Hyaluronate lyases were obtained from two types of naturally isolated bacterial strains Paenibacillus yunnanensis and Paennarthrobacter nicotinovorans.PyHL(form P.yunnanensis)in the culture supernatant of the bacteria...Hyaluronate lyases were obtained from two types of naturally isolated bacterial strains Paenibacillus yunnanensis and Paennarthrobacter nicotinovorans.PyHL(form P.yunnanensis)in the culture supernatant of the bacteria was purified by two steps of column chromatography.The enzyme showed the molecular mass of 74 kDa by SDS-PAGE and the maximal activity at pH 5.0,35℃.PyHL maximally degraded hyaluronate by an endo-type manner,and showed low degradation activity toward chondroitin sulfates.Dermatan sulfate was not the substrate.PnHL(from P.nicotinovorans)in the culture supernatant of the bacteria was purified by two steps of column chromatography.The enzyme showed the molecular mass of 70 kDa by SDS-PAGE and the maximal activity at pH 6.0,30℃.Genomic analysis of P.nicotinovorans on the bases of the internal amino acid sequences of PnHL.展开更多
Abstract: Cis-cinnamic acid (CA) is a naturally occurring compound, presumably converted from trans-CA in higher plants. To investigate the effect of cis-CA on the activity of Arabidopsis phenylalanine ammonia lyase (...Abstract: Cis-cinnamic acid (CA) is a naturally occurring compound, presumably converted from trans-CA in higher plants. To investigate the effect of cis-CA on the activity of Arabidopsis phenylalanine ammonia lyase (PAL), AtPAL1, AtPAL2, and AtPAL4 genes were isolated using reverse transcription poly-merase chain reaction. These genes were fused to a glutathione S-transferase gene and overexpressed in a heterologous prokaryotic system of Escherichia coli. The purified PAL1, PAL2 and PAL4 enzymes were characterized biochemically to determine the effects of cis-CA on the kinetic parameter Km. The results showed that cis-CA is a competitive inhibitor for PAL1, but not PAL2 and PAL4, whereas trans-CA acts as a competitive inhibitor for all three PAL isomers, suggesting that cis- and trans-CA have different effects on the catalytic activity of PAL.展开更多
Green macroalgae,e.g.,Ulva lactuca,are valuable bioactive sources of nutrients;but algae recalcitrant cell walls,composed of a complex cross-linked matrix of polysaccharides,can compromise their utilization as feedstu...Green macroalgae,e.g.,Ulva lactuca,are valuable bioactive sources of nutrients;but algae recalcitrant cell walls,composed of a complex cross-linked matrix of polysaccharides,can compromise their utilization as feedstuffs for monogastric animals.This study aimed to evaluate the ability of pre-selected Carbohydrate-Active enZymes(CAZymes)and sulfatases to degrade U.lactuca cell walls and release nutritive compounds.A databank of 199 recombinant CAZymes and sulfatases was tested in vitro for their action towards U.lactuca cell wall polysaccharides.The enzymes were incubated with the macroalga,either alone or in combination,to release reducing sugars and decrease fluorescence intensity of Calcofluor White stained cell walls.The individual action of a polysaccharide lyase family 25(PL25),an ulvan lyase,was shown to be the most efficient in cell wall disruption.The ulvan lyase treatment,in triplicate measures,promoted the release of 4.54 g/L(P<0.001)reducing sugars,a mono-and oligosaccharides release of 11.4 and 11.2 mmol/100 g of dried alga(P<0.01),respectively,and a decrease of 41.7%(P<0.001)in cell wall fluorescence,in comparison to control.The ability of ulvan lyase treatment to promote the release of nutritional compounds from alga biomass was also evaluated.A release of some monounsaturated fatty acids was observed,particularly the health beneficial 18:1c9(P<0.001).However,no significant release of total fatty acids(P>0.05),proteins(P?0.861)or pigments(P>0.05)was found.These results highlight the capacity of a single recombinant ulvan lyase(PL25 family)to incompletely disrupt U.lactuca cell walls.This enzyme could enhance the bioaccessibility of U.lactuca bioactive products with promising utilization in the feed industry.展开更多
In Gloeobacter violaceus PCC 7421, three possible lyase genes glr1191, glr1182 and gll1188 were selected by Blast sorting. The coded proteins of these three genes were co-expressed with their substrate protein in E. c...In Gloeobacter violaceus PCC 7421, three possible lyase genes glr1191, glr1182 and gll1188 were selected by Blast sorting. The coded proteins of these three genes were co-expressed with their substrate protein in E. coli, respectively, and some chromoproteins were obtained. The fluorescence spectra showed that high fluorescence intensity was observed in the three experimental groups that involved the lyase genes, but little fluorescence intensity was observed in negative control groups. The ratio of relative fluorescence intensity in the experimental group with glr1191 was 64.8%. The result of SDS-PAGE indicated that the molecular weights of the three chromoproteins were 22.0 10 3 , 23.6 10 3 and 22.1 10 3 , respectively. The result of zinc-induced fluorescence re- vealed that the phycobilin in the three chromoproteins was covalently coupled to their apo-proteins. The result also showed that the coded proteins of these three genes (CpeS1 , CpeT1 , CpeY )could cata- lyze the covalent coupling of different phycobilins to their apo- proteins and formed active chromoproteins.展开更多
Background Alginate oligosaccharide(AOS)holds great potential as a novel feed supplement in farm animals.However,the effects of AOS on chicken health and the underlying mechanisms are not fully understood.This study a...Background Alginate oligosaccharide(AOS)holds great potential as a novel feed supplement in farm animals.However,the effects of AOS on chicken health and the underlying mechanisms are not fully understood.This study aimed to optimize the enzymatic preparation of AOS by using bacterial alginate lyases expressed in yeast,investigate the effects of the prepared AOS on the growth performance and gut health of broiler chickens,and reveal the underlying mechanisms.Results Five alginate lyases from bacteria were cloned into Pichia pastoris GS115 and the alginate lyase PDE9 was expressed at relatively high yield,activity and stability in P.pastoris.Animal trials were carried out using 3201-day-old male Arbor Acres broilers(four groups;8 replicates/group×10 chicks/replicate)receiving either a basal diet or the same diet supplemented with 100,200 and 400 mg/kg PDE9-prepared AOS for 42 d.The results showed that dietary supplementation of 200 mg/kg AOS displayed the highest activity in promoting the birds’ADG and ADFI(P<0.05).AOS ameliorated the intestinal morphology,absorption function and barrier function,as indicated by the enhanced(P<0.05)intestinal villus height,maltase activity,and the expression of PEPT,SGLT1,ZNT1,and occludin.AOS also increased serum insulin-like growth factor-1,ghrelin(P<0.05),and growth hormone(P<0.1).Moreover,the concentrations of acetate,isobutyrate,isovalerate,valerate,and total SCFAs in cecum of birds fed AOS were significantly higher than the control birds(P<0.05).Metagenomic analysis indicated that AOS modulated the chicken gut microbiota structure,function,and microbial interactions and promoted the growth of SCFAs-producing bacteria,for example,Dorea sp.002160985;SCFAs,especially acetate,were found positively correlated with the chicken growth performance and growth-related hormone signals(P<0.05).We further verified that AOS can be utilized by Dorea sp.to grow and to produce acetate in vitro.Conclusions We demonstrated that the enzymatically produced AOS effectively promoted broiler chicken growth performance by modulating the chicken gut microbiota structure and function.For the first time,we established the connections among AOS,chicken gut microbiota/SCFAs,growth hormone signals and chicken growth performance.展开更多
