Comparative studies on soluble and immobilized yeast hexokinase

Comparative studies have been carried out on soluble and immobilized yeast hexokinase (ATP: D-hexose 6-phosphotransferase, EC 2.7.1.1). The enzyme was immobilized by covalent attachment to a polyacrylamide type support containing carboxylic functional groups. The effects of immobilization on the catalytic properties and stability of hexokinase were studied. As a result of immobilization, the pH optimum for catalytic activity was shifted in the alkaline direction to ～pH 9.7. The apparent optimum temperature of the immobilized enzyme was higher than that of the soluble enzyme. The apparent K_m value with D-glucose as substrate increased, while that with ATP as substrate decreased, compared with the data for the soluble enzyme. Differences were found in the thermal inactivation processes and stabilities of the soluble and immobilized enzymes. The resistance to urea of the soluble enzyme was higher at alkaline pH values, while that for the immobilized enzyme was greatest at ～pH 6.0.     

中药指纹图谱的研究进展

中药指纹图谱已日渐应用于中药鉴定与质量评价。本文综述了近来的薄层色谱指纹图谱,高效液相指纹图谱,核磁共振指纹图谱,质谱指纹图谱,X-射线衍射指纹图谱,色谱联用指纹图谱及DNA指纹图谱等的研究进展。     

Effects of oxygen and ethanol on recombinant yeast fermentation for hepatitis B virus surface antigen production: modeling and simulation studies

A model was formulated to examine the competitive growth of two phenotypes (Leu(+) and Leu(-)) and the product formation with recombinant Saccharomyces cerevisiae strain DBY-745, which contains the shuttle vector pYGH3-16-s with the foreign gene HBsAg (hepatitis B virus surface antigen) as well as experimental fedbatch fermentation data. The important state variables and the process parameters evaluated include (1) the ratio of the plasmid-free cell concentration to the plasmid-containing cell concentration (rho = X(-)X(+)), (2) the expression of human hepatitis B surface antigen g (CH), (3) the glucose consumption (S), (4) the ethanol production (/), (5) the change of working volume (V) in the fermentor, (6) the different specific growth rates of two phenotype cells, and (7) the plasmid loss frequency coefficient (alpha ). These variables and other parameters were carefully defined, their correlations were studied, and a mathematical model using a set of nonlinear ordinary differential equations (ODEs) for fed-batch fermentation was then obtained based on the theoretical considerations and the experimental results. The extended Kalman filter (EKF) methods was applied for the best estimate of these variables based on the experimentally observable variables: rhoV, and g (CH). Each of these variable was affected by random measuring errors under the different operating conditions. Simulation results presented for verification of the model agreed with our observations and provided useful information relevant to the operation and the control of the fedbatch recombinant yeast fermentation. The method of predicting an optimal profile of the cell growth was also demonstrated under the different dissolved oxygen concentrations. (c) 1993 John Wiley & Sons, Inc.     

Modeling and experimental studies on intermittent starch feeding and citrate addition in simultaneous saccharification and fermentation of starch to flavor compounds

Simultaneous saccharification and fermentation (SSF) is a combined process of saccharification of a renewable bioresource and fermentation process to produce products, such as lactic acid and ethanol. Recently, SSF has been extensively used to convert various sources of cellulose and starch into fermentative products. Here, we present a study on production of buttery flavors, namely diacetyl and acetoin, by growing Lactobacillus rhamnosus on a starch medium containing the enzyme glucoamylase. We further develop a structured kinetics for the SSF process, which includes enzyme and growth kinetics. The model was used to simulate the effect of pH and temperature on the SSF process so as to obtain optimum operating conditions. The model was experimentally verified by conducting SSF using an initial starch concentration of 100 g/L. The study demonstrated that the developed kinetic was able to suggest strategies for improved productivities. The developed model was able to accurately predict the enhanced productivity of flavors in a three stage process with intermittent addition of starch. Experimental and simulations demonstrated that citrate addition can also lead to enhanced productivity of flavors. The developed optimal model for SSF was able to capture the dynamics of SSF in batch mode as well as in a three stage process. The structured kinetics was also able to quantify the effect of multiple substrates present in the medium. The study demonstrated that structured kinetic models can be used in the future for design and optimization of SSF as a batch or a fed-batch process. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

ESR studies on membrane fluidity of Chinese hamster ovary cells grown on microcarriers and in suspension

Electron spin resonance (ESR) spin label methods were used to study membrane fluidity of Chinese hamster ovary (CHO) cells grown on microcarriers and in suspension using 5-doxylstearic acid spin label as a probe. CHO cells grown on microcarriers had a more rigid cell membrane compared to CHO cells grown in suspension culture. CHO cells removed from the surface of the microcarriers by either trypsinization, EDTA treatment or osmotic shock had a membrane fluidity similar to that of CHO cells grown in suspension culture. Conversely, when the cells grown in suspension culture were attached and flattened on the surface of the microcarriers the fluidity decreased. Moreover, membrane fluidity of CHO cells grown on microcarriers changed as a function of the population density, whereas that of the cells in suspension did not. This implies that cell adhesion and/or cell-cell interactions influence the fluidity of the cell surface membrane.     

Microbial inoculant effects on silage and in vitro ruminal fermentation, and microbial biomass estimation for alfalfa, bmr corn, and corn silages

Whole crop third cut alfalfa, brown mid-rib (bmr) corn, and corn were chopped and inoculated with one of four microbial inoculants used. Uninoculated silage was the control treatment. Each crop was ensiled in four mini-silos (1 L glass jars) per treatment. All silos were fermented for 60 days at room temperature (22 °C), and then they were opened and analyzed for fermentation products, fiber constituents and N fractions. A fraction of wet silage was ground with a blender for 30 s. In vitro gas production was measured in 160 ml sealed serum vials at 3, 6, 9, 24, and 48 h using the wet ground silage. At 9 and 48 h, rumen fluid was analyzed for volatile fatty acids (VFA) and microbial biomass yield (MBY). In all the three crops, the four inoculants produced only minor changes in pH and fermentation products during ensiling. Of the variables measured, soluble nonprotein N fractions were the characteristics most often affected by some inoculants. At 9 h incubation, in vitro gas production and VFA did not differ between control and inoculated silages, but MBY did. Among crops, alfalfa and corn silages had higher MBY than did bmr corn silage. Among inoculants, three of the four inoculated silages produced more MBY than did control. At 48 h, alfalfa silage produced higher MBY than did corn or bmr silage, and two of the inoculated silages had more MBY than did the control. There was no inoculant by crop interaction. Results suggest that some silage inoculants are capable of altering rumen fermentation, even in cases where effects on silage fermentation are small, and that this effect may be linked to better preservation of crop protein during ensiling.