Purification and Characterization of Cold-active α-Amylase Excreted by A Strain of Marine Cold-adaptive Penicillia

The filamentous fungi from the Huanghai sea sludge were screened according to their ability to produce cold-active α-amylase. The strain with the highest amylase activity was identified as Penicillium species. The α-amylase purified by ammonium sulphate precipitation and column chromatography on DEAE-sepharose and sephadex G-100 shows a molecular weight of about 55000 and a pI of 4.38. The enzyme is stable in a pH range of 5.5—8.0 and has a maximum activity at pH 6.0. Compared with the α-amylase from mesophiles and thermophiles, the cold-active enzyme shows a high enzyme activity at lower temperatures and a high sensitivity at temperatures higher than 50 ℃. The optimal temperature is 40 ℃ and the activity decreases dramatically at temperatures above 50 ℃. Ca 2+ shows a significant effect on maintaining the structure and the activity of the enzyme. EDTA and Cu 2+ are its inhibitors. The products from the hydrolysis of soluble starch with the cold-active enzyme are maltose and other oligosaccharides.

Comparative analysis of physicochemical properties,ginsenosides content andα-amylase inhibitory effects in white ginseng and red ginsen

Ginseng(Panax ginseng C.A.Meyer)as a common dietary adjunct is widely applied in Traditional Chinese Medicine due to its health-promoting properties,but the differences between white ginseng and red ginseng was rarely studied.In the present study,color parameters and scanning electron microscope(SEM)were determined to evaluate the differences of ginseng color and microstructure induced by processing procedure.Quantitative analysis of multi-components by a single-marker(QAMS)method and anti-α-amylase activity test were used to assess variations of chemical ingredients and pharmacological activity between white and red ginseng.Finally,molecular docking studies were carried out to screen out the most effective compound againstα-amylase.Results indicated that processing had a significant impact on the physicochemical properties and pharmacological activity of white and red ginseng.After processing,the color value of L*declined significantly.Red ginseng sample displayed a compact structure and presented of a gel layer on the surface compared to white ginseng.Additionally,the content of ginsenosides and the activity of anti-α-amylase decreased.The contents of total ginsenosides were positively correlated with the anti-α-amylase activities of ginseng,and ginsenoside Rb1 might be the most effective compound to inhibit the activity ofα-amylase.

Isolation and characterization of glacier VMY22, a novel lytic cold-active bacteriophage of Bacillus cereus

As a unique ecological system with low temperature and low nutrient levels, glaciers are considered a "living fossil" for the research of evolution. In this work, a lytic cold-active bacteriophage designated VMY22 against Bacillus cereus MYB41-22 was isolated from Mingyong Glacier in China, and its characteristics were studied. Electron microscopy revealed that VMY22 has an icosahedral head(59.2 nm in length, 31.9 nm in width) and a tail(43.2 nm in length). Bacteriophage VMY22 was classified as a Podoviridae with an approximate genome size of 18 to 20 kb. A one-step growth curve revealed that the latent and the burst periods were 70 and 70 min, respectively, with an average burst size of 78 bacteriophage particles per infected cell. The pH and thermal stability of bacteriophage VMY22 were also investigated. The maximum stability of the bacteriophage was observed to be at pH 8.0 and it was comparatively stable at p H 5.0–9.0. As VMY22 is a cold-active bacteriophage with low production temperature, its characterization and the relationship between MYB41-22 and Bacillus cereus bacteriophage deserve further study.

In vitro α-amylase inhibitory activity and in vivo hypoglycemic effect of methanol extract of Citrus macroptera Montr.fruit

Objective:To investigate the therapeutic effects of methanol extract of Citrus macroptera Montr,fruit inα-amylase inhibitory activity(in vitro)and hypoglycemic activity in normal and glucose induced hyperglycemic rats(in vivo).Methods:Fruits of Citrus macroptera without rind was extracted with pure methanol following cold extraction and tested for presence of phytochemical constituents,α-amylase inhibitory activity,and hypoglycemic effect in normal rats and glucose induced hyperglycemic rats.Results:Presence of saponin,steroid and terpenoid were identified in the extract.The results showed that fruit extract had moderateα-amylase inhibitory activity[IC_(50)value=(3.638±0.190)mg/mL]as compared to acarbose.Moreover at 500 mg/kg and 1000 mg/kg doses fruit extract significantly(P<0.05 and P<0.01 respectively)reduced fasting blood glucose level in normal rats as compared to glibenclamide(5 mg/kg).In oral glucose tolerance test,500 mg/kg dose significantly reduced blood glucose level(P<0.05)at 2 h but 1000 mg/kg dose significantly reduced blood glucose level at 2 h and 3 h(P<0.05 and P<0.01 respectively)whereas glibenclamide(5 mg/kg)significantly reduced glucose level at every hour after administration.Overall time effect is also considered extremely significant with F value=23.83 and P value=0.0001 in oral glucose tolerance test.Conclusion:These findings suggest that the plant may be a potential source for the development of new oral hypoglycemic agent.

