Immobilization of β-amylase from Bacillus megaterium B6 into gelatin film by cross-linking

β-Amylase (EC 3.2.1.2) from Bacillus megaterium B₆ was immobilized in gelatin by covalent cross-linking with organic hardeners, formaldehyde and gluteraldehyde, of which gluteraldehyde was proved to be better. The effect of temperature, pH, incubation time, substrate concentration, different surfactants and thiol inhibitors on immobilized β-amylase had been investigated. Reuse efficiency and cross-linker concentration was also studied. The data indicated that enhanced resistance to thermal and chemical denaturation, increased temperature optima (from 60° to 65°C) and rapid rate of substrate saturation were achieved after immobilization. The gelatin-bound enzyme could also be reutilized for repeated batch experiments.     

A Gelatin Film Method for Improved Histochemical Localization of Dehydrogenases in Plant Cells

Glucose-6-phosphate and 6-phosphogluconate dehydrogenases diffused from frozen sections of Vicia faba embryos during histochemical incubation. In the liquid incubation medium, the dehydrogenases catalysed the oxidation of substrate and reduction of NADP. NADPH₂ thus formed could lead to artifactual deposition of formazan in frozen sections. The addition of 20% polyvinyl alcohol to the incubation medium was found unsatisfactory in preventing this loss which appeared to be overcome by incorporating the reaction mixture into a gelatin film. Equal volumes of 10% gelatin solution in 0.05 M phosphate buffer at pH 7.8, and the enzyme reaction medium containing twice the normal concentration of substrate (0.014 M), of 0.007 M pyridine nucleotide, of 0.02 M KCN and of 0.0024 M NBT in the buffer, were mixed and layered onto polyethylene, and allowed to set in the dark at room temperature for 30-60 min. The solidified medium and its support were cut into strips and layed onto unfixed, frozen sections of plant tissues which were incubated at 20 C. Evidence is presented to support the supposition that the enzymes are retained in the sections during the reaction.     

The relationship of oocyte diameter and incubation temperature to incubation time in temperate freshwater fish species

Based on the analysis of six egg variables and incubation temperature of 65 temperate freshwater fish species, the possible relationships between oocyte diameter, incubation time and incubation temperature were reassessed and compared to the results obtained from marine fishes. Most freshwater species have eggs (mean ± s . d . 2·19 ± 1·52 mm) larger than marine species, that are chiefly demersal and develop stuck to various substrata, such as plants or rocks. A strong negative relationship was found between incubation time ( t , days) and incubation temperature ( T , ° C): t = 186·23e^{−0·197 T} ( r ²= 0·87). A strong dependence of incubation time on oocyte diameter ( Ø , mm) and incubation temperature was also found and was defined as: log₁₀ t = 3·002 + 0·599 log₁₀ Ø − 1·91 log₁₀ ( T + 2), which explained 92% of the variance of the data set. Five major groups of species were defined based on the principal component analysis (PCA) of four quantitative variables. There were two distinct groups of salmonids, displaying demersal and non-adhesive eggs with a long incubation time at low temperature, the eggs of which required a high number of degree-days. There was a large group of species possessing small, mostly demersal and adhesive eggs developing at high temperature during a short period of time, and requiring a low number of degree-days. Between these two extremes, there was a fourth group displaying intermediate values and a fifth group including three species with large, adhesive and demersal eggs incubating at high temperatures during a short period of time. The burbot Lota lota displayed an unusual combination of variables compared to the remaining species in the data set.     

An evaluation of the use of 4-methylumbelliferyl-β-D-glucuronide (MUG) in different solid media for the detection and enumeration of Escherichia coli in foods

The use of 4-methylumbelliferyl-β- D -glucuronide (MUG) in different solid media for the detection and enumeration of Escherichia coli in foods was evaluated by testing the effects of different substrate concentrations (50 or 100 μg ml⁻¹), incubation temperatures (37 or 41·5°C) and incubation times (8, 12, 24 and 48 h). Different kinds of foods, both naturally and artificially contaminated, were analysed. The use of selective media without differential substances and an incubation time of 24 h seem to be worthy of recommendation. In this case an incubation temperature of 37°C would be preferred and the MUG concentration could be reduced to 50 μg ml⁻¹. Incubation times shorter than 24 h, which may cause a loss of sensitivity, require higher incubation temperatures (41·5°C) and MUG concentration (100 μg ml⁻¹).     

