazophos, fenthion, and deltamethrin were not detected. In addition, procymidone, lambda-cyhalothrin, and cypermethrin were detected in urban areas, while pendimethalin was detected in rural areas. The detection rates of cypermethrin and pendimethalin in rural were 19.8% and 5.4%, respectively, which in urban were at relatively lower levels (13.7% and 1.9%, respectively) (P < 0.05). Compared the differences of pesticide detection rates among five areas of Henan province, we found that there were statistical differences in the detection rates of procymidone, cypermethrin and lambda-cyhalothrin in different regions (all P < 0.05). CONCLUSION: The results have revealed that the pesticide residues are present. Higher detection rates and more types of pesticides were found in rural areas than urban areas. In addition, there were higher detection rates in Eastern Henan. The findings provided valuable information on the current pesticide residues status, which can be a reference of pesticide supervision and management.



        

Title: Encapsulating gold nanoclusters into metal-organic frameworks to boost luminescence for sensitive detection of copper ions and organophosphorus pesticides
Wei D, Li M, Wang Y, Zhu N, Hu X, Zhao B, Zhang Z, Yin D
Ref: J Hazard Mater, 441:129890, 2022 : PubMed
Abstract


ESTHER: Wei_2022_J.Hazard.Mater_441_129890
PubMedSearch: Wei 2022 J.Hazard.Mater 441 129890
PubMedID: 36084467

Abstract

Gold nanoclusters (Au NCs) with luminescence property are emerging as promising candidates in fluorescent methods for monitoring contaminants, but low luminescence efficiency hampers their extensive applications. Herein, GSH-Au NCs@ZIF-8 was designed by encapsulating GSH-Au NCs with AIE effect into metal-organic frameworks, achieving high luminescence efficiency and good stability through the confinement effect of ZIF-8. Accordingly, a fluorescent sensing platform was constructed for the sensitive detection of copper ions (Cu(2+)) and organophosphorus pesticides (OPs). Firstly, the as-prepared GSH-Au NCs@ZIF-8 could strongly accumulate Cu(2+) due to the adsorption property of MOFs, accompanied by a significant fluorescence quenching effect with a low detection limit of 0.016 microM for Cu(2+). Besides, thiocholine (Tch), the hydrolysis product of acetylthiocholine (ATch) by acetylcholinesterase (AchE), could coordinate with Cu(2+) by sulfhydryl groups (-SH), leading to a significant fluorescence recovery, which was further used for the quantification of OPs owing to its inhibition to AChE activity. Furthermore, a hydrogel sensor was explored to accomplish equipment-free, visual, and quantitative monitoring of Cu(2+) and OPs by a smartphone sensing platform. Overall, this work provides an effective and universal strategy for enhancing the luminescence efficiency and stability of Au NCs, which would greatly promote their applications in contaminants monitoring.



        

Title: GSK2256294 Decreases sEH (Soluble Epoxide Hydrolase) Activity in Plasma, Muscle, and Adipose and Reduces F2-Isoprostanes but Does Not Alter Insulin Sensitivity in Humans
Luther JM, Ray J, Wei D, Koethe JR, Hannah L, DeMatteo A, Manning R, Terker AS, Peng D and Brown NJ <4 more author(s)> Luther JM, Ray J, Wei D, Koethe JR, Hannah L, DeMatteo A, Manning R, Terker AS, Peng D, Nian H, Yu C, Mashayekhi M, Gamboa J, Brown NJ (- 4)
Ref: Hypertension, 78:1092, 2021 : PubMed
Abstract


ESTHER: Luther_2021_Hypertension_78_1092
PubMedSearch: Luther 2021 Hypertension 78 1092
PubMedID: 34455816



        

Title: A Lab-in-a-Syringe Device Integrated with a Smartphone Platform: Colorimetric and Fluorescent Dual-Mode Signals for On-Site Detection of Organophosphorus Pesticides
Wei D, Wang Y, Zhu N, Xiao J, Li X, Xu T, Hu X, Zhang Z, Yin D
Ref: ACS Appl Mater Interfaces, :, 2021 : PubMed
Abstract


ESTHER: Wei_2021_ACS.Appl.Mater.Interfaces__
PubMedSearch: Wei 2021 ACS.Appl.Mater.Interfaces
PubMedID: 34623807

Abstract

Herein, a portable lab-in-a-syringe device integrated with a smartphone sensing platform was designed for rapid, visual quantitative determination of organophosphorus pesticides (OPs) via colorimetric and fluorescent signals. The device was chiefly made up of a conjugate pad labeled with cetyltrimethylammonium bromide-coated gold nanoparticles (CTAB-Au NPs) and a sensing pad modified by ratiometric probes (red-emission quantum dots@SiO(2) nanoparticles@green-emission quantum dots, rQDs@SiO(2)@gQDs probe), which was assembled through a disposable syringe and reusable plastic filter. In the detection system, thiocholine (Tch), the hydrolysis product of thioacetylcholine (ATch) by acetylcholinesterase (AchE), could trigger the aggregation of CTAB-Au NPs, resulting in a significant color change from red to purple. Then, CTAB-Au NPs flowed vertically upward and bound to the rQDs@SiO(2)@gQDs probe on the sensing pad, reducing the fluorescence resonance energy transfer effect between CTAB-Au NPs and gQDs. Meanwhile, rQDs embedded in SiO(2) NPs remained stable as internal reference fluorescence, achieving a color transition from red to green. Thus, based on the inhibition of AChE activity by OPs, a colorimetric and fluorescent dual-mode platform was constructed for on-site detection of OPs. Using glyphosate as a model, with the support of a color recognizer application (APP) on a smartphone, the ratio of red and green channel values could be utilized for accurate OP quantitative analysis ranging from 0 to 10 microM with a detection limit of 2.81 nM (recoveries, 90.8-122.4%; CV, 1.2-3.4%). Overall, the portable lab-in-a-syringe device based on a smartphone sensing platform integrated sample monitoring and result analysis in the field, implying great potential for on-site detection of OPs.



