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Microbial Biotechnology (MB)  |  Enzyme, Protein Engineering, and Metabolic Engineering

Microbiol. Biotechnol. Lett. 2021; 49(2): 181-191

Received: April 12, 2019; Accepted: May 30, 2019

Optimization, Purification, and Characterization of Haloalkaline Serine Protease from a Haloalkaliphilic Archaeon Natrialba hulunbeirensis Strain WNHS14

Rania S Ahmed1, Amira M Embaby2, Mostafa Hassan3, Nadia A Soliman1*, and Yasser R Abdel-Fattah1

1Bioprocess Development Department, Genetic Engineering and Biotechnology Research Institute (GEBRI), City for Scientific Research and Technological Applications, Alexandria, Egypt 2Institute of Graduate Studies and Research, Biotechnology Department, 3Institute of Graduate Studies and Research, Environmental Studies Department, Alexandria University, Alexandria 21526, Egypt

The present study addresses isolation, optimization, partial purification, and characterization of a haloalkaline serine protease from a newly isolated haloarchaeal strain isolated from Wadi El Natrun in Egypt. We expected that a two-step sequential statistical approach (one variable at a time, followed by response surface methodology) might maximize the production of the haloalkaline serine protease. The enzyme was partially purified using Hiprep 16/60 sephacryl S-100 HR gel filtration column. Molecular identification revealed the newly isolated haloarchaeon to be Natrialba hulunbeirensis strain WNHS14. Among several tested physicochemical determinants, casamino acids, KCl, and NaCl showed the most significant effects on enzyme production as determined from results of the One-Variable-At-A-time (OVAT) study. The Box- Behnken design localized the optimal levels of the three key determinants; casamino acids, KCl, and NaCl to be 0.5% (w/v), 0.02% (w/v), and 15% (w/v), respectively, obtaining 62.9 U/ml as the maximal amount of protease produced after treatment at 40℃, and pH 9 for 9 days with 6-fold enhancement in yield. The enzyme was partially purified after size exclusion chromatography with specific activity, purification fold, and yield of 1282.63 U/mg, 8.9, and 23%, respectively. The enzyme showed its maximal activity at pH, temperature, and NaCl concentration optima of 10, 75℃, and 2 M, respectively. Phenylmethylsulfonyl fluoride (PMSF, 5 mM) completely inhibited enzyme activity.

Keywords: Protease, Haloalkaliphilic, archaea, Natrialba hulunbeirensis, enzyme production

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