Effects of Different Nitrogen Application Methods on Enzyme Activities in Leaves at Late Growth Stage of Spring Maize

[Objective] This paper aimed at exploring the effects of different nitrogen application methods on the enzyme activities of leaves at ear position in late growth stage of maize. [Method] By pot experiment, the superoxide dismutase （SOD） activity, peroxidase （POD） activity, malonyl dialdehyde （MDA） content and soluble protein content were determined. [Result] At the filling stage and ripening stage, with the increasing of nitrogen application rate, MDA content gradually decreased, while SOD activity, POD activity and soluble protein content increased. MDA content with two topdressing nitrogen was lower than that with one top dressing nitrogen at the same nitrogen application rate, while SOD activity, POD activity and soluble protein content with two topdressing nitrogen were higher than that with one topdressing nitrogen. [Conclusion] Different nitrogen application methods have relatively significant effects on the MDA content, SOD activity, POD activity and soluble protein content, which is of certain directive significance for preventing spring maize prematuration.

Extraction of Pumpkin Polysaccharide by Complex Enzyme Method and Its Antioxidant Research

[Objective] This paper aims to study a new method of extracting pumpkin polysaccharide from pumpkin. Single factor experiments were conducted to examine the effects of extracting time,temperature,the solid-liquid ratio and pH value on the extraction yield of polysaccharide from pumpkin. [Method] The best enzyme ratio and extraction conditions for complex enzymes extraction were determined through orthogonal tests. Scavenging ·OH and O-2 activities of pumpkin polysaccharides were also investigated by salicylic acid and improved self-oxidation of o-pheno methods respectively. [Results] The results showed that the biggest extraction yield of polysaccharide from pumpkin can be got when adding 1% cellulose enzyme,1.5% pectinase,1.0% papain and Na2HPO4-citric acid buffer solution (pH was 4.6),and oscillating for 30 min under water at 40 ℃ with the solid-liquid ratio of 1:30. In addition,pumpkin polysaccharides had a strong activity of eliminating ·OH,but very weak activity to scavenge O-2. [Conclusion] This study provided basic data for research and application of Pumpkin polysaccharide.

Enzyme Inhibition Rate Method for Rapid Detection of Organophosphorus and Carbamate Pesticides in Cowpea

[Objectives ] The paper was to explore enzyme inhibition rate method for rapid detection of organophosphorus and carbamate pesticides in cowpea. [ Methods ] Acetylcholinesterase （ACHE） was added to cowpea extract, to determine the inhibition rate of extract against enzyme. The influences of different sampiing methods and sampling parts on detection results were compared. [ Results] The positive rate of standard sampling was 18.18% higher than that of non-stand- ard sampling, and the positive rate of samples collected from cowpea tail was 16.67% higher than that collected from other parts. [ Condmions] Enzyme inhibi- tion rate method is suitable for rapid detection of organophosphorus and carbamate pesticides in cowpea.

HOMOGENEOUS ENZYME IMMUNOASSAY OF SERUM AFP BY FLUORIMETRIC KINETIC METHOD

The activity of the Ab-bound enzyme(HRP)changed after immunochemical reaction,and can be indicated by a sensitive fluorimetric kinetic method.The finding set the stage for developing sensitive homogeneous immunoassay.AFP was measured as an example.

Study on Process Conditions of Preparation of Microporous Potatoes Starch by Complex Enzyme Method

[Objective] The technology of using c-amylase and glucoamylase to prepare microporeus potato starch was studied.[ Method ] Taking potato starch as raw materials, starch hydrolysis rate and the oil absorption as a measure of index, the influences of the reaction temperature, two enzymes proportion, the quantity of enzyme, the chroma of substrate, buffer solution pH and reaction time on microporous potato starch were inves- tigated. [ Result] The experimental results showed that the best technological conditions were reaction temperature 45 ℃, enzyme ratio （ glucoamy- lase/α-amylase）6, the quantity of enzyme （amount of enzyme and starch quality than） is 1.0%, the substrate quantity chroma of 0.14 g/ml, buffer solution pH 4, the reaction time 8 h. In such process condition, the oil adsorption rate of hydrolyzed potato starch was as high as 70.2%, starch hydrolytic ratio was 34.16%. [ Condmion] The study provided a basis for the development and utilization of microporous starch. Key words Microporous starch; Hydrolysis rate; Oil absorption rate; Preparation; Complex enzyme method; China

