Characterization of ACE Inhibitory Peptides from Mactra veneriformis Hydrolysate by Nano-Liquid Chromatography Electrospray Ionization Mass Spectrometry (Nano-LC-ESI-MS) and Molecular Docking

Food-derived bioactive compounds are gaining increasing significance in life sciences. In the present study, we identified angiotensin I-converting enzyme (ACE)-inhibitory peptides from Mactra veneriformis hydrolysate using a nano-LC-MS/MS method. Mactra veneriformis hydrolysate was first separated into four fractions (F1–F4) based on molecular weight by ultrafiltration. The fraction with molecular weight lower than 1 kDa (F1) showed the highest ACE inhibitory activity. F1 was then analyzed by a high throughput nano-LC-MS/MS method and sequences of peptides in F1 were calculated accordingly. The 27 peptides identified as above were chemically synthesized and tested for ACE-inhibitory activity. The hexapeptide VVCVPW showed the highest potency with an IC₅₀ value of 4.07 μM. We then investigated the interaction mechanism between the six most potent peptides and ACE by molecular docking. Our docking results suggested that the ACE inhibitory peptides bind to ACE via interactions with His383, His387, and Glu411 residues. Particularly, similar to the thiol group of captopril, the cysteine thiol group of the most potent peptide VVCVPW may play a key role in the binding of this peptide to the ACE active site.     

Affinity Purification of Angiotensin Converting Enzyme Inhibitory Peptides from Wakame (Undaria Pinnatifida) Using Immobilized ACE on Magnetic Metal Organic Frameworks

Angiotensin-I-converting enzyme (ACE) inhibitory peptides derived from marine organism have shown a blood pressure lowering effect with no side effects. A new affinity medium of Fe₃O₄@ZIF-90 immobilized ACE (Fe₃O₄@ZIF-90-ACE) was prepared and used in the purification of ACE inhibitory peptides from Wakame (Undaria pinnatifida) protein hydrolysate (<5 kDa). The Fe₃O₄@ZIF-90 nanoparticles were prepared by a one-pot synthesis and crude ACE extract from pig lung was immobilized onto it, which exhibited excellent stability and reusability. A novel ACE inhibitory peptide, KNFL (inhibitory concentration 50, IC₅₀ = 225.87 μM) was identified by affinity purification using Fe₃O₄@ZIF-90-ACE combined with reverse phase-high performance liquid chromatography (RP-HPLC) and MALDI-TOF mass spectrometry. Lineweaver–Burk analysis confirmed the non-competitive inhibition pattern of KNFL, and molecular docking showed that it bound at a non-active site of ACE via hydrogen bonds. This demonstrates that affinity purification using Fe₃O₄@ZIF-90-ACE is a highly efficient method for separating ACE inhibitory peptides from complex protein mixtures and the purified peptide KNFL could be developed as a functional food ingredients against hypertension.     

Multifunctional Cationic Peptide Fractions from Flaxseed Protein Hydrolysates

The aim of this work was to determine the multifunctional properties of flaxseed protein-derived cationic peptide fractions. Alcalase hydrolysis of flaxseed protein fractions liberated cationic peptides, which were separated into two major fractions (FI and FII) by chromatography using a cation-exchange column. Due to their cationic property, the peptide fractions bound and inactivated calmodulin (CaM, a negatively charged enzyme activator protein) with concomitant inhibition of CaM-dependent phosphodiesterase (CaMPDE); this activity was substantially reduced as CaM concentration increased. Enzyme kinetics studies showed competitive inhibition of CaMPDE by FI and FII with enzyme-inhibitor dissociation constants of 0.0202 and 0.0511 mg/ml, respectively. Only the FII peptides showed multifunctional activities by inhibiting CaMPDE, angiotensin converting enzyme (ACE) and renin. Separation of FII peptides by reverse phase HPLC resulted in eight fractions (FII-2 to FII-9) that inhibited the activities of CaMPDE, ACE, and renin but this multifunctional activity was more pronounced in FII-6. From LC-MS analysis, identified peptides present in FII fraction had molecular size range of 330–735 Da, which suggests potential for increased absorption. Potential peptide sequences were identified for each of the HPLC fractions and shown to contain either lysine or arginine as the positively charged amino acid residue. The multifunctional properties of the cationic peptide fractions can potentially enhance their use in targeting multiple symptoms of cardiovascular disease, considering that the excessive levels of CaM, CaMPDE, renin and ACE play important roles in enhancing progression and intensity of chronic human diseases.     

