The goal of this review is to present a comprehensive survey of the many intriguing facets of creatine (Cr) and creatinine metabolism, encompassing the pathways and regulation of Cr biosynthesis an...

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Creatine and Creatinine Metabolism

Proteases represent the class of enzymes which occupy a pivotal position with respect to their physiological roles as well as their commercial applications. They perform both degradative and synthetic functions. Since they are physiologically necessary for living organisms, proteases occur ubiquitously in a wide diversity of sources such as plants, animals, and microorganisms. Microbes are an attractive source of proteases owing to the limited space required for their cultivation and their ready susceptibility to genetic manipulation. Proteases are divided into exo- and endopeptidases based on their action at or away from the termini, respectively. They are also classified as serine proteases, aspartic proteases, cysteine proteases, and metalloproteases depending on the nature of the functional group at the active site. Proteases play a critical role in many physiological and pathophysiological processes. Based on their classification, four different types of catalytic mechanisms are operative. Proteases find extensive applications in the food and dairy industries. Alkaline proteases hold a great potential for application in the detergent and leather industries due to the increasing trend to develop environmentally friendly technologies. There is a renaissance of interest in using proteolytic enzymes as targets for developing therapeutic agents. Protease genes from several bacteria, fungi, and viruses have been cloned and sequenced with the prime aims of (i) overproduction of the enzyme by gene amplification, (ii) delineation of the role of the enzyme in pathogenecity, and (iii) alteration in enzyme properties to suit its commercial application. Protein engineering techniques have been exploited to obtain proteases which show unique specificity and/or enhanced stability at high temperature or pH or in the presence of detergents and to understand the structure-function relationships of the enzyme. Protein sequences of acidic, alkaline, and neutral proteases from diverse origins have been analyzed with the aim of studying their evolutionary relationships. Despite the extensive research on several aspects of proteases, there is a paucity of knowledge about the roles that govern the diverse specificity of these enzymes. Deciphering these secrets would enable us to exploit proteases for their applications in biotechnology.

Molecular and Biotechnological Aspects of Microbial Proteases

Bile salt biotransformations by human intestinal bacteria.

Proteolytic enzymes are ubiquitous in occurrence, being found in all living organisms, and are essential for cell growth and differentiation. The extracellular proteases are of commercial value and find multiple applications in various industrial sectors. Although there are many microbial sources available for producing proteases, only a few are recognized as commercial producers. A good number of bacterial alkaline proteases are commercially available, such as subtilisin Carlsberg, subtilisin BPN′ and Savinase, with their major application as detergent enzymes. However, mutations have led to newer protease preparations with improved catalytic efficiency and better stability towards temperature, oxidizing agents and changing wash conditions. Many newer preparations, such as Durazym, Maxapem and Purafect, have been produced, using techniques of site-directed mutagenesis and/or random mutagenesis. Directed evolution has also paved the way to a great variety of subtilisin variants with better specificities and stability. Molecular imprinting through conditional lyophilization is coming up to match molecular approaches in protein engineering. There are many possibilities for modifying biocatalysts through molecular approaches. However, the search for microbial sources of novel alkaline proteases in natural diversity through the "metagenome" approach is targeting a hitherto undiscovered wealth of molecular diversity. This fascinating development will allow the biotechnological exploitation of uncultured microorganisms, which by far outnumber the species accessible by cultivation, regardless of the habitat. In this review, we discuss the types and sources of proteases, protease yield-improvement methods, the use of new methods for developing novel proteases and applications of alkaline proteases in industrial sectors, with an overview on the use of alkaline proteases in the detergent industry.

Bacterial alkaline proteases: molecular approaches and industrial applications

Publisher Summary This chapter discusses the phosphopeptide mapping and phosphoamino acid analysis by two-dimensional separation on thin-layer cellulose plates. Peptide mapping is a powerful technique used to help determine peptide structure and composition of proteins. Peptide maps or fingerprints of proteolyzed proteins are usually obtained by resolution on either one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), reversed-phase high-performance liquid chromatography (HPLC), or by two-dimensional separation on thin-layer cellulose (TLC) plates. The most common applications of peptide mapping are (1) to compare proteins encoded by the same or related genes, (2) to prepare individual peptides for determining amino acid composition and sequence, and (3) to determine the precise location of amino acid residues that are posttranslationally modified by fatty acid acylation, glycosylation, methylation, acetylation, or phosphorylation.

