Publications by authors named "Nattapol Arunrattanamook"

3 Publications

  • Page 1 of 1

Engineering of β-mannanase from Aspergillus niger to increase product selectivity towards medium chain length mannooligosaccharides.

J Biosci Bioeng 2020 Nov 26;130(5):443-449. Epub 2020 Jul 26.

Enzyme Technology Laboratory, National Center for Genetic Engineering and Biotechnology, 113 Thailand Science Park, Phaholyothin Road, Khlong Luang, Pathumthani 12120, Thailand.

Mannooligosaccharides (MOSs) are one of the most commonly used biomass-derived feed additives. The effectiveness of MOS varies with the length of oligosaccharides, medium length MOSs such as mannotetraose and mannopentaose being the most efficient. This study aims at improving specificity of β-mannanase from Aspergillus niger toward the desirable product size through rational-based enzyme engineering. Tyr 42 and Tyr 132 were mutated to Gly to extend the substrate binding site, allowing higher molecular weight MOS to non-catalytically bind to the enzyme. Hydrolysis product content was analyzed by high-performance anion-exchange chromatography with pulsed amperometric detection. Instead of mannobiose, the enzyme variants yielded mannotriose and mannotetraose as the major products, followed by mannobiose and mannopentaose. Overall, 42% improvement in production yield of highly active mannotetraose and mannopentaose was achieved. This validates the use of engineered β-mannanase to selectively produce larger MOS, making them promising candidates for large-scale MOS enzymatic production process.
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http://dx.doi.org/10.1016/j.jbiosc.2020.07.001DOI Listing
November 2020

Kinetic Characterization of Prenyl-Flavin Synthase from Saccharomyces cerevisiae.

Biochemistry 2018 02 27;57(5):696-700. Epub 2017 Dec 27.

Department of Chemistry, ‡Department of Chemical Engineering, and §Department of Biological Chemistry, University of Michigan , Ann Arbor, Michigan 48109, United States.

We have characterized the kinetics and substrate requirements of prenyl-flavin synthase from yeast. This enzyme catalyzes the addition of an isopentenyl unit to reduced flavin mononucleotide (FMN) to form an additional six-membered ring that bridges N5 and C6 of the flavin nucleus, thereby converting the flavin from a redox cofactor to one that supports the decarboxylation of aryl carboxylic acids. In contrast to bacterial enzymes, the yeast enzyme was found to use dimethylallyl pyrophosphate, rather than dimethylallyl phosphate, as the prenyl donor in the reaction. We developed a coupled assay for prenyl-flavin synthase activity in which turnover was linked to the activation of the prenyl-flavin-dependent enzyme, ferulic acid decarboxylase. The kinetics of the reaction are extremely slow: k = 12.2 ± 0.2 h, and K for dimethylallyl pyrophosphate = 9.8 ± 0.7 μM. The K for reduced FMN was too low to be accurately measured. The kinetics of reduced FMN consumption were studied under pre-steady state conditions. The reaction of FMN was described well by first-order kinetics with a k of 17.4 ± 1.1 h. These results indicate that a chemical step, most likely formation of the carbon-carbon bond between C6 of the flavin and the isopentenyl moiety, is substantially rate-determining in the reaction.
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http://dx.doi.org/10.1021/acs.biochem.7b01131DOI Listing
February 2018

Mechanism of the Novel Prenylated Flavin-Containing Enzyme Ferulic Acid Decarboxylase Probed by Isotope Effects and Linear Free-Energy Relationships.

Biochemistry 2016 05 10;55(20):2857-63. Epub 2016 May 10.

Department of Chemistry, ‡Department of Chemical Engineering, and §Department of Biological Chemistry, University of Michigan , Ann Arbor, Michigan 48109, United States.

Ferulic acid decarboxylase from Saccharomyces cerevisiae catalyzes the decarboxylation of phenylacrylic acid to form styrene using a newly described prenylated flavin mononucleotide cofactor. A mechanism has been proposed, involving an unprecedented 1,3-dipolar cyclo-addition of the prenylated flavin with the α═β bond of the substrate that serves to activate the substrate toward decarboxylation. We measured a combination of secondary deuterium kinetic isotope effects (KIEs) at the α- and β-positions of phenylacrylic acid together with solvent deuterium KIEs. The solvent KIE is 3.3 on Vmax/KM but is close to unity on Vmax, indicating that proton transfer to the product occurs before the rate-determining step. The secondary KIEs are normal at both the α- and β-positions but vary in magnitude depending on whether the reaction is performed in H2O or D2O. In D2O, the enzyme catalyzed the exchange of deuterium into styrene; this reaction was dependent on the presence of bicarbonate. This observation implies that CO2 release must occur after protonation of the product. Further information was obtained from a linear free-energy analysis of the reaction through the use of a range of para- and meta-substituted phenylacrylic acids. Log(kcat/KM) for the reaction correlated well with the Hammett σ(-) parameter with ρ = -0.39 ± 0.03; r(2) = 0.93. The negative ρ value and secondary isotope effects are consistent with the rate-determining step being the formation of styrene from the prenylated flavin-product adduct through a cyclo-elimination reaction.
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http://dx.doi.org/10.1021/acs.biochem.6b00170DOI Listing
May 2016
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