•Production of Hypocrea orientalis cellulase with cassava residues.•Purification and characterization of H. orientalis β-glucosidase.•Inactivation kinetics of fungal β-glucosidase by GuHCl was first investigated.•Active site formed by coiled loops to be obviously flexible.•Substrate presented protective effect on the active site during denaturation.The relationship between unfolding and inactivation of Hypocrea orientalis β-glucosidase has been investigated for the first time. The secretion of β-glucosidase from H. orientalis is induced by raw cassava residues. The enzyme was 75 kD without glycosylation. Guanidine hydrochloride (GuHCl) could reversibly inactivate the enzyme with an estimated IC50 value of 0.4 M. The inactivation kinetics model by GuHCl has been established and the microscopic inactivation rate constants are determined. The values of forward inactivation rate constants of free enzyme are found to be larger than that of substrate–enzyme complex suggesting the enzyme could be protected by substrate during denaturation. Conformational change of the enzyme during denaturation is observed as the intrinsic fluorescence emission peaks appeared red-shift (334–354 nm) with intensity decreased following increase of GuHCl concentrations. Inactivation extent is found to be greater than conformation change of the whole enzyme, indicating that the active site of H. orientalis β-glucosidase might be a more flexible region than the whole enzyme.

Retinoic acid receptor α (RARα) plays important roles in the progression of several cancers such as leukemia, breast cancer, and lung cancer. In this study, we demonstrated that RARα protein was frequently overexpressed in human CRC specimens and CRC cell lines. RARα knockdown decreased cell survival, proliferation, and colony formation in vitro and tumorigenic potential in nude mice. Specifically, RARα knockdown inhibited cell cycle progression at G1 phase through upregulation of cell cycle inhibitor p21, and downregulation of cyclinD1. Furthermore, RARα was directly recruited to the p21 promoter to inhibit the expression of p21. Simultaneously, RARα contributed to the progression of CRC cells in part due to upregulation of cyclinD1 via activation of GSK3β/β-catenin pathway. Molecular mechanism studies revealed RARα interacted with GSK3β and led to decreased expression of GSK3β at ser9, followed by increased β-catenin expression. Taken together, our results signified the importance of RARα in CRC and demonstrated that RARα promotes CRC progression through suppressing p21 transcription and enhancing GSK3β/β-catenin signaling. RARα might become a potential molecular target for the treatment of CRC.

Proteolytic processing of Bacillus thuringiensis (Bt) crystal toxins by insect midgut proteases plays an essential role in their insecticidal toxicities against target insects. In the present study, proteolysis of Bt crystal toxin Cry2Ab by Plutella xylostella L. midgut proteases (PxMJ) was evaluated. Both trypsin and chymotrypsin were identified involving the proteolytic activation of Cry2Ab and cleaving Cry2Ab at Arg139 and Leu144, respectively. Three Cry2Ab mutants (R139A, L144A, and R139A-L144A) were constructed by replacing residues Arg139, Leu144, and Arg139-Leu144 with alanine. Proteolysis assays revealed that mutants R139A and L144A but not R139A-L144A could be cleaved into 50 kDa activated toxins by PxMJ. Bioassays showed that mutants R139A and L144A were highly toxic against P. xylostella larvae, while mutant R139A-L144A was almost non-insecticidal. Those results demonstrated that proteolysis by PxMJ was associated with the toxicity of Cry2Ab against P. xylostella. It also revealed that either trypsin or chymotrypsin was enough to activate Cry2Ab protoxin. This characteristic was regarded as a belt-and-braces approach and might contribute to the control of resistance development in target insects. Our studies characterized the proteolytic processing of Cry2Ab and provided new insight into the activation of this Bt toxin.Keywords: Bacillus thuringiensis; cleavage site; Cry2Ab; insecticidal activity; proteolysis;

•Overexpression of XK/XI markedly boosted the use of xylose.•Recombinant strains partly overcome the inhibitory effect of glucose on xylose use.•The yield for a recombinant strain reached 2.46 mol H2/mol xylose.Biofuels production from lignocellulose hydrolysates by microbe fermentation has merited attention because of the mild reaction conditions involved and the clean nature of the process. In this work, xylulokinase (XK) and xylose isomerase (XI) were overexpressed in Klebsiella oxytoca HP1 to enhance hydrogen production by the fermentation of xylose. The recombinant strains exhibited higher enzyme activity of XI or XK compared with the wild strain. Hydrogen production from pure xylose, xylose/glucose mixtures and bamboo stalk hydrolysate was significantly enhanced with the overexpression of XI and XK in K. oxytoca HP1 in terms of total hydrogen yield (THY), hydrogen yield per mole substrate (HYPM) and hydrogen production rate (HPR). The HYPM of K. oxytoca HP1/xylB and K. oxytoca HP1/xylA reached 1.93 ± 0.05 and 2.46 ± 0.05 mol H2/mol xylose, respectively in pure xylose, while the value for the wild strain was 1.68 ± 0.04 mol H2/mol xylose. The xylose consumption rate (XCR) for the recombinant strain was significantly higher than that for the wild strain, particularly in the early stage of fermentation. Relative to the wild type, hydrogen yield (HY) from 1 g of preprocessed bamboo powder of HP1/xylB and HP1/xylA increased by 33.04 and 41.31%, respectively. It was concluded that overexpression of XK or XI was able to promote hydrogen production from xylose and xylose/glucose mixtures by simultaneously increasing the utilization efficiency of xylose and weakening the inhibitory effect of glucose on xylose use. In addition, the results indicated that overexpression technology was an effective way to further increase hydrogen production from lignocellulosic hydrolysates.

