Purification and properties of an alkaline protease of Aspergillus clavatus

Abstract

An extracellular alkaline serine protease has been purified from a strain of Aspergillus clavatus, to apparent homogeneity, by ammonium sulfate precipitation and chromatography on Sephadex G-75. Its molar mass, estimated by SDS-PAGE, was 35 kDa. Maximum protease activity was observed at pH 9.5 and 40°C. The enzyme was active between pH 6.0 and 11.0 and was found to be unstable up to 50°C. Calcium at 5 mM increased its thermal stability. The protease was strongly inhibited by PMSF and chymostatin as well as by SDS, Tween 80 and carbonate ion. Substrate specificity was observed with N-p-Tos-Gly-Pro-Arg-p-nitroanilide and N-Suc-Ala-Ala-Ala-p-nitroanilide being active substates. Parts of the amino acid sequence were up to 81% homologous with those of several fungal alkaline serine proteases.

Oxidant and solvent stable alkaline protease from Aspergillus flavus and its characterization

“…The thermal stability profile of the enzyme showed an initial retention of 96% activity after 1 h of incubation at 40 °C , whereas, 84 and 53 % of residual activities were scored at 45 and 55 °C , respectively. It also showed that protease from A. flavus was relatively thermostable when compared to other reports for the same organism (Tremacoldi et al 2007, Tunga et al, 2003.…”

“…However, an increase in temperature beyond 55 °C resulted in the inactivation of enzyme. Wang et al (2005) and Tremacoldi et al (2007) also reported alkaline protease from Aspergillus sp. having optimum activity at 40 °C .…”

“…The enzyme produced by the isolated strain was appreciably stable in the presence of non-ionic surfactants like Tween 80 and Triton X-100. The strong anionic surfactant (SDS) at 0.1 % (w/v) caused a moderate inhibition (18 %) in enzyme activity on the other hand Tremacoldi et al (2007) reported that the protease from A. clavatus CCT2759 was strongly inhibited by SDS, Tween 80 and carbonate ion. The stability of the enzyme against SDS was lower than A. parasiticus protease which retained about 97% of its Yadav et al 8639 initial activity after 1 h incubation with 2 % SDS at room temperature (Tunga et al 2003).…”

Purification, characterization and crystallization of an extracellular alkaline protease from Aspergillus nidulans HA‐10

“…1b). The different molecular weight of the alkaline proteases were obtained from other Aspergillus species such as 124 kDa serine protease from A. fumigatus [32], 33 kDa from A. fumigatus [33], 35 kDa from A. clavatus [6], 37 kDa from A. terreus [16], 23 kDa from A. parasiticus [17]. Based on our knowledge, Ansari and Stevens [34] have only isolated two neutral proteases and characterized from A. nidulans and their molecular weights were 30.9 kDa and 30 kDa.…”

Semi Purification and Identifications Molecule Protein Weigh of Alkaline Protease Enzyme from Bacillus cereus LS2B

“…Protein bands that look thin, it is interpreted that the concentration of the enzyme dilution buffers is too high, with 10 times dilution (enzyme 1: buffer 9). Tremacoldi et al, (2007) [20] reported that a mixture of enzyme and buffer from the results of his research is 10:50 or (1:5). Asker et al, (2013) [6] also undergo dialysis to enzyme alkaline protease from Bacillus megatherium with a ratio of enzymes and buffers are (1:2), so that the better comparison between the buffer and the enzyme protein concentrations were stuck in the pockets of dialysis is higher.…”