ACE Inhibitory Peptide from Skin Collagen Hydrolysate of Takifugu bimaculatus as Potential for Protecting HUVECs Injury

Angiotensin-I-converting enzyme (ACE) is a crucial enzyme or receptor that catalyzes the generation of potent vasopressor angiotensin II (Ang II). ACE inhibitory peptides from fish showed effective ACE inhibitory activity. In this study, we reported an ACE inhibitory peptide from Takifugu bimaculatus (T. bimaculatus), which was obtained by molecular docking with acid-soluble collagen (ASC) hydrolysate of T. bimaculatus. The antihypertensive effects and potential mechanism were conducted using Ang-II-induced human umbilical vein endothelial cells (HUVECs) as a model. The results showed that FNLRMQ alleviated the viability and facilitated apoptosis of Ang-II-induced HUVECs. Further research suggested that FNLRMQ may protect Ang-II-induced endothelial injury by regulating Nrf2/HO-1 and PI3K/Akt/eNOS signaling pathways. This study, herein, reveals that collagen peptide FNLRMQ could be used as a potential candidate compound for antihypertensive treatment, and could provide scientific evidence for the high-value utilization of marine resources including T. bimaculatus.     

New angiotensin-converting enzyme inhibitory peptide from Coix prolamin and its influence on the gene expression of renin-angiotensin system in vein endothelial cells

Coix seed, which is a traditional Chinese medicine, has been used to treat hypertension for thousands of years. It has been shown that Coix prolamin peptides display high levels of angiotensin I converting enzyme (ACE) inhibitory activity. Hence, we purified the ACE inhibitory peptides from Coix prolamin hydrolysates and evaluated the influence of the most potent peptide on the renin-angiotensin system (RAS) genes expression in human umbilical vein endothelial cells (HUVECs). In this study, Coix prolamin peptides were sequentially separated by ultrafiltration, ion exchange chromatography, gel filtration chromatography and RP-HPLC, while the peptide structure was analyzed by mass spectrometry. Next, in silico proteolysis, pharmacophore and molecular docking were further applied to screen and optimize the structure of peptides. Finally, a novel ACE inhibitory peptide VDMF was obtained, in which its influence on the gene expression of RAS signaling pathway in AngⅡ-injury HUVECs was evaluated by quantitative real-time PCR. VDMF significantly down-regulated ACE, AngII type 1 receptor (AT1R) and ACE2 mRNA expression in comparation with model group, while up-regulating Mas gene expression. Hence, we obtained a novel antihypertensive candidate that was derived from the Coix peptides, which could involve a multi-modulation mechanism that regulates blood pressure.     

Fate of ACE-inhibitory peptides during the bread-making process: Quantification of peptides in sourdough,bread crumb,steamed bread and soda crackers

This study compared the concentration of angiotensin-converting enzyme (ACE) inhibitory peptides at different stages of the bread-making process, including kneading, proofing, and final products. Steamed bread, baked bread, and soda crackers were produced with 3–20% addition of rye malt sourdoughs to assess products differing in their thermal treatment. Eight tripeptides with known or predicted ACE-inhibitory activity were quantified by LC/MS in multiple reaction monitoring (MRM) mode. In wheat sourdough and rye-malt gluten sourdough, IPP was the predominant tripeptide at 58 and 473 μmol kg⁻¹, respectively, followed by LQP, IQP, and LPP. During the bread-making process, peptide concentrations were modified by enzymatic conversions at the dough stage and by thermal reactions during baking. The concentrations of IPP, LPP and VPP remained stable during dough preparation but decreased during thermal treatment; the concentrations of other peptides were changed at the dough stage but remained relatively stable during baking. The cumulative concentration of 8 ACE-inhibitory peptides in steamed bread and bread crumb exceeded 60 μmol kg⁻¹, while soda crackers contained less than 3 μmol kg⁻¹. The peptide levels in bread thus likely meet in vivo active concentrations.     