•Cys nearby Fe–S clusters have significant effects on O2-tolerance of KoHyd3.•Excellent performance of Gly–Cys exchanges in oxygen tolerance.•Developing an efficient way to generate O2-tolerant KoHyd3 as catalysts.Hydrogenase is the key point of H2-based biotechnology. However, the O2-sensitivity largely hinders its applications in biofuel cells and biological H2 production. Therefore, substantial breakthrough on understanding the molecular basis of O2-sensitivity and developing more O2-tolerant hydrogenases are urgently required. In this study, we found adding extra cysteines to the vicinity of the proximal Fe–S cluster to the NiFe active centre could largely enhance oxygen tolerance of hydrogen-evolving hydrogenase 3 from Klebsiella oxytoca HP1 (KoHyd3), through homologous sequence comparison and site-directed mutagenesis. Ratio of aerobic hydrogen yield to anaerobic hydrogen yield (RHH) of Gly47Cys (Gly47 was replaced with Cys47), Gly50Cys, Gly113Cys, Gly120Cys and Gly50Cys–Gly120Cys (double exchange) were increased by 46.99%, 42.15%, 59.19%, 44.74% and 78.72%, respectively, comparing with that of wild type. Moreover, TiO2-KoHyd3 (Gly47Cys, Gly50Cys, Gly113Cys, Gly120Cys and Gly50Cys–Gly120Cys) particles acted well in UV light-driven H2 production from water. These results revealed that extra cysteines nearby Fe–S clusters had significant effects on oxygen tolerance of KoHyd3. It also provided a promising way to produce O2-tolerant hydrogenase as biocatalysts in biofuel cells or H2 production by photolysis of water.

Cassava residues are byproducts of the starch industry containing abundant cellulose for bioproduction of green fuel. To obtain maximum sugar yields from cassava residues, the optimal conditions for hydrolyzing the residues were determined using cellulase prepared from a novel Hypocrea orientalis strain. The optimal pH value and optimal temperature for the cellulase hydrolysis were 5.0 and 50 °C, respectively. The concentration of NaOH was determined to be 1% for pretreatment of cassava residues to gain enough soluble sugars suitably. The yield of released sugars was 10 mg/mL in the optimal conditions after 24 h of reaction, which was similar to that of bagasse and wheat grass. Inhibition kinetics of H. orientalis β-glucosidase (BG) by glucose was first studied using the progress-of-substrate-reaction method as described by Tsou (Tsou, C. L. Adv. Enzymol. Related Areas Mol. Biol. 1988, 61, 381–436), and the microscopic inhibition rate constants of glucose were determined. The results showed that glucose could inhibit BG reversibly and competitively. The rate constants of forward (k+0) and reverse (k–0) reaction were measured to be 4.88 × 10–4 (mM·s)−1 and 2.7 × 10–4 s–1, respectively. Meanwhile, the inhibition was more significant than that of l-glucose, d-mannose, d-galactose, d-aminoglucose, acetyl-d-glucose, and d-fructose. This work reveals how to increase sugar yields and reduce product inhibition during enzymatic saccharification of cellulose.

Proanthocyanidins were isolated from fruit stone of Chinese hawthorn (Crataegus pinnatifida Bge. var. major N.E.Br.). Their structures were analyzed and elucidated by methods of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and high performance liquid chromatography electrospray ionization mass spectrometry (HPLC-ESI-MS). The results demonstrated that these compounds are complicated mixtures of homo- and heteropolymers consisting of procyanidin/procyanidin gallate and prodelphinidin. They possessed structural heterogeneity in monomer units, polymer length, and interflavan linkage (A-type and B-type). Their antityrosinase and antioxidant activity were then investigated. The results revealed that they can inhibit tyrosinase activities, including the monophenolase activity and the diphenolase activity. In addition, proanthocyanidins possessed potent antioxidant activity. Our studies revealed that proanthocyanidins isolated from fruit stone of Chinese hawthorn may be applied in food, agriculture, pharmaceutical, and cosmetic industries.

To provide information on the structure, activity, and structure–activity relationship of kiwifruit (Actinidia chinensis) pericarp proanthocyanidins (PAs), they were separated into three fractions. These fractions were further identified by MALDI-TOF MS and HPLC-ESI-MS methods. Spectra results revealed that they are complex mixtures of B-type propelargonidins, procyanidins, procyanidins gallate, and prodelphinidins. Enzymatic activity analysis showed that these compounds strongly inhibit the activity of tyrosinase, indicating that they are reversible and mixed-type inhibitors of the enzyme. The results obtained from fluorescence quenching showed PAs inhibit the enzyme activity by interacting with substrate and enzyme. This study confirmed that the mean degree of polymerization (mDP) of PAs produces a positive effect on their anti-tyrosinase activity. In addition, the antioxidant analysis indicated that PAs possess potent antioxidant activity. These conclusions mean kiwifruit pericarp PAs may be explored as insecticides, food preservatives, and cosmetic additives.

Bacillus thuringiensis (Bt) strain FJAT-12 was a novel Bt strain isolated by Agricultural Bio-Resources Institute, Fujian Academy of Agricultural Science. In this study, a new cry2Ab gene was cloned from Bt strain FJAT-12 and named as cry2Ab30 by Bt delta-endotoxin Nomenclature Committee. The sequencing results showed there were two mutations in conservative sites which led to two amino acids modification. Homology modeling indicated that the two changes were located in β-sheet of Domain II. A prokaryotic expression vector pET30a-cry2Ab30 was constructed and the expressed protein was analyzed by western blot using Cry2Ab antibody. The expression conditions including IPTG concentration, revolution and temperature were optimized to get the highest expression level by SDS-PAGE and BandScan. The bioassay results also showed that the Cry2Ab30 toxin had high insecticidal activity against Plutella xylostella and the LC50 value was 0.0103 μg.mL−1. The two mutations in β-sheet of Domain II might contribute to insecticidal activity of Cry2Ab30 toxin against Plutella xylostella.