A NOVEL POLYMER ENZYME LINKED IMMUNOASSAY METHOD AND ITS APPLICATIONS

During the course of study, we found that both poly (N-isopropylacrylamide ) (PNIP) and PNIP-Ab (enzyme-labelled antibody)could be adhere tightly to a cellulose acetale-nitrate membrane, and that the retention of PNIP-Ab on the membrane increased over 30-fold when compared with the unconjugated Ab.Thus we used this characteristic to develop a novel immunoassay method-polymer enzyme linked immunoassay method: homogeneous antigen-antibody reaction and heterogeneous separation process. When applied for detection of human serum HBsAg, this immunoassay system can detect as little as 1 ng/ml of human serum HBsAg.

Studies on Reduced Extent Method for Inhibited Thermokinetics Of Enzyme－Catalyzed Reaction──Inhibition of the laccase－catalyzed oxidation of o－dihydroxybenzene by m－dihydroxybenzene

The thermokinetic reduced extent equations of reversible inhibitions for Michaiels-Menten enzymatic reaction were deduced, and then the criteria for distingushing inhibition type was given and the methods for calculating kinetic parameters, KM,Ki and Urn were suggested. This theory was applied to inverstigate the inhibited thermokinetics of laccase-catalyzed oxidation of o-dihydroxybenzene by m-dihydroxybenzene. The experimental results show the inhibition belongs to reversible competitive type, KM=6.224×10-3 mol L-1, Ki=2. 363 × 10-2 mol. L-1.

Enzyme extraction by ultrasound from sludge flocs

Enzymes play essential roles in the biological processes of sludge treatment. In this article, the ultrasound method to extract enzymes from sludge flocs was presented. Results showed that using ultrasound method at 20 kHz could extract more types of enzymes than that at 40 kHz and ethylenediamine tetraacetic acid （EDTA） methods. The optimum parameters of ultrasound extraction at 20 kHz were duration of 10 min and intensity of 552 W/g TSS. Under the optimum condition, ultrasound could break the cells and extract both the extracellular and a small part of intercellular enzymes. Ultrasound intensity was apparently more susceptive to enzyme extraction than duration, suggesting that the control of intensity during ultrasound extraction was more important than that of duration. The Pearson correlation analysis between enzyme activities and cation contents revealed that the different types of enzymes had distinct cation binding characteristics.

Analytical Solutions of System of Non-Linear Differential Equations in the Single-Enzyme, Single-Substrate Reaction with Non-Mechanism-Based Enzyme Inactivation

A closed form of an analytical expression of concentration in the single-enzyme, single-substrate system for the full range of enzyme activities has been derived. The time dependent analytical solution for substrate, enzyme-substrate complex and product concentrations are presented by solving system of non-linear differential equation. We employ He’s Homotopy perturbation method to solve the coupled non-linear differential equations containing a non-linear term related to basic enzymatic reaction. The time dependent simple analytical expressions for substrate, enzyme-substrate and free enzyme concentrations have been derived in terms of dimensionless reaction diffusion parameters ε, λ1, λ2 and λ3 using perturbation method. The numerical solution of the problem is also reported using SCILAB software program. The analytical results are compared with our numerical results. An excellent agreement with simulation data is noted. The obtained results are valid for the whole solution domain.