Quorum sensing(QS)system can dynamically control the expression of proteins along with the cell growth.The promoting period of QS system has been little focused on until now.In this study,a self-induced dynamic regula...Quorum sensing(QS)system can dynamically control the expression of proteins along with the cell growth.The promoting period of QS system has been little focused on until now.In this study,a self-induced dynamic regulated expression(SIDRE)system was constructed in Escherichia coli.To enable the system suitable for the expression of enzymes,promoter engineering was used to obtain P_(luxI)mutants.To test the SIDRE system,alginate lyase AL493 and esterase Est7 were used as target protein for expression.The enzyme activity of alginate lyase and esterase reached 96.38%and 106.71%of the control strains containing the T7 promoter.In high-density fermentation,the activity of alginate lyase expressed by the SIDRE system with P_(luxI)(T-38C)as promoter was 4.34-fold of that expressed by the T7 promoter.Therefore,the P_(luxI)mutants with different promoting periods and/or different strengths show great potential in both laboratory and industrial scale for protein expression.展开更多
Background: Hepatocellular carcinoma(HCC) is one of the most highly malignant tumors. Liver tumor-initiating cells(LTICs) have been considered to contribute to HCC progression and metastasis. ATP-citrate lyase(ACLY), ...Background: Hepatocellular carcinoma(HCC) is one of the most highly malignant tumors. Liver tumor-initiating cells(LTICs) have been considered to contribute to HCC progression and metastasis. ATP-citrate lyase(ACLY), as a key enzyme for de novo lipogenesis, has been reported to be upregulated in various tumors. However, its expression and role in HCC and LTICs remain unknown. Methods: The expressions of ACLY in HCC tissues were detected by quantitative real-time PCR(q RT-PCR), Western blotting and immunohistochemistry. Kaplan-Meier curves and Chi-square test were used to determine the clinical significance of ACLY expression in HCC patients. A series of assays were performed to determine the function of ACLY on stemness, migration and invasion of HCC cells. Luciferase reporter assay, Western blotting and immunoprecipitation were used to study the regulation of the Wnt/β-catenin signaling by ACLY. Rescue experiments were performed to investigate whether β-catenin was the mediator of ACLY-regulated stemness and migration in HCC cells. Results: ACLY was highly expressed in HCC tissues and LTICs. Overexpression of ACLY was significantly correlated with poor prognosis, progression and metastasis of HCC patients. Knockdown of ACLY remarkably suppressed stemness properties, migration and invasion in HCC cells. Mechanistically, ACLY could regulate the canonical Wnt pathway by affecting the stability of β-catenin, and Lys49 acetylation of β-catenin might mediate ACLY-regulated β-catenin level in HCC cells. Conclusions: ACLY is a potent regulator of Wnt/β-catenin signaling in modulating LTICs stemness and metastasis in HCC. ACLY may serve as a new target for the diagnosis and treatment of HCC.展开更多
In this study, the activities of phenylalanine ammonia lyase (PAL), polyphenoloxidase (PPO), and peroxidase (POD) were assayed in cucumber seedlings (Cucumis sativus L.) at 0, 6, 12, 24, 48, 72, and 96 h after...In this study, the activities of phenylalanine ammonia lyase (PAL), polyphenoloxidase (PPO), and peroxidase (POD) were assayed in cucumber seedlings (Cucumis sativus L.) at 0, 6, 12, 24, 48, 72, and 96 h after they were infested by Bemisia tabaci (Gennadius) using spectrophotometric analysis. The results indicated that herbivore infestation increased the activities of PAL, PPO, and POD. The enzymes showed different activity levels at different times after the infestation. The PAL activity reached the first high peak by 23.1% at 6 h and the highest peak by 29.1% at 48 h compared to the control. The PPO activity reached the first high peak by 22.7% at 6 h and the highest peak by 52.6% at 24 h, and the POD activity reached the highest peak by 213.2% at 6 h and another higher peak value by 135.2% at 96 h. The results suggest that the enhanced activities of the enzymes may contribute to bioprotection of cucumber plants against B. tabaci infestation.展开更多
To exploit peashrub resources in Ordos as fodders, it is very crucial to realize industrial production of cheap cellulase of high activity by optimizing culture technology, especially culture substrate. In this study,...To exploit peashrub resources in Ordos as fodders, it is very crucial to realize industrial production of cheap cellulase of high activity by optimizing culture technology, especially culture substrate. In this study, a new prescription experiment based on uniform design ideal was invented and successfully applied in the solid fermentation of Trichoderma koningii F244, which was performed with two different temperature degrees. The activities of FPA, cotton lyase, CMCase and β-glucosidase were assayed and then mathematical models of enzymatic activities, which were figured out by Unconstraint Mathematical Programming, were developed by Multivariate Regression Program of SPSS10.0. Enzymatic activities of optimized substrate prescriptions corresponding to mathematical models were forecasted to determine an ideal substrate prescription. It is revealed that in solid fermentation, Tween80 has negative effect on cellulase production. Furthermore, the ideal prescription for cellulase complex production by Trichoderma koningii F244 was straw powder 16.9%,wheat bran 26.5%, (NH 4) 2SO 4 9.5% and water 47.1%, whose corresponding cellulase activity was expected to be at the same high level with that of Trichoderma reesei Q9414 on its own recommended substrate. Especially, goats mainly fed on peashrub tissues mixed with cellulase complex of this prescription and culture technology, got an incremental ratio of 0.3 kg/d, which brought a very promising feeding prospect for local peashrub resource. By populization of this cellulase complex, it can integrate living standard, economic construction of local residents into vegetational restoration tightly and thus this paper will be very meaningful to be use for reference for western China like Ordos to realize its sustainable development of economy, society and environment.展开更多
