Purification and characterization of a new neutral metalloprotease from marine Exiguobacterium sp. SWJS2

Abstract

Among the protease-producing bacterial strains isolated from deep-sea sediments, SWJS2 was finally selected and identified as genus Exiguobacterium. Plackett-Burman and orthogonal array designs were applied to optimize the fermentation conditions, and the results are as follows: Glucose 5g, yeast extract 15g, glycerin 2g and CaCl2 ⋅2H2 O 0.5 g dissolved in 1 L artificial seawater; temperature 25 °C, original pH 7, inoculum rate 2%, seed age 12 H, loading volume 25 mL (250-mL Erlenmeyer flask), shaking speed 150 rpm, and fermentation time 44 H. The protease activity production was improved from about 80 to 660 U/mL under the optimized parameters. The protease was purified fourfold with specificity activity of 30,654.1 U/mg protein and a total yield of 16.2%. The protease exhibited the maximum activity at 40-45 °C and pH 7. Moreover, the enzyme activity was found to be inhibited by Cu(2+) , Ba(2+) , Cd(2+) , Hg(2+) , and Al(3+) at 5 mM, whereas it can be increased by Mg(2+) , Mn(2+) , and Ca(2+) at 0.5-5 mM. The enzyme was totally inactivated by 1 or 5 mM ethylenediaminetetraacetic acid but not by phenylmethanesulfonyl fluoride, tyrpsin inhibitor from Glycine max (STI), benzamidine, 5,5'-dithio-bis-(2-nitro benzoic acid), or pepstatin A, suggesting that it belonged to metalloprotease.

Lugdulysin of Staphylococcus lugdunensis, a metalloprotease that inhibits and disrupts protein biofilm of Staphylococcus aureus

“…In the present study, EDTA signi cantly reduced the proteolytic activity, con rming the lugdulysin as a metalloprotease [29,30]. In our attempt to recover its activity after inhibition, there was no observable recovery after adding Ca 2+ , Mg 2+ , Zn 2+ or Mn 2+ .…”

Affinity purification of metalloprotease from marine bacterium using immobilized metal affinity chromatography

“…Metalloprotease, one of the most widely used industrial enzymes, is a class of hydrolases that cleave peptide bonds by action of a water molecule that is activated by bivalent metal ions like zinc, cobalt, manganese, or nickel . It is widely used in detergents, leather processing, food processing, feeds, chemical industry, silver recovery, and waste treatment among others . The purified metalloproteases (more than 90% purity) were also used for pharmaceutical applications.…”

“…Thus far, the Food and Drug Administration of USA has approved 12 protease therapies including two metalloproteases, Botulinum toxin A (Botox) and Botulinum toxin B (Myobloc), for muscle spasms and digestive disorders, respectively . However, it is generally recognized as a challenging task to extract high‐purified metalloprotease with a simple and effective protocol .…”

“…As documented previously, the protocols for the purification of metalloprotease usually consist of three or more different steps, including ultrafiltration, ammonium acetate precipitation, salting out, ion‐exchange chromatography, and hydrophobic interaction chromatography . To the best of our knowledge, a protocol based on affinity purification for metalloprotease has not yet been established.…”

Structure-Based Design and Synthesis of a New Phenylboronic-Modified Affinity Medium for Metalloprotease Purification

“…Currently, there is no straightforward and efficient protocol for the purification of metalloproteases [ 14 , 15 , 16 ]. The traditional protocol that has multiple steps is expensive and results in low recovery [ 17 , 18 , 19 , 20 ]. Although some reports refer to the high-yield purification of metalloproteases (more than 90% purity) in one-step procedure, these protocols were based on immobilized metal affinity chromatography (IMAC) that has its disadvantages [ 21 , 22 ].…”