Analytical Expression for the Concentration of Substrate and Product in Immobilized Enzyme System in Biofuel/Biosensor

In this paper, an approximate analytical method to solve the non-linear differential equations in an immobilized enzyme film is presented. Analytical expressions for concentrations of substrate and product have been derived for all values of dimensionless parameter. Dimensionless numbers that can be used to study the effects of enzyme loading, enzymatic gel thickness, and oxidation/ reduction kinetics at the electrode in biosensor/biofuel cell performance were identified. Using the dimensionless numbers identified in this paper, and the plots representing the effects of these dimensionless numbers on concentrations and current in biosensor/biofuel cell are discussed. Analytical results are compared with simulation results and satisfactory agreement is noted.

Iterative approximate solutions of kinetic equations for reversible enzyme reactions

We study kinetic models of reversible enzyme reactions and compare two techniques for analytic approximate solutions of the model. Analytic approximate solutions of non-linear reaction equations for reversible enzyme reactions are calculated using the Homotopy Perturbation Method (HPM) and the Simple Iteration Method (SIM). The results of the approximations are similar. The Matlab programs are included in appendices.

Analytical expressions of the concentrations of substrate and product in enzyme inhibition process

The initial and boundary value problem in enzyme reactions mechanism for inhabitation process is discussed. Approximate analytical expressions for the concentrations of substrate and product are presented. Approximate analytical solutions of non-linear reaction equations containing non-linear terms related to enzymatic reaction mechanism are solved using Homotopy perturbation method. The relevant analytical expression for the substrate and product concentration profiles is discussed in terms of dimensionless reaction diffusion parameters α, β, γE, and γS. Numerical solution is also obtained using Matlab program. Our analytical expression compared with numerical estimation and good agreement is noted.

Theoretical Analysis of Immobilized Oxidase Enzyme Electrode in the Presence of Two Oxidants

In this paper, mathematical model of Martens and Hall (Analytical chemistry 66, 2763-2770 (1994)) for an immobilized oxidase enzyme electrode is discussed. The model involves the system of non-linear reaction diffusion equations under the steady state conditions. A simple and closed-form of approximate analytical expressions for the concentrations of the immobilization of three enzyme substrates has been derived by solving the system of non-linear reaction diffusion equations using new approach of homotopy perturbation method. Approximate polynomial expression of concentration of substrate, oxygen and oxidized mediator and current was obtained in terms of the Thiele moduli and the small values of parameters Bs, Bo and Bm (normalized surface concentration of substrate, oxygen and oxidized mediator). Furthermore, in this work the numerical simulation of the problem is also reported using Matlab program. An agreement between analytical expressions and numerical results is noted.

酸法和酶法提取对中华草龟皮胶原蛋白结构和理化特性的影响

本研究以中华草龟皮胶原蛋白为研究对象,探究不同提取方法对其结构和理化特性的影响。分别采用酸法和酶法对中华草龟皮胶原蛋白进行提取,并运用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳、高效液相色谱法、圆二色光谱法、差式扫描量热、扫描电镜、溶解性、感官评价及色度等分析酸溶性胶原蛋白(ASC)和酶溶性胶原蛋白(PSC)的组成、结构以及理化特性。结果表明,ASC和PSC的提取率分别为24.50%±0.44%和51.68%±0.06%,酶法提取胶原蛋白的提取率显著高于酸法(P<0.05)。ASC和PSC主要由α1和α2链组成,均为I型胶原蛋白。ASC和PSC中甘氨酸的含量最多,分别是370.17和387.75个残基/1000个氨基酸残基。圆二色光谱表明这两种胶原蛋白二级结构相似,均具有胶原蛋白的特征峰。ASC和PSC的变性温度分别为27.21±0.28℃和28.37±0.23℃。扫描电镜结果表明,ASC具有无序的纤维结构,而PSC的微观结构更为规则。ASC的溶解度整体略低于PSC,PSC的感官品质和色度优于ASC。

Measurement of mouse liver glutathione S-transferase activity by the integrated method