Flamboyant tree, a kind of medicinal plant, was studied as a source of condensed tannins. The antioxidant activities of the condensed tannins from the leaf, fruit, and stem bark of flamboyant tree were screened by ABTS radical and hydroxyl radical scavenging activity methods. The results indicated that these compounds possessed potent antioxidant activity. Their structures were then characterized by high-performance liquid chromatography-electrospray ionization mass spectrometry (HPLC-ESI-MS) after thiolytic degradation. The results showed that the leaf condensed tannins were composed of afzelechin/epiafzelechin, catechin/epicatechin, and gallocatechin/epigallocatechin, while the fruit and stem bark condensed tannins had afzelechin/epiafzelechin and catechin/epicatechin. In addition, the condensed tannins were evaluated for their antityrosinase ability. They were found to have significant antityrosinase activity. The IC50 values were 35 ± 2.0 and 40 ± 0.5 μg/ml for the condensed tannins of fruit and stem bark, respectively. Further, fluorescence quenching and copper interacting techniques were utilized to unravel the molecular mechanisms of the inhibition. The results showed that the hydroxyl group of the condensed tannins could chelate the dicopper center of the enzyme and interact with tryptophan residues. Our studies revealed that condensed tannins might be suitable for use in food, agriculture, cosmetic, nutraceutical, and pharmaceutical applications.

Tyrosinase (EC 1.14.18.1), also known as polyphenol oxidase (PPO), is a key enzyme in pigment biosynthesis of organisms. The inhibitory effects of propyl gallate on the activity of mushroom tyrosinase and effects of propyl gallate on pericarp browning of harvested longan fruits in relation to phenolic metabolism were investigated. The results showed that propyl gallate could potently inhibit diphenolase activity of tyrosinase. The inhibitor concentration leading to 50% activity lost (IC50) was determined to be 0.685 mM. Kinetic analyses showed that propyl gallate was a reversible and mixed type inhibitor on this enzyme. The inhibition constants (KIS and KI) were determined to be 2.135 and 0.661 mM, respectively. Furthermore, the results also showed that propyl gallate treatment inhibited activities of PPO and POD in pericarp of harvested longan fruits, and maintained higher contents of total phenol and flavonoid of longan pericarp. Moreover, propyl gallate treatment also delayed the increases of browning index and browning degree in pericarp of harvested longan fruits. Therefore, application of propyl gallate may be a promising method for inhibiting tyrosinase activity, controlling pericarp browning, and extending shelf life of harvested longan fruits.

In the present study, effects of aqueous extracts from Crocodylus siamensis bile (AE-CB) on SMMC-7721 cell growth, cell cycle, and apoptosis were investigated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay, inverted microscopy, fluorescence microscopy, propidium iodide (PI) single- and fluorescein isothiocyanate (FITC)/PI double-staining flow cytometry, and western blotting. Our data have revealed that AE-CB significantly inhibited the growth of SMMC-7721 cell and arrested cell cycle at G0/G1 phase. SMMC-7721 cells showed typical apoptotic morphological changes after treated with AE-CB for 48 h. Cell death assay indicated that SMMC-7721 cells underwent apoptosis in a dose-dependent manner induced by AE-CB. In addition, AE-CB treatment could downregulate the protein level of Bcl-2 and upregulate the Bax, leading to the increase in the ratio of Bax to Bcl-2 in SMMC-7721 cells. Meanwhile, it was observed that the expression of Survivin and c-Myc decreased, but the expression of P53 increased. All these events were associated with increase of reactive oxygen species. The data indicated that mitochondrial pathway might play an important role in bile extract-induced apoptosis in SMMC-7721 cells. These results provide significant insight into the anticarcinogenic action of bile extract on SMMC-7721 cells.

In this research, the conditions for extraction of phenolics from leaves of Ficus virens were optimized using response surface methodology (RSM). The extraction abilities of phenolics (EAP) and flavonoids (EAF), the 2,2-diphenyl-1-pierylhydrazyl (DPPH) free-radical scavenging potential, and the ferric reducing/antioxidant power (FRAP) were used as quality indicators. The results of single-factor experiments showed that temperature, ethanol concentration, extraction time, and the number of extraction cycles were the main influencing variables, and these provided key information for the central composite design. The results of RSM fitted well to a second degree polynomial model and more than 98% of the variability was explained. The ideal extraction conditions for EAP, EAF, DPPH free-radical scavenging potential, and FRAP were obtained. Considering the four quality indicators overall, the ideal extraction conditions were 58% ethanol at 57 °C for 37 min with three extraction cycles. At the ideal extraction conditions, the values of EAP, EAF, DPPH free-radical scavenging potential, and FRAP were 5.72%, 3.09%, 58.88 mg ascorbic acid equivalent (AAE)/g dry weight (DW), and 15.86 mg AAE/g DW, respectively. In addition, linear correlations were observed between EAP, EAF, and antioxidant potential.

p-Hydroxybenzaldehyde thiosemicarbazone (HBT) and p-methoxybenzaldehyde thiosemicarbazone (MBT) were synthesized and established by 1H NMR and mass spectra. Both compounds were evaluated for their inhibition activities on mushroom tyrosinase and free-cell tyrosinase and melanoma production from B16 mouse melanoma cells. Results showed that both compounds exhibited significant inhibitory effects on the enzyme activities. HBT and MBT decreased the steady state of the monophenolase activity sharply, and the IC50 values were estimated as 0.76 and 7.0 μM, respectively. MBT lengthened the lag time, but HBT could not. HBT and MBT inhibited diphenolase activity dose-dependently, and their IC50 values were estimated as 3.80 and 2.62 μM, respectively. Kinetic analyses showed that inhibition type by both compounds was reversible and their mechanisms were mixed-type. Their inhibition constants were also determined and compared. The research may supply the basis for the development of new food preservatives and cosmetic additives.

