Cells resting

The hydrolysis of nitriles can be carried out with either isolated enzymes or immobilized cells. Eor example, resting cells of P. chlororaphis can accumulate up to 400 g/L of acrylamide in 8 h, provided acrylonitrile is added gradually to avoid nitrile hydratase inhibition (116). The degree of acrylonitrile conversion to acrylamide is 99% without any formation of acryUc acid. Because of its high efficiency the process has been commercialized and currentiy is used by Nitto Chemical Industry Co. on a multithousand ton scale. 

Akinete Thick-walled resting cell of cyanobacteria and algae. 

Synthesis of industrial chemicals by microbial cells may be by fermentation (free, living cells), immobilised growing cells, immobilised resting cells or immobilised dead cells. 

The culture can be used directly for the conversion of phenylpyruvic add to resting cells L-phenylalanine. Therefore, a batch process with resting cells can be carried out, with some glucose added for maintenance (fed-batch fermentation). Another approach is to harvest the cells from the fermentation broth and to use them in a separate bioreactor in higher concentrations than the ones obtained in the cell cultivation. An advantage of the last method can be that the concentration of compounds other than L-phenylalanine is lower, so that downstream processing may be cheaper. 

Due to the large amount of DNA present within the nucleus it must be carefully packaged. In the resting cell DNA is tightly compacted around basic histone proteins, excluding the binding of the enzyme RNA polymerase II, which activates the formation of mRNA. This conformation of the chromatin structure... 

The activation of NFAT is regulated by calcium. In resting cells, NFAT proteins localize in the cytoplasm... 

Intracellular Ca2+-levels are controlled by release into, and removal from, the cytoplasm (Fig. 1). Ca2+-pumps in the plasma membrane and endoplasmic reticulum (ER the Ca2+-store in a cell) keep cytoplasmic Ca2+-levels low (about 0.1 pmol/L in resting cells) and generate a 10,000-fold concentration gradient across membranes (because extracellular Ca2+ is in the millimolar range). Upon stimulation, Ca2+ enters the cytosol of the cell via Ca2+-channels (plasma membrane) or via Ca2+-channels in the ER, leading to the activation of a great variety of Ca2+-dependent processes in the cell. 

Thiamine was biosynthesized by resting cells of S. typhimurium strain thilO/T-ath-383, which can synthesize thiamine from exogenous glucose, AIRs, and thiazole.54 Derepression was achieved by conventional means. The organism was cultivated in the presence of a suboptimal amount of thiamine (20 nM), the washed cells were resuspended in a minimal medium containing glucose (10 mM), thiazole (1-2 mM), and labeled AIRs (10 p,M). During the incubation (1.5 hours 37°C), the level of thiamine diphosphate in the cells had risen from about 0.04 to 0.5 nmol/mg. In work with molecules labeled with stable isotopes, thiamine was extracted and cleaved by ethanethiol to 4-amino-5-(ethyl-... 

Other racemization systems that may be amenable to conversion to deracemization processes in future have recently been reported by Faber and coworkers [33]. Resting cells of L. paracasei have been used for biocatalytic racemization of open-chain and cyclic dialkyl-, alkyl-aryl-, and diaryl-substituted acyloins (29/30) (Figure 5.20). Both... 

In a subsequent study using a resting cell system of N. diaphanozonaria JCM3208, deracemization of 4-substituted-2-thiopropanoic adds (34) were... 

Reaction without 2-hexanol. 1 Reduction of acetophenone in hexane by the ed resting cell ofCeothchumcondWwni and 2-hexanol [18aj. ... 

Resting cell of G. candidum, as well as dried cell, has been shown to be an effective catalyst for the asymmetric reduction. Both enantiomers of secondary alcohols were prepared by reduction of the corresponding ketones with a single microbe [23]. Reduction of aromatic ketones with G. candidum IFO 5 767 afforded the corresponding (S)-alcohols in an excellent enantioselectivity when amberlite XAD-7, a hydro-phobic polymer, was added to the reaction system, and the reduction with the same microbe afforded (R)-alcohols, also in an excellent enantioselectivity, when the reaction was conducted under aerobic conditions (Figure 8.31). 

Fig. 2. Whole cell biocatalytic reactions for four types of recombinant whole cell systems. Bioconversion reactions were performed in resting cell condition. All data were based on unit cell concentration (1 mg-dry cell weight ml ). Each value and error bar represents the mean of two independent experiments and its standard deviation.