Comparisons of phaseolin type and α-amylase inhibitor in common bean(Phaseolus vulgaris L.)in China

The objective of this study was to characterize the phaseolin type and a-amylase(αAI) level in common bean(Phaseolus vidgaris L.) accessions deposited in the Chinese National Genebank.The 40 accessions sampled were common varieties originating in Asia,North America,South America,Europe,and Africa.No Inca(I-) phaseolin was observed in the accessions.Only four accessions contained Tendergreen(T-) phaseolin and the remaining36 contained Sanilac(S-) phaseolin.aAI proteins extracted from nine accessions showed higher a-amylase inhibitory activity than the control(Phase 2,IC_(50) = 0.65 μg).These common bean accessions have potential use as nutraceutical ingredients.

Relationship between malt qualities and β-amylase activity and protein content as affected by timing of nitrogen fertilizer application

The effects of different timing of N fertilizer application at the same rate on grain β-amylase activity, protein con- centration, weight and malt quality of barley were studied. Grain β-amylase activity and protein concentration were significantly higher in treatments where all top-dressed N fertilizer was applied at booting stage only or equally applied at two-leaf stage and booting stage than in the treatment where all top-dressed N fertilizer was applied at two-leaf age stage only. On the other hand, grain weight and malt extract decreased with increased N application at booting stage. There were obvious differences between barley varieties and experimental years in the grain and malt quality response to the timing of N fertilizer application. It was found that grain protein concentration was significantly and positively correlated with β-amylase activity, but significantly and nega- tively correlated with malt extract and Kolbach index. The effect of grain protein concentration on malt quality was predominant over the effect of grain β-amylase activity.

Topographies of Organized Monolayer of α-Amylase Observed by Atomic Force Microscopy

In this paper, (-amylase organized monolayer was assembled on the surface of the PET-CO2- substrate in different conditions. The different topography of the (-amylase/PET monolayer was obtained by AFM in tapping mode.

Cloning and heterologous expression of pro-2127,a gene encoding cold-active protease from Pseudoalteromonas sp.QI-1

The psychrotropic bacterium, Pseudoalteromonas sp. QI-I, which produces extracellular cold-active protease, was isolated from Antarctic seawater. The genomic DNA of this bacterium was used to construct a plasmid genomic library with the goal of screening cold-active protease genes. Gene pro-2127 with an open reading frame of 2127 bp encoding protease PRO-2127 was cloned and sequenced. Alignment of amino acid sequences suggested that the precursor of PRO-2127 was a member of subfamily S8A, and that it might contain four domains： a signal peptide, an N-terminal prosequence, a catalytic domain and a C-terminal extension. Amino acids Asp185, His244 and Ser425 might form a catalytic triad. PRO-2127 showed some structural features common to psychrophilic enzymes, such as a decrease in Arg residues and the Arg/（Arg＋Lys） ratio. Heterologous expression of pro-2127 in Escherichia coli BL21 （DE3） by pColdlII was also successfully observed in this study.

The Dynamics of Changes in Starch and Lipid Droplets and Sub-Cellular Localization of β-Amylase During the Growth of Lily Bulbs

The ultra-structure of mother and outer daughter scales of Lilium Oriental hybrid Sorbonne were studied using transmission electron microscope to examine the sub-cellular localization of starch and lipid droplets during growth and development from shoot emergence to senescence.The contents of starch granules and lipid droplets in the cell of the mother scales decreased significantly from shoot emergence to anthesis,indicating that these scales served as a source for growth and development.After flowering,the number of starch granules and lipid droplets increased dramatically,and finally the cells were filled with the above molecules indicating that the bulb becomes a major sink during bulb enlargement.Ultrastructure observation also showed that symplastic pathway is the main pathway in cells in the exchange and transportation of material during bulb development.The activity of β-amylase,one of the key enzymes catalyzing starch breakdown,showed a similar trend.The enzyme sub-cellular localization via immune-gold electron-microscopy showed that βamylase was predominantly located together with starch granules,while the gold particles were scarcely found in other sub-cellular compartments.The result suggested that this enzyme is compartmented together with its functional substrate supporting its function in catalyzing starch breakdown in living plant cells.