The structures of the condensed tannins isolated from leaf, fruit, and stem bark of Delonix regia (Bojer ex Hook.) Raf. have been investigated with 13C nuclear magnetic resonance (13C NMR) and high performance liquid chromatography electrospray ionization mass spectrometry (HPLC-ESI-MS) coupled with thiolysis and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) analyses. The results showed that these condensed tannins from D. regia possessed structural heterogeneity in monomer units and degree of polymerization. Propelargonidin (PP) and procyanidin (PC) were found in the leaf, fruit, and stem bark of D. regia, while prodelphinidin (PD) was found only in the leaves. The polymer chain lengths of condensed tannins from leaf and fruit organs were detected to be trimers to hexadecamers but from trimers to tridecamers for stem bark. B-type linkages were present in all these compounds. Condensed tannins from different parts of D. regia can be explored as tyrosinase inhibitors and food antioxidants because of their potent antityrosinase and antioxidant activities. The inhibitor concentration leading to 50% enzyme activity (IC50) was estimated to be 38 ± 1, 73 ± 2, and 54 ± 1.5 μg/mL for the condensed tannins of leaf, fruit, and stem bark. Condensed tannins extracted from stem bark exhibited the highest antioxidant activity; the DPPH scavenging activity (IC50) and the FRAP values were 90 ± 2 μg/mL and 5.42 ± 0.09 mmol AAE/g, respectively.

4′-Thiosemicarbazonegriseofulvin, a new thiosemicarbazide derivative of griseofulvin, was synthesized and evaluated for its potential in the control of enzymatic browning and postharvest disease of fruits. Browning on fruits is mainly due to the enzymatic oxidation of phenolic compounds catalyzed by tyrosinase. 4′-Thiosemicarbazonegriseofulvin could effectively inhibit the activity of tyrosinase, and its 50% inhibitory concentration (IC50) against tyrosinase was determined to be 37.8 μM. It was a reversible and noncompetitive inhibitor of tyrosinase, and its inhibition constant (KI) was determined to be 38.42 μM. The antifungal activity of 4′-thiosemicarbazonegriseofulvin was studied against four fungi (Fusarium oxysporum, Fusarium moniliforme, Fusarium solani, and Colletotrichum truncatum) that often cause postharvest diseases of fruits. The results showed that 4′-thiosemicarbazonegriseofulvin could also strongly inhibit the mycelial growth of the four target fungi; the 50% lethal concentration (LC50) values were 5.4, 7.0, 15.3, and 1.5 mM, respectively.

Animal bile is popularly used as a traditional medicine in China, and bile acids are their major bioactive constituents. In the present study, effects of bile extract from crocodile gallbladder on QBC939 cell growth, cell cycle, and apoptosis were investigated by MTT assay, inverted microscopy, fluorescence microscopy, transmission electron microscopy, scanning electron microscopy, PI single- and FITC/PI double-staining flow cytometry, and western blotting. Our data have revealed that bile extract inhibited cells growth significantly, and the cell cycle was arrested in G1 phase. Bile extract induced QBC939 cell apoptosis, which was associated with collapse of the mitochondrial membrane potential and increase of ROS. In bile extract-treated cells, it was observed that the expression of bcl-2 decreased and cytochrome c released to cytosol, but the expression of bax remained unchanged. The data indicated that mitochondrial pathway might play an important role in bile extract-induced apoptosis in QBC939 cells. These results provide significant insight into the anticarcinogenic action of bile extract on cholangiocarcinoma cells.

The inhibition kinetics of 2-phenylethanol, 2-phenylacetaldehyde and 2-phenylacetic acid on the enzyme activity of mushroom tyrosinase have been investigated. The results showed that these aromatic compounds can lead to reversible inhibition of the enzyme; furthermore, 2-phenylacetaldehyde and 2-phenylacetic acid are uncompetitive inhibitors and 2-phenylethanol is a mixed-type inhibitor. The inhibition constants have been determined and the inhibiting ability was: 2-phenylacetaldehyde > 2-phenylacetic acid > 2-phenylethanol, indicating that the functional group on the benzene ring group played an important role in the inhibition of the enzyme. In addition, 2-phenylacetic acid and 2-phenylethanol were found to have effective antibacterial activities, and 2-phenylacetic acid was more effective against Escherichia coli and Ralstonia solanacearum than 2-phenylethanol, but 2-phenylacetaldehyde lacked of antibacterial activity.

In order to optimize the cellulase (from Aspergillus glaucus) hydrolysis of pretreated rice straw, the effects of varying enzyme concentration, temperature, and pH were studied. The best experimental conditions found to degrade the pretreated rice straws were 24 h of incubation at 55 °C and pH 5.0, with an enzyme concentration of 48 mg/L. Urea is one of the important nitrogen sources used in fungi culture, but it is also a denaturant. The model of denaturation of endoglucanase (EG) in urea solutions was established. The denaturation was a slow, reversible reaction. Determination of microscopic rate constants showed k+0 > k′+0, indicating that EG was protected by the substrate to a certain extent during denaturation. Comparison with the results from fluorescence emission spectroscopy revealed that the inactivation of EG occurred before the marked conformational changes could be detected.

In insects, tyrosinase plays important roles in normal developmental processes, such as cuticular tanning, scleration, wound healing, production of opsonins, encapsulation and nodule formation for defense against foreign pathogens. Thus, tyrosinase may be regarded as a potential candidate for novel bioinsecticide development. A family of alkyl 3,4-dihydroxybenzoates (C6–C9), new tyrosinsase inhibitors, were synthesized. Their inhibitory effects on the activity of tyrosinase have been investigated. The results showed all of them could inhibit the activity of tyrosianse effectively. The order of potency was nonyl 3,4-dihydroxybenzoate (C9DB) > octyl 3,4-dihydroxybenzoate(C8DB) > heptyl 3,4-dihydroxybenzoate(C7DB) > hexyl 3,4-dihydroxybenzoate (C6DB). The kinetic analysis of these four compounds on tyrosinase was taken to expound their inhibitory mechanism. The research of the control of insects in agriculture was taken as C6DB for example. C6DB could inhibit the development and molting of Plutella xylostella effectively. To clarify its insecticidal mechanism, we researched the expression of tyrosinase in the P. xylostella treated with C6DB by real-time quantitative PCR. The results showed C6DB could inhibit the expression of tyrosinase in the P. xylostella as expected.