The soybean cyst nematode, Heterodeara glycines, is a serious pathogen of soybean, and reported to be the host of a wide range of Fabaceae. In the present study, the host specificity and reproductivity of two populati...The soybean cyst nematode, Heterodeara glycines, is a serious pathogen of soybean, and reported to be the host of a wide range of Fabaceae. In the present study, the host specificity and reproductivity of two populations of H. glycines collected from soybean and tobacco were identified and characterized. The comparative identity between β-1,4-endoglucanase, pectate lyase and chorismate mutase of H. glycines parasitizing on soybean and tobacco were 99, 97 and 98%, respectively. The qR T-PCR analysis indicated that the expression of pectate lyase 2 gene was significantly higher in second-stage juveniles of H. glycines Henan population parasitizing on tobacco than that of H. glycines Shanxi population parasitizing on soybean. In addition, the pectic acid content of cell wall was significantly higher(45%) in the roots of tobacco than the roots of soybean. Our results indicate that the changes in transcript parasitism genes may be a result of long-term evolution illustrating how a plant-parasitic nematode adapts to the host environment for optimal infestation and survival.展开更多
CO2 fixation pathway of Caminibacter profundus, a chemolithoautotrophic e-Proteobacteria from deep-sea hydrothermal vent, was determined and characterized by genetic and enzymatic analyses. Gene expression of key enzy...CO2 fixation pathway of Caminibacter profundus, a chemolithoautotrophic e-Proteobacteria from deep-sea hydrothermal vent, was determined and characterized by genetic and enzymatic analyses. Gene expression of key enzymes for CO2 fixation in response to salinity, pH and O2 in Medium 829 were also investigated. The results demonstrate that C. profundus contained aclB, porA and oorA, the genes encoding key enzymes of reductive tricarboxylic acid (rTCA) cycle. However, genes fragments of cbbL and cbbMencoding key enzyme of Calvin cycle were not recovered. Key enzymatic activities of ATP citrate lyase (ACL), pyruvate: ferredoxin oxidoreductase (POR) and 2-oxoglutarate: ferredoxin oxidoreductase (OOR) were also present in C. profun- dus. The combination of genetic and enzymatic analyses confirm that C. profundus adopted rTCA cycle for carbon assimilation. The results of aclB and oorA relative expressions of C. profundus demonstrate that the ranges of environmental factors for high genes expression were sea salt 3.0%-5.0% (optimum 3.0%), pH 5.0-6.5(optimum pH 6.5), anaerobic to microaerobic conditions (optimum 1.0% 02). Gene expression pat- terns under different conditions show similar patterns with bacterial growth, revealing that key rTCA cycle genes provided molecular basis for bacterial growth and propagation. Our results suggest that C. profun- dus could regulate key genes of rTCA cycle for carbon assimilation and energy metabolism in response to environmental fluctuations in hydrothermal vent.展开更多
When devoured by macrophages,Mycobacterium tuberculosis remains persistent in macrophages and gains energy through the glyoxylate bypass to maintain its long-term existence in host cells.Therefore it is possible to st...When devoured by macrophages,Mycobacterium tuberculosis remains persistent in macrophages and gains energy through the glyoxylate bypass to maintain its long-term existence in host cells.Therefore it is possible to stop persistent infections by interdicting the glyoxylate bypass in which the isocitrate lyase(ICL) is the key rate-limiting enzyme and a persistence factor.ICL is the target of anti-TB(TB:tubercular) drugs,which could screen ICL out and effectively inhibit the activity of ICL in Mycobacterium tuberculosis,and because of this,anti-TB drugs can be used to kill persistent Mycobacterium tuberculosis.In this study,the ICL gene of the Mycobacterium tuberculosis H37Rv was cloned successfully and recombinant protein with bioactivity was obtained through the enzyme characteristic appraisal.The specific activity of the recombined ICL is 24μmol·mg-1·min-1.The recombined ICL protein was used as the target,and phages which can specifically combine to ICL were screened in the phage 7 peptide library.According to the results of the ELISA and DNA sequence detection,eventually three 7-peptide chains were synthesized.Then the peptide chains were reacted with ICL,respectively,to detect their inhibitory effects on ICL.The results show that all the three 7-peptide chains possessed varying inhibitory effects on the activity of ICL.This study provided lead compounds for the research and development of new peptide anti-TB drugs.展开更多
A.americana L.is a crop with very little genetic variability.In order to evaluate the effect of ethyl methanesulfonate(EMS)to induce variability in in vitro plantlets of A.americana,different explants(meristems,leaves...A.americana L.is a crop with very little genetic variability.In order to evaluate the effect of ethyl methanesulfonate(EMS)to induce variability in in vitro plantlets of A.americana,different explants(meristems,leaves and roots)were evaluated for the production of callus.MS medium supplemented with ANA(2.68μM)and BAP(2.68μM)was used.Callus obtained from apical meristem were treated with 15 mM EMS for two hours after which shoot formation was induced using 2,4-D(0.11μM)and BAP(44μM).The EMS induced variations in the morphometric and morphological parameters of the plantlets obtained,with 60%of the plantlets presenting differences such as dwarfism and different leaf forms,without the presence of spines,as well as an increase in fructan content of 30%with respect to the control plantlets.PAL was increased and this activity is related with higher anthocyanins concentration in A.americana L.plantlets.展开更多
Combinatorial chemistry has been a focus of research activity in modern drug discovery and biotechnology. It is a concept by which a vast library of molecular diversity is synthesized and screened for target propertie...Combinatorial chemistry has been a focus of research activity in modern drug discovery and biotechnology. It is a concept by which a vast library of molecular diversity is synthesized and screened for target properties. This report is to illustrate the application of enzyme technology using the concept of combinatorial chemistry as a novel approach for the bioconversion of plant fibers. Citrus pectin was subjected to combinatorial enzyme digestion to create libraries of pectic oligosaccharides with diverse structural variants. Repeated cycles of fractionation and screening resulted in the isolation and identification of an active oligoGalA species with antimicrobial activity.展开更多