Multifunctional bioactive peptides derived from quinoa protein hydrolysates: Inhibition of α-glucosidase,dipeptidyl peptidase-IV and angiotensin I converting enzymes

The study aimed to characterize and identify anti-diabetic and anti-hypertensive bioactive peptides generated upon enzymatic hydrolysis of quinoa protein isolates. Different quinoa protein hydrolysates (QPHs) were produced using food grade enzymes like Bromelain, chymotrypsin and Pronase E at a hydrolysis interval of 2 h up to 6 h. QPHs were characterized for their physicochemical properties using degree of hydrolysis, SDS-PAGE, and their anti-diabetic properties via inhibition of dipeptidyl peptidase-IV (DPP-IV) and α-glucosidase (AG), and anti-hypertensive property via inhibition of angiotensin converting enzyme (ACE) were explored. IC₅₀ for DPP-IV, AG and ACE inhibitory activities of QPHs were in the range of 0.72–1.12, 1.00–1.86 and 0.18–0.31 mg/mL, respectively. The chymotrypsin derived 6 h hydrolysate (QC6) was sequenced for peptides identification and 136 peptides were identified among which 35 peptides were predicted as potential bio-active peptides (BAPs) based on their Peptide Ranker score. Results showed that identified peptides were predicted to possess high potential in inhibiting the DPP-IV, AG and ACE. In particular, QHPHGLGALCAAPPST was found to bind to the highest number of active hotspots of the target enzymes that are involved in their enzymatic activities. In conclusion, quinoa protein hydrolysates were identified as potential sources of BAPs with inhibitory properties towards key enzymes involved in the control of type 2 diabetes and hypertension.     

Characterization of ACE Inhibitory Peptides Prepared from Pyropia pseudolinearis Protein

More than 7000 red algae species have been classified. Although most of them are underused, they are a protein-rich marine resource. The hydrolysates of red algal proteins are good candidates for the inhibition of the angiotensin-I-converting enzyme (ACE). The ACE is one of the key factors for cardiovascular disease, and the inhibition of ACE activity is related to the prevention of high blood pressure. To better understand the relationship between the hydrolysates of red algal proteins and the inhibition of ACE activity, we attempted to identify novel ACE inhibitory peptides from Pyropia pseudolinearis. We prepared water soluble proteins (WSP) containing phycoerythrin, phycocyanin, allophycocyanin, and ribulose 1,5-bisphosphate carboxylase/oxygenase. In vitro analysis showed that the thermolysin hydrolysate of the WSP had high ACE inhibitory activity compared to that of WSP. We then identified 42 peptides in the hydrolysate by high-performance liquid chromatography and mass spectrometry. Among 42 peptides, 23 peptides were found in chloroplast proteins. We then synthesized the uncharacterized peptides ARY, YLR, and LRM and measured the ACE inhibitory activity. LRM showed a low IC₅₀ value (0.15 μmol) compared to ARY and YLR (1.3 and 5.8 μmol). In silico analysis revealed that the LRM sequence was conserved in cpcA from Bangiales and Florideophyceae, indicating that the novel ACE inhibitory peptide LRM was highly conserved in red algae.     

Angiotensin I Converting Enzyme Inhibitory Peptides Derived from Phycobiliproteins of Dulse Palmaria palmata

We examined the inhibitory activity of angiotensin I converting enzyme (ACE) in protein hydrolysates from dulse, Palmaria palmata. The proteins extracted from dulse were mainly composed of phycoerythrin (PE) followed by phycocyanin (PC) and allophycocyanin (APC). The dulse proteins showed slight ACE inhibitory activity, whereas the inhibitory activity was extremely enhanced by thermolysin hydrolysis. The ACE inhibitory activity of hydrolysates was hardly affected by additional pepsin, trypsin and chymotrypsin treatments. Nine ACE inhibitory peptides (YRD, AGGEY, VYRT, VDHY, IKGHY, LKNPG, LDY, LRY, FEQDWAS) were isolated from the hydrolysates by reversed-phase high-performance liquid chromatography (HPLC), and it was demonstrated that the synthetic peptide LRY (IC₅₀: 0.044 μmol) has remarkably high ACE inhibitory activity. Then, we investigated the structural properties of dulse phycobiliproteins to discuss the origin of dulse ACE inhibitory peptides. Each dulse phycobiliprotein possesses α-subunit (Mw: 17,477–17,638) and β-subunit (Mw: 17,455–18,407). The sequences of YRD, AGGEY, VYRT, VDHY, LKNPG and LDY were detected in the primary structure of PE α-subunit, and the LDY also exists in the APC α- and β-subunits. In addition, the LRY sequence was found in the β-subunits of PE, PC and APC. From these results, it was suggested that the dulse ACE inhibitory peptides were derived from phycobiliproteins, especially PE. To make sure the deduction, we carried out additional experiment by using recombinant PE. We expressed the recombinant α- and β-subunits of PE (rPEα and rPEβ, respectively), and then prepared their peptides by thermolysin hydrolysis. As a result, these peptides showed high ACE inhibitory activities (rPEα: 94.4%; rPEβ: 87.0%). Therefore, we concluded that the original proteins of dulse ACE inhibitory peptides were phycobiliproteins.     