2-Chlorobenzaldehyde thiosemicarbazone (2-Cl-BT) and 4-chlorobenzaldehyde thiosemicarbazone (4-Cl-BT) were synthesized, and their inhibitory kinetics on the activity of mushroom tyrosinase were investigated. Results showed that these compounds exhibited significant inhibitory potency on both monophenolase activity and diphenolase activity of tyrosinase. For the monophenolase activity, both compounds could decrease the steady-state activity of the enzyme sharply, without any influence on the lag period. The IC50 values of them were estimated to be 15.4 μM and 6.7 μM, respectively. For the diphenolase activity, both compounds belonged to reversible inhibitors, but their mechanisms were different: 2-Cl-BT was a noncompetitive type inhibitor, while 4-Cl-BT was a mixed-type inhibitor. Their inhibition constants were determined and compared.

An endoglucanase (EG) from Aspergillus glaucus XC9 grown on 0.3% sugar cane bagasse as a carbon source was purified from the culture filtrate using ammonium sulfate, an anion exchange DEAE Sepharose fast flow column, and a Sephadex G-100 column, with a purification fold of 21.5 and a recovery of 22.3%. The ideal time for EG production is on the fourth day at 30 °C using bagasse as a substrate. Results obtained indicate that the enzyme was a monomer protein, and the molecular weight was determined to be 31 kDa. The optimum pH and temperature of EG for the hydrolysis of carboxymethylcellulose sodium (CMC-Na) were pH 4.0 and 50 °C, respectively. EG was stable over the pH range from 3.5 to 7.5 and at temperatures below 55 °C. Kinetic behavior of EG in the hydrolysis of CMC-Na followed Michaelis−Menten kinetics with constant Km of 5.0 mg/mL at pH 4.0 and 50 °C. The enzyme activity was stimulated by Fe2+ and Mn2+ but inhibited by Cd2+, Pb2+, and Cu2+. The EDC chemical modification suggested that at least one carboxyl group probably acted as a proton donor in the enzyme active site.

The effects of betaine on prawn β-N-acetyl-d-glucosaminidase (NAGase) activity for the hydrolysis of p-nitrophenyl-N-acetyl- β-d-glucosaminide (pNP-NAG) have been studied. The results showed that appropriate concentrations of betaine could lead to reversible inhibition against NAGase, and the IC50 value was estimated to be 15.00 ± 0.30 mM. The inhibitory kinetics assay showed that betaine was a mixed type inhibitor with a KI value of 9.17 ± 0.85 mM and a KIS value of 45.58 ± 2.52 mM. The inhibitory model was set, and the microscopic rate constants were determined using the kinetic method of the substrate reaction. The time course of the hydrolysis of pNP-NAG catalyzed by NAGase in the presence of different betaine concentrations showed that at each betaine concentration, the rate decreased with an increase in time until a straight line was approached, indicating that the inhibition of NAGase by betaine is a slow, reversible reaction with fractional residual activity. The fact that k+0 is much larger than k+0′ indicated that the free enzyme molecule is more fragile than the enzyme−substrate complex against betaine. It is suggested that the presence of the substrate offers marked protection of NAGase against inhibition by betaine.

Heavy metal pollution such as chromium and zinc in the seawater has been increasing in recent years in the China Sea. Marine shellfish such as prawn and crab are sensitive to this pollution. β-N-Acetyl-d-glucosaminidase (NAGase, EC 3.2.1.52) catalyzes the cleavage the oligomers of N-acetylglucosamine (NAG) into the monomer. In this study, taking p-nitrophenyl-N-acetyl-β-d-glucosaminide (pNP-NAG) as substrate, the effects of Zn2+ on NAGase from green crab (Scylla serrata) have been studied. The results showed that appropriate concentrations of zinc could lead to reversible inhibition on the enzyme, and the IC50 has been estimated to be 0.5 ± 0.012 mM. Furthermore, it has been shown that Zn2+ could reduce the thermal stability of NAGase depending on the concentration of Zn2+. The inhibitory kinetics of zinc on the enzyme in the appropriate concentrations has been studied using the kinetic method of substrate reaction. The inhibition model has been set up, and the rate constants have been determined. The results showed that Zn2+ was a mixed-type inhibitor of NAGase and that it could combine at the free enzyme and the enzyme−substrate active sites.

Cardol triene was first purified from cashew (Anacardium occidentale L.) nut shell liquid and identified by gas chromatography coupled to mass spectroscopy and nuclear magnetic resonance. The effects of this compound on the activity of mushroom tyrosinase were studied. The results of the kinetic study showed that cardol triene was a potent irreversible competitive inhibitor and the inactivation was of the complexing type. Two molecules of cardol triene could bind to one molecule of tyrosinase and lead to the complete loss of its catalytic activity. The microscopic rate constants were determined for the reaction of cardol triene with the enzyme. The anti-tyrosinase kinetic research of this study provides a comprehensive understanding of inhibitory mechanisms of resorcinolic lipids and is beneficial for the future design of novel tyrosinase inhibitors.

The effects of fatty acids, octanoic acid, (2E, 4E)-hexa-2,4-dienoic acid, hexanoic acid, (2E)-but-2-enoic acid, and butyric acid on the activities of mushroom tyrosinase have been investigated. The results showed that the fatty acids can potently inhibit both monophenolase activity and diphenolase activity of tyrosinase, and that the unsaturated fatty acids exhibited stronger inhibitory effect against tyrosinase than the corresponding saturated fatty acids, and the inhibitory effects were enhanced with the extendability of the fatty acid chain. For the monophenolase activity, the fatty acids could not only lengthen the lag period, but also decrease the steady-state activities. For the diphenolase activity, fatty acids displayed reversible inhibition. Kinetic analyses showed that octanoic acid and hexanoic acid were mixed-type inhibitors and (2E,4E)-hexa-2,4-dienoic acid and (2E)-but-2-enoic acid were noncompetitive inhibitors. The inhibition constants have been determined and compared.

