Release of β-galactosidase by Permeabilization of Indigenously Isolated Lactobacillus acidophilus Using Lysozyme

There is a constant increase in the demand for biomolecules produced by microorganisms for a wide range of commercial applications. Although many such microbial enzymes and proteins are produced extracellularly, an equally high proportion of such potentially useful products are retained within the cells.1–2 One such enzyme, β-galactosidase (EC 3.2.1.23), has found wide application for lactose hydrolysis in the dairy industry. We have earlier reported an indigenous strain of gram positive Lac to ba cillus acidophilus isolated from Eleusine co racana to be a potential producer of intracellular β-galactosidase.3–4 Intracellular location (periplasmic, cytoplasmic or cell-wall bound) can hinder recovery of such biomolecules and increase complications in their downstream processing. It therefore becomes necessary to select an efficient cell disruption or permeabilization method that ensures high enzyme recovery and cost-effective processing.4 Various physical, chemical, enzymatic, or mechanical methods are available for cell disruption to release intracellular enzymes and proteins.1,5 Widely used mechanical methods such as bead milling, high-pressure homogenization and ultrasonication cause non-specific disruption of the entire cell wall structure to small fragments and may cause product loss due to high heat generation associated with such processes.6 Chemical and enzymatic permeabilization, on the other hand, through interaction with specific cell wall components cause selective release without generation of small cell fragments.1,7 Enzymatic lysis involves use of enzymes that offer advantages like high specificity in their lysis mechanism and mild reaction conditions. The permeabilization process thereby ensures minimal damage and maximum stability of the released protein with the residual cell debris being large enough to assist easy separation of the protein from the cell mass.1,5 To achieve this, the selection of an appropriate enzyme for permeabilization plays a key role, which, in turn is largely influenced by the type of microorganism (Gram-positive or Gram-negative) and intracellular location of the desired product. Lysozyme (EC 3.2.1.17), also known as muramidase or N-acetylmuramide glycanhydrolase, are glycoside hydrolases that specifically permeabilize bacterial cell wall by catalyzing hydrolysis of 1,4-beta-linkages between N-acetylmuramic acid and N-acetyl-D-glucosamine residues in their peptidoglycan layer. The presence of a pronounced peptidoglycan layer (50–80 % of the cell wall) and lack of the outer membrane make Gram-positive bacteria highly susceptible to permeabilization with lysozyme. Moreover, lysozyme derived from egg white is relatively cheap and currently is the only bacteriRelease of β-galactosidase by Permeabilization of Indigenously Isolated Lactobacillus acidophilus Using Lysozyme