Seven bacterial clones with alginate-utilizing activity were isolated from rotten kelp. By activity test, the Vibrio sp. QD-5 with the potential alginate-degrading capability was chosen to carry out the draft genome s...Seven bacterial clones with alginate-utilizing activity were isolated from rotten kelp. By activity test, the Vibrio sp. QD-5 with the potential alginate-degrading capability was chosen to carry out the draft genome sequencing, and the result showed that the Vibrio sp. QD-5 containing an alginate lyase gene cluster. One of these genes, aly-IV, was cloned and characterized for the first time. After overexpression, Aly-IV, with a molecular mass of about 62 kDa and a theoretical isoelectric point (pI) of 5.12, was purified to a specific activity of 1 256.78 U/mg and showed highest activity at 35°C in the Tris-HCl buffer at pH of 8.9. Moreover, the enzyme activity was enhanced by the metal ions of Na+, K+ and Mg2+ under certain concentration. Aly-IV degraded favorably polyG blocks in an endo-type, yielding monomer and dimer as the main products. Due to its high substrate specificity, Aly-IV could be used as a potential tool for production of polyG oligosaccharides with low degree of polymerization (DP) and for determining the fine structure of alginate.展开更多
Dear Editor,In recent years,owing to the fact that antibiotic-resistant bacteria have become more and more prevalent,there has been a resurgence of interest in the use of bacteriophages.However,bacteriophage therapy r...Dear Editor,In recent years,owing to the fact that antibiotic-resistant bacteria have become more and more prevalent,there has been a resurgence of interest in the use of bacteriophages.However,bacteriophage therapy remains an underutilized option in modern medicine due to technical hurdles such as limited host range,narrow spectrum of展开更多
Hydrogen sulfide(H2S)is found to be the third endogenous gaseous transmitter and plays an important role in many systems of organism. In cardiovascular system,H2S is produced endogenously by cystathionine gamma-lyase(...Hydrogen sulfide(H2S)is found to be the third endogenous gaseous transmitter and plays an important role in many systems of organism. In cardiovascular system,H2S is produced endogenously by cystathionine gamma-lyase(CSE).The CSE /H2S system executes the physiological function of vasorelaxation,inhibition of vascular remodeling and cardioprotection.These results suggest that H2S may be a novel cardiovascular functional regulator.ATM :To study whether hydrogen展开更多
We investigated the expression of hydrogen sulphide (H2S) in human and rat lower urinary tract (including bladder, prostate and urethra) tissues, and we sought to determine whether H2S induces relaxation of human ...We investigated the expression of hydrogen sulphide (H2S) in human and rat lower urinary tract (including bladder, prostate and urethra) tissues, and we sought to determine whether H2S induces relaxation of human and Sprague-Dawley (SD) rat bladder strips. Human normal lower urinary tract tissue was obtained for the evaluation of endogenous H2S productivity using a sulphide-sensitive electrode and for the analysis of the expression levels of all three synthases of endogenous H2S, cystathionine β-synthase (CBS), cystathionine y lyase (CSE) and 3-mercaptopyruvate sulphur transferase (MPST, as known as 3-MST) by Western blot assay. CBS, CSE and MPST were located in human sample slides by immunohistochemistry. Human and male adult SD rat bladder strips were tested for H2S function with a transducer and recorded. All experiments were repeated six times. The endogenous H2S productivity and the H2S synthases had various distributions in the human and rat lower urinary tract tissues and were located in both epithelial and stromal sections. L-cysteine (L-Cys, a substrate of CBS, CSE and MPST) elicited relaxation in a dose-dependent manner on human bladder strips ere-contracted by acetylcholine chloride. This effect could be diminished by the ATe-sensitive potassium ion (KATe) channel blocker glibenclamide (GLB), the CSE inhibitor DL-propargylglycine (PEG) and the CBS inhibitor hydroxylamine (HA). H2S and its three synthases were present in the human and rat lower urinary tract tissues and relaxed human and rat bladder strips, which implied that endogenous H2S might play a role in physiological function and pathological disorders of the lower urinary tract symptoms (LUTS) or overactive bladder (OAB).展开更多
Objective:To isolate,purify,and characterize gossypol from the fruits of Thespesia populnea(L)Sol.ex Correa,test its antidermatophytic activity,identify its targets on the dermatophyte,and confirm the binding of gossy...Objective:To isolate,purify,and characterize gossypol from the fruits of Thespesia populnea(L)Sol.ex Correa,test its antidermatophytic activity,identify its targets on the dermatophyte,and confirm the binding of gossypol with the fungal target by molecular docking study.Methods:Gossypol from Thespesia populnea was characterized by high performance liquid chromatography,liquid chromatographmass spectrometry,Fourier transform infrared spectroscopy,and nuclear magnetic resonance.The anti-dermatophytic activity of gossypol was tested against four different dermatophytes,viz.Trichophyton mentagrophytes,Trichophyton rubrum,Microsporum canis,and Microsporum gypseum.Trichophyton mentagrophytes was selected for further studies.The inhibitory mode of action of gossypol on Trichophyton mentagrophytes was determined by analyzing the modulation of gene expression in various pathways of the dermatophyte.Results:Gossypol inhibited all the dermatophytes.The minimum inhibitory concentrations were 12.5μg/mL for Trichophyton mentagrophytes and Microsporum canis and 25μg/mL for Trichophyton rubrum and Microsporum gypseum.The minimum fungicidal concentrations were 50μg/mL for Trichophyton mentagrophytes,100μg/mL for Microsporum canis and Trichophyton rubrum,and 200μg/mL for Microsporum gypseum.Gossypol inhibited the mRNA expression of metalloprotease(MEP4)and isocitrate lyase(ICL).The binding of gossypol with the enzymes was confirmed by molecular docking studies.The best docking poses were found and the low binding energies were recorded with the two target enzymes.Conclusions:Gossypol is a potential antifungal agent and can be further explored as an anti-dermatophytic drug.展开更多
文摘Hyaluronate lyases were obtained from two types of naturally isolated bacterial strains Paenibacillus yunnanensis and Paennarthrobacter nicotinovorans.PyHL(form P.yunnanensis)in the culture supernatant of the bacteria was purified by two steps of column chromatography.The enzyme showed the molecular mass of 74 kDa by SDS-PAGE and the maximal activity at pH 5.0,35℃.PyHL maximally degraded hyaluronate by an endo-type manner,and showed low degradation activity toward chondroitin sulfates.Dermatan sulfate was not the substrate.PnHL(from P.nicotinovorans)in the culture supernatant of the bacteria was purified by two steps of column chromatography.The enzyme showed the molecular mass of 70 kDa by SDS-PAGE and the maximal activity at pH 6.0,30℃.Genomic analysis of P.nicotinovorans on the bases of the internal amino acid sequences of PnHL.