        

Title: Benzisothiazolinone derivatives as potent allosteric monoacylglycerol lipase inhibitors that functionally mimic sulfenylation of regulatory cysteines
Castelli R, Scalvini L, Vacondio F, Lodola A, Anselmi M, Vezzosi S, Carmi C, Bassi M, Ferlenghi F and Piomelli D <11 more author(s)> Castelli R, Scalvini L, Vacondio F, Lodola A, Anselmi M, Vezzosi S, Carmi C, Bassi M, Ferlenghi F, Rivara S, Moller IR, Rand KD, Daglian J, Wei D, Dotsey EY, Ahmed F, Jung KM, Stella N, Singh S, Mor M, Piomelli D (- 11)
Ref: Journal of Medicinal Chemistry, 63:1261, 2020 : PubMed
Abstract


ESTHER: Castelli_2020_J.Med.Chem_63_1261
PubMedSearch: Castelli 2020 J.Med.Chem 63 1261
PubMedID: 31714779
Inhibitor(s) related to this paper: Octhilinone, Octyl-Benzisothiazolinone, Benzisothiazolinone-13

Abstract

We describe a set of benzisothiazolinone (BTZ) derivatives that are potent inhibitors of monoacylglycerol lipase (MGL), the primary degrading enzyme for the endocannabinoid 2-arachidonoyl-sn-glycerol (2-AG). Structure-activity relationship studies evaluated various substitutions on the nitrogen atom and the benzene ring of the BTZ nucleus. Optimized derivatives with nanomolar potency allowed to investigate the mechanism of MGL inhibition. Site-directed mutagenesis and mass spectrometry experiments showed that BTZs interact in a covalent reversible manner with regulatory cysteines, Cys201 and Cys208, causing a reversible sulfenylation known to modulate MGL activity. Metadynamics simulations revealed that BTZ adducts favor a closed conformation of MGL that occludes substrate recruitment. The BTZ derivative 13 protected neuronal cells from oxidative stimuli and increased 2-AG levels in mouse brain. The results identify Cys201 and Cys208 as key regulators of MGL function, and point to the BTZ scaffold as a useful starting point for the discovery of allosteric MGL inhibitors.



        

Title: A Novel Targeted and High-Efficiency Nanosystem for Combinational Therapy for Alzheimer's Disease
Yang H, Mu W, Wei D, Zhang Y, Duan Y, Gao JX, Gong XQ, Wang HJ, Wu XL and Chang J <1 more author(s)> Yang H, Mu W, Wei D, Zhang Y, Duan Y, Gao JX, Gong XQ, Wang HJ, Wu XL, Tao H, Chang J (- 1)
Ref: Adv Sci (Weinh), 7:1902906, 2020 : PubMed
Abstract


ESTHER: Yang_2020_Adv.Sci.(Weinh)_7_1902906
PubMedSearch: Yang 2020 Adv.Sci.(Weinh) 7 1902906
PubMedID: 33042734

Abstract

Alzheimer's disease (AD) remains the most prevalent neurodegenerative disease, and no effective treatment is available yet. Metal-ion-triggered aggregates of amyloid-beta (Abeta) peptide and acetylcholine imbalance are reported to be possible factors in AD pathogenesis. Thus, a combination therapy that can not only inhibit and reduce Abeta aggregation but also simultaneously regulate acetylcholine imbalance that can serve as a potential treatment for AD is needed. Here, clioquinol (metal-ion chelating agent) and donepezil (acetylcholinesterase (AChE) inhibitor) co-encapsulated human serum albumin (HSA) nanoparticles (dcHGT NPs) are designed, which are modified with transcriptional activator protein (TAT) and monosialotetrahexosylganglioside (GM1). The GM1 lipid and TAT peptide endow this drug delivery nanosystem with high brain entry efficiency and long-term retention capabilities through intranasal administration. It is found that dcHGT NPs can significantly inhibit and eliminate Abeta aggregation, relieve acetylcholine-related inflammation in microglial cells, and protect primary neurons from Abeta oligomer-induced neurotoxicity in vitro. The alleviation of Abeta-related inflammation and AChE-inhibited effect further synergistically adjust acetylcholine imbalance. It is further demonstrated that dcHGT NPs reduce Abeta deposition, ameliorate neuron morphological changes, rescue memory deficits, and greatly improve acetylcholine regulation ability in vivo. This multifunctional synergetic nanosystem can be a new candidate to achieve highly efficient combination therapy for AD.



        

Title: Hydroxy-alpha-sanshool isolated from Zanthoxylum bungeanum attenuates learning and memory impairments in scopolamine-treated mice
Zhang M, Xie M, Wei D, Wang L, Hu M, Zhang Q, He Z, Peng W, Wu C
Ref: Food Funct, 10:7315, 2019 : PubMed
Abstract


ESTHER: Zhang_2019_Food.Funct_10_7315
PubMedSearch: Zhang 2019 Food.Funct 10 7315
PubMedID: 31637395

Abstract

Learning and memory impairments are common symptoms of dementia in neurodegenerative disorders. Occasionally, we found that Zanthoxylum bungeanum pericarps (ZBP) significantly activated the spontaneous activity of the hippocampus (HIPP) and paraHIPP (P < 0.001, uncorrected), implying the potential ability of ZBP to improve cognitive impairments. Thus, this study aimed to investigate the improving effect of hydroxy-alpha-sanshool (HAS), a characteristic ingredient of ZBP, against scopolamine (1 mg kg(-1), i.p.)-induced learning and memory deficits. HAS (5 mg kg(-1), p.o.) markedly reversed scopolamine-induced cognitive impairments, as indicated by its performance in the passive avoidance test and Morris water maze test (P < 0.01). Furthermore, HAS (2.5 and 5.0 mg kg(-1), p.o.) also dose-dependently prevented changes in hippocampal neuronal morphology and apoptosis, inhibited acetylcholinesterase (AChE) activity, increased the acetylcholine (ACh) content, and increased the protein and mRNA expression of brain-derived neurotrophic factor (BDNF) and phospho-cAMP response element-binding (p-CREB) compared with those in the model group (P < 0.05 & P < 0.01). These findings demonstrated that HAS attenuated scopolamine-induced cognitive impairments mainly by enhancing the activity of the cholinergic system and increasing the CREB/BDNF signalling pathway.



        

Title: Association between FASN gene polymorphisms ultrasound carcass traits and intramuscular fat in Qinchuan cattle
Raza SHA, Gui L, Khan R, Schreurs NM, Xiaoyu W, Wu S, Mei C, Wang L, Ma X and Zan L <5 more author(s)> Raza SHA, Gui L, Khan R, Schreurs NM, Xiaoyu W, Wu S, Mei C, Wang L, Ma X, Wei D, Guo H, Zhang S, Wang X, Kaleri HA, Zan L (- 5)
Ref: Gene, 645:55, 2018 : PubMed
Abstract


ESTHER: Raza_2018_Gene_645_55
PubMedSearch: Raza 2018 Gene 645 55
PubMedID: 29273553
Gene_locus related to this paper: bovin-fas

Abstract

Fatty acid synthase (FASN) is an enzyme involved with fat deposition and fatty acid composition in cattle. This study was conducted to detect single nucleotide polymorphisms (SNPs) of the FASN gene and explore their relationships with ultrasound carcass traits in order to assess the potential use of the FASN gene for the breeding selection of Qinchuan cattle for desirable carcass traits. The frequencies of SNP g.12740C>T, g.13192T>C and g.13232C>T were identified in 525 individual Qinchuan cattle which were also assessed for backfat depth, eye muscle area and intramuscular fat by ultrasound. According to the PIC values, g.13192T>C possessed an intermediate polymorphism (0.25T, g.12740C>T possessed low polymorphism (PIC<0.25). Chi-square tests showed that g.13192T>C were in Hardy-Weinberg disequilibrium (c2C was associated with a greater eye muscle area and the TT genotype at g.13232C>T was associated with greater intramuscular fat. When these genotypes were combined there was no difference in eye muscle area and intramuscular fat between the diplotypes. The H2H2 diplotype was associated with carcass traits that are likely to provide economic advantage in Qinchuan cattle. Variations in the FASN genes and their corresponding genotypes may be considered as molecular markers for economic traits in cattle breeding.



