Clarified enzyme product

Enzymes can be expressed as either intracellular or extracellular products. Isolation of intracellular molecules often involves the separation of complex biological mixtures. On the other hand, extracellular molecules are generally released into the medium containing fewer other components. 

Depending on the cell separation equipment used, the cell removal can be facilitated by inducing self-flocculation of cells (Stratford, 1989), or by adding flocculation polymers to the fermentation broth. Flocculants are added to create larger particles 

FIGURE 6.5 Simplified recovery schematic for a clarified enzyme recovery process. 

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In the process of CGTase production by fermentation, after the termination of fermentation, in addition to enzymes and other metabolites, the fermentation broth also contains large amounts of bacteria and medium residues. The purification of the enzyme cannot be completed without filtration and separation processes of the broth. If the CGTase prepared without this process is directly applied to the production of CD, the yield and quality of CD would greatly be reduced. Thus, the broth must be processed to obtain clarified enzyme solution. In the preparation of CGTase, bacteria and some medium residues are usually removed by centrifugal separation. 

In order to clarify the potential physiological function of RJ, the gastrointestinal enzyme production of antihypertensive peptides from RJ was studied, showing that protease N-treated RJ (ProRJ) and peptides from ProRJ (Ile-Tyr (lY), Val-Tyr (VY), and Ile-Val-Tyr (IVY)) inhibited angiotensin I-converting enzyme (ACE) activity, and they have an antihypertensive effect in repeated oral administration for 28 days on spontaneously hypertensive rats (SHR). These results suggested that peptides contributed to the antihypertensive effect of ProRJ, which was computed to be about 38%. Therefore, it is considered that intake of RJ (and its peptides), as a functional food, would be beneficial for improving blood pressure in people with hypertension [81-84]. 

The structure of the complex between native enzyme and tyrosyl adenylate has been elucidated (Rubin and Blow, 1981). Tyrosyl adenylate is one of the products of the first step in the enzyme mechanism (47), and is the substrate for the second step of the reaction (48). Eleven possible hydrogen bonds are identified and the structure is given in [88]. Previously, the structure of the complex between the enzyme and the competitive inhibitor tyrosinyl adenylate had been clarified (Monteilhet and Blow, 1978). 

The reaction path of thiamine-dependent catalysis is essentially unchanged in the presence of an apoenzyme, except that the enzyme active site residues increase reaction rates and yields and influence the substrate and product specificity. The X-ray crystal structures of TDP-dependent enzymes have clarified this view and permitted an understanding of the roles of the individual amino acids of the active site in activating and controlling the thiazolium reactivity [36-40]. 

Despite the large amount of biochemical and structural studies of sirtuins in complex with various substrates, cofactors and reaction products, the catalytic mechanism of this class of enzymes is still a matter of debate. SN -like [56] and SN -like [60] mechanisms have been inferred from structural studies but further biochemical and possibly structural studies will be required to clarify which mechanism is used by sirtuins. It should also be noted that another matter of debate concerns the mode of noncompetitive inhibition of sirtuins by the reaction product nicotinamide [62], various structural studies having highlighted different binding pockets for this molecule [63, 64]. 

When a racemic substance is hydrogenated or when the reduction leads to the production of centers of asymmetry, the phytochemical reduction will take at first a completely or partially asymmetric course. Examples of such asymmetric reactions are the conversions of pure racemic valeraldehyde, acetaldol, furoin and furil, diacetyl and acetyl-methylcarbinol to optically active alcohols. Occasionally meso forms also arise, as for example in the case off glycols (p. 84). The reasons for the stereochemical specificity of these reactions have not been clarified. This type of phenomenon has frequently been observed in the related intramolecular dismutation of keto aldehydes, especially if enzyme materials of differing origins are used. 

Hydroxymellein production by the synthase was also observed when NADH was employed instead of NADPH. However, the effect of NADPH on enzyme activity was not fully replicated by NADH, and the activity of NADH-dependent 6-hydroxymellein production of the synthase was usually 50 - 70% of that for the corresponding NADPH-mediated reaction [80]. To clarify the biochemistry of the lower yield using NADH, the kinetic parameters of the reactions were determined under various reaction conditions. The Km value of 6-hydroxymellein synthase for NADPH was estimated to be 70 pM, while for NADH it was 10 pM, so that the affinity of the enzyme protein for NADPH is appreciably lower than for NADH. It follows that the difference in affinities of NADPH and NADH for the enzyme protein is not responsible for the low efficiency of NADH-mediated product formation. 

The quality of extracted citrus juices depends on enzyme reactions that occur not only in the fruit during the development period, but also in the juice during processing. When juice is extracted from citrus fruit, enzymes are released from their normal restraint in the cell. Several of these enzymes catalyze reactions that adversely affect taste and appearance of the juice. Unless the reactions are controlled, the juice products will not meet the standards of quality set up by the USDA Food Safety and Quality Service. The two reactions of commercial importance are the hydrolysis of pectin to pectic acid, which clarifies juice, and the lactonization of limonoic acid A-ring lactone to the bitter compound, limonin. Research efforts to identify and characterize the reactions, to isolate and purify the enzymes, and to develop methods to control the reactions are described in this review. 

The metabolic study, considered separately, consists of treatment of the animal with the radiolabeled compound followed by chemical analysis of all metabolites formed in vivo and excreted via the lungs, kidneys, or bile. Although reactive intermediates are unlikely to be isolated, the chemical structure of the end products may provide vital clues to the nature of the intermediates involved in their formation. The use of tissue homogenates, subcellular fractions, and purified enzymes may serve to clarify events occurring during metabolic sequences leading to the end products. 

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