Objective: The integrated method was investigated to measure Vm/Km of mouse liver glutathione S-transfer-ase (GST) activity on GSH and 7-Cl-4-nitrobenzofurazozan. Methods: Presetting concentration of one substrate twenty-fold above the other's and taking maximum product absorbance Am as parameter while Km as constant, Vm/Km was obtained by nonlinear fitting of GST reaction curve to the integrated Michaelis-Menten equation In [Am/(Am -Ai)] + Ai/ ( ξ× Km ) = ( Vm/Km )×ti (1). Results: Vm/Km for GST showed slight dependence on initial substrate concentration and data range, but it was resistant to background absorbance, error in reaction origin and small deviation in presetting Km. Vm/Km was proportional to the amount of GST with upper limit higher than that by initial rate. There was close correlation between Vm/Km and initial rate of the same GST. Consistent results were obtained by this integrated method and classical initial rate method for the measurement of mouse liver GST. Conclusion: With the concentration of one substrate twenty-fold above the other's, this integrated method was reliable to measure the activity of enzyme on two substrates , and substrate concentration of the lower one close to its apparent Km was able to be used.

Antibiotic-Resistant Bacterial Group in Field Soil Evaluated by a Newly Developed Method Based on Restriction Fragment Length Polymorphism Analysis

Spreading of antibiotic resistant bacteria into environment is becoming a major public health problem, implicating affair of the indirect transmission of antibiotic resistant bacteria to human through drinking water, or vegetables, or daily products. Until now, the risk of nosocomial infection of antibiotic resistant bacteria has mainly been evaluated using clinical isolates by phenotypic method. To evaluate a risk of community-acquired infection of antibiotic resistant bacteria, a new method has been developed based on PCR-RFLP without isolation. By comparing restriction fragment lengths of the 16S rDNA gene from bacterial mixture grown under antibiotic treatment to those simulated from the DNA sequence, bacterial taxonomies were elucidated using the method of Okuda and Watanabe [1] [2]. In this study, taxonomies of polymyxin B resistant bacteria group in field soils, paddy field with organic manure and upland field without organic manure were estimated without isolation. In the both field soils, the major bacteria grown under the antibiotic were B. cereus group, which had natural resistance to this antibiotic. In field applied with organic manure, Prevotella spp., and the other Cytophagales, which were suggested to be of feces origin and to acquire resistance to the antibiotic, were detected. When numbers of each bacterial group were roughly estimated by the most probable number method, B. cereus group was enumerated to be 3.30 × 106 MPN/g dry soil in paddy field soil and 1.32 × 106 MPN/g dry soil in upland filed. Prevotella spp. and the other Cytophagales in paddy field were enumerated to be 1.31 × 106 MPN, and 1.07 × 106 MPN·g-1 dry soil.

Sampling Small Volumes of Saliva for Determination of the Stress Hormone α-Amylase: A Comparative Methodological Study

Two sampling devices that allow saliva collection through absorption to a cotton roll (Salivette?-method) or to small cotton pellets (VectaSpinTM Micro [VSM]-method) were studied. Any loss of salivary alpha-amylase (sAA) activity in relation to the saliva volume absorbed and harvested by centrifugation was examined. A pooled saliva sample prepared from stimulated whole saliva (collected by drooling) of 30 subjects was used. Three different saliva volumes (2.9 ml, 1.5 ml, and 0.8 ml) were tested on cotton rolls and two (0.03 ml, and 0.015 ml) on cotton pellets. The sample sAA activity was determined from the hydrolysis of 2-chloro-4-nitrophenyl-α-D-maltotrioside. In comparison with the original drooling sample, no sAA loss was observed in 1.5 ml samples tested with Salivette, while a significant decrease of activity was recorded with smaller volumes. VSM collected samples showed a non-volume dependent decrease of sAA activity of about 25%. Salivette requires large saliva volumes to allow an accurate sAA estimation. With cases of limited saliva access, VSM may be a suitable sampling device.