大豆分离蛋白生产降血压肽酶解技术研究

选用6种蛋白酶水解大豆分离蛋白,根据ACE抑制率高低,最终选择胰蛋白酶为最佳蛋白酶。通过单因素试验和L16(45)正交试验研究底物浓度、水解时间、酶与底物比、温度和pH值对酶解液降血压活性的影响,确定了水解最优条件组合。结果表明:底物浓度5%,水解时间6 h,酶和底物比为0.08,温度为50℃,pH值为8.0时,所得水解液ACE抑制率最高,为76.8%。     

A Multistrategic Approach in the Development of Sourdough Bread Targeted Towards Blood Pressure Reduction

Rising prevalence of hypertension is pushing food industry towards the development of innovative food products with antihypertensive effects. The aim was to study the effect of reduced sodium content and 21 % addition of wholemeal wheat sourdough (produced by Lactobacillus brevis CECT 8183 and protease) on proximate composition, γ-aminobutyric acid (GABA) and peptide content of wheat bread. Angiotensin converting enzyme I (ACE) inhibitory and antioxidant activities were also evaluated. Sodium replacement by potassium salt did not affect chemical composition and biological activities of bread. In contrast, GABA and peptides <3 kDa contents in sourdough bread (SDB) were 7 and 3 times higher, respectively, than the observed in control. ACE inhibitory and antioxidant activities of the peptide fraction?<?3 kDa from SDB was 1.7 and 2.6-3.0 times higher than control. Therefore, the combination of reduced sodium content with enriched concentrations of bioactive compounds in bread making may provide interesting perspectives for development of innovative breads towards blood pressure reduction.

     

A Novel Angiotensin-I-Converting Enzyme (ACE) Inhibitory Peptide from Takifugu flavidus

Alcalase, neutral protease, and pepsin were used to hydrolyze the skin of Takifugu flavidus. The T. flavidus hydrolysates (TFHs) with the maximum degree of hydrolysis (DH) and angiotensin-I-converting enzyme (ACE)-inhibitory activity were selected and then ultra-filtered to obtain fractions with components of different molecular weights (MWs) (<1, 1–3, 3–10, 10–50, and >50 kDa). The components with MWs < 1 kDa showed the strongest ACE-inhibitory activity with a half-maximal inhibitory concentration (IC₅₀) of 0.58 mg/mL. Purification and identification using semi-preparative liquid chromatography, Sephadex G-15 gel chromatography, RP-HPLC, and LC–MS/MS yielded one new potential ACE-inhibitory peptide, PPLLFAAL (non-competitive suppression mode; IC₅₀ of 28 μmmol·L⁻¹). Molecular docking and molecular dynamics simulations indicated that the peptides should bind well to ACE and interact with amino acid residues and the zinc ion at the ACE active site. Furthermore, a short-term assay of antihypertensive activity in spontaneously hypertensive rats (SHRs) revealed that PPLLFAAL could significantly decrease the systolic blood pressure (SBP) and diastolic blood pressure (DBP) of SHRs after intravenous administration. These results suggested that PPLLFAAL may have potential applications in functional foods or pharmaceuticals as an antihypertensive agent.     