Alvarez-Cohen L, McCarty PL. 1991b. Product toxicity and cometabolic competitive inhibition modeling of chloroform and trichloroethylene transformation by methanotrophic resting cells. Appl Environ Microbiol 57 1031-1037. 

These results led us to reexamine the published work on nitrosation in the presence of bacteria (Table II) In two of the studies (, 10) the amount of NDMA formed is actually less than the amount that can be predicted from theoretical consideration of the uncatalyzed chemical reaction alone (15) In another study (12), the yield of NDMA is again slightly lower than the predicted chemical yield at the final pH (6 0) of the growing culture The work by Kunisaki and Hayashl (13), on the other hand, does indicate that resting cells of E. coli B catalyze... 

Recently, the distribution of 2,3-dihydroxybenzoate decarboxylase has been found in a variety of fungal strains (unpubhshed data), and the carboxylation activity for catechol is confirmed by the reaction using resting cells (or cell-free extract) in the presence of 3M KHCO3. The detailed comparative studies of enzyme structures and catalytic properties between 2,3-dihydroxybenzoate decarboxylase and 3,4-dihyroxybenzoate decarboxylase might explain how the decarboxylases catalyze the regioselective carboxylation of catechol. 

The carboxylafion of indole into indole-3-carboxylate was observed by the purified indole-3-carboxylate decarboxylase as well as by the whole cells. For the carboxylafion reaction, temperatures over 30°C were not appropriate. The activities at 10, 20, and 30°C were about the same. The activity was maximal at pH 8.0 (Tris-HCl buffer, 100 mM). As shown in Fig. 10, the resting cells of A. nicotianae F11612 also catalyzed the carboxylafion of indole efficiently in the reaction mixture containing 20 mM indole, 3M KHCO3, 100mM potassium phosphate buffer (pH 6.0) in a tightly closed reaction vessel. By 6h, 6.81 mM indole-3-carboxylic acid accumulated in the reaction mixture with a molar conversion yield of 34%. Compared to the carhoxylation of pyrrole by pyrrole-2-carboxylate decarboxylase, the lower value compared might derive from the lower solubility of indole in the reaction mixture. 

Resting cells of A. nicotianae FI1612 also catalyzed the carboxylafion of 2-methylindole and quinoxaline. The activities toward 2-methylindole and quinoxaline were 37 and < 1% of the activity toward indole, respectively. The reaction products of the reverse carboxylafion, indole-3-carboxylic acid and 2-methylindole-3-carboxylic acid, were isolated and identified through physicochemical analyses with the authentic compounds as reference. 

The dehydration reaction of aldoxime to form nitriles using the resting cells of Rhodococcus sp. YH3-3 was optimized. We found that the enzyme was induced by aldoxime and catalyzed the stoichiometric synthesis of nitriles from aldoximes at pH 7.0 and 30°C. Phenylacetonitrile once synthesized from phenylacetaldoxime was hydrolyzed to phenylacetic acid, since the strain has nitrile degradation enzymes such as nitrile hydratase and amidase. We have been successful in synthesizing phenylacetonitrile and other nitriles stoichiometrically by a selective inactivation of nitrile hydratase by heating the cells at 40°C for 1 h. Various nitriles were synthesized under optimized conditions from aldoximes in good yields. 

The data reported in table 1 show that the pectin fi action in active cells is rich in methyl-esterified galacturonans, whereas the resting cells contain mostly low-ester acidic pectins. 

These acidic molecules might result from either the lack of PMT activity or the action of PME, known to be present in most plant cell walls. Pectin methyltransferases and pectin methylesterases extracted from active and resting cells were therefore characterized. 

Pectin methyltransferases from active and resting cells of flax. 

Table 2. PME activity solubilized from cell walls of active and resting cells. Activities as neq.

As illustrated in Fig. 3, in both poplar stems and mung bean hypocotyls, basic isoforms became prevalent in mature, resting cells whereas in young, growing cells, neutral isoforms were predominant. 

Figure 3. PME isoform patterns in cell wall extracts fiom active and resting cells, a cell wall extracts from successive segments (A, B, C and D) sectioned along mui bean hypocotyb and exhibiting decreasing elongation rates a, and y m c the main PME isoforms present in the extracts, tteir pi are respectively around 7.5, S.5 and above 9.1. b cell wall extracts obtained from poplar cambium and inner bark tissues during cambial active (may) and rest (december and march) periods 1, 2 and 3 represent the activity of 3 groups of PME isoforms with pi around 5-6, 7.5 and above 9.1. Activities expressed as percent of total PME activity present in each extract.

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