电场强化淀粉酶解反应超滤膜分离一体化的研究(三)

以淀粉水解为研究对象 ,研究电场强度作用下 ,相关因素 ,如压力差、基质浓度、反应物温度等对酶膜反应器透过通量的影响 .结果表明 ,在其他因素对透过通量作用的基础上 。

发芽小麦α-淀粉酶活性的研究

通过对在不同发芽温度和不同发芽时间下培养的芽麦粉中α 淀粉酶活性、降落值、还原糖及非还原糖含量的测定 ,研究了发芽小麦的α 淀粉酶活性、降落值、还原糖及非原糖含量与芽麦的发芽温度和发芽时间的关系。并通过响应面分析法 ,进行中心组合设计 ,得出了它们的二元二次回归方程。并对植酸作为α 淀粉酶抑制剂的抑制效果进行了研究 .通过测定不同植酸添加量下的α 淀粉酶活性及降落值 ,表明植酸对α 淀粉酶的抑制效果十分明显 。

Chemical Constituents of Pu-erh Tea and Its Inhibition Effect on α-amylase in vitro

[Objective]The aim was to clarify the chemical substance basis of hypoglycemic and lipid-lowering effects of Pu-erh Tea. [Method]Pu-erh Tea was extracted with 95% ethanol,followed by petroleum ether,chloroform,ethyl acetate and n-butanol extraction,after the further purification and through the NKA-9 macroporous resin and many times of Sephadex column chromatography,two compounds were isolated,in the same time,the effect of Uracil and Gallic acid on α-amylase was studied. [Result]The Uracil and Gallic acid were isolated and identified respectively from Pu-erh Tea and the Uracil was firstly isolated from Pu-erh Tea; Gallic acid had strong inhibition on α-amylase. [Conclusion]It could provide some theories on the hypoglycemic and lipid-lowering effects of Pu-erh Tea.

Analysis of α-amylase Inhibitor Content Change in Pu-erh Tea During Pile-fermentation Process

The study was to investigate the changes of α-amylase inhibitor content in Pu-erh tea during pile-fermentation process. Pu-erh tea samples from two regions of Shuangjiang County and Jinggu Dai and Yi Autonomous County of Yunnan Province at various fermentation stages were used as experimental materials to investigate the effect of different fermentation stages on the inhibitory effect to α-amylase; and the change law of the inhibitory effect of c-amylase inhibitor during processing was meanwhile studied by determining the contents of tea polyphenol and amino acid. The results showed that crude meterial of Pu-erh tea presented strong inhibitory effect to α-amylase; this inhibitory effect assumed a de： creasing trend to the minimum at the middle stage of fermentation, whereafter it increased to some extent. Made tea also showed a strong inhibitory effect to α-amylase. During whole processing period, contents of tea polyphenol and amino acid generally assumed a remarkably decreasing trend. Our results provided references for further isolating co-amylase inhibitor from Pu-erh tea and discussing the mechanism of its health care function.

Activities, Quantitative Changes and Subcellular Localization of α-Amylase During Development of Apple Fruit

Starch degradation in cells is closely associated with cereal seed germination, photosynthesis in leaves, carbohydrate storage in tuberous roots, and fleshy fruit development. α_Amylase is considered as one of the key enzymes catalyzing starch breakdown, but up to date its role in starch breakdown in living cells remains unclear because the enzyme was often shown extrachloroplastic in living cells. The present experiment showed that α_amylase activity was progressively increasing concomitantly with the decreasing starch concentrations during the development of apple ( Malus domestica Borkh cv. Starkrimson) fruit. The apparent amount of α_amylase assessed by Western blotting also increased during the fruit development, which is consistent with the seasonal changes in the enzyme activity. The enzyme subcellular_localization studies via immunogold electron_ microscopy technique showed that α_amylase visualized by gold particles was predominantly located in plastids, but the gold particles were scarcely found in other subcellular compartments. A high density of the enzyme was observed at the periphery of starch granules during the middle and late developmental stages. These data proved that the enzyme is compartmented in its functional sites in the living cells of the fruit. The predominantly plastid_distributed pattern of α_amylase in cells was shown unchanged throughout the fruit development. The density of gold particles (α_amylase) in plastids was increasing during the fruit development, which is consistent with the results of Western blotting. So it is considered that α_amylase is involved in starch hydrolysis in plastids of the fruit cells.

