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

Microbial alkaline proteases: From a bioindustrial viewpoint

A mathematical model for gluconic acid fermentation by Aspergillus niger

Transforming growth factor-β (TGF-β) has been reviewed for its sources, types of isoforms, biochemical effects on cartilage formation/repair, and its possible clinical applications. Purification of three isoforms (TGF-β-1, β-2 and β-3) and their biochemical characterization revealed mainly their homo-dimer nature, with heterodimers in traces, each monomer comprised of 112 amino acids and MW. of 12 500 Da. While histo-chemical staining by a variety of dyes has revealed precise localization of TGF-β in tissues, immune-blot technique has thrown light on their expression as a function of age (neonatal vs. adult), as also on its quantum in an active and latent state. X-ray crystallographic studies and nuclear magnetic resonance (NMR) analysis have unraveled mysteries of their three-dimensional structures, essential for understanding their functions. Their similarities have led to interchangeability in assays, while differences have led to their specialized clinical applicability. For this purpose, their latent (inactive) form is changed to an active form through enzymatic processes of phosphorylation/glycosylation/transamination/proteolytic degradation. Their functions encompass differentiation and de-differentiation of chondrocytes, synthesis of collagen and proteoglycans (PGs) and thereby maintain homeostasis of cartilage in several degenerative diseases and repair through cell cycle signaling and physiological control. While several factors affecting their performance are already identified, their interplay and chronology of sequences of functions is yet to be understood. For its success in clinical applications, challenges in judicious dealing with the factors and their interplay need to be understood. J. Cell. Physiol. 226: 3094–3103, 2011. © 2011 Wiley Periodicals, Inc.

An update on transforming growth factor‐β (TGF‐β): Sources, types, functions and clinical applicability for cartilage/bone healing

Glucoamylase (EC 3.2.1.3) hydrolyzes polysaccharides from the nonreducing chain ends by cleaving α-1,4 and α-1,6 glycosidic bonds consecutively. Glucoamylases are used mainly in the production glucose syrup, high fructose corn syrup, and in whole grain and starch hydrolysis for alcohol production. This paper reviews the status of glucoamylases with respect to microbial sources, biochemical and physical properties. Methods used to assay glucoamylase activity are also compared, with reference being made to the specificity of spectrophotometric methods in detecting end products of the enzyme action. Commercial glucoamylases and development of immobilization techniques are discussed. Also, structural analysis of glucoamylase and the main amino acids involved in catalysis and starch binding are emphasized. The cloning of the glucoamylase gene in Saccharomyces cerevisiae for the brewing of low calorie beer is presented. This review highlights the use of glucoamylase in the food industry.

Glucoamylases: microbial sources, industrial applications and molecular biology — a review

An economical, upgraded, stabilized and efficient preparation of amyloglucosidase

A phenotypic requirement for cysteine was introduced inBacillus licheniformis producing alkaline protease to facilitate its isolation from poor or nonproducerBacillus species. This facilitated purification of the strain in cases of cross-contamination, preparation of good inocula for commercial production and stabilization of alkaline protease harvest values, alleviating economic losses incurred through cross-contamination.

Isolation of Bacillus licheniformis mutants for stable production profiles of alkaline protease

Optimized economical procedures for the submerged batchwise (250-L scale) production and recovery of injectable-grade calcium gluconate are devised. While the optimal parameters of production envisaged the use of Aspergillus niger, salts-fortified dextrose-rich production medium, pH 6.5±0.1, 29±1°C, 250±10 rpm, 1.0-1.5 vol of air (vol of medium) −1 min −1 aeration rate over 24±2 h duration, and intermittent neutralization with calcium carbonate, the optimal recovery procedure consisted of charcoal decolorization, vacuum filtration, methanol-aided precipitation, centrifugation, vacuum drying, and pulverization to provide 85% recovery of calcium gluconate, passing Indian/British pharmacopeial tests for the injectable-grade preparation

Fermentative production of injectable-grade calcium gluconate

In view of the soaring prices of corn and tapioca starch, use of their hydrolysate in the production of 70% sorbitol became less remunerative. Therefore, an economical alternative is explored by using hydrolysates of cereal flours, namely, rice (Oryzae sativa), wheat (Triticum aestivum), jowar (Sorghum vulagare) and bajra (Pennisetum typhoideum). A protocol is devised to a) prepare their high DE hydrolysates, b) purify it after saccharification, c) monitor the chemical characteristics of concentrated hydrolysate, as fedstock for Raney nickel catalyzed pressure hydrogenation and d) finally prepare 70% sorbitol. Merits and demerits of hydrolysates of these cereal flours are discussed in terms of operational limitations and percentage recovery, the governing factors for their industrial acceptability. Rice flour hydrolysate appears to be an alternative substrate, operationally and economically.



Alternative wirtschaftliche starkehaltige Substrate fur die Herstellung von 70%igem Sorbitol.



Im Hinblick auf die steigenden Preise fur Mais- und Tapiokastarke wird die Verwendung ihrer Hydrolysate zur Herstellung von 70%igem Sorbitol immer weniger lohnend. Daher wurde eine wirtschaftliche Alternative durch Verwendung der Hydrolysate von Getreidemehlen, namlich Reis (Oryzae sativa), Weizen (Triticum aestivum), Jowar (Sorghum vulgare) und Bajra (Pennisetum typhoideum), erprobt. Es wurde ein Verfahren entwickelt, um a) ihre Hoch-DE-Hydrolysate herzustellen, b) diese nach der Verzuckerung zu reinigen, c) die chemischen Charakteristika der konzentrierten Hydrolysate festzustellen als Grundlage fur die mit Raney-Nickel katalysierte Druckhydrierung und d) schlieslich 70%igen Sorbitol darzustellen. Vorzuge und Nachteile der genannten Mehle werden diskutiert hinsichtlich verfahrensmasiger Grenzen und prozentualer Ausbeute, den beherrschenden Faktoren fur einen industriellen Einsatz. Reismehlhydrolysat durfte sowohl verfahrenstechnisch als auch wirtschaftlich ein alternatives Substrat darstellen.

Deepak N. Shah

Papers

An economical, upgraded, stabilized and efficient preparation of amyloglucosidase

Isolation of Bacillus licheniformis mutants for stable production profiles of alkaline protease

Fermentative production of injectable-grade calcium gluconate

Alternate Economical Starchy Substrates for the Production of 70% Sorbitol