The effects of α-cyano-4-hydroxycinnamic acid (HCCA) on the activity of mushroom tyrosinase have been studied. Results showed that HCCA could inhibit both the monophenolase activity and diphenolase activity of mushroom tyrosinase. For the monophenolase activity, the lag phase was obviously lengthened, and the steady-state activity of the enzyme decreased sharply. When the concentration of HCCA reached to 80 μM, the lag time was lengthened from 20 s to 150 s and the steady-state activity was lost by about 75%. The IC50 value was estimated to be 48 μM. For the diphenolase activity, the inhibitory effect of HCCA was also dose-dependent and the IC50 value was estimated to be 2.17 mM. The kinetic analyses showed that the inhibition of HCCA on the diphenolase activity was reversible and competitive with the inhibition constants (KI) determined to be 1.24 mM.

Tyrosinase (EC 1.14.18.1) is a key enzyme in pigment biosynthesis of organisms. trans-Cinnamaldehyde thiosemicarbazone, a derivative of benzaldehyde thiosemicarbazone, was synthesized as an inhibitor of tyrosinase. The inhibitory effects of this compound on the activity of mushroom tyrosinase were investigated. The results showed that trans-cinnamaldehyde thiosemicarbazone could potently inhibit both monophenolase activity and diphenolase activity of tyrosinase. For monophenolase activity, trans-cinnamaldehyde thiosemicarbazone could not only lengthen the lag time but also decrease the steady-state rate. For diphenolase activity, the IC50 value was determined to be 5.72 μM. Kinetic analyses showed that trans-cinnamaldehyde thiosemicarbazone was a reversible and mixed type inhibitor on this enzyme. The inhibition constants (KI and KIS) were determined to be 4.45 and 8.85 μM, respectively. Furthermore, the antibacterial activity against Bacillus subtilis, Escherichia coli, Staphyloccocus aureus, Pseudomonas aeruginosa, Klebsiella pneumonia, and Agrobacterium tumefaciens was investigated. The results showed that trans-cinnamaldehyde thiosemicarbazone was more effective against B. subtilis and S. aureus with the same minimum inhibitory concentration (MIC) of 50 μg/mL and with the same minimum bactericidal concentration (MBC) of 50 μg/mL.

The control of food browning and growth of disease-causing microorganisms is critical to maintaining the quality and safety of food. Tyrosinase is the key enzyme in food browning. The inhibitory effect of methyl trans-cinnamate on the activity of tyrosinase has been investigated. Methyl trans-cinnamate can strongly inhibit both monophenolase and diphenolase activity of mushroom tyrosinase. When the concentration of methyl trans-cinnamate reached 2.5 mM, the lag time was lengthened from 32 to 160 s and the steady-state activity was lost about 65%. The IC50 value was 1.25 mM. For the diphenolase activity, the inhibition of methyl trans-cinnamate displayed a reversible and noncompetitive mechanism. The IC50 value was 1.62 mM, and the inhibition constant (KI) was determined to be 1.60 mM. Moreover, the antibacterial activity against Escherichia coli, Bacillus subtilis and Staphyloccocus aureus and antifungal activity against Candida albicans were tested. The results showed that methyl trans-cinnamate possessed an antimicrobial ability.

The inhibitory effects of 4-chlorosalicylic acid on the activity of mushroom tyrosinase have been investigated. The results showed that 4-chlorosalicylic acid could strongly inhibit both monophenolase activity and diphenolase activity. The IC50 values were estimated as 1.89 mM and 1.10 mM for monophenolase and diphenolase activities, respectively. For the monophenolase activity, 4-chlorosalicylic acid could not only lengthen the lag time, but also decrease the steady-state rate. For the diphenolase activity, kinetic analyses showed that the inhibition by 4-chlorosalicylic acid was reversible and its mechanism was mixed-II type, which is different from salicylic acid. The inhibition constants (KI and KIS) were determined to be 1.51 mM and 0.82 mM, respectively. Furthermore, the antibacterial activity against Escherichia coli, Bacillus subtilis and Staphyloccocus aureus and antifungal activity against Aspergillus niger and Candida boidinii were investigated. The results showed that 4-chlorosalicylic acid was the most effective against E. coli with the MIC of 250 μg/ml and with the MBC of 500 μg/ml.

Tyrosinase can catalyze the oxidation of o-diphenols to o-quinones. In this paper, some o-diphenols were used as mushroom tyrosinase substrates to study the catalyzed specificity of the enzyme. The enzyme kinetic analysis of substrate specificities and the substrate analogues towards mushroom tyrosinase has been investigated. Taking l-3,4-dihydroxyphenylalanine (I), 3,4-dihydroxyhydrocinnamic acid (II), 3,4-dihydroxycinnamic acid (III) and 1,2,4-benzenetriol (IV) as substrates, the results of specificity studies showed that the oxidation reaction of tested o-diphenols by mushroom tyrosinase followed Michaelis–Menten kinetics. The Michaelis–Menten constants for these four substrates were determined to be 0.615, 1.238, 0.331 and 1.886 mM, respectively. The values of Vm/Km, which denotes the affinity of the enzyme to the substrate, were determined and compared, and the results showed that the affinity of the enzyme to these substrates followed the order: compound IV > III > I > II. Furthermore, mushroom tyrosinase cannot catalyze the oxidation of 3,4-dihydroxybenzonitrile (a), 3,4-dihydroxybenzaldehyde (b), 3,4-dihydroxybenzoic acid (c) and 2,3-dihydroxybenzoic acid (d). On the contrary, compounds a, b and c can inhibit the activity of tyrosinase for the oxidation of DOPA, while compound d had no effects on enzyme activity. The results show that compounds a and b are reversible non-competitive inhibitors.

