title: Immobilization of keratinolytic metalloprotease from Chryseobacterium sp. strain kr6 on glutaraldehyde-activated chitosan.
abstract: Keratinases are exciting keratin-degrading enzymes; however, there have been relatively few studies on their immobilization. A keratinolytic protease from Chryseobacterium sp. kr6 was purified and its partial sequence determined using mass spectrometry. No significant homology to other microbial peptides in the NCBI database was observed. Certain parameters for immobilization of the purified keratinase on chitosan beads were investigated. The production of the chitosan beads was optimized using factorial design and surface response techniques. The optimum chitosan bead production for protease immobilization was a 20 g/l chitosan solution in acetic acid [1.5% (v/v)], glutaraldehyde ranging from 34 g to 56 g/l, and an activation time between 6 and 10 h. Under these conditions, above 80% of the enzyme was immobilized on the support. The behavior of the keratinase loading on the chitosan beads surface was well described using the Langmuir model. The maximum capacity of the support (qm) and dissociation constant (Kd) were estimated as 58.8 U/g and 0.245 U/ml, respectively. The thermal stability of the immobilized enzyme was also improved around 2-fold, when compared with that of the free enzyme, after 30 min at 65 degrees C. In addition, the activity of the immobilized enzyme remained at 63.4% after it was reused five times. Thus, the immobilized enzyme exhibited an improved thermal stability and remained active after several uses.
authors.affiliation: Laboratory of Biochemistry and Applied Microbiology, Department of Food Science, Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil.
authors.forename: Silvana Terra
authors.initials: ST
authors.lastname: Silveira
authors.forename: Sabrine
authors.initials: S
authors.lastname: Gemelli
authors.forename: Jeferson
authors.initials: J
authors.lastname: Segalin
authors.forename: Adriano
authors.initials: A
authors.lastname: Brandellli
mesh_terms: D000595:Amino Acid Sequence; D001709:Biotechnology / Q000379:methods; D048271:Chitosan / Q000378:metabolism*; D045247:Chryseobacterium / Q000201:enzymology*; D004795:Enzyme Stability; D004800:Enzymes, Immobilized / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D005976:Glutaral / Q000378:metabolism; D007633:Keratins / Q000378:metabolism*; D013058:Mass Spectrometry; D045726:Metalloproteases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D011485:Protein Binding; D017386:Sequence Homology, Amino Acid; D013696:Temperature
chemical_list: D004800:Enzymes, Immobilized; D007633:Keratins; D048271:Chitosan; D045726:Metalloproteases; D005976:Glutaral