Cloning and Expression of Islandisin, a New Thermostable Subtilisin from Fervidobacterium islandicum , in Escherichia coli

Abstract

A gene encoding a subtilisin-like protease, designated islandisin, from the extremely thermophilic bacterium Fervidobacterium islandicum (DSMZ 5733) was cloned and actively expressed in Escherichia coli. The gene was identified by PCR using degenerated primers based on conserved regions around two of the three catalytic residues (Asp, His, and Ser) of subtilisin-like serine protease-encoding genes. Using inverse PCR regions flanking the catalytic residues, the gene could be cloned. Sequencing revealed an open reading frame of 2,106 bp. The deduced amino acid sequence indicated that the enzyme is synthesized as a proenzyme with a putative signal sequence of 33 amino acids (aa) in length. The mature protein contains the three catalytic residues (Asp177, His215, and Ser391) and has a length of 668 aa. Amino acid sequence comparison and phylogenetic analysis indicated that this enzyme could be classified as a subtilisin-like serine protease in the subgroup of thermitase. The whole gene was amplified by PCR, ligated into pET-15b, and successfully expressed in E. coli BL21(DE3)pLysS. The recombinant islandisin was purified by heat denaturation, followed by hydroxyapatite chromatography. The enzyme is active at a broad range of temperatures (60 to 80°C) and pHs (pH 6 to 8.5) and shows optimal proteolytic activity at 80°C and pH 8.0. Islandisin is resistant to a number of detergents and solvents and shows high thermostability over a long period of time (up to 32 h) at 80°C with a half-life of 4 h at 90°C and 1.5 h at 100°C.Hyperthermophiles that grow optimally between 80 and 110°C belong to phylogenetically distant groups. Due to the low solubility of oxygen at high temperature, biotopes of hyperthermophiles are mainly anoxic (13). The eubacterial order Thermotogales, including the genera Thermotoga, Thermosipho, Fervidobacterium, Geotoga, Petrotoga, Thermopallium, and Marinitoga, comprises the most extremely thermophilic eubacteria presently known. Phylogenetically, this order represents the deepest branch within the bacteria (39). Members of the order Thermotogales are able to grow on various substrates such as proteins, starch, cellulose, and xylan. They are all capable of growing at temperatures above 60°C with an optimum of about 80°C. The cells of Thermotogales are characteristically surrounded by a "toga," a sheath-like envelope containing regularly arranged porin-like proteins (12).Enzymes from thermophilic microorganisms are called thermozymes. They can potentially be used in several industrial processes, in which they replace mesophilic enzymes or chemical catalysts. The main advantages of performing processes at higher temperatures are a reduced risk of microbial contamination, lower viscosity, improved transfer rates, and improved solubility of substrates (7). To date, the mechanisms that are responsible for the thermostability of proteins are not well understood. Often it is a combination of intrinsic properties, such as increased number of Van der Waals interactions, hydrogen bonds and ion-pairs, which lea...

Purification and characterization of cloned alkaline protease gene of Geobacillus stearothermophilus

“…One of the significant outcome of the present study was the expression of G. stearothermophilus B-1172, that showed stability after subjecting it to a range of temperature (50-90°C) under alkaline pH 7-11, for 3 h pre-incubation period. The pH and temperature stability of alkaline serine protease discovered in this study was reasonably higher than those reported for other proteases purified from B. subtilis, B. mojavensis, B. licheniformis, B. linens, islandisin from Fervidobacterium islandicum and nattokinases from B. subtilis that showed stability under similar conditions but only for 1 h of preincubation [39][40][41][42]. It is known that thermostable enzymes have rigid structure and higher temperature introduces flexibility and fluidity in their structural features [43,44].…”

Keratinases and sulfide from Bacillus subtilis SLC to recycle feather waste

“…A differential for this keratinase from B. subtilis SLC is that it exhibited proteolytic activity above 240 U ml -1 in the 2.0 to 5.0 pH range. The subtilisin like serine peptidases isolated from other Bacillus have optimal proteolytic activity at a basic pH (7-10), due to the requirement of a deprotonated histidine (Godde et al 2005;Radha and Gunasekara 2009). The optimal temperature for B. subtilis SLC keratinase activities was 60°C.…”

Cloning, expression and characterization of the serine protease gene fromChaetomium thermophilum

“…the protease from Microsporum canis has an optimum temperature around 55°C but it was only stable at 37°C (Mignon et al 1998); the protease from Stenotrophomonas maltophilia was found to be stable up to 40°C for only 5 min (Miyaji et al 2005); an extracellular serine proteinase purified from psychrotrophic Vibrio species (strain PA-44) undergoes autoproteolytic cleavage under mild heat treatment (30 min at 40°C) (Kristjansson et al 1999); the proteolytic activity of the purified proteinase from Ophiostoma piceae was determined to be optimal at pH 7-9 and 40°C (Abraham and Breuil 1996). It is noteworthy that although the optimum reaction temperature of this protease from C. thermophilum is still lower than that of proteases isolated from hyperthermophilic archaea (Voorhorst et al 1997;Matsumi et al 2005;Lee et al 2006Lee et al , 2007 or other extremely thermophile bacterium (Jang et al 2002;Wu et al 2004;Godde et al 2005). Some of them have the optimum temperature at 80-90°C, as most applications of enzymatic action occur at 40-60°C, the thermostable enzymes from thermophilic fungi may be more suitable for commercial applications than those from hyperthermophiles (Maheshwari et al 2000).…”