In the present study the structure of proanthocyanidins from Polyalthia longifolia leaves was characterized with 13C nuclear magnetic resonance, high performance liquid chromatography electrospray ionization mass spectrometry, and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analyses. The results showed that the proanthocyanidins were mixture of homopolymers of B-type procyanidins with degree of polymerization up to 14-mer. Furthermore, the antioxidant activity of the proanthocyanidins was studied through 2,2-diphenyl-1-picrylhydrazyl and 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) free-radical scavenging activities, and ferric reducing/antioxidant power assays. In addition, antityrosinase activity of the proanthocyanidins was investigated. The IC50 for 2,2-diphenyl-1-picrylhydrazyl and 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) free-radical scavenging activity of the proanthocyanidins were 89.32 ± 12.07 and 76.79 ± 5.88 μg/mL, respectively; the ferric reducing/antioxidant power value was 710.54 ± 142.82 mg ascorbic acid equivalent/g dry weight. The IC50 for antityrosinase activity was 773.09 ± 1.47 μg/mL. In conclusion, the proanthocyanidins from P. longifolia leaves exhibited potent antioxidant and antityrosinase activities. This research would provide scientific evidence for the use of proanthocyanidins from P. longifolia leaves as antioxidant and antityrosinase agents.

4-Dimethylaminobenzaldehyde-thiosemicarbazone (DABT) and 4-dimethylaminobenzaldehyde-N-phenyl-thiosemicarbazone (DABPT) were synthesized and established by 1H and 13C NMR and mass spectrum. Both compounds were evaluated for their inhibition activities on mushroom tyrosinase and their anti-tyrosinase kinetics was investigated. The results showed that both compounds exhibited significant inhibitory effects on activity of monophenolase and diphenolase; DABT and DABPT decreased the steady-state rate with 1.54 μM and 1.78 μM as their IC50 values respectively. The inhibitory effects of diphenolase activity exhibited sharp in a dose-dependent manner and their IC50 values were estimated as 2.01 μM and 0.80 μM, respectively. Kinetic analysis showed that their inhibition mechanism was reversible. The inhibition type of DABT was mix-type with inhibition constants KI = 1.77 μM and KIS = 6.49 μM, while that of DABPT displays non-competitive with the inhibition constant KI = 0.77 μM.

•The benzylideneacetone, benzylacetone, and 4-phenyl-2-butanol was firstly used as antityrosinase agents.•The benzylideneacetone, benzylacetone, and 4-phenyl-2-butanol has already been used as food and cosmetic additives. So they have low toxicity.•Benzylideneacetone, benzylacetone, and 4-phenyl-2-butanol of different concentrations were firstly assayed for their effects on the oxidation of L-DOPA catalyzed by mushroom tyrosinase.•These three aromatic compounds' monophenolase inhibition mechanism and inhibition type were firstly justified and their inhibition constants were determined.Tyrosinase (EC 1.14.18.1) is the key enzyme of melanin synthesis and fruit-vegetable browning. The inhibition of benzylideneacetone, benzylacetone, and 4-phenyl-2-butanol on mushroom tyrosinase was first investigated. The results shown that these three compounds could effectively inhibit the enzyme activity sharply and the inhibitory effects were determined to be reversible. Their inhibitor concentrations leading to 50% activity lost values were determined to be 1.5, 2.8, and 1.1 mM for monophenolase and 2.0, 0.6, and 0.8 mM for diphenolase, respectively. For the monophenolase activity, all of these three compounds were mixed-type inhibitors, however, only 4-phenyl-2-butanol obviously lengthened the lag time. For the diphenolase activity, benzylideneacetone and benzylacetone were mixed-type inhibitors, while 4-phenyl-2-butanol was a noncompetitive type inhibitor. In conclusion, these compounds exhibited potent antityrosinase activities. This research would provide scientific evidence for the use of benzylideneacetone, benzylacetone, and 4-phenyl-2-butanol as antityrosinase agents.

Tyrosinase (EC 1.14.18.1) is the key enzyme of most food enzymatic oxidation. Tyrosinase inhibitors are important in food industry. In the present paper, 2-chlorcinnamic acid and 2,4-dichlorocinnamic acid were synthesized and the inhibitory kinetics on mushroom tyrosinase were investigated. The results showed that both compounds synthesized could inhibit tyrosinase activity. For monophenolase activity, both chlorocinnamic acids could extended the lag time and decrease the steady-state activities, 2-chlorcinnamic acid extended the lag time just by 5%, and 2,4-dichlorcinnamic acid extended the lag time more than by 30.4%. For diphenolase activity, the IC50 values of 2-chlorcinnamic acid and 2,4-dichlorocinnamic acid were determined to be 0.765 mM and 0.295 mM, respectively. The inhibition kinetics showed that 2-chlorcinnamic acid and 2,4-dichlorocinnamic acid displayed a reversible and uncompetitive mechanism. The inhibition constants were determined to be 0.348 mM and 0.159 mM, respectively. The research may supply the basis for designing new tyrosinase inhibitors.

