title: Enzyme dehydration using Microglassification™ preserves the protein's structure and function.
abstract: Controlled enzyme dehydration using a new processing technique of Microglassification™ has been investigated. Aqueous solution microdroplets of lysozyme, α-chymotrypsin, catalase, and horseradish peroxidase were dehydrated in n-pentanol, n-octanol, n-decanol, triacetin, or butyl lactate, and changes in their structure and function were analyzed upon rehydration. Water solubility and microdroplet dissolution rate in each solvent decreased in the order: butyl lactate > n-pentanol > triacetin > n-octanol > n-decanol. Enzymes Microglassified™ in n-pentanol retained higher activity (93%-98%) than n-octanol (78%-85%) or n-decanol (75%-89%), whereas those Microglassified™ in triacetin (36%-75%) and butyl lactate (48%-79%) retained markedly lower activity. FTIR spectroscopy analyses showed α-helix to β-sheet transformation for all enzymes upon Microglassification™, reflecting a loss of bound water in the dried state; however, the enzymes reverted to native-like conformation upon rehydration. Accelerated stressed-storage tests using Microglassified™ lysozyme showed a significant (p < 0.01) decrease in enzymatic activity from 46,560 ± 2736 to 31,060 ± 4327 units/mg after 3 months of incubation; however, it was comparable to the activity of the lyophilized formulation throughout the test period. These results establish Microglassification™ as a viable technique for enzyme preservation without affecting its structure or function.
authors.affiliation: Department of Mechanical Engineering and Materials Science, Duke University, Durham, North Carolina, 27708.
authors.lastname: Aniket
authors.forename: David A
authors.initials: DA
authors.lastname: Gaul
authors.forename: Deborah L
authors.initials: DL
authors.lastname: Bitterfield
authors.forename: Jonathan T
authors.initials: JT
authors.lastname: Su
authors.forename: Victoria M
authors.initials: VM
authors.lastname: Li
authors.forename: Ishita
authors.initials: I
authors.lastname: Singh
authors.forename: Jackson
authors.initials: J
authors.lastname: Morton
authors.forename: David
authors.initials: D
authors.lastname: Needham
mesh_terms: D000818:Animals; D002374:Catalase / Q000737:chemistry* / Q000502:physiology; D002417:Cattle; D002645:Chickens; D002918:Chymotrypsin / Q000737:chemistry* / Q000502:physiology; D003890:Desiccation / Q000379:methods*; D004789:Enzyme Activation / Q000502:physiology; D005612:Freeze Drying / Q000379:methods; D005898:Glass; D006735:Horseradish Peroxidase / Q000737:chemistry* / Q000502:physiology; D055616:Microtechnology / Q000379:methods*; D009113:Muramidase / Q000737:chemistry* / Q000502:physiology
chemical_list: D006735:Horseradish Peroxidase; D002374:Catalase; D009113:Muramidase; D002918:Chymotrypsin; C117608:alpha-chymotrypsin
keywords: FTIR; dehydration; drying; enzymes; microparticles; protein formulation; protein structure