        

Title: Crius: A novel fragment-based algorithm of de novo substrate prediction for enzymes
Yao Z, Jiang S, Zhang L, Gao B, He X, Zhang JZH, Wei D
Ref: Protein Science, 27:1526, 2018 : PubMed
Abstract


ESTHER: Yao_2018_Protein.Sci_27_1526
PubMedSearch: Yao 2018 Protein.Sci 27 1526
PubMedID: 29722450

Abstract

The study of enzyme substrate specificity is vital for developing potential applications of enzymes. However, the routine experimental procedures require lot of resources in the discovery of novel substrates. This article reports an in silico structure-based algorithm called Crius, which predicts substrates for enzyme. The results of this fragment-based algorithm show good agreements between the simulated and experimental substrate specificities, using a lipase from Candida antarctica (CALB), a nitrilase from Cyanobacterium syechocystis sp. PCC6803 (Nit6803), and an aldo-keto reductase from Gluconobacter oxydans (Gox0644). This opens new prospects of developing computer algorithms that can effectively predict substrates for an enzyme.



        

Title: Autotransporter domain-dependent enzymatic analysis of a novel extremely thermostable carboxylesterase with high biodegradability towards pyrethroid pesticides
Cai X, Wang W, Lin L, He D, Huang G, Shen Y, Wei W, Wei D
Ref: Sci Rep, 7:3461, 2017 : PubMed
Abstract


ESTHER: Cai_2017_Sci.Rep_7_3461
PubMedSearch: Cai 2017 Sci.Rep 7 3461
PubMedID: 28615636

Abstract

The EstPS1 gene, which encodes a novel carboxylesterase of Pseudomonas synxantha PS1 isolated from oil well-produced water, was cloned and sequenced. EstPS1 has an open reading frame of 1923 bp and encodes the 640-amino acid carboxylesterase (EstPS1), which contains an autotransporter (AT) domain (357-640 amino acids). Homology analysis revealed that EstPS1 shared the highest identity (88%) with EstA from Pseudomonas fluorescens A506 (NCBI database) and belonged to the carboxylesterase family (EC 3.1.1.1). The optimum pH and temperature of recombinant EstPS1 were found to be 8.0 and 60 degrees C, respectively. EstPS1 showed high thermostability, and the half-lives (T1/2 thermal inactivation) at 60, 70, 80, 90, and 100 degrees C were 14 h, 2 h, 31 min, 10 min, and 1 min, respectively. To understand the role of the AT domain in carboxylesterase, AT domain-truncated carboxylesterase (EstPS1DeltaAT) was generated. EstPS1DeltaAT showed a clearly decreased secretion rate, owing to the AT domain strongly improved secretory expression in the heterogeneous system. EstPS1 degraded various pyrethroid pesticides, and hydrolysis efficiencies were dependent on the pyrethroid molecular structure. EstPS1 degraded all the tested pyrethroid pesticides and hydrolysed the p-nitrophenyl esters of medium-short-chain fatty acids, indicating that EstPS1 is an esterase with broad specificity.



        

Title: A novel esterase from a marine mud metagenomic library for biocatalytic synthesis of short-chain flavor esters
Gao W, Wu K, Chen L, Fan H, Zhao Z, Gao B, Wang H, Wei D
Ref: Microb Cell Fact, 15:41, 2016 : PubMed
Abstract


ESTHER: Gao_2016_Microb.Cell.Fact_15_41
PubMedSearch: Gao 2016 Microb.Cell.Fact 15 41
PubMedID: 26892801

Abstract

BACKGROUND: Marine mud is an abundant and largely unexplored source of enzymes with unique properties that may be useful for industrial and biotechnological purposes. However, since most microbes cannot be cultured in the laboratory, a cultivation-independent metagenomic approach would be advantageous for the identification of novel enzymes. Therefore, with the objective of screening novel lipolytic enzymes, a metagenomic library was constructed using the total genomic DNA extracted from marine mud.
RESULTS: Based on functional heterologous expression, 34 clones that showed lipolytic activity were isolated. The five clones with the largest halos were identified, and the corresponding genes were successfully overexpressed in Escherichia coli. Molecular analysis revealed that these encoded proteins showed 48-79 % similarity with other proteins in the GenBank database. Multiple sequence alignment and phylogenetic tree analysis classified these five protein sequences as new members of known families of bacterial lipolytic enzymes. Among them, EST4, which has 316 amino acids with a predicted molecular weight of 33.8 kDa, was further studied in detail due to its strong hydrolytic activity. Characterization of EST4 indicated that it is an alkaline esterase that exhibits highest hydrolytic activity towards p-nitrophenyl butyrate (specific activity: 1389 U mg(-1)) at 45 degrees C and pH 8.0. The half-life of EST4 is 55 and 46 h at 40 and 45 degrees C, respectively, indicating a relatively high thermostability. EST4 also showed remarkable stability in organic solvents, retaining 90 % of its initial activity when incubated for 12 h in the presence of hydrophobic alkanes. Furthermore, EST4 was used as an efficient whole-cell biocatalyst for the synthesis of short-chain flavor esters, showing high conversion rate and good tolerance for high substrate concentrations (up to 3.0 M). These results demonstrate a promising potential for industrial scaling-up to produce short-chain flavor esters at high substrate concentrations in non-aqueous media.
CONCLUSIONS: This manuscript reports unprecedented alcohol tolerance and conversion of an esterase biocatalyst identified from a marine mud metagenomic library. The high organic solvent tolerance and thermostability of EST4 suggest that it has great potential as a biocatalyst.



