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Chemical Modification of Saccharomycopsis fibuligera R64 α-Amylase to Improve its Stability Against Thermal, Chelator, and Proteolytic Inactivation

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Abstract

α-Amylase catalyzes hydrolysis of starch to oligosaccharides, which are further degraded to simple sugars. The enzyme has been widely used in food and textile industries and recently, in generation of renewable energy. An α-amylase from yeast Saccharomycopsis fibuligera R64 (Sfamy) is active at 50 °C and capable of degrading raw starch, making it attractive for the aforementioned applications. To improve its characteristics as well as to provide information for structural study ab initio, the enzyme was chemically modified by acid anhydrides (nonpolar groups), glyoxylic acid (GA) (polar group), dimethyl adipimidate (DMA) (cross-linking), and polyethylene glycol (PEG) (hydrophilization). Introduction of nonpolar groups increased enzyme stability up to 18 times, while modification by a cross-linking agent resulted in protection of the calcium ion, which is essential for enzyme activity and integrity. The hydrophilization with PEG resulted in protection against tryptic digestion. The chemical modification of Sfamy by various modifiers has thereby resulted in improvement of its characteristics and provided systematic information beneficial for structural study of the enzyme. An in silico structural study of the enzyme improved the interpretation of the results.

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Abbreviations

Aotamy:

Aspergillus oryzae taka-amylase

CC:

Cyanuric chloride

DEAE:

Diethylaminoethyl

DMA:

Dimethyl adipimidate

DTT:

Dithiothreitol

EDTA:

Ethylenediaminetetraacetate

HIC:

Hydrophobic interaction chromatography

PAGE:

Polyacrylamide gel electrophoresis

PEG:

Polyethylene glycol

PEGylation:

Modification with polyethylene glycol

SDS:

Sodium dodecyl sulfate

Sfamy:

Saccharomycopsis fibuligera α-amylase

TIM:

Triosephosphate isomerase

TNBS:

Trinitrobenzene sulfonate

TPCK:

L-1-Tosylamido-2-phenylethyl chloromethyl ketone

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Acknowledgments

The research was financed by the Indonesian Directorate General of Higher Education (DIKTI), Ministry of National Education, Republic of Indonesia (competitive grant batch IX/1-3 entitled Protein engineering of α-amylase from Saccharomycopsis fibuligera R64, for WTI). We thank Prof. J.J. Beintema for his valuable advice and discussion and Dr. H.J. Doddema for editing the manuscript.

Author information

Author notes

  1. Wangsa Tirta Ismaya

    Present address: Institute for Bioengineering and Biosciences, Georgia Institute of Technology, 901 Atlantic Drive, 30332, Atlanta, GA, USA

  2. Khomaini Hasan

    Present address: Loschmidt Laboratories, Department of Experimental Biology and Research Center for Toxic Compounds in the Environment, Faculty of Science Masaryk University, Kamenice 5/A13, 625 00, Brno, Czech Republic

Authors and Affiliations

  1. Biochemistry Laboratory, Department of Chemistry, Faculty of Mathematics and Natural Sciences, Padjadjaran University, Jalan Singaperbangsa No. 2, 40133, Bandung, Indonesia

    Wangsa Tirta Ismaya, Khomaini Hasan, Idar Kardi, Amalia Zainuri, Rinrin Irma Rahmawaty, Satyawisnu Permanahadi, Baiq Vera El Viera, Shabarni Gaffar, Toto Subroto & Soetijoso Soemitro

  2. Biochemistry Research Division, Faculty of Mathematics and Natural Sciences, and Center for Life Sciences, Bandung Institute of Technology, Jalan Ganesa No. 10, 40132, Bandung, Indonesia

    Gunawan Harinanto & Dessy Natalia

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  1. Wangsa Tirta Ismaya
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Correspondence to Wangsa Tirta Ismaya or Soetijoso Soemitro.

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Ismaya, W.T., Hasan, K., Kardi, I. et al. Chemical Modification of Saccharomycopsis fibuligera R64 α-Amylase to Improve its Stability Against Thermal, Chelator, and Proteolytic Inactivation. Appl Biochem Biotechnol 170, 44–57 (2013). https://doi.org/10.1007/s12010-013-0164-8

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