Optimization of culture conditions for the production of haloalkaliphilic thermostable protease from an extremely halophilic archaeon Halogeometricum sp. TSS101

Abstract

Aims: Isolation and screening of extreme halophilic archaeon producing extracellular haloalkaliphilic protease and optimization of culture conditions for its maximum production. Methods and Results: Halogeometricum sp. TSS101 was isolated from salt samples and screened for the secretion of protease on gelatin and casein plates containing 20% NaCl. The archaeon was grown aerobically in a 250 ml flask containing 50 ml of (w/v) NaCl 20%; MgCl 2 1%; KCl 0AE5%; trisodium citrate 0AE3%; and peptone 1%; pH 7AE2 at 40°C on rotary shaker. The production of enzyme was investigated at various pH, temperatures, NaCl concentrations, metal ions and different carbon and nitrogen sources. The partially purified protease had activity in a broad pH range (7AE0-10AE0) with optimum activity at pH 10AE0 and a temperature (60°C). The enzyme was thermostable and retained 70% initial activity at 80°C. Maximum protease production occurred at 40°C in a medium containing 20% NaCl (w/v) and 1% skim milk powder after 84 h in shaking culture. Enzyme secretion was observed at a broad pH range of 7AE0-10AE0. Addition of CaCl 2 (200 mmol) to the culture medium enhanced the production of protease. Protein rich flours proved to be cheap and good alternative source for enzyme production. Different osmolytes were tested for the growth and production of haloalkaliphilc protease and found that betaine and glycerol enhanced growth without secretion of the protease. Immobilization studies showed that whole cells immobilized in 2% alginate beads were stable up to 10 batches and able to secrete the protease, which attained maximum production within 60 h under shaking conditions. Conclusions: Halogeometricum sp. TSS101 secreted an extracellular haloalkaliphilic and thermostable protease. The optimum conditions required for maximum production are 20% NaCl, 1% skim milk powder and temperature at 40°C. Addition of CaCl 2 (200 mmol) enhanced the enzyme production. Immobilization of whole cells in absence of NaCl proved to be useful for continuous production of haloalkaliphilic protease. Significance and Impact of the Study: The low cost protein rich flours were used as an alternative carbon and nitrogen sources for enzyme production. Immobilization of halophilic cells in alginate beads can be used in continuous production of halophilic enzyme. The halophilic and thermostable protease from Halogeometricum sp. TSS101 is good source for industrial applications and can be a suitable source for preparation of fish sauce.

Enzymes from Halophilic Archaea: Open Questions

“…Whole haloarchaeal cells have been used for the degradation of organic pollutants and in the treatment of concentrated textile waste waters, particularly the first dye bath liquor from the dying process, which contains a high salt load (Margesin and Schinner 2001), but only few studies have been reported on their enzymes. Thus, few biochemical studies have been carried out with amylases (Good and Hartman 1970;Pérez-Pomares et al 2003 or proteases (Vidyasagar and Prakash 2006) from haloarchaea. These enzymes could keep high activity and stability in high salt environments and could have potential application value.…”

Enhanced production of histamine dehydrogenase by <i>Natrinema gari</i> BCC 24369 in a non-sterile condition

“…Halophilic archaea have a wide range of temperature preferences depending upon the nature of adaptation and salt requirements (Wejse et al, 2003). Most halophilic archaea and their enzyme production have rather high temperature optima, in the range between 35 and 50°C (Shand and Perez, 1999;Vidyasagar et al, 2006). In this study, Nnm.…”

“…The commonly used organic components included casamino acid, yeast extract and tryptone (Oren, 2006). In addition to medium components, the initial pH of the cultivation medium and the incubation temperature were found to play an important role in enzyme production (Vidyasagar et al, 2006). Another problem typically encountered in fermentation is the contamination of fermentation mixtures.…”

“…Among various medium components, NaCl and organic components including complex nitrogen and carbon sources were the key factors having an extreme influence on the growth of cells and the enzyme production of halophilic archaea (Manikandan et al, 2009;Vidyasagar et al, 2006;Wejse et al, 2003). The commonly used organic components included casamino acid, yeast extract and tryptone (Oren, 2006).…”

The Haloprotease CPI Produced by the Moderately Halophilic Bacterium Pseudoalteromonas ruthenica Is Secreted by the Type II Secretion Pathway

“…Considerable attention has been focused on enzymes of moderately halophilic bacteria, since they have substantial biotechnological potential (29,56). While several proteases from extreme halophiles, members of the halophilic archaea, have been characterized (12,15,19,36,44,49,50,51,57), fewer proteases from moderately halophilic bacteria have been purified and studied in depth (9,14,18,20,21,33).The moderately halophilic bacterium Pseudoalteromonas ruthenica CP76 was isolated from a saltern located in Isla Cristina (Huelva, Spain). This strain was selected for its ability to produce an extracellular protease, haloprotease CPI, which has a molecular mass of 38.0 kDa (46, 47).…”