Phase equilibria and gelation in gelatin/maltodextrin systems — Part III: phase separation in mixed gels

Co-gels of potato maltodextrin Paselli SA-6 with gelatin were prepared by rapid quenching of mixed solutions from 90°C. At fixed setting temperature and fixed concentration of gelatin, the time required to form a self-supporting network showed an initial steady decrease with increasing concentration of SA-6 (as expected from polymer exclusion), but then increased dramatically before again decreasing. The interpretation of this behaviour as phase inversion from a gelatin-continuous network with SA-6 inclusions to a (more slowly-forming) SA-6 network with gelatin inclusions was confirmed by differential scanning calorimetry (showing both components melting separately, with no evidence of specific interaction), mechanical spectroscopy (showing that the mixed gel network was destroyed completely by melting of the gelatin component at low concentrations of SA-6, but only weakened at SA-6 concentrations above the inversion point) and by light microscopy (showing the expected changes in distribution of the two polymers).

In similar studies using the faster-gelling potato maltodextrin Paselli SA-2, microscopy and gel-melting profiles again showed phase-inversion from a gelatin-continuous network at low concentrations of SA-2 to a maltodextrin-continuous network at higher concentrations. Inversion, however, occurred at a lower concentration of maltodextrin than in the gelatin/SA-6 systems, and the accompanying change in gelation rate was confined to a sharp decrease in the dependence of gel-time on SA-2 concentration.     

Urease-gelatin interdigitated microelectrodes for the conductometric determination of protease activity

Conductometric microbiosensors for the determination of trypsin were elaborated via the modification of microfabricated interdigitated gold electrodes by a cross-linked urease/BSA coating covered by a gelatin film. The resulting microelectrodes were exposed to different trypsin concentrations ranging from 100pg/mL to 1mg/mL (1mU/mL to 10,000U/mL) for selective proteolytic degradation of the gelatin film. Then, the conductometric response of the microbiosensors to urea (33muM) was recorded as a function of the trypsin concentration, the gelatin amount (8-80ng) and the incubation time (40s, 100min). The optimum incubation time for each trypsin concentration was determined leading to a detection limit of 100pg/mL (1mU/mL). In these optimized conditions, the proof of concept of this sensitive, disposable, low-cost and label-free trypsin biosensors based on a conductometric transducer was demonstrated for the first time.     

The effect of incubation temperature on steady-state concentrations of hydrogen and volatile fatty acids during anaerobic degradation in slurries from wetland sediments

Abstract Increasing the incubation temperature of two swamp slurries from 2°C to37°C resulted in a 8- to 18-fold increase in the H₂ partial pressure. The concentration of volatile fatty acids remained fairly constant except for butyrate, which decreased with increasing temperature. Calculation of Gibbs free energies of syntrophic degradation of butyrate and propionate, and of methanogenesis from acetate and H₂ revealed that these reactions were exergonic after the slurries had stabilized at the incubation temperatures. The changes in H₂ partial pressure and butyrate concentration with temperature were found important to render the processes exergonic within the tested temperature range.     

Phase equilibria and gelation in gelatin/maltodextrin systems — Part II: polymer incompatibility in solution

The effect of thermodynamic incompatibility in mixed solutions of gelatin and Paselli maltodextrins SA-6 and SA-2 has been studied at a temperature (45°C) where the individual polymers are stable as disordered coils. Concentrated mixtures of SA-6 and gelatin showed classic phase separation into two co-existing liquid layers, with compositions lying along a well-defined binodal. On decreasing SA-6 concentration below the binodal, however, a substantial proportion (up to 60%) of the maltodextrin was precipitated, with normal single-phase solutions occurring only at much lower concentrations of both polymers. SA-2 showed a more extreme version of the same behaviour, with precipitation of up to 100% of the maltodextrin and no evidence of co-existing liquid phases at any accessible concentrations. In both cases, the amount of maltodextrin precipitated was proportional to the square of its initial concentration and to the first power of gelatin concentration, indicating that gelatin drives self-association and aggregation of maltodextrin when both polymers are present in a single liquid phase. ¹H NMR showed the precipitated maltodextrin to be higher in molecular weight and in degree of branching than the material remaining in solution, and particlesize analysis indicated that the volume of the individual maltodextrin particles increased linearly with the total mass precipitated.     