醋制对黑豆蛋白含量及多肽ACE抑制活性的影响

为考察醋制对黑豆蛋白含量及多肽ACE抑制活性的影响,为对黑豆醋浸过程中蛋白组分及含量变化进行分析,并对相应ACE抑制肽活性进行测定。黑豆经醋浸不同时间后,采用顺序抽提法提取清蛋白、球蛋白、醇溶蛋白和谷蛋白,并采用凯氏定氮法及SDS-PAGE电泳进行定量和定性分析;各蛋白组分经胃蛋白酶水解,经超滤(截留分子量3 k D)后收集滤液,获得多肽组分,RP-HPLC法进行ACE抑制活性评价。结果表明:经醋浸14 d后,黑豆总蛋白含量变化幅度小于±0.5%;清蛋白含量由55.13%(0 d)降低至9.61%(14 d);球蛋白由7.04%(0 d)增加到20.34%(14 d);醇溶蛋白由0.81%(0 d)增加到1.72(14 d);谷蛋白由21.11%(0 d)增加到59.45%(14 d),含量最高。醋浸处理降低了清蛋白源多肽的ACE抑制活性,但提高了球蛋白、醇溶蛋白及谷蛋白多肽的ACE抑制活性,其中,谷蛋白多肽抑制率由18.18%(0 d)增加至37.58%(14 d),抑制活性升高。醋浸可改变黑豆各蛋白组分的含量和对应多肽的ACE抑制活性,其中,谷蛋白含量及其多肽ACE抑制活性均有增加。研究结果表明可进一步采用分离纯化技术从谷蛋白多肽中获得高活性降压肽。     

Screening of Human CYP1A2 and CYP3A4 Inhibitors from Seaweed In Silico and In Vitro

Phenolic compounds and carotenoids are potential inhibitors of cytochrome P450s. Sixteen known compounds, phenolic compounds and carotenoids from seaweed were examined for potential inhibitory capacity against CYP1A2 and CYP3A4 in silico and in vitro. Morin, quercetin, and fucoxanthin inhibited the enzyme activity of CYP1A2 and CYP3A4 in a dose-dependent manner. The IC₅₀ values of morin, quercetin, and fucoxanthin were 41.8, 22.5, and 30.3 μM for CYP1A2 and 86.6, 16.1, and 24.4 μM for CYP3A4, respectively. Siphonaxanthin and hesperidin did not show any significant effect on CYP1A2, but they slightly inhibited CYP3A4 activity at high concentrations. In silico modeling of CYP’s binding site revealed that the potential inhibitors bound in the cavity located above the distal surface of the heme prosthetic group through the 2a or 2f channel of CYPs. This study presents an approach for quickly predicting CYP inhibitory activity and shows the potential interactions of compounds and CYPs through in silico modeling.     

Preparation, physicochemical and texture properties of texturized rice produce by Improved Extrusion Cooking Technology

Using broken rice and rice bran as raw material, texturized rice (TR) was prepared by Improved Extrusion Cooking Technology (IECT) in which gelatinization is formed by means of low temperature and high pressure. The expansion of extrudate was hardly changed so that TR showed similar texture properties and shape with polished rices. The effect of rice bran addition (0% and 4%) and IECT conditions, including feed moisture content (26.6-33.4%), screw speed (20.1-32.6 rpm) and shearing compression metering zone temperature (SCMT, 69.8-120.2 °C) on the physicochemical, texture and nutritional characteristics of TR, were investigated by response surface methodology using Central Composite Design. When the bran addition was 4%, feed moisture content was 30%, screw speed was 26.6 rpm, SCMT was 95 °C, prepared TR contained 16.61 ± 0.02% of total dietary fiber, 9.40 ± 0.04% of protein, 3.68 ± 0.03% of fat, 2.42 ± 0.02 μg/g of thiamin, 0.52 ± 0.01 μg/g of riboflavin and 16.07 ± 0.12 mg/100 g of γ-oryzanol (dry matter content). The content increase of TDF for TR was 15.81% and the content increases of nutrients for thiamin, riboflavin, and γ-oryzanol were 1.39 μg/g, 0.24 μg/g, and 8.99 mg/g dry matter content, respectively, compared with those of polished rice.     