Optimization of Fermentation Conditions of Acid Resistant α-amylase Producing Strain

[Objective] The aim was to optimize the fermentation conditions of acid resistant α-amylase producing strain. [Method] Based on the selection of an acid resistant α-amylase producing strain,the fermentation conditions including C,N contents and initial pH of culture medium,seed age,inoculum size,rotation speed of shake flask and fermentation temperature were optimized. [Result] The optimum fermentation conditions for acid resistant α-amylase producing strain were：seed age 14 h,inoculum size 8%,initial pH 5.5,fermentation temperature 35 ℃,rotation speed 150 r/min,the volume of inoculum broth 25 ml,C content 1.0% and N content 0.5%. [Conclusion] Under the optimum fermentation conditions,α-amylase activity reached 31.4 U/ml,which was 65.3 % higher than that before optimization.

Studies on the Isolation and Screening of α-amylase Producing Strain and Their Enzymatic Properties

[Objective] The aim was to obtain α-amylase producing strains with some excellent properties like high temperature resistance,strong acid resistance,strong alkali resistance,etc..[Method] α-amylase producing strains were isolated and screened; furthermore their enzymatic properties were studied.[Result] 10 strains with an obvious starch hydrolysis cycle were screened out from starch screening plate coated by diluted sample,from which 3 strains with higher α-amylase activity were screened out,that was X6,X8 and X10.As for X6,X8 and X10,their optimum pH values all belonged to neutral,and their optimum temperatures were all 60 ℃.Meanwhile,Ca^2＋ could increase their enzyme thermal stability.When the concentration of Ca^2＋ was 0.02-0.04 mol/L,the enzyme thermal stability of X6 and X8 reached the highest; When the concentration of Ca^2＋ was 0.03-0.04 mol/L,that of X10 reached the highest; When the concentration of Ca^2＋ was increased continuously,those of the 3 strains all decreased.[Conclusion] The research provides theoretical basis for satisfying the demands of different industries for α-amylase with different characteristics.

Germination and Expression of α-Amylase of KF0680-1 and KF0680-2(Cultivars of Sweet Sorghum) under Salt Stress

[Objective] The aim was to study on germination and expression of α-amy- lase of KF0680-1 and KF0680-2 （cultivars of sweet sorghum） under salt stress. [Method] In the research, KF0680-1 and KF0680-2, cultivars of sweet sorghum, were used to measure related indices of germination and expression of co-amylase to discuss effect of salt stress on two cultivars. [Result] Germination of sweet sorghum would be promoted if treated with salt in low concentration and inhibited if treated in high concentration. In the latter condition, roots and seedlings were smaller than that of control group in length, but expression of s-amylase in the two cultivars was pro- moted under proper salt stress. [Conclusion] The research indicates that KF0680-1 and KF0680-2 are inhibited in growth by salt stress and the latter is stronger in salt resis- tance than the former under low salt concentration. In addition, expression of α-amy- lase could be promoted by salt in proper volume. The results provide references for selection of sweet sorghum which could be planted in northwestern areas.

Effects of Ionic Surfactants on Bacterial Luciferase and α-Amylase

In order to study the effects of ionic surfactants on bacterial luciferase,the cationic surfactant dodecyltrimethylammonium biomide (DTAB) and anionic surfactant sodium dodecylsulfate (SDS) were chosen.For comparison with bacterial luciferase,α-amylase was used since these two enzymes have similar electrostatic potential and charged active sites.After the enzymes were treated with the surfactants,the catalytic properties of bacterial luciferase andα-amylase were assayed,and fluorescence spectroscopy and circular dichroism (CD) were used to analyze the alteration of the protein structure.The results showed that when the DTAB concentration was low,the cationic surfactant DTAB enhanced the enzymatic activities of bacterial luciferase andα-amylase.On the other hand,the anionic surfactant SDS did not alter the enzymatic activity.The main interaction of cationic surfactant DTAB and the negatively charged surface of the proteins was the ionic interaction,which could alter the environment for the enzyme to work when the DTAB/enzyme molar ratio was low.However,at high cationic surfactant concentration,the ionic interaction and hydrophobic interaction might destroy the secondary and tertiary structures of the proteins,leading to the loss of enzymatic activities.

Effect of Static Magnetic Field on α-Amylase Activity and Enzymatic Reaction

The effect of magnetic field on a-amylase was studied. Under the experimental conditions, a-amylase solution was treated by 0.15 T, 0.30 T and 0.45 T static magnetic fields for a known period of time, then the activity, kinetic parameters, and the secondary conformation were investigated. The results showed that there was a considerable effect of the magnetic exposure on the α-amylase. The activity was increased by 27%, 34.1%, 37.8% compared with the control. It was also found that both kinetic parameters Km and Vm could be decreased due to the increasing magnetic field, Km decreased from 2.20×10^2 to 0.87×10^2, whereas Vm decreased from 2.0×10^3 g/min to 1.1 ×10^3 g/min. At the same time, there were some irregular changes in a-amylase secondary conformation.

