Extraction of cephalosporin C from whole broth and separation of desacetyl cephalosporin C by aqueous two-phase partition

Cephalosporin C was extracted from diluted or whole broth by PEG/salt aqueous two-phase systems. Parameters such as PEG molecular weight, salt type, pH, and salt concentration were investigated for finding a suitable extraction system. In PEG 600/ammonium sulfate or phosphate systems, K(c) (partition coefficienct of cephalosporin C) was observed to be larger than 1, with K(d) (partition coefficient of desacetyl cephalosporin C) being smaller than 1. The particular values of these coefficients would imply that the difficult separation of cephalosporin C and desacetyl cephalosporin C could possibly be achieved via the aqueous two-phase extraction. The addition of surfactants, water-miscible solvents, and neutral salts for enhancement of the separation efficiency was also investigated. The addition of surfactants to the system did not affect the separation efficiency substantially. K(c) would increase whereas K(d) decreased as a result of the addition of acetone, MeOH, EtOH, IPA, and n-BuOH. Meanwhile both K(c) and K(d) would decrease whenever neutral salts, NaCl, KCl, Kl, or KSCN, were added. The partitioning behavior of cephalosporin C and desacetyl cephalosporin C in filtered, whole, and different batches of broth was notably quite similar to that of diluted broth. The recovery yield of cephalosporin C in whole broth extraction was observed to be a function of centrifugal force used in phase separation. (c) 1994 John Wiley & Sons, Inc.     

Development of the cephalosporin C fermentation taking into account the instability of cephalosporin C

Summary Cephalosporin C undergoes chemical hydrolysis during fermentation at 25°C at a rate of 0.48% per hour to give increasing amounts of 2-(D-4-amino-4-carboxybutyl)thiazole-4-carboxylic acid (compound X). Although the cephalosporin C titer in a fermentation levels off at 160–170h, the total of C+X rises almost linearly up to 200h. Cells, therefore, biosynthesize cephalosporin C at an almost constant rate up to 200h. This instability greatly influences attempts to improve the cephalosporin strain by genetic manipulation, or the process by alterations in technology.     

Quantification of Moraxella bovis haemagglutinating adhesins with monoclonal antibodies

Six monoclonal antibodies (MAbs) against Moraxella bovis GF 9 were used to quantify haemagglutinating adhesins of 16 strains of this organism. The amount of each MAb necessary to inhibit one haemagglutinating unit of each strain varied between 4 and 0.007 times that required by strain GF 9. Five strains reacted with six MAbs, one with five, two with four, one with three, two with two and three with none. The procedures used enabled to detect dominant strains candidates for vaccines.     

Separation of cephalosporin C and desacetyl cephalosporin C by high speed counter-current chromatography in aqueous two-phase systems

Summary In the recovery of cephalosporin C (CPC) from fermentation broth, the separation of desacetyl cephalosporin C (DAC) is a major concern. Multistage extraction in aqueous two-phase systems, mainly PEG/ammonium sulfate systems, proved to be promising. In preparative scale operation, high speed counter-current chromatography (HSCCC) with eccentric columns was used with aqueous two-phase systems to obtain baseline resolution of CPC and DAC. Solvents (e.g. 5% acetone) or neutral salts (e.g. 1.45% KSCN) added into aqueous two-phase systems enhanced the separation efficiency. Operation parameters of HSCCC such as rotational speed and mobile phase flow rate can affect the retention of the stationary phase and HETP.     

Stromal-dependent tumor promotion by MIF family members

Solid tumors are composed of a heterogeneous population of cells that interact with each other and with soluble and insoluble factors that, when combined, strongly influence the relative proliferation, differentiation, motility, matrix remodeling, metabolism and microvessel density of malignant lesions. One family of soluble factors that is becoming increasingly associated with pro-tumoral phenotypes within tumor microenvironments is that of the migration inhibitory factor family which includes its namesake, MIF, and its only known family member, D-dopachrome tautomerase (D-DT). This review seeks to highlight our current understanding of the relative contributions of a variety of immune and non-immune tumor stromal cell populations and, within those contexts, will summarize the literature associated with MIF and/or D-DT.     

Cybernetic modeling of the cephalosporin C fermentation process by Cephalosporium acremonium

A cybernetic mathematical model has been developed to describe the production of cephalosporin C. In developing the model, diauxic behavior of substrate consumption, morphological differentiation of cells, and catabolite repression of cephalosporin C production by the preferred substrate, glucose, were considered. The proposed model was tested on the experimental data from the literature and could adequately describe the morphological differentiation of cells, the sequential utilization of carbon sources and the production of cephalosporin C. It could be a useful tool to optimize the production of cephalosporin C by Cephalosporium acremonium in batch, fed-batch or continuous operations.     

Deacylation activity of cephalosporin acylase to cephalosporin C is improved by changing the side-chain conformations of active-site residues

Semisynthetic cephalosporins are primarily synthesized from 7-aminocephalosporanic acid (7-ACA), mainly by environmentally toxic chemical deacylation of cephalosporin C (CPC). Thus, the enzymatic conversion of CPC to 7-ACA by cephalosporin acylase (CA) would be very interesting. However, CAs use glutaryl-7-ACA (GL-7-ACA) as a primary substrate and the enzymes have low turnover rates for CPC. The active-site residues of a CA were mutagenized to various residues to increase the deacylation activity of CPC, based on the active-site conformation of the CA structure. The aim was to generate sterically favored conformation of the active-site to accommodate the D-alpha-aminoadipyl moiety of CPC, the side-chain moiety that corresponds to the glutaryl moiety of GL-7-ACA. A triple mutant of the CA, Q50betaM/Y149alphaK/F177betaG, showed the greatest improvement of deacylation activity to CPC up to 790% of the wild-type. Our current study is an efficient method for improving the deacylation activity to CPC by employing the structure-based repetitive saturation mutagenesis.     

Control of proliferation by Bcl-2 family members

The anti-proliferative effect of Bcl-2 acts mainly at the level of the G0/G1 phase of the cell cycle. Deletions and point mutations in the bcl-2 gene show that the anti-proliferative activity of Bcl-2, can in some cases, be dissociated from its anti-apoptotic function. This indicates that the effect of Bcl-2 on cell cycle progression can be a direct effect and not only a consequence of its anti-apoptotic activity. Bcl-2 appears to mediate its anti-proliferative effect by acting on both signal transduction pathways (NFAT, ERK) and on specific cell cycle regulators (p27, p130).