Phase separation and gel formation in kinetically trapped gelatin/maltodextrin gels

The kinetics of phase separation and gel formation of gelatin/maltodextrin mixtures have been studied using confocal laser scanning microscopy (CLSM), transmission electron microscopy (TEM), stereological image analysis and rheology. The quantified microstructural parameters were the volume-weighted mean volume and the interfacial area. The temperature of phase separation was defined as the temperature where the first signs of phase separation were observed in CLSM. The gelatin concentration varied between 4 (wt.) and 5% and the maltodextrin concentration varied between 2 and 6%. Maltodextrin was labelled covalently with RITC to improve the contrast between the gelatin phase and the maltodextrin phase. The solutions were cooled from 60 to 10 degrees C, and the cooling rates used were 0.4, 1 and 3 degrees C/min. All systems were found to be gelatin continuous under the experimental conditions used. The results showed that the temperature of phase separation (TPS) increased both with the gelatin concentration and the maltodextrin concentration, but particularly with the former. The size of the maltodextrin inclusions increased with TPS, and the interfacial area decreased with increasing TPS. The diameter of the maltodextrin inclusions varied between 1.6 and 8.5 microm at 1 degrees C/min. The size of the maltodextrin inclusions was found to increase with decreasing cooling rate and was largest at 0.4 degrees C/min. The TPS was compared with the gelation temperature (Tgel) at three different concentrations of gelatin and maltodextrin (4/3, 4/5, 5/5%). CLSM micrographs and TEM micrographs showed that these three concentrations of gelatin and maltodextrin had different microstructures. At a TPS above Tgel (5/5%), the phase separation proceeded faster than the gel formation and the microstructure had few, large maltodextrin inclusions and a clean continuous gelatin phase. At a TPS comparable with Tgel (4/5%), phase separation occurred during gel formation, which led to a varying microstructure and competition between the phase separation and the gel formation. At a TPS below Tgel (4/3%), gel formation proceeded faster than the phase separation and the microstructure had many, small inclusions and a diffuse microstructure, and the phase separation was incomplete. It was established that the microstructure was determined by the relative rates of the phase separation and the gel formation. Three different zones of phase separation could be distinguished based on comparisons of TPS and Tgel, and results from CLSM, TEM and image analysis.     

黄土高原不同植被带人工刺槐林土壤氮磷转化酶动力学参数及其温度敏感性

土壤酶是有机质降解的催化剂,其动力学特征是表征酶催化性能的重要指标,对评价土壤健康质量有重要作用。本研究选择黄土高原3种植被带下人工刺槐林土壤为对象,探讨了土壤酶动力学参数对温度变化的响应及其温度敏感性(Q₁₀)的变化特征。结果表明: 随着培养温度的升高,土壤丙氨酸转氨酶、亮氨酸氨基肽酶和碱性磷酸酶的潜在最大反应速率(V_max)和半饱和常数(K_m)均呈线性增加,且V_max呈现出森林带>森林草原带>草原带的地带性规律。V_max的温度敏感性(Q10(V_max))为1.14～1.62,K_m的温度敏感性(Q_10(Km))为1.05～1.47,且两者在森林草原带的值均低于其他植被带。在低、高温区,不同土壤酶的Q₁₀在各植被带间的变化也不尽相同。冗余分析显示,Q₁₀与环境变量尤其是土壤养分有显著的相关关系,这表明Q₁₀可能还受到除温度以外其他环境因子的影响。     

Compositional mapping of mixed gels using FTIR microspectroscopy

We have developed a technique to produce compositional maps of phase-separated protein/polysaccharide mixed gels using Fourier transform infrared (FTIR) microspectroscopy. The maps plot out the composition of either the protein, the polysaccharide or the water as a function of position in the sample and are presented in the form of two-dimensional contour plots. Our technique is completely general in nature, since it simply relies on there being some measurable spectral difference between the two biopolymers. However, in this paper we use our technique to study the particular case of aqueous gelatin/ amylopectin gels.

