Application of empirical design methodologies to the study of the influence of reaction conditions and N-alpha-protecting group structure on the enzymatic X-Phe-Leu-NH(2) dipeptide synthesis in buffer/dimethylformamide solvents systems

The influence of five different N-terminal protecting groups (For, Ac, Boc, Z, and Fmoc) and reaction conditions (temperature and dimethylformamide content) on the alpha-chymotrypsin-catalyzed synthesis of the dipeptide derivative X-Phe-Leu-NH(2) was studied. Groups such as For, Ac, Boc, and Z always rendered good peptide yields (82% to 85%) at low reaction temperatures and DMF concentrations, which depended on the N-alpha protection choice. Boc and Z were the most reactive N-alpha groups and, in addition, the most suitable for peptide synthesis. On the other hand, the use of empirical design methodologies allowed, with minimal experimentation and by multiple regression, to deduce an equation, which correlates the logarithm of the first order kinetic constant (log k') with reaction temperature, DMF concentration, and hydrophobicity (log P values) of the different protecting groups. The predictive value of the equation was tested by comparing the performance of another protective group, such as Aloc, with the performance predicted by said equation. Experimental and calculated k' values were found to be in good agreement.     

Utilization of the tricarboxylic acid cycle, a reactor design criterion for the microaerobic production of 2,3-butanediol

A new parameter, the relative utilization of tricarboxylic acid (TCA) cycle beta, is introduced to quantitatively account for the involvement of fermentation pathways and TCA cycle in the utilization of oxygen under oxygen-limiting (microaerobic) conditions. With the facultative anaerobe Enterobacter aerogenes, which produces 2,3-butanediol, a method is proposed to calculate beta from measurement of metabolites and exhaust gas. In continuous culture beta was found to be small under oxygen limitation, indicating that the fermentation pathways were preferred over the TCA cycle and oxygen was almost entirely consumed through oxidation of reduced nicotinamide adenine dinucleotide (NADH(2)) released by fermentation under these conditions. The increase of beta at high oxygen supply revealed a saturation of oxygen utilization through fermentation pathways. It could be concluded that, for the optimal performance of a microaerobic culture, oxygen uptake rate must be kept at such a level that as much NADH(2) as possible from fermentation pathways is oxidized by oxygen, and at the same time the utilization of TCA cycle is kept at a minimum. As the dynamics of the microaerobic culture can be fast, a significant effect of reactor hydrodynamics, i.e., mixing, on the overall performance can be expected. This was confirmed experimentally, and the parameter beta proved to be a useful reactor design criterium for the microaerobic cultivation. (c) 1992 John Wiley & Sons, Inc.     