文摘Abstract: Cis-cinnamic acid (CA) is a naturally occurring compound, presumably converted from trans-CA in higher plants. To investigate the effect of cis-CA on the activity of Arabidopsis phenylalanine ammonia lyase (PAL), AtPAL1, AtPAL2, and AtPAL4 genes were isolated using reverse transcription poly-merase chain reaction. These genes were fused to a glutathione S-transferase gene and overexpressed in a heterologous prokaryotic system of Escherichia coli. The purified PAL1, PAL2 and PAL4 enzymes were characterized biochemically to determine the effects of cis-CA on the kinetic parameter Km. The results showed that cis-CA is a competitive inhibitor for PAL1, but not PAL2 and PAL4, whereas trans-CA acts as a competitive inhibitor for all three PAL isomers, suggesting that cis- and trans-CA have different effects on the catalytic activity of PAL.
基金Fundaçao para a Ciencia e Tecnologia(FCT,Lisbon,Portugal)through grant PTDC/CAL-ZOO/30238/2017 associated post-doc contract to MMC,CIISA(Project UIDB/00276/2020)a PhD fellowship to DFC(SFRH/BD/126198/2016).
文摘Green macroalgae,e.g.,Ulva lactuca,are valuable bioactive sources of nutrients;but algae recalcitrant cell walls,composed of a complex cross-linked matrix of polysaccharides,can compromise their utilization as feedstuffs for monogastric animals.This study aimed to evaluate the ability of pre-selected Carbohydrate-Active enZymes(CAZymes)and sulfatases to degrade U.lactuca cell walls and release nutritive compounds.A databank of 199 recombinant CAZymes and sulfatases was tested in vitro for their action towards U.lactuca cell wall polysaccharides.The enzymes were incubated with the macroalga,either alone or in combination,to release reducing sugars and decrease fluorescence intensity of Calcofluor White stained cell walls.The individual action of a polysaccharide lyase family 25(PL25),an ulvan lyase,was shown to be the most efficient in cell wall disruption.The ulvan lyase treatment,in triplicate measures,promoted the release of 4.54 g/L(P<0.001)reducing sugars,a mono-and oligosaccharides release of 11.4 and 11.2 mmol/100 g of dried alga(P<0.01),respectively,and a decrease of 41.7%(P<0.001)in cell wall fluorescence,in comparison to control.The ability of ulvan lyase treatment to promote the release of nutritional compounds from alga biomass was also evaluated.A release of some monounsaturated fatty acids was observed,particularly the health beneficial 18:1c9(P<0.001).However,no significant release of total fatty acids(P>0.05),proteins(P?0.861)or pigments(P>0.05)was found.These results highlight the capacity of a single recombinant ulvan lyase(PL25 family)to incompletely disrupt U.lactuca cell walls.This enzyme could enhance the bioaccessibility of U.lactuca bioactive products with promising utilization in the feed industry.
基金Supported by the National Natural Science Foundation of China (30870519 and 30870541)
文摘In Gloeobacter violaceus PCC 7421, three possible lyase genes glr1191, glr1182 and gll1188 were selected by Blast sorting. The coded proteins of these three genes were co-expressed with their substrate protein in E. coli, respectively, and some chromoproteins were obtained. The fluorescence spectra showed that high fluorescence intensity was observed in the three experimental groups that involved the lyase genes, but little fluorescence intensity was observed in negative control groups. The ratio of relative fluorescence intensity in the experimental group with glr1191 was 64.8%. The result of SDS-PAGE indicated that the molecular weights of the three chromoproteins were 22.0 10 3 , 23.6 10 3 and 22.1 10 3 , respectively. The result of zinc-induced fluorescence re- vealed that the phycobilin in the three chromoproteins was covalently coupled to their apo-proteins. The result also showed that the coded proteins of these three genes (CpeS1 , CpeT1 , CpeY )could cata- lyze the covalent coupling of different phycobilins to their apo- proteins and formed active chromoproteins.
基金funded by the National Key Research and Development Program of China(2021YFD1800400)the Beijing Natural Science Foundation(6222032)the Starting Grants Program for Young Talents at China Agricultural University,the 2115 Talent Development Program of China Agricultural University and Chinese Universities Scientific Fund.