Semi-quantitative compositional maps were generated in the first instance by simply plotting the area of the infrared amide II absorption band from the gelatin. Fully quantitative compositional maps, in terms of actual weight percentage concentration of gelatin, amylopectin and water, were also produced by analysing a particular region of the spectra with the method of partial least-squares (PLS).

We recently showed how PLS analysis can be used in conjunction with FTIR spectroscopy to plot the phase diagram of bulk phase-separated solutions which are held above the gel temperature of both components. Thus, our mapping technique allows the concentrations in a gel to be directly compared with those reached at equilibrium in the bulk phase-separated solution, using the same molecular probe, namely, infrared radiation.     

Organosolv pulping of wheat straw by use of phenol

A central composite design was used to investigate the influence of the cooking conditions (time, temperature and phenol concentration) for wheat straw with phenol-water mixtures on the properties of the pulp obtained (yield and holocellulose, -cellulose, lignin and ethanol-benzene extractable contents) and the pH of the resulting wastewater. A second-order polynomial model consisting of three independent process variables was found to accurately describe the organosolv pulping of wheat straw. The equations derived predict the yield, the holocellulose, -cellulose, lignin and ethanol-benzene extractable contents of the pulp, and the pH of the wastewater with multiple-R, R² and adjusted-R² high values. The process variables must be set at low variables in order to ensure a high yield and pH. Conversely, if high holocellulose and -cellulose contents, and low lignin and ethanol-benzene extractable contents are desired, then a high temperature (200°C), long cooking time (120 min), and intermediate phenol concentration (65%) must be used.     

玉米和大豆秸秆还田初期对黑土CO2排放的影响

通过恒温培养试验,研究了不同类型秸秆还田后的土壤CO₂排放特征及其与秸秆C、N含量的关系,以明晰黑土区不同类型秸秆还田后的分解特征,探明还田秸秆的C、N含量对固碳效果的影响.结果表明： 在61 d的培养试验中,土壤CO₂排放速率随时间呈现出“下降 稳定 增大(出现‘较高值’) 下降”的过程.不同类型秸秆还田后土壤CO2排放速率随时间变化的特征存在明显差异,主要体现在“较高值”出现和持续的时间不同.秸秆类型对土壤CO₂累积排放量具有显著影响,前21 d和前61 d的土壤CO2累积排放量对秸秆添加的响应不同.在前21 d,玉米根、玉米茎下部、玉米叶、大豆叶的CO₂累积排放量(约160 μmol·g^-1)显著大于其他秸秆;而除大豆叶外,大豆秸秆61 d的CO₂累积排放量均比玉米秸秆大.前21 d CO₂累积排放量与秸秆含碳量的比值(CR)和秸秆的C/N、含氮量之间均呈显著的线性相关;而61 d的CO₂累积排放量与秸秆的C、N含量之间不存在线性关系.综上,在还田条件下,秸秆类型对土壤CO₂的排放有明显影响;大豆秸秆比玉米秸秆容易分解,但与长时间分解不同,大豆秸秆还田最初阶段的分解速率小于玉米秸秆;秸秆的C/N、含氮量只对还田最初阶段的土壤CO₂排放有较大影响.     

Preparation and characterizations of self-assembled PEGylated gelatin nanoparticles

