An assay for the measurement of the protein content of cells immobilized in carrageenan

Abstract

A reliable and reproducible method for the estimation of the protein content of fungal cells immobilized in a carrageenan gel is described. The procedure depends upon the acid lability of the polysaccharide gel at 90 degrees C and on the acetone solubility of accumulated phenolics. Freeze-dried gel beads (2-3 mm) containing entrapped cells of Penicillium urticae were ground to a fine powder and samples of powder (approximately 20 mg) were sequentially extracted with hot 1 N HCl - 0.9% NaCl and acetone. The precipitated residue contained the cell protein, which was then solubilized with 1 N NaOH at 90 degrees C and quantitated by the Folin-Lowry method. Interferences from both carrageenan and phenols were thus eliminated. The presence of carrageenan (20-25 mg) did not affect the recovery of varying amounts (0-2500 micrograms) of bovine serum albumin. The recovery of radiolabeled protein from immobilized cells was parallel to that of Folin-Lowry positive material over a range of 0-60 beads (0-60 mg powder). Cycloheximide (0-100 micrograms/mL) was shown to progressively inhibit the incorporation of L-[U-14C]leucine so that the radioactivity present in the initial HCl-NaCl extract (i.e., [14C]leucine) increased as that in the final NaOH extract (i.e., 14C-labeled protein) decreased. Using this new assay for cell protein, free and immobilized cell cultures were found to exhibit virtually identical kinetics of glucose utilization, growth, and patulin production. In addition to providing a means of comparing the specific productivity of free versus immobilized cell preparations, this assay accurately measures the incorporation of [14C]leucine into cellular protein and could be used as a measure of cell viability.

The production of the antibiotic patulin in a three phase fluidized bed reactor: II. Longevity of the biocatalyst

“…In Part 1 of this study (Berk et al, 1983), we have shown that the batch production of patulin by immobilized Penicillium urricae cells in the initial stages follows a Type 111 fermentation. Moreover by developing a medium which retarded growth, it was possible to produce patulin in a three phase fluidized bed by immobilized P .…”

“…The growth of the organism, the operation of the continuous reactors and the analytical methods are described in Part 1 of this study (Berk et al, 1983). The composition of the medium that was used in all runs is shown in Table 1.…”

“…The composition of the medium that was used in all runs is shown in Table 1. The total protein content in the reactor was determined by measuring the average protein content per bead (Jones et al, 1983) and multiplying by the total number of beads (approximately 20 000) that were present in the reactor.…”

The production of the antibiotic patulin in a three phase fluidized bed reactor I. Effect of medium composition

“…The effluent samples were directly analyzed for glucose content by the Worthington glucostat enzymatic assay procedure. The protein content of the beads was determined by an assay developed by Jones et al, 1983. In this assay, the freeze-dried beads containing the immobilized cells are ground to a fine powder, and samples of this powder (-20 mg) are extracted first with hot (90°C) 1 N HCI + 0.9% NaCl solution then with acetone.…”

Manganese and antibiotic biosynthesis. III. The site of manganese control of patulin production in Penicillium urticae

“…The protein content of the crude filtered homogenate was assayed by the method of Lowry et al (195 1) after sample preparation as described by Jones et al (1984).…”