Characterization of Bacillus cereus isolated from fermented cabbage and conventional optimization of extracellular protease production

摘要

A strain of Bacillus cereus, isolated from fermented cabbage, was tested a laboratory scale in order to evaluate the production of extracellular protease on batch cultivation in Modified Basal Medium without agar (MB). Furthermore optimization of culture medium and cultivation conditions were carried out too.B. cereus spp. presented a typical growth kinetic with a short lag phase. The best conditions for B. cereus spp. growth were found at pH 7, 32oC and in MB with glucose. Respecting to protease production, the best expression were at pH 8 and 32oC. At this temperature the addition of modifiers in the fermentation media did not produce a significant improvement in the protease production. The novel properties of the protease liberated by this strain could have important benefic for the biotechnology industry. Introduction The biosynthesis of proteolytic enzymes by microorganisms is not only of scientific but also of great practical importance. Bacteria, moulds and yeast are some of the microorganisms that are able to produce proteases. Proteases are the most important kind of enzymes from an industrial point of view; they execute a large variety of functions and have important biotechnological applications. They could be useful in leather processing, laundry detergents, producing of protein hydrolysates and food processing (Salem S.R. et al., 2009). Proteases are commonly classified according to their optimum pH: acidic protease, neutral protease and alkaline protease. There have been extensive researches on the properties and functionalities of acidic or alkaline proteases (Dunn-Coleman N. et al.). Among bacteria, Bacillus strains are the most important producers of commercial proteases (Ghorbel-Frikha B. et al., 2005), being specific producers of extracellular protease. Strains of Bacillus cereus possess high proteolytic potential, expressed by their ability to synthesize and excrete protease into the medium. This spore forming, mesophilic and relatively anaerobic rod is potent in terms of biosynthesis of various hydrolytic enzymes, including proteases, alpha- and beta-glucanases or lipases. Proteolytic enzymes containing serine proteinases (Shaheen M. et al., 2008), Ca2+- and Zn2+- dependent metaloproteinases (Karbalaei-Heidari H.R. et al., 2007), keratinases (Ghosh A. et al., 2008; Brandelli A. et al., 2005) and collagenases (Nip W.K. et al., 2006) are known to be produced by B. cereus. Microbial proteases account for approximately 60% of the total enzyme sales in the world (Banik R.M. et al., 2004). One of the several challenges faced by industrial application of microbial proteases is to obtain its optimal activity and stability. Each organism or strain has its own special conditions for maximum enzyme production. Extracellular protease production by microorganisms is highly influenced by media components, variation in carbonitrogen ratio, presence of some easily metabolically sugars, such as glucose (Beg Q.A.K., 2003) and presence of metal ions (Secades P. et al., 1999). Several others factors, such as aeration, inoculums density, pH, temperature and incubation time also affect the amount of protease production and their interaction plays an important role in the synthesis of these enzymes (Puri S. et al., 2002). Zambare et al. (2007) described an extracellular protease produced by Bacillus cereus that grow on a medium containing starch, wheat bran and soya flour (SWS).Taken into account the potential uses of the highly demanded Bacillus proteases, there is a need for the research of new strains of bacteria that produce enzymes with novel properties and the development of low cost industrial medium formulations. In commercial practice, the optimization of medium composition is done to maintain a balance between the various medium components, thus minimizing the amount of unutilized components at the end of fermentation. Recently, in a previous work (Pérez Borla et al., 2009) through the utilization of a simple and re