Root biomass fraction as a function of growth degree days in wheat

Root, underground and above-ground biomass were measured on various wheat cultivars from 1986 to 1988 in the south-east of France. The results are expressed as root: total (f _r) or underground: total (f _u) biomass fractions. Observed f _r and f _u values are in good agreement with previous results. f _r and f _u decrease steadily from emergence to maturity, with an exponential tendency. When using cumulative growth degree days since emergence relative to cumulative growth degree days until ear emergence () as time scale, f _r and f _u can be expressed as simple functions of % MathType!MTEF!2!1!+-% feaafiart1ev1aaatCvAUfeBSjuyZL2yd9gzLbvyNv2CaerbuLwBLn% hiov2DGi1BTfMBaeXatLxBI9gBaerbd9wDYLwzYbItLDharqqtubsr% 4rNCHbGeaGqiVu0Je9sqqrpepC0xbbL8F4rqqrFfpeea0xe9Lq-Jc9% vqaqpepm0xbba9pwe9Q8fs0-yqaqpepae9pg0FirpepeKkFr0xfr-x% fr-xb9adbaqaaeGaciGaaiaabeqaamaabaabaaGceaqabeaacaWGMb% addaWgaaqaaiaadkhaaeqaamaabmaabaGccqaH4oqCdaahaaWcbeqa% aiaacQcaaaaamiaawIcacaGLPaaakiabg2da9iaaicdacaGGUaGaaG% imaiaaiwdacqGHRaWkcaaIWaGaaiOlaiaaiwdacaaI4aGaamyzamaa% CaaaleqabaGaeyOeI0IaaGymaiaac6cacaaI0aGaaGioaiabeI7aXn% aaCaaameqabaGaaiOkaaaaaaaakeaacaWGMbaddaWgaaqaaiaadwha% aeqaamaabmaabaGccqaH4oqCdaahaaWcbeqaaiaacQcaaaaamiaawI% cacaGLPaaakiabg2da9iaaicdacaGGUaGaaGymaiaaikdacqGHRaWk% caaIWaGaaiOlaiaaiIdacaaI4aGaamyzamaaCaaaleqabaGaeyOeI0% IaaGOmaiaac6cacaaIYaGaaGioaiabeI7aXnaaCaaameqabaGaaiOk% aaaaaaaaaaa!610D!\[\begin{gathered} f_r \left( {\theta ^* } \right) = 0.05 + 0.58e^{ - 1.48\theta ^* } \hfill \\ f_u \left( {\theta ^* } \right) = 0.12 + 0.88e^{ - 2.28\theta ^* } \hfill \\ \end{gathered} \]The incremental root biomass partitioning coefficient, % MathType!MTEF!2!1!+-% feaafiart1ev1aaatCvAUfeBSjuyZL2yd9gzLbvyNv2CaerbuLwBLn% hiov2DGi1BTfMBaeXatLxBI9gBaerbd9wDYLwzYbItLDharqqtubsr% 4rNCHbGeaGqiVu0Je9sqqrpepC0xbbL8F4rqqrFfpeea0xe9Lq-Jc9% vqaqpepm0xbba9pwe9Q8fs0-yqaqpepae9pg0FirpepeKkFr0xfr-x% fr-xb9adbaqaaeGaciGaaiaabeqaamaabaabaaGcbaGaeqySde2aaS% baaSqaaiaadkhaaeqaaOGaeyypa0JaaiikaiaadsgacaWGxbWaaSba% aSqaaiaadkhaaeqaaOGaai4laiaadsgacaWG0bGaaiykaiaac+caca% GGOaGaamizaiaadEfadaWgaaWcbaGaamiDaaqabaGccaGGVaGaamiz% aiaadshacaGGPaaaaa!4834!\[\alpha _r = (dW_r /dt)/(dW_t /dt)\], which describes the net increase in root biomass dW _r over time dt relative to the increase in total biomass (dW _r) over the same time period, has been derived from f and the relative growth rate. Its time course is accurately represented by% MathType!MTEF!2!1!+-% feaafiart1ev1aaatCvAUfeBSjuyZL2yd9gzLbvyNv2CaerbuLwBLn% hiov2DGi1BTfMBaeXatLxBI9gBaerbd9wDYLwzYbItLDharqqtubsr% 4rNCHbGeaGqiVu0Je9sqqrpepC0xbbL8F4rqqrFfpeea0xe9Lq-Jc9% vqaqpepm0xbba9pwe9Q8fs0-yqaqpepae9pg0FirpepeKkFr0xfr-x% fr-xb9adbaqaaeGaciGaaiaabeqaamaabaabaaGcbaGaeqySdegdda% WgaaqaaiaadkhaaeqaamaabmaabaGccqaH4oqCdaahaaWcbeqaaiaa% cQcaaaaamiaawIcacaGLPaaakiabg2da9iabgkHiTiaaicdacaGGUa% GaaGymaiaaiwdacqGHRaWkcaaIWaGaaiOlaiaaiAdacaaIZaGaamyz% amaaCaaaleqabaGaeyOeI0IaaGimaiaac6cacaaI5aGaaGioaiabeI% 7aXnaaCaaameqabaGaaiOkaaaaaaaaaa!4D15!\[\alpha _r \left( {\theta ^* } \right) = - 0.15 + 0.63e^{ - 0.98\theta ^* } \]Under our experimental conditions, with no severe water stresses or nutrient deficiencies, and for our sampling frequency, around 2 weeks, the development scale , is the main factor governing the time courses of f _r, f _u and _r.     

