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Effect of introducing a disulphide bond between the A and C domains on the activity and stability of Saccharomycopsis fibuligera R64 α-amylase
Natalia D.a, Vidilaseris K.a, Ismaya W.T.b,c, Puspasari F.a, Prawira I.a, Hasan K.a, Fibriansah G.b,d, Permentier H.P.b, Nurachman Z.a, Subroto T.c, Dijkstra B.W.b, Soemitro S.c
a Biochemistry Research Division, Faculty of Mathematics and Natural Sciences, Bandung Institute of Technology, Bandung, 40132, Indonesia
b Laboratory of Biophysical Chemistry, University of Groningen, AG Groningen, 9747, Netherlands
c Biochemistry Laboratory, Department of Chemistry, Faculty of Mathematics and Natural Sciences, Padjadjaran University, Bandung, 40133, Indonesia
d Laboratory of Virus Structure and Function, Duke-NUS Graduate Medical School, Singapore, 169857, Singapore
[vc_row][vc_column][vc_row_inner][vc_column_inner][vc_separator css=”.vc_custom_1624529070653{padding-top: 30px !important;padding-bottom: 30px !important;}”][/vc_column_inner][/vc_row_inner][vc_row_inner layout=”boxed”][vc_column_inner width=”3/4″ css=”.vc_custom_1624695412187{border-right-width: 1px !important;border-right-color: #dddddd !important;border-right-style: solid !important;border-radius: 1px !important;}”][vc_empty_space][megatron_heading title=”Abstract” size=”size-sm” text_align=”text-left”][vc_column_text]© 2014 Elsevier B.V.Native enzyme and a mutant containing an extra disulphide bridge of recombinant Saccharomycopsis fibuligera R64 α-amylase, designated as Sfamy01 and Sfamy02, respectively, have successfully been overexpressed in the yeast Pichia pastoris KM71H. The purified α-amylase variants demonstrated starch hydrolysis resulting in a mixture of maltose, maltotriose, and glucose, similar to the wild type enzyme. Introduction of the disulphide bridge shifted the melting temperature (TM) from 54.5 to 56°C and nearly tripled the enzyme half-life time at 65°C. The two variants have similar kcat/KM values. Similarly, inhibition by acarbose was only slightly affected, with the IC50 of Sfamy02 for acarbose being 40±3.4μM, while that of Sfamy01 was 31±3.9μM. On the other hand, the IC50 of Sfamy02 for EDTA was 0.45mM, nearly two times lower than that of Sfamy01 at 0.77mM. These results show that the introduction of a disulphide bridge had little effect on the enzyme activity, but made the enzyme more susceptible to calcium ion extraction. Altogether, the new disulphide bridge improved the enzyme stability without affecting its activity, although minor changes in the active site environment cannot be excluded.[/vc_column_text][vc_empty_space][vc_separator css=”.vc_custom_1624528584150{padding-top: 25px !important;padding-bottom: 25px !important;}”][vc_empty_space][megatron_heading title=”Author keywords” size=”size-sm” text_align=”text-left”][vc_column_text]Disulphide bonds,Disulphide bridge,Enzyme half-life time,Enzyme stability,Pichia Pastoris,Saccharomycopsis,Starch Hydrolysis,Wild-type enzymes,alpha-Amylases,Disulfides,Enzyme Stability,Fungal Proteins,Models, Molecular,Pichia,Protein Structure, Tertiary,Recombinant Proteins,Saccharomycopsis[/vc_column_text][vc_empty_space][vc_separator css=”.vc_custom_1624528584150{padding-top: 25px !important;padding-bottom: 25px !important;}”][vc_empty_space][megatron_heading title=”Indexed keywords” size=”size-sm” text_align=”text-left”][vc_column_text]Disulphide bond,Enzyme stability,Pichia pastoris,Saccharomycopsis fibuligera R64,α-Amylase[/vc_column_text][vc_empty_space][vc_separator css=”.vc_custom_1624528584150{padding-top: 25px !important;padding-bottom: 25px !important;}”][vc_empty_space][megatron_heading title=”Funding details” size=”size-sm” text_align=”text-left”][vc_column_text]The research was supported by the Ministry of Research and Technology of the Republic of Indonesia (Riset Unggulan Terpadu XII), the Koninklijke Nederlandse Academie van Wetenschapen of The Kingdom of the Netherlands (the SPIN program), and the Directorate General for Higher Education of the Ministry of Education and Culture of the Republic of Indonesia (Beasiswa Unggulan).[/vc_column_text][vc_empty_space][vc_separator css=”.vc_custom_1624528584150{padding-top: 25px !important;padding-bottom: 25px !important;}”][vc_empty_space][megatron_heading title=”DOI” size=”size-sm” text_align=”text-left”][vc_column_text]https://doi.org/10.1016/j.jbiotec.2014.12.002[/vc_column_text][/vc_column_inner][vc_column_inner width=”1/4″][vc_column_text]Widget Plumx[/vc_column_text][/vc_column_inner][/vc_row_inner][/vc_column][/vc_row][vc_row][vc_column][vc_separator css=”.vc_custom_1624528584150{padding-top: 25px !important;padding-bottom: 25px !important;}”][/vc_column][/vc_row]