Abstract
The kinetically stable alkaline serine protease from Nocardiopsis sp.; NprotI, possessing polyproline II fold (PPII) was characterized for its pH stability using proteolytic assay, fluorescence and Circular Dichroism (CD) spectroscopy, and Differential Scanning Calorimetry (DSC). NprotI was found to be functionally stable when incubated at pH 1.0, even after 24 h, while after incubation at pH 10.0, drastic loss in the activity was observed. The enzyme showed enhanced activity after incubation at pH 1.0 and 3.0, at higher temperature (50–60 °C). NprotI maintained the overall PPII fold in broad pH range as seen using far UV CD spectroscopy. The PPII fold of NprotI incubated at pH 1.0 remained fairly intact up to 70 °C. Based on the isodichroic point and Tm values revealed by secondary structural transitions, different modes of thermal denaturation at pH 1.0, 5.0 and 10.0 were observed. DSC studies of NprotI incubated at acidic pH (pH 1.0–5.0) showed Tm values in the range of 74–76 °C while significant decrease in Tm (63.8 °C) was observed at pH 10.0. NprotI could be chemically denatured at pH 5.0 (stability pH) only with guanidine thiocynate. NprotI can be classified as type III protein among the three acid denatured states. Acid tolerant and thermostable NprotI can serve as a potential candidate for biotechnological applications.
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Abbreviations
- PPII:
-
Polyproline II
- CD:
-
Circular Dichroism
- DSC:
-
Differential Scanning Calorimetry
- NprotI:
-
Nocardiopsis protease I
- GuSCN:
-
Guanidine thiocyanate
- GdnHCl:
-
Guanidine hydrochloride
- MRE:
-
Mean residual ellipticity
- ANS:
-
1-Anilino 8-napthalene sulfonic acid
- Napase:
-
Nocardiopsis alba protease
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Acknowledgments
The authors thank Dr. M. Fernandes, Organic Chemistry Division, NCL, for allowing the use of CD spectropolarimeter. SR, SD and PKN were supported by Senior Research Fellowships from the CSIR, New Delhi, India. The MicroCal VP-DSC equipment used in this study was supported by a UPE grant from the UGC (India) to the University of Hyderabad.
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Figure S3: Activity profile of NprotI incubated with GuSCN (0-6 M). Experiment was performed as described in Materials and Methods section (DOC 67 kb)
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Rohamare, S., Javdekar, V., Dalal, S. et al. Acid Stability of the Kinetically Stable Alkaline Serine Protease Possessing Polyproline II Fold. Protein J 34, 60–67 (2015). https://doi.org/10.1007/s10930-014-9597-3
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DOI: https://doi.org/10.1007/s10930-014-9597-3