Optimal nonsingular control of fed-batch fermentation

Presented is a new simple method for multidimensional optimization of fed-batch fermentations based on the use of the orthogonal collocation technique. Considered is the problem of determination of optimal programs for fermentor temperature, substrate concentration in feed, feeding profile, and process duration. By reformulation of the state and control variables is obtained a nonsingular form of the optimization problem which has considerable advantage over the singular case since a complicated procedure for determination of switching times for feeding is avoided. The approximation of the state variables by Lagrange polynomials enables simple incorporation of split boundary conditions in the approximation, and the use of orthogonal collocations provides stability for integration of state and costate variables. The interpolation points are selected to obtain highest accuracy for approximation of the objective functional by the Radau-Lobatto formula. The control variables are determined by optimization of the Hamiltonian at the collocation points with the DFP method. Constraints are imposed on state and control variables.The method is applied for a homogeneous model of fermentation with volume, substrate, biomass, and product concentrations as the state variables. Computer study shows considerable simplicity of the method, its high accuracy for low order of approximation, and efficient convergence.     

Enhanced glucan formation of filamentous fungi by effective mixing,oxygen limitation and fed-batch processing

Summary Glucan formation ofSchizophyllum commune andSclerotium glucanicum were investigated. Process data obtained during batch cultivation are presented. Glucan release can be improved by oxygen limitation. Thus, growth and glucan release are influenced by oxygen in opposite ways. Possible pathways of this oxygen-dependent regulation are discussed. A draft-tube/propeller system, rushtonturbine-, fan- and helicon-ribbon-impeller as well as a fundaspi and intermig agitator were tested. The 4-bladed fan impeller withd ^*=0.64 yielded the best results, since effective bulk mixing is much more important than bubble break up (micromixing) with regard to this system. Fed-batch cultivation always resulted in higher rates of glucan formation than the batch process.     

Some factors influencing the proportion of periplasmic hepatitisB virus pre-S2 antigen in the recombinant yeastHansenula polymorpha

Summary A central composite design (CCD) was used to evaluate, for the purpose of future process optimization, the influence of pH, yeast extract and ammonium chloride concentrations on the proportion of periplasmic hepatitisB pre-S2 antigen in the recombinant yeastHansenula polymorpha. Each factor was tested at five levels, and a second order polynomial model for the proportion of periplasmic antigen was fitted to the treatment combinations. pH showed the greatest effect: the proportion of periplasmic antigen was greatly increased at the higher pH levels. At the higher pH levels used, the proportion of periplasmic antigen was enhanced by a high concentration of ammonium chloride. Additional experiments have confirmed both the validity of the selected model and the optimal conditions found. A significant correlation was found between the proportion of periplasmic antigen and the total yield of antigen. These results indicated that is should be possible to modulate the distribution of the pre-S2 antigen between the periplasm and the cytoplasm of the yeast.     

Biochemical differences between cytoplasmic malate dehydrogenase allozymes of Drosophila virilis

Two allozymes (MDH^f and MDH^s) of cytoplasmic malate dehydrogenase of Drosophila virilis were partially purified and their biochemical properties were compared. MDH^f has a pH optimum of 9.75 and MDH^s one of 9.25 for malate oxidation. Optimal pH for oxaloacetate reduction is 6.75 and 8.0 for MDH^f and MDH^s, respectively. The K_m value for oxaloacetate of MDH^s is approximately twice as that of MDH^f. No differences were found with respect to thermostability and K_m's for malate, NAD⁺, or NADH. These results are discussed in terms of the physiological role of cytoplasmic malate dehydrogenase of D. virilis.This work was supported in part by grants from the Ministry of Education, Japan, Nos. 134050 and 154205.