Efficiency of Oryza punctata extract on glucose regulation: Inhibition of α-amylase and α-glucosidase activities

Red rice(Oryza punctata) is a type of unpolished rice which has higher nutritional value compared to white rice or even polished rice. Owing to higher nutritive content and metabolites, dieticians strongly advise red rice for peoples with metabolic disorders including diabetics. However, the mechanism of action and contents of secondary metabolites in Indian red rice variety not reported scientifically. Therefore, the present study aimed to evaluate its mechanism of action through inhibitory effect of α-amylase and α-glucosidase. Initially, the whole grain of red rice was macerated with methanol at room temperature for 2 weeks. Then, the dried and powdered, samples at different concentrationfi(2.5, 10, 40, and 80 μg/m L) were employed to nd out in vitro inhibitory effects on α-amylase and α-glucosidase. In addition, an enzyme kinetics of effective extract was calculated by Line-weaver Burk(LWB) plot analysis. Moreover,the valuable metabolites in the efficient methanolic extract were quantified using high performance liquid chromatography(HPLC). The results demonstrated that red rice methanolic extract(RRMEt) possess strong inhibitory activity onα-amylase and α-glucosidase compared with acarbose(P < 0.01). The IC50 values of RRMEt was found to be 29.7 ±7.43 μg/m L for α-amylase and 20.4 ± 0.25 μg/m L for α-glucosidase. LWB indicated that RRMEt is an uncompetitive inhibitor. Further, HPLC analysis revealed protocatechuic acid, catechin, and chlorogenic acids were more abundant in RRMEt among the fourteen metabolites. We conclude, the efficiency of enzyme inhibition through the influence of phenolic compounds in RRMEt.

Synthesis and Characterization of Zinc Oxide and Zinc Oxide Doped with Chlorine Nanoparticles as Novel <i>α</i>-Amylase Inhibitors

In this study we used a chemical solution method from oxalic acid (OX. acid) and zinc acetate (ZnAc) to prepare Zinc Oxide nanoparticles (ZnONPs) and Zinc Oxide nanoparticles doped with Chlorine (Cl:ZnONPs). The characterizations (FTIR, X-ray, SEM, TEM) of ZnONPs and Cl:ZnONPs were determined. Amylase inhibitors of ZnONPs and Cl:ZnONPs also were determined. SEM indicated that the ZnONPs and Cl:ZnONPs have an average particle size of 46.65 - 74.64 nm. TEM images of the ZnONPs and Cl:ZnONPs showed the round shaped. Compounds b, d and e exhibited significant inhibitory activity against amylase enzyme (from 69.21 ± 1.44 to 76.32 ± 0.78), respectively, and were comparable with that of acarbose (86.32 ± 0.63) at 1000 μg, thereby, projecting ZnONPs and Cl:ZnONPs as α-amylase inhibitors.

Inhibition of α-amylase and α-glucosidase activities by ethanolic extract of Telfairia occidentalis (fluted pumpkin) leaf

Objective:To investigate the inhibitory effect of Telfairia occidentalis Hook f.(Curcubitaceae)(T.occidentalis)leaf on key enzyme linked to type-2 diabetes(α-amylase andα-glucosidase)as well as assess the effect of blanching(a commonly practiced food processing technique)of the vegetable on these key enzymes.Methods:Fresh leaves of T.occidentalis were blanched in hot water for 10 minutes,and the extracts of both the fresh and blanched vegetables were prepared and used for subsequent analysis.The inhibitory effect of the extract onα-amylase andα-glucosidase activities as well as some antioxidant parameter was determined in vitro.Results:The result revealed that unprocessed T.occidentalis leaf reduce Fe^(3+)to Fe^(2+)and also inhibitedα-amylase andα-glucosidase activities in a dose dependent manner.However,blanching of the leafy vegetables caused a significant(P<0.05)increase in the antioxidant properties but decrease their ability to inhibitα-amylase andα-glucosidase activities.Conclusions:This antioxidant properties and enzyme inhibition could be part of the mechanism by which they are used in the treatment/prevention of type-2 diabetes.However,the blanched vegetable reduces their ability to inhibit bothα-amylase andα-glucosidase activity in vitro.

