Premium
Cloning and characterization of a new ribitol dehydrogenase from Providencia alcalifaciens RIMD 1656011
Author(s) -
Hassanin Hinawi AM,
Wang Xiao,
Mu Wanmeng,
Zhang Tao,
Jiang Bo
Publication year - 2016
Publication title -
journal of the science of food and agriculture
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.782
H-Index - 142
eISSN - 1097-0010
pISSN - 0022-5142
DOI - 10.1002/jsfa.7589
Subject(s) - ribitol , chemistry , molecular mass , dehydrogenase , enzyme kinetics , enzyme , tetramer , biochemistry , gel electrophoresis , substrate (aquarium) , chromatography , biology , active site , ecology
BACKGROUND A new ribitol dehydrogenase gene was cloned from Providencia alcalifaciens RIMD 1656011 and expressed in Escherichia coli BL21 . This study aimed to purify and characterize the ribitol dehydrogenase from P. alcalifaciens RIMD 1656011 and investigate its substrate specificity for potential use as an industrial enzyme. RESULTS The protein was purified by nickel affinity chromatography. The molecular mass of the purified enzyme was determined as ∼25 000 and 26 650 Da through sodium dodecyl sulfate polyacrylamide gel electrophoresis and liquid chromatography/mass spectrometry respectively. The result for native molecular mass (104 kDa ) suggested that the enzyme functions as a tetramer. Optimum activity of the enzyme was determined at pH 10.0 and a temperature of 35 °C. Regarding its thermal stability, the enzyme retained 72, 72, 48 and 0% of its initial activity after 4 h at 25, 30, 40 and 50 °C respectively. The K m , k cat and k cat / K m values of the enzyme for the substrate ribitol were determined as 13.9 mmol L −1 , 10.0 s −1 and 0.71 L mmol −1 s −1 respectively. The K m of NAD + was 0.042 mmol L −1 . CONCLUSION The substrate specificity indicated that the ribitol dehydrogenase from P. alcalifaciens RIMD 1656011 can be used for direct production of allitol from d ‐fructose without any by‐product formation. © 2015 Society of Chemical Industry