        

Title: Functional characterization of an alpha-esterase gene involving malathion detoxification in Bactrocera dorsalis (Hendel)
Wang LL, Lu XP, Meng LW, Huang Y, Wei D, Jiang HB, Smagghe G, Wang JJ
Ref: Pestic Biochem Physiol, 130:44, 2016 : PubMed
Abstract


ESTHER: Wang_2016_Pestic.Biochem.Physiol_130_44
PubMedSearch: Wang 2016 Pestic.Biochem.Physiol 130 44
PubMedID: 27155483
Gene_locus related to this paper: bacdo-a0a034w7m1
Inhibitor(s) related to this paper: TPHP

Abstract

Extensive use of insecticides in many orchards has prompted resistance development in the oriental fruit fly, Bactrocera dorsalis (Hendel). In this study, a laboratory selected strain of B. dorsalis (MR) with a 21-fold higher resistance to malathion was used to examine the resistance mechanisms to this organophosphate insecticide. Carboxylesterase (CarE) was found to be involved in malathion resistance in B. dorsalis from the synergism bioassay by CarE-specific inhibitor triphenylphosphate (TPP). Molecular studies further identified a previously uncharacterized alpha-esterase gene, BdCarE2, that may function in the development of malathion resistance in B. dorsalis via gene upregulation. This gene is predominantly expressed in the Malpighian tubules, a key insect tissue for detoxification. The transcript levels of BdCarE2 were also compared between the MR and a malathion-susceptible (MS) strain of B. dorsalis, and it was significantly more abundant in the MR strain. No sequence mutation or gene copy changes were detected between the two strains. Functional studies using RNA interference (RNAi)-mediated knockdown of BdCarE2 significantly increased the malathion susceptibility in the adult files. Furthermore, heterologous expression of BdCarE2 combined with cytotoxicity assay in Sf9 cells demonstrated that BdCarE2 could probably detoxify malathion. Taken together, the current study bring new molecular evidence supporting the involvement of CarE-mediated metabolism in resistance development against malathion in B. dorsalis and also provide bases on functional analysis of insect alpha-esterase associated with insecticide resistance.



        

Title: A Semiautomated Structure-Based Method To Predict Substrates of Enzymes via Molecular Docking: A Case Study with Candida antarctica Lipase B
Yao Z, Zhang L, Gao B, Cui D, Wang F, He X, Zhang JZ, Wei D
Ref: J Chem Inf Model, 56:1979, 2016 : PubMed
Abstract


ESTHER: Yao_2016_J.Chem.Inf.Model_56_1979
PubMedSearch: Yao 2016 J.Chem.Inf.Model 56 1979
PubMedID: 27529495

Abstract

The discovery of unique substrates is important for developing potential applications of enzymes. However, the experimental procedures for substrate identification are laborious, time-consuming, and expensive. Although in silico structure-based approaches show great promise, recent extensive studies have shown that these approaches remain a formidable challenge for current biocomputational methodologies. Here we present an open-source, extensible, and flexible software platform for predicting enzyme substrates called THEMIS, which performs in silico virtual screening for potential catalytic targets of an enzyme on the basis of the enzyme's catalysis mechanism. On the basis of a generalized transition state theory of enzyme catalysis, we introduce a modified docking procedure called "mechanism-based restricted docking" (MBRD) for novel substrate recognition from molecular docking. Comprising a series of utilities written in C/Python, THEMIS automatically executes parallel-computing MBRD tasks and evaluates the results with various molecular mechanics (MM) criteria such as energy, distance, angle, and dihedral angle to help identify desired substrates. Exhaustive sampling and statistical measures were used to improve the robustness and reproducibility of the method. We used Candida antarctica lipase B (CALB) as a test system to demonstrate the effectiveness of our computational prediction of (non)substrates. A novel MM score function for CALB substrate identification derived from the near-attack conformation was used to evaluate the possibility of chemical transformation. A highly positive rate of 93.4% was achieved from a CALB substrate library with 61 known substrates and 35 nonsubstrates, and the screening rate has reached 103 compounds/day (96 CPU cores, 100 samples/compound). The performance shows that the present method is perhaps the first reported scheme to meet the requirement for practical applicability to enzyme studies. An additional study was performed to validate the universality of our method. In this verification we employed two distinct enzymes, nitrilase Nit6803 and SDR Gox2181, where the correct rates of both enzymes exceeded 90%. The source code used will be released under the GNU General Public License (GPLv3) and will be free to download. We believe that the present method will provide new insights into enzyme research and accelerate the development of novel enzyme applications.



        

Title: Overexpression of two alpha-esterase genes mediates metabolic resistance to malathion in the oriental fruit fly, Bactrocera dorsalis (Hendel)
Wang LL, Huang Y, Lu XP, Jiang XZ, Smagghe G, Feng ZJ, Yuan GR, Wei D, Wang JJ
Ref: Insect Molecular Biology, 24:467, 2015 : PubMed
Abstract


ESTHER: Wang_2015_Insect.Mol.Biol_24_467
PubMedSearch: Wang 2015 Insect.Mol.Biol 24 467
PubMedID: 25940547

Abstract

Esterase has been reported to be involved in malathion resistance in the oriental fruit fly, Bactrocera dorsalis (Hendel). However, the underlying molecular mechanism of the esterase-mediated resistance remains largely unknown in this species. Here, with the use of a strain selected for malathion resistance in the laboratory (MR), we found that two overexpressed alpha-esterase genes, namely BdCarE4 and BdCarE6, predominant in the adult midgut and fat body, function in conferring malathion resistance in B. dorsalis. Notably, these two genes were found to be mostly close to the esterase E3, which are usually implicated in detoxifying organophosphate insecticides. The transcript levels of BdCarE4 and BdCarE6 were investigated and compared between the MR and a susceptible (MS) strain of B. dorsalis. Both genes were significantly up-regulated in the MR strain, which was consistent with the enhanced esterase activity in the MR strain. However, no changes in either the coding sequence or gene copy number were observed between the two strains. Subsequently, heterologous expression combined with cytotoxicity assay in Sf9 cells demonstrated that BdCarE4 and BdCarE6 can probably detoxify malathion. Furthermore, RNA interference-mediated knockdown of each of these two genes significantly increased malathion susceptibility in the MR strain adults. In conclusion, these results expand our molecular understanding of the important role of alpha-esterases during the development of resistance to organophosphorous insecticides in B. dorsalis.