Molecular Cloning and Characterization of a New Cold-active Extradiol Dioxygenase from a Metagenomic Library Derived from Polychlorinated Biphenyl-contaminated Soil

To find new extradiol dioxygenases（EDOs,EC 1.13.11.2）,a metagenomics library was constructed from polychlorinated biphenyl-contaminated soil and was screened for some dioxygenase with aromatic ring cleavage activity.A novel EDO,designated as BphC_A,was identified and heterologously expressed in Escherichia coli.The deduced amino acid sequence of BphC_A exhibited a homology of less than 60% with other known EDOs.Phylogenetic analysis of BphC_A suggests that the protein is a novel member of the EDO family.The enzyme exhibits higher substrate affinity and catalytic efficiency toward 3-methylcatechol than toward 2,3-dihydroxybiphenyl or catechol,the preferred substrate of other known EDOs.The optimum activity of purified BphC_A occurred at pH=8.5 and 35 °C,and BphC_A showed more than 40% of its initial activity at 5 °C.The activity of purified BphC_A was significantly induced by Mn^2＋ and slightly reduced by Al^3＋,Cu^2＋ and Zn^2＋.

The relationship between psychological distress with salivary <i>α</i>-amylase and stress related symptoms among breast cancer survivors

Background and Aims: This study examined the stress levels, the stressors and biomarker such as Alpha (α)-Amylase enzyme which is secreted under distress conditions. The aim was to determine the relationship between these three variables. Methods: The study respondents were made up of cancer outpatients from 3 hospitals namely the Hospital Kuala Lumpur, Seberang Jaya and Johor Baru, Pantai Ipoh and Putrajaya. The Personal Stress Inventory (PSI) questionnaire was used to identify stressors, while the Hospital Anxiety and Depression Scale (HADS) were used to determine the psychological distress levels. The Salimentary Oral Swab (SOS) Technique was used to collect the saliva and then the Salivary α-Amylase Assay Kit was used to analyse for α amylase. Results: The majority of respondents were stressed due to their sensitive emotion and nervousness in their daily lives (68.7%), they had poor memory and short attention spans of carrying out job tasks (67.3%) as well as they were emotionally depressed (65.3%). Their poor memory and short attention span (p = 0.037), heavy work load and poor task delivery (p = 0.008) were predictors for distress using HADS. The salivary α-amylase concentration was significantly related to the stress levels (p = 0.002). Conclusion: Breast cancer respondents with musculoskeletal system related symptoms, with heavy work load and poor task delivery, as well as had poor memory and short attention span were at higher risk of experiencing psychological distress. The salivary α-amylase which had significant relationships with psychological distress was therefore, a potential biological indicator for distress, while the musculoskeletal system related symptoms from the PSI questionnaire were predictors for distress.

Salivary alpha-amylase activity—An indicator of relaxation response in perioperative patients

The psychological stress of most perioperative patients has been reduced by many interventions. In order to evaluate the effects of these interventions, biomarkers are being considered as objective assessment tools in addition to the subjective assessment tools. It has been thought that salivary α-amylase (sAA) activity is associated with sympathetic nervous system activity, which reflects psychological stress. We examined the relationship between sAA, self-reported data, and autonomic nervous system activity to assess the reliability of sAA as a marker for relaxing response in surgery-related stress. The sAA, the State anxiety, Visual Analogue Scale (VAS) of pain, and heart rate variability were used to assess prior to and after the relaxation on the day before surgery, and on postoperative days (POD) 1, 2, 3, and 7. A total of 75 patients participated and 301 relaxing response readings were obtained. We found that the sAA significantly decreased depending upon the pain and anxiety by relaxation, but they were unrelated to sympathetic nervous system activity. There was weak correlation between the change in sAA, and anxiety and VAS pain. Thus, sAA appears to be an easy-to-use, non-invasive and good marker for relaxing response within a short period in surgery-related stress patients.

Kinetics of medium-temperature α-amylase hydrolyzed <i>Huai yam</i>powder

In order to learn the enzymatic characteristics of Huai yam powder with medium-temperatureα-amylase, effects of substrate concentration,enzyme concentration, pH and temperature wereinvestigated. The Michealis-Menten equationwas used to fit the kinetics of the hydrolysis reaction. Experimental results indicate that maximum rate (Vm) is 3.1588 mg/mL·min under the condition of 70&#176;C, pH 7.0 and 0.0200 mg/mL of enzyme concentration. The Michealis constant (Km) is 6.6641 mg/mL. The kinetic model, including the factors such as substrate concentration, enzyme concentration and temperature, was established for the hydrolysis reaction under the temperature range from 40&#176;C - 70&#176;C.