The PEGylated gelatin nanoparticles were prepared by self-assembling method and characterized. The gelatin drug carrier was proposed as a targeting drug delivery system with the hypothesis that the gelatin carrier could be degraded by the matrix metalloprotease (MMP) and release the anticancer drug loaded inside carriers around the cancer site. The gelatin nanoparticles proposed in this study were composed of deoxycholic acid (DOCA), monomethoxy polyethylene glycol (MPEG), and gelatin. The carboxyl groups of DOCA and carboxylated MPEG were coupled with amine group of gelatin by dichlorohexylcarbodiimide (DCC) method. One molecule of gelatin coupled with 205 molecules of MPEG and 275 molecules of DOCA. The synthesized gelatin/DOCA/MPEG conjugates (GDM) were ultrasonicated to produce self-assembled nanoparticles. DOCA acted as the hydrophobic core, thereby aggregating gelatin molecules and hydrophilic MPEG chains located at the surface of the nanoparticles. The concentration of GDM, intensity of sonication, sonication time and temperature, all affected to control the particle size in the ultrasonication. The optimum condition was obtained as 1.0 mg/mL of GDM, 28 W for sonication intensity, 3 min of sonication time, and room temperature. The size distribution of particle was found to be 100–1000 nm in this condition. The particles which had a broad size distribution were filtered by 0.2 μm membrane. The product yield of particles having below 200 nm of size was about 30%. After filtration, an average diameter of GDM nanoparticle was 176 nm (155–200 nm).     

玉米和大豆秸秆还田初期对黑土CO2排放的影响

通过恒温培养试验,研究了不同类型秸秆还田后的土壤CO₂排放特征及其与秸秆C、N含量的关系,以明晰黑土区不同类型秸秆还田后的分解特征,探明还田秸秆的C、N含量对固碳效果的影响.结果表明： 在61 d的培养试验中,土壤CO₂排放速率随时间呈现出“下降 稳定 增大(出现‘较高值’) 下降”的过程.不同类型秸秆还田后土壤CO2排放速率随时间变化的特征存在明显差异,主要体现在“较高值”出现和持续的时间不同.秸秆类型对土壤CO₂累积排放量具有显著影响,前21 d和前61 d的土壤CO2累积排放量对秸秆添加的响应不同.在前21 d,玉米根、玉米茎下部、玉米叶、大豆叶的CO₂累积排放量(约160 μmol·g^-1)显著大于其他秸秆;而除大豆叶外,大豆秸秆61 d的CO₂累积排放量均比玉米秸秆大.前21 d CO₂累积排放量与秸秆含碳量的比值(CR)和秸秆的C/N、含氮量之间均呈显著的线性相关;而61 d的CO₂累积排放量与秸秆的C、N含量之间不存在线性关系.综上,在还田条件下,秸秆类型对土壤CO₂的排放有明显影响;大豆秸秆比玉米秸秆容易分解,但与长时间分解不同,大豆秸秆还田最初阶段的分解速率小于玉米秸秆;秸秆的C/N、含氮量只对还田最初阶段的土壤CO₂排放有较大影响.     

An Egg-yolk Agar Diffusion Assay for Monitoring Phospholipase C in Cultures of Clostridium welchii

An egg-yolk agar diffusion assay of phospholipase C has been developed and investigated. The results demonstrate the necessity for strict control of the pH and volume of the egg-yolk agar and of the temperature and duration of incubation of the assay plates. The method has been used to monitor phospholipase C production in cultures of Clostridium welchii sparged continuously with CO₂ in N₂ and shows that maximal concentration occurs after ca. 6-8 h incubation at 37°C. The concentration falls slowly during subsequent anaerobic incubation supporting the opinion that Cl. welchii may elaborate a proteinase which destroys phospholipase C.     

The role of the chloroplast coupling factor in the inactivation of thylakoid membranes at low temperatures

Thylakoids isolated from spinach leaves ( Spinacia oleracea L. cv. Monatol) were exposed to variable low temperatures under non-freezing conditions. After incubation, changes in the activities of several photochemical reactions and physical properties of the membranes were measured at room temperature.
Cyclic photophosphorylation was strictly dependent on the temperature and the electrolyte concentration: decrease in temperature and increase in NaCl concentration enhanced membrane damage. Inactivation of photophosphorylation was accompanied by stimulation of non-cyclic electron transport, increase in proton permeability and decrease in δpH. When dicyclohexylcarbodiimide was added, the proton gradient became completely restored. The temperature- and salt-dependent breakdown of photophosporylation was closely related to the release of the chloroplast coupling factor (CF₁) from the membranes. The addition of Mg²⁺, very low concentrations of ATP or ADP, or higher concentrations of low-molecular-weight polyols prior to temperature treatment prevented thylakoid damage.
The data indicate that inactivation of photophosphorylation of thylakoids at low temperatures is determined to a considerable extent by the cold lability of the CF₁. As a consequence, it must be concluded that damage of biomembranes caused by freezing is not due solely to changes resulting from the ice formation but additionally by temperature-dependent alterations of cold-labile proteins. Moreover, the data explain the mechanism of non-colligative cryoprotection of isolated thylakoid membranes.     