Phenolic compounds in raw and cooked rice (Oryza sativa L.) and their inhibitory effect on the activity of angiotensin I-converting enzyme

Whole rice has been widely studied due to the abundance of bioactive compounds in its pericarp. Some of the beneficial effects of these compounds on human health have been attributed to their antioxidant and other biological activities, such as enzyme inhibition. In this work, we evaluated the contents of total, soluble and insoluble phenolic compounds of 6 red and 10 non-pigmented genotypes of whole rice as well as their inhibitory effect on the activity of angiotensin I-converting enzyme (ACE). The effects of cooking on phenolics and their inhibitory activities were also investigated. Red genotypes showed high content of phenolics, mainly soluble compounds, at an average of 409.7 mg ferulic acid eq./100 g, whereas overall lower average levels (99.4 mg ferulic acid eq./100 g) at an approximate soluble/insoluble compound ratio of 1:1 were observed in non-pigmented rice. Pigmented rice displayed a greater inhibitory effect on ACE than non-pigmented rice. In fact, a significant correlation between the content of soluble phenolics and ACE inhibition was observed (r = 0.8985, p < 0.05). In addition to significantly reducing the levels of total phenolics and ACE inhibition, cooking altered the soluble/insoluble compound ratio, especially among red rice genotypes.     

Purification and Identification of Novel Xanthine Oxidase Inhibitory Peptides Derived from Round Scad (Decapterus maruadsi) Protein Hydrolysates

The objective of the present study was to investigate the xanthine oxidase (XO) inhibitory effects of peptides purified and identified from round scad (Decapterus maruadsi) hydrolysates (RSHs). In this study, RSHs were obtained by using three proteases (neutrase, protamex and alcalase). Among them, the RSHs of 6-h hydrolysis by neutrase displayed the strongest XO inhibitory activity and had an abundance of small peptides (<500 Da). Four novel peptides were purified by immobilized metal affinity chromatography and identified by nano-high-performance liquid chromatography mass/mass spectrometry. Their amino acid sequences were KGFP (447.53 Da), FPSV (448.51 Da), FPFP (506.59 Da) and WPDGR (629.66 Da), respectively. Then the peptides were synthesized to evaluate their XO inhibitory activity. The results indicated that the peptides of both FPSV (5 mM) and FPFP (5 mM) exhibited higher XO inhibitory activity (22.61 ± 1.81% and 20.09 ± 2.41% respectively). Fluorescence spectra assay demonstrated that the fluorescence quenching mechanism of XO by these inhibitors (FPSV and FPFP) was a static quenching procedure. The study of inhibition kinetics suggested that the inhibition of both FPSV and FPFP was reversible, and the type of their inhibition was a mixed one. Molecular docking revealed the importance of π-π stacking between Phe residue (contained in peptides) and Phe⁹¹⁴ (contained in the XO) in the XO inhibitory activity of the peptides.     

Preparation and bioactivity evaluation of low salt peptide from sunf lower seed meal

Sunflower seed meal peptide as one sort of bioactive peptide has intensively application prospects. However, preparation of low salt peptide from sunflower seed meal with high efficiency remains a challenge. In this study, single and compound proteases were optimized to hydrolyze protein. Results showed that hydrolysis at pH 7.0 by proteases resulted in ash content in the range of 5.66%-7.37% and small peptides. Among all hydrolysis processes, sequential hydrolysis of Alcalase with Flavourzyme and Alcalase with Protamex showed higher nitrogen recovery ratio (67.66% and 66.49%, respectively). Furthermore, biological activities of peptides were investigated by testing their ABTS (2,2-azinobis (3-ethylben-zothiazoline-6-sulfonic acid) diammonium salt) radical scavenging activity, DPPH (2,2-diphenyl-1-picrylhydrazil) radical scavenging activity and angiotensin converting enzyme (ACE) inhibitory activity. Peptide hydrolyzed by Alcalase with Papain presented the highest antioxidant activity, followed by Alcalase with Protamex, with ABTS scavenging rate as 63.01% and 31.75%, and DPPH scavenging rate as 56.04% and 28.06%, respectively. Synchronously, peptide hydrolyzed by Alcalase with Protamex and Alcalase with Alcalase had the highest ACE inhibitory activity (56.74%, 56.76%). In conclusion, hydrolysis by proteases Alcalase with Protamex at pH 7.0 was the most effective method for the preparation of low salt peptide from sunflower seed meal, which could be an alternative for anti-oxidants and anti-vasoconstrictor.     