Introducing New Peptide Extracts from Saccharomyces cerevisiae and Achatina achatina Fluids with Strong Inhibitory Activities on Human α-Amylase

This study aimed at exploring for new natural peptides with strong inhibitory capabilities on α-amylase, the main metabolic enzyme that regulates mellitus diabetes, in order to contribute in controlling this global pandemic. It has consisted in heat shock (to 60&deg;C, 70&deg;C, 80&deg;C, 90&deg;C and 100&deg;C for 10, 20 and 30 minutes) of crude proteins extracted from biomass and extracellular parts of Saccharomyces cerevisiae under cultivation, and from the digestive fluid of the giant snail Achatina achatina, and in-vitro assays of the resulting solutions, as effectors, in human α-amylase catalyzing reactions. The results showed that whatever the temperature and time of treatment, an increase (from 2.65 to 3.98-fold) in proteins concentration was noticed. When blended up to 75 microliters in reaction mixtures, the three peptide extracts showed beyond 11% of inhibition of initial α-amylase activity. By reducing samples volume, only 5 microliters of the studied peptide extracts representing 4.70 μg of S. cerevisiae biomass peptides, 0.55 μg of S. cerevisiae extracellular peptides or 1.05 μg of peptides from the digestive fluid A. achatina were quite sufficient to induce complete (100%) inhibition of the human α-amylase activity. Compared to the inhibitory effect obtained from 2.50 μg of acarbose, a renowned antidiabetic, the studied peptide effectors showed more pronounced inhibitory activities. So, we can positively state that S. cerevisiae as well as A. achatina are both capable of synthesizing proteins made up of small inhibitory peptides which deserve purification and structural analysis for potential exploitation as healthy antidiabetic drugs.

An Optimization Study of α-Amylase Production by Aspergillus niger ATCC 16404 Grown on Orange Waste Powder

In this work, sequential optimization strategy, based on statistical designs, was employed to enhance the production of α-amylase by Aspergillus niger ATCC 16404. This statistical study consists of optimizing the factors that influence the production of α-amylase of A. niger ATCC 16404. Indeed, another statistical study has allowed the selection of 5 factors (pH, starch, yeast extract, “corn steep liquor”, CaCl2 and salts) affecting both the development of mould (biomass) and that of the enzyme production. The central composite design allows the determination of the optimum of these selected factors and a quadratic model explains the factor reaction. Thus, the “ridge analysis” method, has led to maximizing the experimental reaction. The results indicate that the production rate of α-amylase is maximized in the presence of starch at 8.97 g/l, yeast extract at 2.86 g/l, CaCl2 at 1.224 g/l, salts (composed of 25% FeSO4, 7H2O, 25% MnSO4 and 50% MgCl2, 6H2O): FeSO4, 7H2O, MnSO4 0.1518 g/l and MgCl2, 6H2O at 0.3036 g/l. As for the pH, it is maintained at the rate of 5.68.

In silico study of wine anthocyanins and their polymeric pigments on human pancreatic α-amylase and salivary α-amylase

Polyphenols,such as procyanidins and anthocyanins,as naturalα-amylase inhibitors,have been extremely studied about their inhibitory activity in vitro.In this work,molecular docking was used to explore the efficacy of wine anthocyanins and their polymeric pigments to inhibit human pancreaticα-amylase(HPA)and salivaryα-amylase(HSA).Three residues,ASP197,GLU233 and ASP300 were proposed as main interacting residues with both HPA and HSA.Hydrogen bonds,π-πstacking,hydrophobic interaction and electrostatic interaction played important roles in binding.Polymeric pigments showed stronger affinity with HPA and might be a potentialα-amylase inhibitor.Moreover,according to the docking result of HSA,polymeric pigments exhibited more impact on astringency than anthocyanins.

Distance-basedα-amylase biosensor fabricated with amylopectin-coated mesoporous membrane

Paper-based biosensors are widely employed in point-of-care testing(POCT)due to their convenience,portability,low cost,and ease of use.This study reports an integrated distance-based paper biosensor fabricated with a mesoporous membrane coated with stimuli-responsive polymer.The detection ofα-amylase(AMY)using amylopectin-coated mesoporous membrane is demonstrated as an example.After introducing the AMY solution,it is observed that the aqueous solution flows along the paper strip due to AMY-catalyzed hydrolysis of amylopectin.The flow distance is proportional to the concentration of AMY with a detection limit as low as 4 mU/mL.In addition,the detection of AMY is demonstrated in human serum.Furthermore,the inhibitory effect of acarbose on AMY is evaluated.This reagent-free and disposable biosensor allows single-step rapid detection of the analyte.This approach is very promising for the development of user-friendly,equipment-free,and cost-effective biosensors with remarkable sensitivity and excellent selectivity for disease diagnosis and hypoglycemic drug screening.