Reliable Method for Steady-State Concentrations and Current over the Diagnostic Biosensor Transducers

A mathematical modelling of diagnostic biosensors system at three basic types of enzyme kinetics is discussed in the presence of diffusion. Enzyme kinetics is adopted to be first order, Michaelis-Menten and ping-pong mechanism. In this paper, approximate analytical solutions are obtained for the non-linear equations under steady-state conditions by using the new Homotopy perturbation method. Simple and closed forms of analytical expressions for concentrations of substrate, product and co-substrate and corresponding current response have been derived for all possible values of parameters. Furthermore, the numerical simulation of the problem is also reported here by using Matlab program. Good agreement between analytical and numerical results is noted.

Bacterial Groups Concerned with Maturing Process in Manure Production Analyzed by a Method Based on Restriction Fragment Length Polymorphism Analysis

Composting is a biological aerobic decomposition process consisted from different phases. Although the Japanese Standards for manure recommended that it took at least 6 months to complete the maturing phase, there was no reliable ground. In order to find out shortening method of the maturing phase, the microorganisms concerned with a progress of the maturing was determined by using the most probable number method (MPN) and PCR-RFLP of the 16S rDNA, which was found effective to provide numbers and taxonomy of polymyxin B resistant bacterial groups in the former paper [1]. Compared to the numbers after thermophilic phase, those of Actinobacteria, δ-proteobacteria, and the other gram negative bacteria increased to 50 times, 20 times, and 105 times, respectively, after maturing phase, while those of Bacillus spp., and α and β-proteobacteria decreased to 1/10, and 1/105 after maturing phase. Numbers of the other Fumicutes, and γ-proteobacteria remained in the same revel. Actinobacteria, δ-proteobacteria, and the other gram negative bacteria might be concerned with a progress of the maturing phase, because these bacterial groups were detected and enumerated due to their proliferation ability. Although number of Acitinobacteria might be underestimated because of a PCR bias, the method was found effective for the purpose to monitor bacteria actively proliferated in culture medium.

Evaluation of the Method Based on Restriction Fragment Length Polymorphism Analysis as Simple Analysis Method of Lactic Acid Bacteria in Foods

Lactic acid bacteria have not only been used to produce various kinds of fermented food, but also used as probiotic products. As lactic acid bacterial group was consisted from diverse genera, a simple inspection method by which numbers and contained microorganisms could be automatically analyzed without any preliminary information was required to use them more effectively. In this manuscript, lactic acid bacterial groups in commercial products of kimuchi, komekouji-miso, and yoghurt were identified and enumerated by our newly developed method [1]-[3], to evaluate whether the method could be used as an inspection method of various food samples. In kimuchi, numerically dominant bacteria were Lactobacillus sakei, and L. casei (1.4 × 104 MPN g-1) and Leuconostoc spp. (l.4 × 104 MPN). In kouji-miso, numerically dominant bacteria was Bacillus spp. (3 × 103 MPN), which mainly included B. subtilis group and B. cereus group. Lactic acid bacteria such as Lactobacillus spp., or Lactococcus spp., included in the komekouji-miso, could be enumerated after 3 days incubation (1.24 × 104 MPN), but not detected after 7 days incubation. In yoghurt A and C, Lactococcus lactis was detected as numerically dominant lactic acid bacteria (3.0 × 105 MPN). In yoghurt B, Lactobacillus spp., or Lactococcus spp., was detected not only by a culturebased method but also by an unculture-based method, although there was a difference between the both estimated numbers. The present results suggested that the method might become useful as a simple inspection method of food microorganisms, because time and labor of the analysis could be reduced by using an unculture-based method and MCE-202 MultiNA. In this study, Bifidobacteriium spp. was not detected in B and C yoghurt, in spite of indicating their existence, and numbers of lactic acid bacteria were lower than the level of the daily product regulation, because 16S rDNA of Bifidobacteriium spp. might not be amplified by the used PCR condition. The PCR condition must be changed so as to amplify Bifidobacterium spp., before the method will be used as an inspection method for lactic acid bacteria.