文摘Background Alginate oligosaccharide(AOS)holds great potential as a novel feed supplement in farm animals.However,the effects of AOS on chicken health and the underlying mechanisms are not fully understood.This study aimed to optimize the enzymatic preparation of AOS by using bacterial alginate lyases expressed in yeast,investigate the effects of the prepared AOS on the growth performance and gut health of broiler chickens,and reveal the underlying mechanisms.Results Five alginate lyases from bacteria were cloned into Pichia pastoris GS115 and the alginate lyase PDE9 was expressed at relatively high yield,activity and stability in P.pastoris.Animal trials were carried out using 3201-day-old male Arbor Acres broilers(four groups;8 replicates/group×10 chicks/replicate)receiving either a basal diet or the same diet supplemented with 100,200 and 400 mg/kg PDE9-prepared AOS for 42 d.The results showed that dietary supplementation of 200 mg/kg AOS displayed the highest activity in promoting the birds’ADG and ADFI(P<0.05).AOS ameliorated the intestinal morphology,absorption function and barrier function,as indicated by the enhanced(P<0.05)intestinal villus height,maltase activity,and the expression of PEPT,SGLT1,ZNT1,and occludin.AOS also increased serum insulin-like growth factor-1,ghrelin(P<0.05),and growth hormone(P<0.1).Moreover,the concentrations of acetate,isobutyrate,isovalerate,valerate,and total SCFAs in cecum of birds fed AOS were significantly higher than the control birds(P<0.05).Metagenomic analysis indicated that AOS modulated the chicken gut microbiota structure,function,and microbial interactions and promoted the growth of SCFAs-producing bacteria,for example,Dorea sp.002160985;SCFAs,especially acetate,were found positively correlated with the chicken growth performance and growth-related hormone signals(P<0.05).We further verified that AOS can be utilized by Dorea sp.to grow and to produce acetate in vitro.Conclusions We demonstrated that the enzymatically produced AOS effectively promoted broiler chicken growth performance by modulating the chicken gut microbiota structure and function.For the first time,we established the connections among AOS,chicken gut microbiota/SCFAs,growth hormone signals and chicken growth performance.
基金supported by the National Natural Science Foundation of China(31922072)Natural Science Foundation of Shandong Province(ZR2020JQ15)+3 种基金Project of Shandong Province Higher Educational Science and Technology Program(2019KJF012)Taishan Scholar Project of Shandong Province(tsqn201812020)China Agriculture Research System(CARS-48)Qingdao Science and Technology Demonstration and Guidance Project for Benefiting the People(20-3-4-28-nsh)。
文摘Quorum sensing(QS)system can dynamically control the expression of proteins along with the cell growth.The promoting period of QS system has been little focused on until now.In this study,a self-induced dynamic regulated expression(SIDRE)system was constructed in Escherichia coli.To enable the system suitable for the expression of enzymes,promoter engineering was used to obtain P_(luxI)mutants.To test the SIDRE system,alginate lyase AL493 and esterase Est7 were used as target protein for expression.The enzyme activity of alginate lyase and esterase reached 96.38%and 106.71%of the control strains containing the T7 promoter.In high-density fermentation,the activity of alginate lyase expressed by the SIDRE system with P_(luxI)(T-38C)as promoter was 4.34-fold of that expressed by the T7 promoter.Therefore,the P_(luxI)mutants with different promoting periods and/or different strengths show great potential in both laboratory and industrial scale for protein expression.
基金supported by grants from the National Natu-ral Science Foundation of China (81972779)Ministry of Education (MOE) Key Laboratory on signaling Regulation and Targeting Therapy of Liver Cancer,and Shanghai Key Laboratory of Hepato-biliary Tumor Biology,Chinese National Key Project (2018ZX10723204-006-003)。
文摘Background: Hepatocellular carcinoma(HCC) is one of the most highly malignant tumors. Liver tumor-initiating cells(LTICs) have been considered to contribute to HCC progression and metastasis. ATP-citrate lyase(ACLY), as a key enzyme for de novo lipogenesis, has been reported to be upregulated in various tumors. However, its expression and role in HCC and LTICs remain unknown. Methods: The expressions of ACLY in HCC tissues were detected by quantitative real-time PCR(q RT-PCR), Western blotting and immunohistochemistry. Kaplan-Meier curves and Chi-square test were used to determine the clinical significance of ACLY expression in HCC patients. A series of assays were performed to determine the function of ACLY on stemness, migration and invasion of HCC cells. Luciferase reporter assay, Western blotting and immunoprecipitation were used to study the regulation of the Wnt/β-catenin signaling by ACLY. Rescue experiments were performed to investigate whether β-catenin was the mediator of ACLY-regulated stemness and migration in HCC cells. Results: ACLY was highly expressed in HCC tissues and LTICs. Overexpression of ACLY was significantly correlated with poor prognosis, progression and metastasis of HCC patients. Knockdown of ACLY remarkably suppressed stemness properties, migration and invasion in HCC cells. Mechanistically, ACLY could regulate the canonical Wnt pathway by affecting the stability of β-catenin, and Lys49 acetylation of β-catenin might mediate ACLY-regulated β-catenin level in HCC cells. Conclusions: ACLY is a potent regulator of Wnt/β-catenin signaling in modulating LTICs stemness and metastasis in HCC. ACLY may serve as a new target for the diagnosis and treatment of HCC.
文摘In this study, the activities of phenylalanine ammonia lyase (PAL), polyphenoloxidase (PPO), and peroxidase (POD) were assayed in cucumber seedlings (Cucumis sativus L.) at 0, 6, 12, 24, 48, 72, and 96 h after they were infested by Bemisia tabaci (Gennadius) using spectrophotometric analysis. The results indicated that herbivore infestation increased the activities of PAL, PPO, and POD. The enzymes showed different activity levels at different times after the infestation. The PAL activity reached the first high peak by 23.1% at 6 h and the highest peak by 29.1% at 48 h compared to the control. The PPO activity reached the first high peak by 22.7% at 6 h and the highest peak by 52.6% at 24 h, and the POD activity reached the highest peak by 213.2% at 6 h and another higher peak value by 135.2% at 96 h. The results suggest that the enhanced activities of the enzymes may contribute to bioprotection of cucumber plants against B. tabaci infestation.
