High-alkaline protease from Bacillus PB92 entrapped in calcium alginate gel. Physicochemical and microscopic studies.
High-alkaline protease (HAP) has been entrapped in Manugel DMB (an alginate gel) and assayed with two sizes and types of substrates: neutral protein casein and synthetic chromogenic tripeptide substrate, Z-Gly-Pro-Cit-PNA. Increasing the concentration of calcium chloride used for capsule formation decreased the measured enzyme activity with both substrates. Capsules were found to be stable in water for long periods of time, but they dissolved in both phosphate and carbonate-bicarbonate buffers. The pH vs activity profiles of encapsulated enzyme showed pH optima between 10 and 11 with both substrates. The calcium alginate matrix surrounding the enzyme was quite effective in stabilizing the enzyme at 20-25 degrees C and even more so at 4 degrees C. Enzyme stability at 50 degrees C was quite impressive, some enzyme activity being evident even after remaining for 1 wk at this temperature in water. Increasing concentrations of sodium dodecyl sulfate (SDS) were also found to inhibit the protease progressively, whereas a polyhexamethylene biguanidium chloride (PHMBH+Cl-) and SDS:PHMBH+Cl- combination showed the opposite effect. Optical microscopy, especially polarized light microscopy, provided a sensitive physical means of ascertaining some of the structural properties (sphericity, disorganization or organization, distinct layer enveloping the capsules, intensity of the maltese cross) of the capsules with and without enzyme before and after different chemical treatments and the presence or absence of the substrate.
D000464:Alginates / Q000378:metabolism*; D000595:Amino Acid Sequence; D001407:Bacillus / Q000201:enzymology*; D001645:Biguanides / Q000494:pharmacology; D002214:Capsules; D002364:Caseins / Q000737:chemistry; D003902:Detergents / Q000378:metabolism*; D004202:Disinfectants / Q000494:pharmacology; D004795:Enzyme Stability / Q000187:drug effects; D004800:Enzymes, Immobilized / Q000378:metabolism*; D005782:Gels; D020723:Glucuronic Acid; D006603:Hexuronic Acids; D006863:Hydrogen-Ion Concentration; D008863:Microspheres; D008969:Molecular Sequence Data; D012697:Serine Endopeptidases / Q000378:metabolism*; D012967:Sodium Dodecyl Sulfate / Q000494:pharmacology; D013379:Substrate Specificity; D013381:Subtilisins; D013696:Temperature; D014783:Viscosity
D000464:Alginates; D001645:Biguanides; D002214:Capsules; D002364:Caseins; D003902:Detergents; D004202:Disinfectants; D004800:Enzymes, Immobilized; D005782:Gels; D006603:Hexuronic Acids; C031233:polihexanide; D012967:Sodium Dodecyl Sulfate; D020723:Glucuronic Acid; D012697:Serine Endopeptidases; D013381:Subtilisins; C106091:serine protease PB92; C032923:microbial serine proteinases