        

Title: Efficient kinetic resolution of phenyl glycidyl ether by a novel epoxide hydrolase from Tsukamurella paurometabola
Wu K, Wang H, Sun H, Wei D
Ref: Applied Microbiology & Biotechnology, 99:9511, 2015 : PubMed
Abstract


ESTHER: Wu_2015_Appl.Microbiol.Biotechnol_99_9511
PubMedSearch: Wu 2015 Appl.Microbiol.Biotechnol 99 9511
PubMedID: 26088175
Gene_locus related to this paper: tsupd-d5us69

Abstract

Enantioselective hydrolysis of racemic epoxides mediated by epoxide hydrolases (EHs) is one of the most promising approaches to obtain enantiopure epoxides. In this study, we identified and characterized a novel EH (TpEH1) from Tsukamurella paurometabola by analyzing the conserved catalytic residues of EH. TpEH1 was overexpressed and purified, and its catalytic properties were studied using racemic phenyl glycidyl ether (PGE) and its derivatives as substrates. TpEH1 showed excellent enantioselectivity to the substrates PGE, 3-methylPGE, and 3-nitroPGE. The highest enantioselectivity (E > 100) was achieved when 3-nitroPGE was used as the substrate. The recombinant Escherichia coli TpEH1 demonstrated high substrate tolerance toward PGE and could hydrolyze PGE at concentrations of up to 400 mM (60 g/L) with high enantioselectivity (E = 65), giving (R)-PGE with enantiomeric excess of more than 99 % ee and 45 % yield within 1 h. This concentration of PGE is the highest reported concentration catalyzed by native EHs to date. Thus, the easily available and highly active E. coli TpEH1 showed great potential for the practical preparation of optically pure (R)-PGE.



        

Title: A method to rationally increase protein stability based on the charge-charge interaction, with application to lipase LipK107
Zhang L, Tang X, Cui D, Yao Z, Gao B, Jiang S, Yin B, Yuan YA, Wei D
Ref: Protein Science, 23:110, 2014 : PubMed
Abstract


ESTHER: Zhang_2014_Protein.Sci_23_110
PubMedSearch: Zhang 2014 Protein.Sci 23 110
PubMedID: 24353171
Gene_locus related to this paper: promi-c2lfd0

Abstract

We report a suite of enzyme redesign protocol based on the surface charge-charge interaction calculation, which is potentially applied to improve the stability of an enzyme without compromising its catalytic activity. Together with the experimental validation, we have released a suite of enzyme redesign algorithm Enzyme Thermal Stability System, written based on our model, for open access to meet the needs in wet labs. Lipk107, a lipase of a versatile industrial use, was chosen to test our software. Our calculation determined that four residues, D113, D149, D213, and D253, located on the surface of LipK107 were critical to the stability of the enzyme. The model was validated with mutagenesis at these four residues followed by stability and activity tests. LipK107 mutants D113A and D149K were more resistant to thermal inactivation with approximately 10 degrees C higher half-inactivation temperature than wild-type LipK107. Moreover, mutant D149K exhibited significant retention in residual activity under constant heat, showing a 14-fold increase in the half-inactivation time at 50 degrees C. Activity tests showed that these mutants retained the equal or higher specific activity, among which noteworthy was the mutant D253A with as much as 20% higher activity. We suggest that our protocol could be used as a general guideline to redesign protein enzymes with increased stabilities and enhanced activities.



        

Title: Structure, mechanism, and enantioselectivity shifting of lipase LipK107 with a simple way
Zhang L, Gao B, Yuan Z, He X, Yuan YA, Zhang JZ, Wei D
Ref: Biochimica & Biophysica Acta, 1844:1183, 2014 : PubMed
Abstract


ESTHER: Zhang_2014_Biochim.Biophys.Acta_1844_1183
PubMedSearch: Zhang 2014 Biochim.Biophys.Acta 1844 1183
PubMedID: 24602769
Gene_locus related to this paper: promi-c2lfd0

Abstract

Because of the complex mechanisms of enzymatic reactions, no precise and simple method of understanding and controlling the chiral selectivity of enzymes has been developed. However, structure-based rational design is a powerful approach to engineering enzymes with desired catalytic activities. In this work, a simple, structure-based, large-scale in silico design and virtual screening strategy was developed and successfully applied to enzyme engineering. We first performed protein crystallization and X-ray diffraction to determine the structure of lipase LipK107, which is a novel family I.1 lipase displaying activity for both R and S isomers in chiral resolution reactions. The catalytic mechanism of family I.1, which includes LipK107, was ascertained first through comparisons of the sequences and structures of lipases from other families. The binding states of LipK107, including the energy and the conformation of complexes with the R and S enantiomers, have been evaluated by careful biocomputation to figure out the reason for the higher S selectivity. Based on this study, a simple strategy for manipulating the chiral selectivity by modulating a crucial distance in the enzyme-substrate complex and judging virtual mutations in silico is recommended. Then, a novel electrostatic interaction analysis protocol was used to design LipK107 mutants to validate our strategy. Both positive and negative mutations determined using this theoretical protocol have been implemented in wet experiments and were proved to produce the desired enantioselectivity, showing a 176% increase or 50% decrease in enantioselectivity as desired. Because of its accuracy and versatility, the strategy is promising for practical applications.



        

Title: t-AUCB, an improved sEH inhibitor, suppresses human glioblastoma cell growth by activating NF-kappaB-p65
Li J, Liu H, Xing B, Yu Y, Wang H, Chen G, Gu B, Zhang G, Wei D and Hu W <2 more author(s)> Li J, Liu H, Xing B, Yu Y, Wang H, Chen G, Gu B, Zhang G, Wei D, Gu P, Li M, Hu W (- 2)
Ref: J Neurooncol, 108:385, 2012 : PubMed
Abstract


ESTHER: Li_2012_J.Neurooncol_108_385
PubMedSearch: Li 2012 J.Neurooncol 108 385
PubMedID: 22382785
Inhibitor(s) related to this paper: t-AUCB

Abstract

Although sEH inhibitors are well studied in inflammatory and cardiovascular diseases, their effects on gliomas are unclear. In this study, we investigated the effects of t-AUCB, a more potent and selective sEH inhibitor, on U251 and U87 human glioblastoma cell lines and the HepG2 human hepatocellular carcinoma cell line. Our results showed that t-AUCB efficiently inhibited sEH activities in all three cell lines (the inhibition rate was more than 80% in each) and suppressed U251 and U87 cell growth in a dose-dependent manner, but exhibited no cell growth inhibition on HepG2. We detected high levels of phosphorylated NF-kappaB-p65 (Ser536) in t-AUCB-treated U251 and U87 cells, and then found that the NF-kappaB inhibitor PDTC can completely abolish t-AUCB-induced growth inhibition. This indicated that t-AUCB suppresses U251 and U87 cell growth by activating NF-kappaB-p65. Moreover, we found that t-AUCB induces cell-cycle G0/G1 phase arrest by regulating Cyclin D1 mRNA and protein levels and CDC2 (Thr161) phosphorylation level. We propose to further test this promising reagent for its anti-glioma activity in clinical relevant orthotopic brain glioma models.