Phosphopeptide mapping and phosphoamino acid analysis by two-dimensional separation on thin-layer cellulose plates.

Post-proline cleaving enzyme. Synthesis of a new fluorogenic substrate and distribution of the endopeptidase in rat tissues and body fluids of man.

Several peptides and peptide derivatives were tested for their inhibitory effect on prolyl endopeptidase and possible properties as anti-amnesic agents. Among the compounds tested, Z-Gly-Pro-CH2Cl, Z-Val-prolinal, Boc-Pro-prolinal, Z-Pro-prolinal, aniracetam and pramiracetam inhibited the enzyme activities at Ki values in the order of nM to microM, and the effect of the prolinal-containing peptide derivatives was specific for prolyl endopeptidase. Z-Pro-prolinal was the most effective inhibitor in vitro (Ki = 5 nM) and in vivo (50 to 70% inhibition in various organs of rat at a dose of 1 mumol/animal i.p.). Regional differences were observed in the effect of inhibitors on the brain enzyme activities: most active in mesencephalon, followed by striatum, cerebellum, hippocampus, hypothalamus; and inactive in cerebral cortex and medulla oblongata. In the passive avoidance learning test using rats, pretreatment with Z-Pro-prolinal prevented the induction of amnesia by scopolamine at the dose of 1 mumol/animal, i.p. Z-Val-prolinal, Z-Pyr-prolinal and Z-Gly-Pro-CH2Cl were also effective in the retention test at 24 and 48 h after the training trial. The antiamnesic effect of these compounds was approximately parallel to the in vitro inhibitory activities on prolyl endopeptidase. These results suggest the possibility that the inhibitors exhibit their anti-amnesic effect through the regulation of the enzyme activity in the brain.

Specific inhibitors for prolyl endopeptidase and their anti-amnesic effect.

Post-proline cleaving enzyme and post-proline dipeptidyl aminopeptidase. Comparison of two peptidases with high specificity for proline residues.

Proline-specific endopeptidase from Flavobacterium. Purification and properties.

A rat serum enzyme that catalyzes the conversion of a pro-drug, 7-ethyl-10-[4-(1-piperidino)-1-piperidino] carbonyloxycamptothecin (CPT-11), to an anticancer drug, 7-ethyl-10-hydroxycamptothecin (SN-38), was purified and its properties were characterized. The enzyme was purified by column chromatography on diethylaminoethyl Toyopearl 650M, QAE-Sephadex, Sephadex G-150, Con A-Sepharose and high performance liquid chromatography with an ion-exchanger column. It was most active at pH 7.5 and was stable at pH 4-9 for 1 h at 30 degrees C. The molecular weight was estimated to be 60 and 57 kDa by gel filtration and sodium dodecylsulfate-polyacrylamide gel electrophoresis methods, respectively, and the isoelectric point was 4.6, as determined by isoelectric focusing. The Km value for CPT-11 was 0.28 microM. This enzyme was inhibited by diisopropyl phosphorofluoridate (DFP) and phenylmethanesulfonyl fluoride (PMSF) but insensitive to eserine, p-chloromercuribenzoate (PCMB) and ethylenediaminetetraacetate (EDTA). The enzyme also hydrolyzed p-nitrophenylacetate (p-NPA), a commonly used substrate for esterases, but was not active toward acetylcholine, suggesting that the enzyme is a carboxylesterase[EC 3.1.1.1]. During the hydrolyses of CPT-11 and p-NPA, an initial burst phenomenon similar to that found in the alpha-chymotrypsin-catalyzed hydrolysis of p-NPA was observed. Kinetic analysis revealed that the deacylation of the enzyme is the rate-limiting step in substrate hydrolysis. This enzyme was found to also split other ester derivatives of SN-38 besides CPT-11.

Tadashi Yoshimoto

Papers

Post-proline cleaving enzyme. Synthesis of a new fluorogenic substrate and distribution of the endopeptidase in rat tissues and body fluids of man.

Specific inhibitors for prolyl endopeptidase and their anti-amnesic effect.

Post-proline cleaving enzyme and post-proline dipeptidyl aminopeptidase. Comparison of two peptidases with high specificity for proline residues.

Proline-specific endopeptidase from Flavobacterium. Purification and properties.

CPT-11 Converting Enzyme from Rat Serum : Purification and Some Properties