•Confirmation of the remarkable anti-tyrosinase activities of cardanols.•Dynamic and quantitative observation on the interaction of tyrosinase and cardanols.•Model and hypothesis development on cardanol structures and their functions.Cashew nut shell liquid (CNSL), extracted from cashew nut shell, is an abundant natural resource. Cardanols were the major phenolic components isolated from CNSL. In this research, we reported on the inhibitory mechanism of cardanols on tyrosinase for the first time. We studied the functions of cardanols and revealed the underlying mechanism of cardanols as tyrosinase inhibitors. Cardanol triene, cardanol diene and cardanol monoene could decrease the steady-state rate of the tyrosinase diphenolase activity efficiently. The IC50 values of three cardanol compounds were determined to be 40.5 ± 3.7, 52.5 ± 3.2 and 56.0 ± 3.6 μM (n = 3), respectively. Meanwhile, the kinetic analysis and the intrinsic/ANS-binding fluoresecence-quenching showed that one cardanol might enter into one tyrosinase. The characteristic values further revealed that cardanols could interact with tyrosinase. Besides, computational study with molecular docking implied that cardanols might affect the amino acid residues of the tyrosinase active site. Collectively, cardanols could moderate inhibitory activities on tyrosinase effectively.Download full-size image

•The total phenolic content of Ficus altissima leaves was determined.•The concentration of extractable and bound condensed tannins was measured.•The structure was characterized by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.•Reversed-phase high-performance liquid chromatography electrospray ionization mass spectrometry was also used.•Condensed tannins exhibited remarkable antioxidant and antityrosinase activity.The total phenolic content and the concentration of extractable and bound condensed tannins of Ficus altissima leaves were determined. The antioxidant activity of crude extracts and condensed tannins of F. altissima leaves was evaluated using 2,2-diphenyl-1-pichydrazyl (DPpH), 2,2-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), and ferric-reducing antioxidant power (FRAP) methods. The results showed that condensed tannins possessed higher free radical scavenging power. The structure of condensed tannins was characterized using high-performance liquid chromatography electrospray ionization mass spectrometry coupled with thiolysis and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The findings showed that condensed tannins from F. altissima leaves were mixtures of procyanidins, prodelphinidins, and propelargonidins with a degree of polymerization up to 30-mer. In addition, condensed tannins effectively protected plasmid DNA against free radical damage and alleviated t-butylhydroperoxide-induced cytotoxicity on human hepatocyte LO2 cells. Condensed tannins exhibited remarkable inhibitory effects on both monophenolase and diphenolase activities of tyrosinase. The IC50 values were estimated to be 256.7 ± 0.3 and 41.3 ± 0.2 μg/mL, respectively. For the diphenolase activity, inhibition by condensed tannins was reversible and of mixed-type. Condensed tannins from leaves of F. altissima were indicated to possess significant antioxidant and antityrosinase activities, suggesting that F. altissima might be a good source of proanthocyanidins with biological activity.Download high-res image (168KB)Download full-size image

Polyphenol oxidase (PPO) is the enzyme responsible for enzymatic browning during the growth of insects. It is also involved in defense reactions and is related with immunities in insects. PPO, a metalloenzyme oxidase, catalyzes the oxidation of o-diphenol to o-quinone. The present paper describes the effects of benzaldehyde and its p-substituted derivatives on the activity of PPO from the fifth instar of Pieris rapae L. PPO from the fifth instar of Pieris rapae L. was purified using ammonium sulfate fractionation and chromatography on Sephadex G-100. The enzyme kinetics was characterized using L-3,4-dihydroxyphenylalanine (L-DOPA) as substrate. The results show that benzaldehyde, p-hydroxybenzaldehyde, p-chlorobenzaldehyde, and p-cyanobenzaldehyde can inhibit the PPO activity for the oxidation of L-DOPA. The inhibitor concentration leading to 50% activity lost, IC50, was estimated to be 5.90, 5.62, 2.83, and 2.91 mmol/L for the four tested inhibitors, respectively. Kinetic analyses show that the inhibitory effects of these compounds are reversible. Benzaldehyde, p-hydroxybenzaldehyde, and p-chlorobenzaldehyde are noncompetitive inhibitors while p-cyanobenzaldehyde is a mixed-type inhibitor. The inhibition constants were determined for all four inhibitors. p-chlorobenzaldehyde and p-cyanobenzaldehyde were more potent inhibitors than the other compounds. These results provide a basis for developing PPO inhibition-based pesticides.

Tyrosinase (EC 1.14.18.1) catalyzes both the hydroxylation of tyrosine into o-diphenols and the oxidation of o-diphenols into o-quinones that form brown or black pigments. In the present paper, cefotaxime, a cephalosporin antibacterial drug, was tested as an inhibitor of tyrosinase. The results show that cefotaxime inhibits both the monophenolase and diphenolase activities of tyrosinase. For the monophenolase activity, cefotaxime increased the lag time and decreased the steady-state activity with an IC50 of 3.2 mM. For the diphenolase activity, the inhibition by cefotaxime is reversible and mix-I type with an IC50 of 0.14 mM. The inhibition constants (KI and KIS) were determined to be 0.14 and 0.36 mM, respectively. The molecular mechanism of inhibition of tyrosinase by cefotaxime was determined by fluorescence quenching and molecular docking. The results demonstrated that cefotaxime was a static quencher of tyrosinase and that cefotaxime could dock favorably in the active site of tyrosinase. This research may offer a lead for designing and synthesizing novel and effective tyrosinase inhibitors in the future.

•We detect aberrant expression proteins during apoptosis of Mz-ChA-1 cell.•The localization of PHB shifted from cytoplasm to the nucleus.•PHB co-localized with AIF, Rb, p53, and c-Fos and the regions of co-localization were altered.•We examine a direct interaction between PHB and both p53 and Rb.In this study, we aimed to investigate the aberrant expression and shift in localization of prohibitin (PHB) during apoptosis of human cholangiocarcinoma cells. Our study demonstrated that PHB was expressed primarily in the cytoplasm and only a little in the nucleus. However, PHB expression significantly decreased, and its localization shifted from the cytoplasm to the nucleus during apoptosis. PHB co-localized with AIF, Rb, p53, and c-Fos, but the region of co-localization was altered after treatment. Meanwhile, we detected a direct interaction between PHB and both p53 and Rb in Mz-ChA-1 cells. These results suggest that the altered localization and expression of PHB, as well as its co-localization with related oncogenes and tumor suppressor genes, can affect the apoptosis of Mz-ChA-1 cells.