Purification, Characterization and Thermodynamic Assessment of an Alkaline Protease by Geotrichum Candidum of Dairy Origin

Abstract

Background Alkaline proteases is the important group of enzymes having numerous industrial applications including dairy food formulations. Objectives The current study deals with the purification and characterization of an alkaline serine protease produced by Geotrichum candidum QAUGC01, isolated from indigenous fermented milk product, Dahi. Material and Methods In total twelve G. candidum strains were screened for their proteolytic activity by using standard protease assay. The protease production from G. candidum QAUGC01 was optimized by varying physio-chemical conditions. The protease was purified by using two-step method: ammonium sulfate precipitation and gel filtration chromatography. Protease was further characterized by studying various parameter like temperature, pH, modulators, metal ions and organic solvent. A thermodynamic study was also carried out to explore the half-life of protease. Results The G. candidum grew profusely at 25 °C and at an initial pH of 4.0 for 72 h of incubation producing 26.21 U/ml maximum extracellular protease. Protease revealed that V max and K m was 26.25 U.ml -1 .min -1 and 0.05 mg.mL -1 , respectively using casein as substrate. The enzyme was stable at a temperature range (25–45 °C) and pH (8–9). Residual enzyme activity was strongly inhibited in the presence of PMSF (7.5%). The protease could hydrolyze proteinaceous substrates, casein (98%) and BSA (95%). The thermodynamic studies explored that the half-life of the enzyme that was 106.62 min, 38.72 min and 15.71 min at 50, 60 and 70 °C, respectively. Conclusions Purified protease from G. candidum GCQAU01 is an ideal candidate for industrial application.

Synthesis of flavor esters by a novel lipase from Aspergillus niger in a soybean-solvent system

“…This is similar to the behavior of lipase from B. methylotrophicus PS3 (Sharma et al 2017). A lipase from G. candidum was inhibited by Ca 2+ , Mn 2+ , Cu 2+ , Mg 2+ , Hg 2+ , Cd 2+ , and Zn 2+ (Muhammad et al 2017), whereas the lipase from Marinobacter sp. EMB5 was slightly stimulated by Ca 2+ (Hemamalini and Khare 2016).…”

“…The enzyme showed excellent stability over a wide pH range from acidic to alkaline (Fig. 3d), which is similar to the lipases of Geotrichum candidum (Muhammad et al 2017). This broad-range stability may render it useful for industrial processes carried out in both acidic and alkaline environments.…”

Realm of Thermoalkaline Lipases in Bioprocess Commodities

“…RSJ-1, B. thermoleovorans ID-1, Bacillus LBN2, Cohnella sp. A01, B. licheniformis Ht7, and B. sonorensis 4R which were 67.0 kDa, 60.0 kDa, 59.0 kDa, 57.0 kDa, 50.0 kDa, 45.0 kDa, 40.0 kDa, 39.0 kDa, 37 kDa, 34.0 kDa, 33.0 kDa, 29.5 kDa, 22.0 kDa, and 21.87 kDa, respectively, as determined using sodium dodecyl sulfate- (SDS-) polyacrylamide gel electrophoresis (PAGE) [ 4 , 9 , 11 , 18 , 23 , 26 , 27 , 29 – 33 , 54 , 69 – 71 ].…”

“…It is well-accepted that enzyme resistant to thermodenaturation is also resistant to extreme pH [ 68 ]. Thermostable lipase from Geotrichum candidum showed stability at varying range of pH (5–12) and thermostability (15–65°C) [ 71 ]. The lipase LBN2 was found to be stable in the pH range of 8–11 and retained 90% activity at 60°C for 1 h [ 9 ].…”

Characterization of Geobacillus stearothermophilus protease for detergent industry

“…Although high yielding alkaline protease strains have been widely studied, but bleach-stable enzymes are still not available where only few studies have been reported (Mei and Jiang, 2005;Srinubabu et al, 2007;Lakshmi et al, 2014;Ochi et al, 2017;Bano et al, 2019). Due to their broad specificity, numerous alkaline proteases have been identified and purified from many Bacilli strains (Bhaskar et al, 2007;Doddapaneni et al, 2007;Muhammad et al, 2019;Osire et al, 2019), such as subtilisin, which has been produced by Bacillus licheniformis (Jacobs 1985) and Bacillus amyloliquefaciens (Wells et al, 1983). However, production of thermostable enzymes has been the focus of attention for many years (Chen et al, 2004), because of their high stability and sustainability at elevated temperature.…”