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α‐Amylase from malted Sorghum bicolor : Conformational stability
Author(s) -
Kumar Siva Sai R.,
Singh Sridevi Annapurna,
Rao Appu A. G.
Publication year - 2007
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.21.5.a639-b
Subject(s) - chemistry , urea , maltose , thermal stability , enzyme , denaturation (fissile materials) , amylase , sorghum , enzyme assay , ionic strength , chromatography , crystallography , biochemistry , nuclear chemistry , organic chemistry , aqueous solution , forestry , geography
Malted sorghum, a source of α‐amylase can be an integral part of supplementary foods. The α‐amylase is more thermostable compared to other cereal amylases. The digestion of gelatinized starch by enzyme results in maltose, glucose and maltotriose, which decreases its water absorption capacity, thereby increasing nutrient density. Insights of the basis of stability, which could be valuable in protein engineering of the molecule, are obtained from conformational studies. The conformational stability measurements are followed by unfolding induced by thermal and chemical denaturants. The unfolding of the molecule by chemical denaturants is a two step transition N ↔ U . Δ G U‐N values for GuHCl and urea are 3.6 kcal mol −1 and 5.8 kcal mol −1 , respectively. The enzyme is more stable in urea being [Urea] 1/2 = 6.4 M compared to [GuHCl] 1/2 = 1.35 M, specifying the role of ionic interactions in stability. Enzyme regaines structure and activity when renatured at pH 7.0 in presence of DTT. Thermal denaturation followed at 222nm by CD, absorbance at 280nm and activity measurements indicates a T M of 71.5°C, revealing the simultaneous loss of structure and activity. Ca 2+ affords stability for enzyme as revealed by resistance to the thermal inactivation and proteolysis. Masked Cys residues play role in enzyme activity. Cys accessibility increased from 1.3 mol of SH/mol of protein to 2.5 for Ca 2+ depleted sample. (Supported by CSIR‐INDIA)