Purification and characterization of a thermostable alkaline protease from alkalophilic Bacillus pumilus.
OBJECTIVE
An investigation was carried out on the purification and characterization of an alkaline protease from Bacillus pumilus MK6-5.


RESULTS
An alkalophilic Bacillus pumilus MK6-5 was grown in a laboratory fermenter containing 1% reverse osmosis concentrated cheese whey powder, 0.25% corn steep liquor, 1% glucose, 0.5% tryptone, 1% sodium citrate, 0.02% MgSO4.7H2O and 0.65% Na2CO3 at 35 degrees C and pH 9.6, agitation at 250 rev min(-1) and aeration of 1 vvm for 60 h. When the enzyme was purified using ammonium sulphate precipitation, ion exchange and gel filtration chromatographies, a 26.2% recovery of enzyme with 36.6-fold purification was recorded. The purified protease was found to be homogenous by SDS-PAGE with molecular mass estimate of 28 kDa. The enzyme was optimally active at pH 11.5 and temperature of 55-60 degrees C. The Km and kcat values observed with synthetic substrates at 37 degrees C and pH 8.0 were 1.1 mmol l(-1) and 624 s(-1) for Glu-Gly-Ala-Phe-pNA and 3.7 mmol l(-1) and 826 s(-1) for Glu-Ala-Ala-Ala-pNA, respectively. The kinetic data revealed that small aliphatic and aromatic residues were the preferred residues at the P1 position. Inhibition profile exhibited by PMSF suggested the B. pumilus protease to be an alkaline serine protease.


CONCLUSIONS
Bacillus pumilus MK6-5 produced a calcium-dependent, thermostable alkaline serine protease.


CONCLUSIONS
The thermostable alkaline protease from Bacillus pumilus MK6-5 will be extremely useful in ultrafiltration membrane cleaning due to its ability to work in broad pH and temperature ranges, and tolerance to detergents, unlike the mesophilic proteases which face these limitations.
Kumar
C G
CG
Molekularbiologisches Zentrum, Bundesforschungsanstalt für Ernährung, Haid und Neu Strasse 9, D-76131 Karlsruhe, Germany.
D001407:Bacillus / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D012697:Serine Endopeptidases / Q000302:isolation & purification* / Q000378:metabolism; D013379:Substrate Specificity; D013696:Temperature
D012697:Serine Endopeptidases; C032923:microbial serine proteinases