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

Alkaline prorcasex are of considcruhlc interest in view of their activity and stability at alkaline pll. This review describes the protcascs that can resist extreme alkaline environments produced by a wide range of ulk alophilic microorg anisms. Different isol.uion methods which enable the screening and selection of promising organisms 1'01' industrial production are discussed. The various nutritional and cnvironmcntul parameters affecting the production of alkaline protcuscs are dclinctucd. The production of prorcuscs by free and immobilized whole cells is discussed. The purification. properties. and applications of these pretenses are also discussed.

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A Review on Microbial Alkaline Proteases

The efficiency of any fermentation process depends largely on downstream processing which ensures the purity and quality of the biochemicals. Since many biomolecules have narrow tolerance limits of pH, temperature, osmotic pressure, surface charges, etc. the extraction and isolation techniques should be specific and compatible to the product. Therefore, it is not surprising that about 60-90% of the cost of a biological process is that expended on downstream processing. Aqueous two-phase systems (ATPS) result from the incompatibility or immiscibility of polymers, either between two polymers in water or a polymer solution with a salt solution. ATPS contain about 80-90% water and thereby can provide an excellent environment for cells, cell organelles and biologically active substances. 'Extractive fermentation' is an emerging technique that involves the use of ATPS-based in situ fermentation processes. The advantages of such a system include rapid mass transfer due to low-interfacial tension, ease of operation under continuous mode, rapid and selective separation, biocompatibility, separation at room temperature, easy and reliable scale-up of bench scale results to production scale, ecofriendliness, suitability for systems with product inhibition and high yield of biomolecules. In this review, we will consider the basic principles involved and factors affecting extractive fermentation, technical aspects and technological developments like use of ligands and surfactants to improve separation, recent trends and future prospects of extractive fermentation systems. This review will also focus on the parameters responsible for better separation for in situ fermentation.

Technological aspects of extractive fermentation using aqueous two-phase systems

Proteases or peptidases constitute the largest group of enzymes in bio-industry with a long array of uses. They play an invincible role in industrial biotechnology, especially in detergent, food and pharmaceutical arena. This focused review encompasses an overview on alkaline proteases, mainly of microbial sources in a handy module. Following an introduction and general classification with evolutionary insight, major sources of proteases (animal, plant and microbial including fungal, bacterial), their general properties with mechanism of action and molecular masses are discussed. Proteases fromBacillusspp. have been given special attention. In addition to this, an overview on the applications of proteases in detergent, tannery, food, metal recovery and waste treatment industries is also addressed briefly.

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Versatility of microbial proteases

Alkaline protease production by Bacillus thuringiensis H 14 was studied in aqueous two-phase systems composed of PEG X (X = 9000, 6000, 4000) and potassium phosphate. The top phase is continuous and rich in PEG while the bottom phase is dispersed and is rich in phosphate, microbial cells being mainly retained in the bottom phase. The alkaline protease produced during fermentation, partitioned into the upper phase (about 80%) and total protease produced were about 2·80 and 2·26 times higher than that of homogeneous fermentation when the fermentations were carried out in aqueous two-phase systems from the beginning and made after 45 h of inoculation respectively. © 1997 SCI.

Srinivas Hotha

Papers

Production of Alkaline Protease by Bacillus thuringiensis H 14 in Aqueous Two‐Phase Systems