        

Title: In vivo functional expression of a screened P. aeruginosa chaperone-dependent lipase in E. coli
Wu X, You P, Su E, Xu J, Gao B, Wei D
Ref: BMC Biotechnol, 12:58, 2012 : PubMed
Abstract


ESTHER: Wu_2012_BMC.Biotechnol_12_58
PubMedSearch: Wu 2012 BMC.Biotechnol 12 58
PubMedID: 22950599
Gene_locus related to this paper: pseae-llipa

Abstract

BACKGROUND: Microbial lipases particularly Pseudomonas lipases are widely used for biotechnological applications. It is a meaningful work to design experiments to obtain high-level active lipase. There is a limiting factor for functional overexpression of the Pseudomonas lipase that a chaperone is necessary for effective folding. As previously reported, several methods had been used to resolve the problem. In this work, the lipase (LipA) and its chaperone (LipB) from a screened strain named AB which belongs to Pseudomonas aeruginosa were overexpressed in E. coli with two dual expression plasmid systems to enhance the production of the active lipase LipA without in vitro refolding process. RESULTS: In this work, we screened a lipase-produced strain named AB through the screening procedure, which was identified as P. aeruginosa on the basis of 16S rDNA. Genomic DNA obtained from the strain was used to isolate the gene lipA (936 bp) and lipase specific foldase gene lipB (1023 bp). One single expression plasmid system E. coli BL21/pET28a-lipAB and two dual expression plasmid systems E. coli BL21/pETDuet-lipA-lipB and E. coli BL21/pACYCDuet-lipA-lipB were successfully constructed. The lipase activities of the three expression systems were compared to choose the optimal expression method. Under the same cultured condition, the activities of the lipases expressed by E. coli BL21/pET28a-lipAB and E. coli BL21/pETDuet-lipA-lipB were 1300 U/L and 3200 U/L, respectively, while the activity of the lipase expressed by E. coli BL21/pACYCDuet-lipA-lipB was up to 8500 U/L. The lipase LipA had an optimal temperature of 30 degreesC and an optimal pH of 9 with a strong pH tolerance. The active LipA could catalyze the reaction between fatty alcohols and fatty acids to generate fatty acid alkyl esters, which meant that LipA was able to catalyze esterification reaction. The most suitable fatty acid and alcohol substrates for esterification were octylic acid and hexanol, respectively. CONCLUSIONS: The effect of different plasmid system on the active LipA expression was significantly different. pACYCDuet-lipA-lipB was more suitable for the expression of active LipA than pET28a-lipAB and pETDuet-lipA-lipB. The LipA showed obvious esterification activity and thus had potential biocatalytic applications. The expression method reported here can give reference for the expression of those enzymes that require chaperones.



        

Title: Cardiac abnormalities in severe acute dichlorvos poisoning
He X, Li C, Wei D, Wu J, Shen L, Wang T
Ref: Critical Care Medicine, 39:1906, 2011 : PubMed
Abstract


ESTHER: He_2011_Crit.Care.Med_39_1906
PubMedSearch: He 2011 Crit.Care.Med 39 1906
PubMedID: 21516037

Abstract

OBJECTIVE: Patients with organophosphorus poisoning sometimes die suddenly during rigorous treatment, possibly from myocardial injury. This study sought to elucidate the mechanisms underlying organophosphorus poisoning-induced cardiotoxicity. DESIGN: Prospective observational study. SETTING: Urban, tertiary teaching hospital emergency intensive care unit with 10 beds. PATIENTS: Forty-one patients with severe acute dichlorvos poisoning were consecutively enrolled (n = 92) at emergency intensive care unit and followed for 3 months. MEASUREMENTS AND MAIN RESULTS: Levels of serum creatine kinase isoenzyme myocardium, cardiac troponin I, acetylcholinesterase, acetylcholine, epinephrine, and norepinephrine were tested on hospital days 1, 3, and 5 and on discharge day. Electrocardiography was recorded on admission and then every other day. Transthoracic echocardiography was performed at admission, in the acute phase, before discharge, and during follow-up. Technetium 99m-sestamibi myocardial single photon emission computed tomography was conducted in four patients. Thirty-seven (90.2%) patients survived and four (9.8%) patients died during treatment. We observed sinus tachycardia in 37 (90.2%) patients and ST-T changes in 33 (80.4%) patients. Creatine kinase isoenzyme myocardium and cardiac troponin I levels peaked at day 3 postadmission and then decreased to normal levels. Serum acetylcholine, epinephrine, and norepinephrine peaked at day 1 after admission and then decreased. Echocardiography revealed marked decreases in wall motion of the interventricular septum and left ventricle in the acute phase but returned to normal in the recovery phase. The left ventricular ejection fraction improved significantly from 42 +/- 5% to 59 +/- 4% (p = .001). Single photon emission computed tomography showed abnormal left ventricle perfusion. CONCLUSION: Severe acute dichlorvos poisoning is associated with reversible myocardial dysfunction, possibly through an increase in catecholamine levels.



        

Title: Co-expression of an organic solvent-tolerant lipase and its cognate foldase of Pseudomonas aeruginosa CS-2 and the application of the immobilized recombinant lipase
Peng R, Lin J, Wei D
Ref: Appl Biochem Biotechnol, 165:926, 2011 : PubMed
Abstract


ESTHER: Peng_2011_Appl.Biochem.Biotechnol_165_926
PubMedSearch: Peng 2011 Appl.Biochem.Biotechnol 165 926
PubMedID: 21720839

Abstract

The genes of CS-2 lipase and its cognate foldase were cloned from Pseudomonas aeruginosa CS-2. A stop codon was not found in the lipase gene. The amino acid sequence deduced from the lipase gene from P. aeruginosa CS-2 showed 97.8%, 71.3%, and 71.2% identity with lipases from P. aeruginosa LST-03, P seudomonas mendocina ymp, and Pseudomonas stutzeri A1501, respectively. The co-expression of CS-2 lipase and its cognate foldase of P. aeruginosa CS-2 in E scherichia coli BL21 (DE3) resulted in the formation of a soluble lipase. The recombinant lipase and foldase were purified to homogeneity using nickel affinity chromatography and about 10.2-fold with 40.9% recovery was achieved for the purification of the recombinant lipase. The molecular masses of the lipase and the foldase were estimated to be 35.7 and 38.3 kDa in SDS-PAGE, respectively. The recombinant lipase showed stability in the presence of some organic solvents. The recombinant CS-2 lipase was immobilized and subsequently used for the synthesis of butyl acetate in heptane. The conversion of substrate decreased from 98.2% to 87.4% after 5 cycles in reuse of the immobilized lipase.