Bienzyme system immobilized in biomimetic silica for application in antifouling coatings

Antifouling coatings are used extensively on vessels and underwater structures. Conventional antifouling coatings contain toxic biocides and heavy metals, which may induce unwanted adverse effects such as toxicity to non-target organisms, imposex in gastropods and increased multiresistance among bacteria. Therefore,enzyme-based coatings could be a new alternative solution. A H2O2-producing bienzyme system was developed in this study. H2O2 can be produced from starch by the cooperation of α-amylase and glucose oxidase, which promotes the hydrolysis of polymeric chain and oxidizes the glucose to produce H2O2, respectively. The encapsulated bienzyme(A-G@BS) exhibits enhanced stabilities of thermal, pH, recycling and tolerance of xylene. The A-G@BScontaining coating releases H2O2 at rates exceeding a target of 36 nmol·cm-2·d-1for 90 days in a laboratory assay. The results demonstrate that the method is a promising coating technology for entrapping active enzymes,presenting an interesting avenue for enzyme-based antifouling solutions.

Kinetic Parameter of Alfa-amylase and Glucoamylase Enzymatic Reaction on the Glucose Yield from Hydrolyzed Processes of Tapioca Solid Waste

The increasing of tapioca production nowadays effected the production of waste. The waste of tapioca industries consists of two kinds, which were liquid waste and solid waste. Further more, tapioca solid waste treatment was ineffective. Weather solid waste produced from the extraction process still contains high concentration of starch that can be used to produce high quality product, for example, bio ethanol or other alternative energy sources. Objective of these experimental work was utilizing solid waste of tapioca industries and looking for the exactly composition of n-amylase and gluco-amylase enzymes on the hydrolysis processes of the solid waste of tapioca. The exact composition from both enzymes can be expected to increase the yield of glucose. Variables of cx-amylase enzyme for this research were 0.3% （w/w） and 0.5% （w/w） with liquefaction time were 1 hour and 1.5 hours, and variables of glucoamylase enzyme were 0.3% （w/w） and 0.5% （w/w）. To achieve these goals, the experimental work was held in laboratory scale with batch process. Firstly, tapioca solid waste was pretreated at 90 ~C and added u-amylase enzyme for 1 hour and 1.5 hours （variable of liquefaction time）. Then, substrate was cooled down to 60 ~C added with proposed variables of glucoamylase enzyme, and was analysed 24 hours after added. This experiment showed the best ratio between a-amylase and glucoamylase enzymes 0.5%：0.5% with 1 hour of liquefaction time. The highest glucose reaches 8.468% and yields 0.892 （g glucose/g starch） with starch conversion of 59.94%. KM = 0.0468 g/mL and rmax = 0.311 g/mL·h,

Study on chemical constituents and biological activities of Sanghuangporus vaninii under static magnetic field

The chemical composition and biological activity of Sanghuangporus vaninii solid culture treated by static magnetic field were studied.The compounds were isolated and purified using ultrasonic extraction and semi-preparative liquid chromatography.Their structures were identified through spectral data,and the inhibitory activities againstα-amylase,α-glucosidase and pancreatic lipase were evaluated.Three compounds,5,7-dihydroxy-3,4'-dimethoxyflavone(1),D-(+)-Trehalose(2),pinolenic acid(3),were extracted from the petroleum ether layer.Comparison of peak heights indicated a significantly higher content of compounds subjected to a 4 mT static magnetic field compared to those untreated.Activity tests revealed that compounds 1 and 2 exhibited strong anti-α-amylase and anti-α-glucosidase activities.Specifically,compound 1 had inhibition rates of(65.37±0.05)%and(73.81±0.12)%after 4 mT treatment,compared to(57.26±0.11)%and(65.33±0.14)%without.Compound 2 showed inhibition rates of(68.61±0.12)%and(65.38±0.09)%with the magnetic field,versus(60.71±0.06)%and(56.18±0.02)%without.Compound 3 displayed a notable inhibitory effect on pancreatic lipase,with rates of(60.83±0.03)%after 4 mT treatment and(53.77±0.09)%without.This study demonstrates that the static magnetic field enhances the chemical content and bioactivity of Sanghuangporus vaninii solid culture,providing a basis for its further development and application.