文摘To exploit peashrub resources in Ordos as fodders, it is very crucial to realize industrial production of cheap cellulase of high activity by optimizing culture technology, especially culture substrate. In this study, a new prescription experiment based on uniform design ideal was invented and successfully applied in the solid fermentation of Trichoderma koningii F244, which was performed with two different temperature degrees. The activities of FPA, cotton lyase, CMCase and β-glucosidase were assayed and then mathematical models of enzymatic activities, which were figured out by Unconstraint Mathematical Programming, were developed by Multivariate Regression Program of SPSS10.0. Enzymatic activities of optimized substrate prescriptions corresponding to mathematical models were forecasted to determine an ideal substrate prescription. It is revealed that in solid fermentation, Tween80 has negative effect on cellulase production. Furthermore, the ideal prescription for cellulase complex production by Trichoderma koningii F244 was straw powder 16.9%,wheat bran 26.5%, (NH 4) 2SO 4 9.5% and water 47.1%, whose corresponding cellulase activity was expected to be at the same high level with that of Trichoderma reesei Q9414 on its own recommended substrate. Especially, goats mainly fed on peashrub tissues mixed with cellulase complex of this prescription and culture technology, got an incremental ratio of 0.3 kg/d, which brought a very promising feeding prospect for local peashrub resource. By populization of this cellulase complex, it can integrate living standard, economic construction of local residents into vegetational restoration tightly and thus this paper will be very meaningful to be use for reference for western China like Ordos to realize its sustainable development of economy, society and environment.
基金supported by the Special Fund for Agro-scientific Research in the Public Interest in China (201503114)
文摘The soybean cyst nematode, Heterodeara glycines, is a serious pathogen of soybean, and reported to be the host of a wide range of Fabaceae. In the present study, the host specificity and reproductivity of two populations of H. glycines collected from soybean and tobacco were identified and characterized. The comparative identity between β-1,4-endoglucanase, pectate lyase and chorismate mutase of H. glycines parasitizing on soybean and tobacco were 99, 97 and 98%, respectively. The qR T-PCR analysis indicated that the expression of pectate lyase 2 gene was significantly higher in second-stage juveniles of H. glycines Henan population parasitizing on tobacco than that of H. glycines Shanxi population parasitizing on soybean. In addition, the pectic acid content of cell wall was significantly higher(45%) in the roots of tobacco than the roots of soybean. Our results indicate that the changes in transcript parasitism genes may be a result of long-term evolution illustrating how a plant-parasitic nematode adapts to the host environment for optimal infestation and survival.
基金China Ocean Mineral Resources R&D Association under contract No. DYXM-115-02-2-19Basic Scientific Fund for National Public Research Institutes of China under contract No. 2011T04
文摘CO2 fixation pathway of Caminibacter profundus, a chemolithoautotrophic e-Proteobacteria from deep-sea hydrothermal vent, was determined and characterized by genetic and enzymatic analyses. Gene expression of key enzymes for CO2 fixation in response to salinity, pH and O2 in Medium 829 were also investigated. The results demonstrate that C. profundus contained aclB, porA and oorA, the genes encoding key enzymes of reductive tricarboxylic acid (rTCA) cycle. However, genes fragments of cbbL and cbbMencoding key enzyme of Calvin cycle were not recovered. Key enzymatic activities of ATP citrate lyase (ACL), pyruvate: ferredoxin oxidoreductase (POR) and 2-oxoglutarate: ferredoxin oxidoreductase (OOR) were also present in C. profun- dus. The combination of genetic and enzymatic analyses confirm that C. profundus adopted rTCA cycle for carbon assimilation. The results of aclB and oorA relative expressions of C. profundus demonstrate that the ranges of environmental factors for high genes expression were sea salt 3.0%-5.0% (optimum 3.0%), pH 5.0-6.5(optimum pH 6.5), anaerobic to microaerobic conditions (optimum 1.0% 02). Gene expression pat- terns under different conditions show similar patterns with bacterial growth, revealing that key rTCA cycle genes provided molecular basis for bacterial growth and propagation. Our results suggest that C. profun- dus could regulate key genes of rTCA cycle for carbon assimilation and energy metabolism in response to environmental fluctuations in hydrothermal vent.
基金Supported by the Technology Development Funds of Education Department of Jilin Province,China(No.2008110)
文摘When devoured by macrophages,Mycobacterium tuberculosis remains persistent in macrophages and gains energy through the glyoxylate bypass to maintain its long-term existence in host cells.Therefore it is possible to stop persistent infections by interdicting the glyoxylate bypass in which the isocitrate lyase(ICL) is the key rate-limiting enzyme and a persistence factor.ICL is the target of anti-TB(TB:tubercular) drugs,which could screen ICL out and effectively inhibit the activity of ICL in Mycobacterium tuberculosis,and because of this,anti-TB drugs can be used to kill persistent Mycobacterium tuberculosis.In this study,the ICL gene of the Mycobacterium tuberculosis H37Rv was cloned successfully and recombinant protein with bioactivity was obtained through the enzyme characteristic appraisal.The specific activity of the recombined ICL is 24μmol·mg-1·min-1.The recombined ICL protein was used as the target,and phages which can specifically combine to ICL were screened in the phage 7 peptide library.According to the results of the ELISA and DNA sequence detection,eventually three 7-peptide chains were synthesized.Then the peptide chains were reacted with ICL,respectively,to detect their inhibitory effects on ICL.The results show that all the three 7-peptide chains possessed varying inhibitory effects on the activity of ICL.This study provided lead compounds for the research and development of new peptide anti-TB drugs.
基金SJ R-Z and ML R-M recibed a grant by‘ConsejoNacional de Ciencia y Tecnología’(CONACyT,México).
文摘A.americana L.is a crop with very little genetic variability.In order to evaluate the effect of ethyl methanesulfonate(EMS)to induce variability in in vitro plantlets of A.americana,different explants(meristems,leaves and roots)were evaluated for the production of callus.MS medium supplemented with ANA(2.68μM)and BAP(2.68μM)was used.Callus obtained from apical meristem were treated with 15 mM EMS for two hours after which shoot formation was induced using 2,4-D(0.11μM)and BAP(44μM).The EMS induced variations in the morphometric and morphological parameters of the plantlets obtained,with 60%of the plantlets presenting differences such as dwarfism and different leaf forms,without the presence of spines,as well as an increase in fructan content of 30%with respect to the control plantlets.PAL was increased and this activity is related with higher anthocyanins concentration in A.americana L.plantlets.
