1. Stabilization of Bacillus Licheniformis ATCC alkaline protease by immobilization and modification Samia A. Ahmed, Shireen A. Saleh and Ahmed F. Abdel-Fattah Department of Chemistry of Natural and Microbial Products, NRC, Cairo, Egypt.
Introduction
Proteases execute a large variety of functions and have important biotechnological applications in detergents, leather, food, pharmaceutical industries and bioremediation processes (Gupta et al., 2002). Stabilization against thermal inactivation can be performed in several ways such as immobilization and chemical modification (Ben Ammar et al., 2002; Fernandez et al., 2002). For industrial applications, the immobilization of protease on a solid support can offer several advantages, including repeated usage of enzyme, ease of product separation, improvement of enzyme stability and continuous operation in packed-bed reactors ((Abdel-Naby et al., 1998).
Materials and methods
Strain: B. licheniformis ATCC was obtained from the American Type Culture Collection, USA.
Enzyme assay: was determined as reported earlier Bergkvist (1963)
Alkaline protease modification: was carried out according to Ben Ammar (2002).
Immobilization methods: were carried out according to Abdel-Naby et al., 1998.
Results
Alkaline protease from B. licheniformis ATCC21415 was partially purified by fractional precipitation at 70% ethanol which showed 6.2-fold purification. The enzyme was immobilized on different carriers by different methods. Physical adsorption of enzyme on loofa (as a new carrier) had the highest immobilization yield (70.5%).Chemical modification of the enzyme by covalent coupling with sodium -periodate activated amylopectin retained (78.3%) of the original activity. The catalytic properties and the stability of the immobilized and modified enzymes were compared to those of the native enzyme. The crude enzyme digested some natural proteins and was able to extract collagen from chicken skin.
Effect of crude enzyme on some natural proteins
Thermal stability of B. licheniformis ATCC alkaline protease
References
Gupta R, Beg,QK and Lorenz P. Applied Microbiology and Biotechnology (2002); 59(1):
Ben Ammar Y, Matsubara T, Ito K, Iizuka M and Minamiura N. Enzyme and Microbial Technology,(2002); 30:
Fernandez M, Villalonga ML, Cao R, Alex F and Villalonga R. Biotechnology, (2002);36:
Abdel-Naby MA, Ismail A-MS, Ahmed SA and Abdel-Fattah AF. Bioresource Technology, (1998); 64:
Bergkvist R. Acta Chemica Scandinavica, (1963); 17:
Temperature
(ºC)
Time of
heating (min)
Residual activity (%)
Free enzyme
Immobilized enzyme
Modified enzyme 50 60 70 45
86.4
25.8
1.3
90.3
70.8
39.6
92.1
88.0
41.5
83.1
19.4
0.0
88.7
59.9
18.2
90.4
76.1
19.6 75
81.5
9.8
86.3
50.5
2.6
87.8
63.6
3.9 90
69.2
84.7
42.8
86.0
51.2