Two alkaloids as α-amylase inhibitors: enzyme kinetics and molecular modeling investigations

In the present study, we studied the inhibitory effects of chelidonine and rutaecarpin on porcine pancreatic a-amylase （PPA） catalyzed hydrolysis using 2-chloro-4-nitrophenyl-4-O-β-D-galactopyranosylmaltoside （Gal-G2-α-CNP）. We, for the first time, provided kinetic report and detailed inhibitory effects of both compounds on PPA. Lineweaver-Burk plot revealed that the inhibition was a mixed-noncompetitive type, and only one molecule of inhibitor bound to the enzyme or to the enzyme-substrate complex. Kinetic constants calculated from secondary plots were in millimole range. Dissociation constants of enzyme-inhibitor complex （KEI） were 0.9 mM and 3.5 mM, respectively. Moreover, dissociation constants of enzyme-inhibitor-substrate complex （KESI） were 0.04 mM and 0.31 mM, respectively. These data indicated that the inhibition was more inclined to competitive to Gal-G2-α-CNP hydrolysis. Further molecular docking study manifested that hydrogen bonding formed between acarbose and aspartic acid （Asp300）, histidine （His305） and glycine （Gly3-6）, while hydrogen bonding was observed between chelidonine and glutamic acid （Glu233）, lysine （Lys200） and His305. In addition, rutaecarpine had only one hydrogen bond with Lys200. Our data indicated that chelidonine and rutaecarpine were two promising drug candidates, and chelidonine possessed stronger inhibitory effect compared with rutaecarpine.

Initial exploration of the mechanism underlying H2O2-induced root horizontal bending in pea

It was reported that exogenous hydrogen peroxide （H2O2） can induce primary root bend in Arabidopsis and pea. However, the mechanism remains unclear. Here we explored the mechanism underlying this phenomenon by using the pea （Pisum sativum L.） variety ＂longwan No. 1＂ The results showed that the endogenous indole-3-acetic acid （IAA） content decreased and gibberellin A3 （GA3） content increased in the curving primary pea root induced by H2O2. Meanwhile, both of the two hormones asymmetrically distributed in the inside and outside parts of the curving root. Also, the starch content decreased due to the increased a-amylase activity in this process. However, exogenous Ca2＋ can relieve the horizontal bending of pea root induced by H2O2 and altered the contents of endogenous IAA and GA3. A working model was proposed： Exogenous H2O2 causes the increase in GA3 content, and GA3 stimulates the activity of or-amylase, which leads to the hydrolysis of starch, and then the root lost the gravity perceiving. The asymmetric distribution of IAA and GA3 in two sides of curving root may cause the horizontal bending.Exogenous Ca^2＋ can relieve root bending through altering the endogenous IAA and GA3 contents.

Influences of AMY1 gene copy number and protein expression on salivary alpha-amylase activity before and after citric acid stimulation in splenic asthenia children

OBjECTIVE:To compare the correlations between salivary alpha-amylase(sAA) activity and amylase,alpha 1(salivary) gene(AMY1) copy number or its gene expression between splenic asthenia and healthy children,and investigate the reasons of attenuated sAA activity ratio before and after citric acid stimulation in splenic asthenia children.METHODS:Saliva samples from 20 splenic asthenia children and 29 healthy children were collected before and after citric acid stimulation.AMY1 copy number,sAA activity,and total sAA and glycosylated sAA contents were determined,and their correlations were analyzed.RESULTS:Although splenic asthenia and healthy children had no differences in AMY1 copy number,splenic asthenia children had positive correlations between AMY1 copy number and sAA activity before or after citric acid stimulation.Splenic asthenia children had a higher sAA glycosylated proportion ratio and glycosylated sAA content ratio,while their total sAA content ratio and sAA activity ratio were lower compared with healthy children.The glycosylated sAA content ratio was higher than the total sAA content ratio in both groups.Splenic asthenia and healthy children had positive correlations between total sAA or glycosylated sAA content and sAA activity.However,the role played by glycosylated sAA content in sAA activity in healthy children increased after citric acid stimulation,while it decreased in splenic asthenia children.CONCLUSION:Genetic factors like AMY1 copy number variations,and more importantly,sAA glycosylation abnormalities leading to attenuated sAA activity after citric acid stimulation,which were the main reasons of the attenuated sAA activity ratio in splenic asthenia children compared with healthy children.