        

Title: Purification and characterization of an organic solvent-tolerant lipase from Pseudomonas aeruginosa CS-2
Peng R, Lin J, Wei D
Ref: Appl Biochem Biotechnol, 162:733, 2010 : PubMed
Abstract


ESTHER: Peng_2010_Appl.Biochem.Biotechnol_162_733
PubMedSearch: Peng 2010 Appl.Biochem.Biotechnol 162 733
PubMedID: 19936633

Abstract

An extracellular lipase secreted by Pseudomonas aeruginosa CS-2 was purified to homogeneity about 25.5-fold with an overall yield of 45.5%. The molecular mass of the lipase was estimated to be 33.9 kDa by SDS-PAGE and 36 kDa by gel filtration. The optimum temperature and pH were 50 degrees C and 8.0. The lipase was found to be stable at pH 4-10 and below 50 degrees C. Its hydrolytic activity was highest against p-nitrophenyl palmitate (p-NPP) among p-nitrophenyl esters of fatty acids with various chain lengths. The lipase was activated in the presence of Ca(2+), while it was inactivated by other metal ions more or less. EDTA significantly reduced the lipase activity, indicating the lipase was a metalloenzyme. Gum Arabic and polyvinyl alcohol 124 enhanced lipase activity but Tween-20, Tween-80, and hexadecyltrimethyl ammonium bromide strongly inhibited the lipase. It exhibited stability in some organic solvents. The lipase was activated in the presence of acetonitrile. Conversely, it was drastically inactivated by methanol and ethanol.



        

Title: Template-based modeling of a psychrophilic lipase: conformational changes, novel structural features and its application in predicting the enantioselectivity of lipase catalyzed transesterification of secondary alcohols
Xu T, Gao B, Zhang L, Lin J, Wang X, Wei D
Ref: Biochimica & Biophysica Acta, 1804:2183, 2010 : PubMed
Abstract


ESTHER: Xu_2010_Biochim.Biophys.Acta_1804_2183
PubMedSearch: Xu 2010 Biochim.Biophys.Acta 1804 2183
PubMedID: 20828637
Gene_locus related to this paper: promi-c2lfd0

Abstract

In order to fully explore the structure-function relationship of a Proteus lipase (LipK107) that was screened from the soil in our previous study, we have modeled the three-dimensional (3-D) structures of the enzyme in its active and inactive conformations on the basis of crystal structures of Burkholderia glumae and Pseudomonas aeruginosa lipases in the present study. Both homology models suggested that LipK107 possessed a catalytic triad (Ser79-Asp232-H254), an oxyanion hole (Leu13 and Gln80) which was used to stabilize the reaction tetrahedral intermediates, and a lid substructure that controlled the access of the substrate to the active site. The existence of the lid was further verified by carrying out the interfacial activation experiment. The conformational change of LipK107 which was caused by lid opening action was predicted by superimposing the two theoretical models for the first time. Finally, both 3-D structures were used to predict the enantioselectivity of LipK107 when the enzyme was used to catalyze the resolution of racemic 1-phenylethanol. Lid-open model of LipK107 identified the R-enantiomer as the preferred enantiomer, while lid-closed mode showed that the S-enantiomer was more favored. However, only the lid-open conformational model could led to predictions that agreed with the following the experimental result of real biocatalysis reaction of 1-phenylethanol.



        

Title: Development of recombinant Escherichia coli whole-cell biocatalyst expressing a novel alkaline lipase-coding gene from Proteus sp. for biodiesel production
Gao B, Su E, Lin J, Jiang Z, Ma Y, Wei D
Ref: J Biotechnol, 139:169, 2009 : PubMed
Abstract


ESTHER: Gao_2009_J.Biotechnol_139_169
PubMedSearch: Gao 2009 J.Biotechnol 139 169
PubMedID: 19007827
Gene_locus related to this paper: promi-c2lfd0

Abstract

A lipase-producing bacterium K107 was isolated from soil samples of China and identified to be a strain of Proteus sp. With genome-walking method, the open reading frame of lipase gene lipK107, encoding 287 amino acids, was cloned and expressed in a heterologous host, Escherichia coli BL21 (DE3). The recombinant lipase was purified and characterized, and the optimum pH of the purified LipK107 was 9, at 35 degrees C. The recombinant E. coli expressing lipK107 was applied in biodiesel production in the form of whole-cell biocatalyst. Activity of the biocatalyst increased significantly when cells were permeabilized with 0.3% (w/v) cetyl-trimethylammoniumbromide (CTAB). This transesterification was carried out efficiently in a mixture containing 5M equivalents of methanol to the oil and 100% water by weight of the substrate. It was the first time to use E. coli whole-cell biocatalyst expressing lipase in biodiesel production, and the biodiesel reached a yield of nearly 100% after 12h reaction at the optimal temperature of 15 degrees C, which was the lowest temperature among all the known catalyst in biodiesel production.



        

Title: Structure-based substrate screening for an enzyme
Xu T, Zhang L, Wang X, Wei D, Li T
Ref: BMC Bioinformatics, 10:257, 2009 : PubMed
Abstract


ESTHER: Xu_2009_BMC.Bioinformatics_10_257
PubMedSearch: Xu 2009 BMC.Bioinformatics 10 257
PubMedID: 19695105

Abstract

BACKGROUND: Nowadays, more and more novel enzymes can be easily found in the whole enzyme pool with the rapid development of genetic operation. However, experimental work for substrate screening of a new enzyme is laborious, time consuming and costly. On the other hand, many computational methods have been widely used in lead screening of drug design. Seeing that the ligand-target protein system in drug design and the substrate-enzyme system in enzyme applications share the similar molecular recognition mechanism, we aim to fulfill the goal of substrate screening by in silico means in the present study. RESULTS: A computer-aided substrate screening (CASS) system which was based on the enzyme structure was designed and employed successfully to help screen substrates of Candida antarctica lipase B (CALB). In this system, restricted molecular docking which was derived from the mechanism of the enzyme was applied to predict the energetically favorable poses of substrate-enzyme complexes. Thereafter, substrate conformation, distance between the oxygen atom of the alcohol part of the ester (in some compounds, this oxygen atom was replaced by nitrogen atom of the amine part of acid amine or sulfur atom of the thioester) and the hydrogen atom of imidazole of His224, distance between the carbon atom of the carbonyl group of the compound and the oxygen atom of hydroxyl group of Ser105 were used sequentially as the criteria to screen the binding poses. 223 out of 233 compounds were identified correctly for the enzyme by this screening system. Such high accuracy guaranteed the feasibility and reliability of the CASS system. CONCLUSION: The idea of computer-aided substrate screening is a creative combination of computational skills and enzymology. Although the case studied in this paper is tentative, high accuracy of the CASS system sheds light on the field of computer-aided substrate screening.