Fungitoxicity and synergism of mixtures of fluopicolide and pyraclostrobin against Phytophthora infestans

Fluopicolide and pyraclostrobin were new systemic fungicides with highly inhibitory activity on a broad spectrum of oomycetes, but so far the fungitoxicity of their mixtures on the different developmental stages of Phytophthora infestans and whether synergism exists have not been investigated. The joint-toxicity of the mixtures of fluopicolide with pyraclostrobin was determined against the different developmental stages (mycelial growth, zoospore release, cystospore germination and sporangial germination) of P. infestans and late blight on the leaf discs and the potted plants of potato, and whether the synergistic interaction exists in the mixtures of the two fungicides were evaluated in vitro and in vivo based on the synergistic ratios calculated with the Wadley formulas. The protective activity and curative activity against P. infestans and the efficacy duration in controlling potato late blight of the synergistic mixtures of the two fungicides were examined in this study. The results showed that mixtures of fluopicolide (F) and pyraclostrobin (P) at the ratios of 10:1 and 1:4 (F:P) exhibited synergistic interactions and had excellent inhibitory activity against almost all developmental stages of P. infestans. Synergistic interactions of the mixtures at the ratios of 1:4 and 10:1 (F:P) against the mycelial growth of metalaxyl-resistant isolates were more obvious than against the mycelial growth of metalaxyl-sensitive isolates. Synergistic interactions of the mixtures at the ratio of 1:4 (F:P) against the mycelial growth of the most of the tested isolates were more obvious than those of the mixtures at the ratio of 10:1 (F:P). The highest activity of the mixtures was observed against zoospore release at the ratio of 1:4 (F:P) with EC₅₀ value of 0.0044 μg ml⁻¹. The strongest synergistic interaction of the mixtures was observed against zoospore release at the ratio of 10:1 (F:P) with the synergistic ratio of 5.27. The mixtures of fluopicolide and pyraclostrobin at the ratios of 1:4 and 10:1 (F:P) showed synergistic interactions against P. infestans on the leaf discs and intact plants of potato. The EC₅₀ values for protective activity and curative activity of the mixture at the ratio of 10:1 (F:P) were 0.63 μg ml⁻¹ and 0.87 μg ml⁻¹, and EC₅₀ values of protective activity and curative activity of the mixture at the ratio of 1:4 (F:P) were 0.27 μg ml⁻¹ and 0.44 μg ml⁻¹, respectively. At 14 days after spraying of fungicide solutions, the disease control efficacy of the mixtures at the ratios of 10:1 and 1:4 (F:P) was still over 80% at the dosages of 50 μg ml⁻¹ and 100 μg ml⁻¹, and the disease control efficacy of the two mixtures remained at about 97% at the dosages of 200 μg ml⁻¹, indicating long-lasting effective control efficacy of the two fungicides against potato late blight.     

Identification of a GDSL-motif carboxylester hydrolase from rice bran (Oryza sativa L.)

A major esterase (designated OsEST1) showing high activity using 1-naphthyl acetate as a substrate was identified from rice bran and purified approximately 239-fold to near-homogeniety. The purified enzyme migrated as a single polypeptide band on native and SDS-polyacrylamide gels and had a molecular mass of 25 kDa under denaturing conditions. Analysis of its tryptic peptides by MALDI-TOF-MS and subsequent data mining identified a corresponding cDNA OsEST1 consisting of 714 nucleotides and encoding a 238 amino acid protein. Analysis of its primary sequence indicated that OsEST1 is a GDSL-motif carboxylester hydrolase belonging to the SGNH protein subfamily in containing the putative catalytic triad of Ser¹¹, Asp¹⁸⁷, and His¹⁹⁰. OsEST1 showed the highest catalytic activity at approximately pH 8.0–8.5 and at 45 °C with K_m and V_max values for 1-naphthyl acetate of 172 μM and 63.7 μmol/min/mg protein, respectively. However, OsEST1 showed no activity with triacylglycerol. Alignment of the primary sequence of OsEST1 and other rice GDSL-motif esterases/lipases showed that OsEST1 aligns with a specific family of plant SGNH esterases involved in response to dehydration and cuticle formation. These results suggest that OsEST1 is not a lipase but an esterase activity which has some other function in rice, especially during seed development.