Thiol-dependent serine proteinase from Paecilomyces lilacinus: Purification and catalytic properties

Abstract

An extracellular thiol-dependent serine proteinase was isolated from culture medium filtrate of the microscopic fungus Paecilomyces lilacinus with a yield of 33%. The enzyme is inactivated by specific inhibitors of serine proteinases, phenylmethylsulfonyl fluoride, as well as by chloromercuribenzoate and mercury acetate, but is resistant to chelating agents. The proteinase has broad specificity, hydrolyzes proteins and p-nitroanilides of N-acylated tripeptides, exhibiting maximal activity in hydrolysis of substrates containing long hydrophobic and aromatic residues (norleucine, leucine, phenylalanine) as well as arginine at the P1 position. The enzyme has a molecular weight of 33 kD. The enzyme is most active at pH 10.0-11.5; it is thermostable and is characterized by broad optimum temperature range (30-60 degrees C), displaying about 25% of maximal activity at 0 degrees C. The N-terminal sequence of the enzyme (Gly-Ala-Thr-Thr-Gln-Gly-Ala-Thr-Gly/Ile-Xxx-Gly) has no distinct homology with known primary structures of serine proteinases from fungi and bacilli. Based on its physicochemical and enzymatic properties, the serine proteinase from P. lilacinus can be classified as a thiol-dependent subtilisin-like enzyme.

Enhanced Thermostability of a Fungal Alkaline Protease by Different Additives

“…The protective effect of 5 mM Ca 2+   was similar to that with 10 mM Mg 2+ , but effect of 20 mM Mg 2+ was less than 10 mM Ca 2+ . Similar pattern of increase in thermostability by Ca 2+ was also observed in alkaline proteases from P. lilacinus , Aspergillus oryxae , A. tamarii, and Conidiobolus coronatus [13, 18–20]. 5 mM CaCl 2 offered maximum protection against thermal inactivation of A. tamarii protease increasing the half-life at 55°C from 20 to 140 min.…”

“…But the protective effect gradually decreased with increasing CaCl 2 concentration, while the addition of other salts such as MgCl 2 , MnCl 2 , and NaCl did not offer any protection [20]. Addition of 1 mM Ca 2+ increased the stability of P. lilacinus proteinase at 60°C by 2.5 times [18]. Ca 2+ ion played a vital role in maintaining the active confirmation of the enzyme at higher temperatures [21].…”

Isolation of a thiol-dependent serine protease in peanut and investigation of its role in the complement and the allergic reaction

“…Moreover, proteinase K is inhibited by mercury ions (Müller and Saenger, 1993). More recently, other thiol-dependent serine proteases from Bacillus mojavensis (Beg and Gupta, 2003) and a subtilisin-like enzyme from Paecilomyces lilacinus (Kotlova et al, 2007) were also described. In those cases, the binding of the mercury ion, either covalently or by complexation of the cysteine residue located in the vicinity of the catalytic site of these proteases, affects their proteolytic activity.…”

Bioprocessing of “Hair Waste” byPaecilomyces lilacinusas a Source of a Bleach-Stable, Alkaline, and Thermostable Keratinase with Potential Application as a Laundry Detergent Additive: Characterization and Wash Performance Analysis

“…These results indicate that the enzyme is stable in a wide range of pH and temperatures, preserving more than 60% of its activity after 2 h of incubation at pH 11 and 45°C. A serine proteinase purified from P. lilacinus (Thom) Samson VKM F-3891 displayed 40% of residual activity after 3 h incubation at 60°C [29]. However, in other cases, such as the serine proteinase from Aspergillus chrysogenum , enzyme stability in the alkaline range is substantially reduced.…”