        

Title: Efficient display of active lipase LipB52 with a Pichia pastoris cell surface display system and comparison with the LipB52 displayed on Saccharomyces cerevisiae cell surface
Jiang Z, Gao B, Ren R, Tao X, Ma Y, Wei D
Ref: BMC Biotechnol, 8:4, 2008 : PubMed
Abstract


ESTHER: Jiang_2008_BMC.Biotechnol_8_4
PubMedSearch: Jiang 2008 BMC.Biotechnol 8 4
PubMedID: 18221563

Abstract

BACKGROUND: For industrial bioconversion processes, the utilization of surface-displayed lipase in the form of whole-cell biocatalysts is more advantageous, because the enzymes are displayed on the cell surface spontaneously, regarded as immobilized enzymes.
RESULTS: Two Pichia pastoris cell surface display vectors based on the flocculation functional domain of FLO with its own secretion signal sequence or the alpha-factor secretion signal sequence were constructed respectively. The lipase gene lipB52 fused with the FLO gene was successfully transformed into Pichia pastoris KM71. The lipase LipB52 was expressed under the control of the AOX1 promoter and displayed on Pichia pastoris KM71 cell surface with the two Pichia pastoris cell surface display vectors. Localization of the displayed LipB52 on the cell surface was confirmed by the confocal laser scanning microscopy (CLSM). The LipB52 displayed on the Pichia pastoris cell surface exhibited activity toward p-nitrophenol ester with carbon chain length ranging from C10 to C18, and the optimum substrate was p-nitrophenol-caprate (C10), which was consistent with it displayed on the Saccharomyces cerevisiae EBY100 cell surface. The hydrolysis activity of lipase LipB52 displayed on Pichia pastoris KM71-pLHJ047 and KM71-pLHJ048 cell surface reached 94 and 91 U/g dry cell, respectively. The optimum temperature of the displayed lipases was 40 degrees C at pH8.0, they retained over 90% activity after incubation at 60 degrees C for 2 hours at pH 7.0, and still retained 85% activity after incubation for 3 hours. CONCLUSION: The LipB52 displayed on the Pichia pastoris cell surface exhibited better stability than the lipase LipB52 displayed on Saccharomyces cerevisiae cell surface. The displayed lipases exhibited similar transesterification activity. But the Pichia pastoris dry cell weight per liter (DCW/L) ferment culture was about 5 times than Saccharomyces cerevisiae, the lipase displayed on Pichia pastoris are more suitable for whole-cell biocatalysts than that displayed on Saccharomyces cerevisiae cell surface.



        

Title: Characterization of two novel lipase genes isolated directly from environmental sample
Jiang Z, Wang H, Ma Y, Wei D
Ref: Applied Microbiology & Biotechnology, 70:327, 2006 : PubMed
Abstract


ESTHER: Jiang_2006_Appl.Microbiol.Biotechnol_70_327
PubMedSearch: Jiang 2006 Appl.Microbiol.Biotechnol 70 327
PubMedID: 16003556
Gene_locus related to this paper: 9psed-q66wt1, psefl-q7wzt7

Abstract

Two novel lipase genes (lipJ02, lipJ03) were isolated directly from environmental DNA via genome-walking method. Lipase gene lipJ02 contained an open reading frame (ORF) of 1,425 bp and encoded a 474-amino acids lipase protein, while lipase gene lipJ03 contained an ORF of 1,413 bp and encoded a 470-amino acids lipase protein. The lipase genes were cloned into expression vector pPIC9K and successfully integrated into a heterologous fungal host, Pichia pastoris KM71, and the recombinant P. pastoris were screened via a high-throughput method. The recombinants were induced by methanol to secrete active lipases into cultural medium. The recombinant lipases were also purified and characterized. The optimum temperature for the purified lipase LipJ02 and LipJ03 was 30 and 35 degrees C, respectively, at pH 8.0. They exhibited similar thermostability, but LipJ02 exhibited better pH stability than LipJ03.



        

Title: High-level secretion and purification of recombinant acetylcholinesterase from human cerebral tissue in P. pastoris and identification by chromogenic reaction
Ma X, Tan J, Wei D, Zhu P, Sun M
Ref: Applied Microbiology & Biotechnology, 72:316, 2006 : PubMed
Abstract


ESTHER: Ma_2006_Appl.Microbiol.Biotechnol_72_316
PubMedSearch: Ma 2006 Appl.Microbiol.Biotechnol 72 316
PubMedID: 16397771

Abstract

The gene encoding human cerebral tissue acetylcholinesterase (AChE) was cloned from an 18-week fetal cerebral tissue and expressed in Pichia pastoris. Twenty-two positive transformants were obtained by Mut(+)/Mut(s) phenotypes screening in MD/MM medium and polymerase chain reaction amplification, and four recombinant P. pastoris strains that could secrete active AChE at high level were identified by simple and specific development reaction with indoxyl acetate as the chromogenic substrate. In shake-flask culture induced with methanol, the recombinant human AChE (rhAChE) content was about 76% of the total secreted proteins, and rhAChE activity in supernatant was 40 U/ml. The enzyme was purified through anion-exchange and affinity chromatography. Purity of the rhAChE was up to 96% after the simple purification procedure. The enzymatic activity reached 200 U/mg.



        

Title: Cloning and expression of a novel lipase gene from Pseudomonas fluorescens B52
Jiang Z, Zheng Y, Luo Y, Wang G, Wang H, Ma Y, Wei D
Ref: Mol Biotechnol, 31:95, 2005 : PubMed
Abstract


ESTHER: Jiang_2005_Mol.Biotechnol_31_95
PubMedSearch: Jiang 2005 Mol.Biotechnol 31 95
PubMedID: 16170209
Gene_locus related to this paper: psefl-q6gx93

Abstract

A novel lipase gene (lipB52) was isolated directly from the genomic DNA of Pseudomonas fluorescens B52 with the genome-walking method, an effective method for isolating lipase gene from bacteria. There was an open reading frame (ORF) of 1854 bp, which encoded 617 amino acids. The lipase gene (lipB52) was cloned into expression vector pPIC9K and successfully integrated into a heterologous fungal host, Pichia pastoris KM71, and the recombinant Pichia pastoris were screened with a high throughput method. The recombinant was induced by methanol to secrete active lipase into the culture medium. The recombinant lipase LipB52 was also purified and characterized. The optimum temperature for the purified lipase LipB52 was 40 degrees C at pH 8.0. It exhibited better thermostability and pH stability than its homologs.



        

Title: Studies on a novel carbon source and cosolvent for lipase production by Candida rugosa
Wei D, Zhang LY, Song Q
Ref: J Ind Microbiol Biotechnol, 31:133, 2004 : PubMed
Abstract


ESTHER: Wei_2004_J.Ind.Microbiol.Biotechnol_31_133
PubMedSearch: Wei 2004 J.Ind.Microbiol.Biotechnol 31 133
PubMedID: 15069604

Abstract

Oleic acid esters were shown to be the best carbon source for both cell growth and lipase production by Candida rugosa. Use of a cosolvent, dodecane, in fermentations improved the solubility of solid substrates and increased oxygen solubility. This resulted in the highest lipase activity in batch fermentation with glycerol trioleate and dodecane. Lipase activity reached 77.1 units ml(-1).