Nickel resistance in Escherichia coli V38 is dependent on the concentration used for induction

Strain Escherichia coli V38 resistant to 4 mM NiCl₂ was isolated from the city sewage sludge. It showed low nickel accumulation by cells and nickel ion efflux. Cells were pregrown (induced) overnight in the presence of Ni²⁺, then the culture was kept on ice for 20–30 min and transferred to 37°C for further incubation. When the Ni²⁺ concentration during growth was the same as during incubation, there was no noticeable accumulation of Ni²⁺. When the Ni²⁺ concentration during incubation was higher than that used for induction, uptake of ⁶³Ni²⁺ and delayed efflux were seen. The uptake and delay of both efflux and growth were directly proportional to the difference between the concentrations used for induction and incubation. Active nickel ion uptake was seen in cells taken from cultures in the delayed efflux period.     

Influence of egg vitamin A status and egg incubation temperature on subsequent development of the early vertebral column in Atlantic salmon fry

The effect of egg vitamin A (VA) status and egg incubation temperature on the development of spinal disorders was investigated in Atlantic salmon Salmo salar fry. Atlantic salmon eggs were sorted into two groups with high VA (3·3 ± 0·1 μg retinol g⁻¹ dry mass) and low VA (2·2 ± 0·3 μg retinol g⁻¹ dry mass) status before fertilization and incubated at high (14° C) or low (8° C) temperature from 133 day degrees until the onset of feeding. High egg incubation temperatures increased the concentration of retinol in the eggs: the high VA and high temperature group displayed a significantly higher retinol concentration than the high VA and low temperature group ( P  = 0·001). After hatching, all experimental groups increased their retinol concentration. The source of the increased retinol levels was probably retinal, although astaxanthin may also be a VA precursor after hatching. Atlantic salmon fry incubated at high temperatures had increased amounts of notochord tissue. When measuring morphogenic activity in the notochord using the expression of sonic hedgehog ( shh , mRNA), however, no significant difference was found between the experimental groups. No clear effect of VA status or incubation temperature could be found on the formation of the early vertebral column although Atlantic salmon fry incubated at low temperatures had less regular constrictions of the prospective vertebral column than fry incubated at high temperatures.     

The distribution of phospholipids on the mitochondrial inner membrane from the brains of goldfish acclimated at 5 and 30°C

1. 1.|The instantaneous addition of phospholipase C to goldfish brain inner mitochondrial vesicles resulted in a partial loss of ferricyanide reduction. The retained activity rose when the phospholipase concentration and incubation period were increased.

2. 2.|No impairment of ferricyanide reduction was observed when Antimycin A-treated inner membrane vesicles were incubated with the phospholipase at 0.05 and 0.2 U mg⁻¹ to 20 min. However, higher concentrations of the hydrolytic enzyme caused a considerable elevation in reduction and is attributed to a deterioration in vesicular integrity.

3. 3.|The hydrolysis of phospholipids from the inner membrane revealed that the phospholipase, at concentrations above 0.2 U mg⁻¹ (minimum of 10 min incubation), resulted in removal of over 50% lipid phosphate. It was suggested that the phospholipase, at such levels, is accessible to the interior of the vesicles.

4. 4.|With the exception of cardiolipin and phosphatidyl inositol, the phospholipase was observed to be non-specific to the phospholipids from mitochondrial inner membrane treated with Triton X-100.

5. 5.|The phospholipids are asymmetrically arranged across the inner membrane and their distribution is dependent on acclimation temperature.

6. 6.|It is suggested that the compositional redistribution of the phospholipids, when acclimation temperature was changed, is necessary to stabilize the bilayer conformation of the membrane in concurrence with maintaining fluidity and functional capacity.