文摘Combinatorial chemistry has been a focus of research activity in modern drug discovery and biotechnology. It is a concept by which a vast library of molecular diversity is synthesized and screened for target properties. This report is to illustrate the application of enzyme technology using the concept of combinatorial chemistry as a novel approach for the bioconversion of plant fibers. Citrus pectin was subjected to combinatorial enzyme digestion to create libraries of pectic oligosaccharides with diverse structural variants. Repeated cycles of fractionation and screening resulted in the isolation and identification of an active oligoGalA species with antimicrobial activity.
基金The Marine Public Welfare Project of SOA under contract No.201505032the Scientific and Technological Innovation Project financially of Qingdao National Laboratory for Marine Science and Technology under contract No.2016ASKJ14
文摘Seven bacterial clones with alginate-utilizing activity were isolated from rotten kelp. By activity test, the Vibrio sp. QD-5 with the potential alginate-degrading capability was chosen to carry out the draft genome sequencing, and the result showed that the Vibrio sp. QD-5 containing an alginate lyase gene cluster. One of these genes, aly-IV, was cloned and characterized for the first time. After overexpression, Aly-IV, with a molecular mass of about 62 kDa and a theoretical isoelectric point (pI) of 5.12, was purified to a specific activity of 1 256.78 U/mg and showed highest activity at 35°C in the Tris-HCl buffer at pH of 8.9. Moreover, the enzyme activity was enhanced by the metal ions of Na+, K+ and Mg2+ under certain concentration. Aly-IV degraded favorably polyG blocks in an endo-type, yielding monomer and dimer as the main products. Due to its high substrate specificity, Aly-IV could be used as a potential tool for production of polyG oligosaccharides with low degree of polymerization (DP) and for determining the fine structure of alginate.
基金supported by the Natural Science Foundation of China(30571637)
文摘Dear Editor,In recent years,owing to the fact that antibiotic-resistant bacteria have become more and more prevalent,there has been a resurgence of interest in the use of bacteriophages.However,bacteriophage therapy remains an underutilized option in modern medicine due to technical hurdles such as limited host range,narrow spectrum of
基金Supported by the National Natur Science Foundation of China(No.30770852No.30800480No.30872710)
文摘Hydrogen sulfide(H2S)is found to be the third endogenous gaseous transmitter and plays an important role in many systems of organism. In cardiovascular system,H2S is produced endogenously by cystathionine gamma-lyase(CSE).The CSE /H2S system executes the physiological function of vasorelaxation,inhibition of vascular remodeling and cardioprotection.These results suggest that H2S may be a novel cardiovascular functional regulator.ATM :To study whether hydrogen
基金We thank Professor Jun-Bao Du for providing experimental suggestions and advice, and our study was supported by the National Natural Science Foundation of China (No. 30571851 to Jie Jill, No. 81201527 to Hui Guo).
文摘We investigated the expression of hydrogen sulphide (H2S) in human and rat lower urinary tract (including bladder, prostate and urethra) tissues, and we sought to determine whether H2S induces relaxation of human and Sprague-Dawley (SD) rat bladder strips. Human normal lower urinary tract tissue was obtained for the evaluation of endogenous H2S productivity using a sulphide-sensitive electrode and for the analysis of the expression levels of all three synthases of endogenous H2S, cystathionine β-synthase (CBS), cystathionine y lyase (CSE) and 3-mercaptopyruvate sulphur transferase (MPST, as known as 3-MST) by Western blot assay. CBS, CSE and MPST were located in human sample slides by immunohistochemistry. Human and male adult SD rat bladder strips were tested for H2S function with a transducer and recorded. All experiments were repeated six times. The endogenous H2S productivity and the H2S synthases had various distributions in the human and rat lower urinary tract tissues and were located in both epithelial and stromal sections. L-cysteine (L-Cys, a substrate of CBS, CSE and MPST) elicited relaxation in a dose-dependent manner on human bladder strips ere-contracted by acetylcholine chloride. This effect could be diminished by the ATe-sensitive potassium ion (KATe) channel blocker glibenclamide (GLB), the CSE inhibitor DL-propargylglycine (PEG) and the CBS inhibitor hydroxylamine (HA). H2S and its three synthases were present in the human and rat lower urinary tract tissues and relaxed human and rat bladder strips, which implied that endogenous H2S might play a role in physiological function and pathological disorders of the lower urinary tract symptoms (LUTS) or overactive bladder (OAB).
文摘Objective:To isolate,purify,and characterize gossypol from the fruits of Thespesia populnea(L)Sol.ex Correa,test its antidermatophytic activity,identify its targets on the dermatophyte,and confirm the binding of gossypol with the fungal target by molecular docking study.Methods:Gossypol from Thespesia populnea was characterized by high performance liquid chromatography,liquid chromatographmass spectrometry,Fourier transform infrared spectroscopy,and nuclear magnetic resonance.The anti-dermatophytic activity of gossypol was tested against four different dermatophytes,viz.Trichophyton mentagrophytes,Trichophyton rubrum,Microsporum canis,and Microsporum gypseum.Trichophyton mentagrophytes was selected for further studies.The inhibitory mode of action of gossypol on Trichophyton mentagrophytes was determined by analyzing the modulation of gene expression in various pathways of the dermatophyte.Results:Gossypol inhibited all the dermatophytes.The minimum inhibitory concentrations were 12.5μg/mL for Trichophyton mentagrophytes and Microsporum canis and 25μg/mL for Trichophyton rubrum and Microsporum gypseum.The minimum fungicidal concentrations were 50μg/mL for Trichophyton mentagrophytes,100μg/mL for Microsporum canis and Trichophyton rubrum,and 200μg/mL for Microsporum gypseum.Gossypol inhibited the mRNA expression of metalloprotease(MEP4)and isocitrate lyase(ICL).The binding of gossypol with the enzymes was confirmed by molecular docking studies.The best docking poses were found and the low binding energies were recorded with the two target enzymes.Conclusions:Gossypol is a potential antifungal agent and can be further explored as an anti-dermatophytic drug.