Mutagenesis for improvement of activity and thermostability of amylomaltase from Corynebacterium glutamicum

Aim: To express amylomaltase from Thermus filiformis (TfAM) in a generally recognized as safe (GRAS) organism and to use the enzyme in starch modification. Methods and Results: TfAM was expressed in Saccharomyces cerevisiae, using 2% (w/v) galactose inducer under GAL1 promoter. The enzyme was thermostable with high disproportionation and cyclization activities. The main large-ring cyclodextrin (CD) products were CD24-CD29, with CD26 as maximum at all incubation times. TfAM was used to modify cassava and pea starches, the amylose content decreased 18% and 30%, respectively, when 5% (w/v) starch was treated with 0Á5 U TfAM g À1 starch. The increase in short branched chain (DP, degree of polymerization, 1-5) and the broader chain length distribution pattern which extended to the longer chain (DP40) after TfAM treatment were observed. The thermal property was changed, with an increase in retrogradation of starch as suggested by a lower enthalpy. Conclusions: TfAM was successfully expressed in S. cerevisiae and was used to make starches with new functionality. Significance and Impact of the Study: This is the first report on the expression of AM in the GRAS yeast and the production of a modified starch gel from pea starch to improve the versatility of starch for food use.

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“…The LR‐CD hydrolysis, disproportionation and coupling activities of Tf AM were assayed as described for Cg AM (Nimpiboon et al 2016…”

Section: Methodsmentioning

confidence: 99%

“…The LR‐CD products were analysed by HPAEC‐PAD (ICS 5000 system (Dionex, Sunnyvale, CA) using a Carbopac PA‐100 column (4 × 250 mm) as described (Nimpiboon et al 2016…”

Section: Methodsmentioning

confidence: 99%

Amylomaltase fromThermus filiformis: expression inSaccharomyces cerevisiaeand its use in starch modification

Nimpiboon

Tumhom

Nakapong

et al. 2020

J. Appl. Microbiol.

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“…Similarly, the mutated A460V enzyme from Corynebacterium glutamicum (CgAM) exhibited 5 • C increase in optimum temperature which made it more thermostable compared to the wild type. The mutation resulted in increased catalytic efficiency and gave 46% higher product yields at 40 • C [32]. However, mutation of the same CgAM at Tyr172 to produce Y172A mutant did not change the optimum temperature, but altered the LR-CD product pattern when compared to that of the wild type enzyme [33].…”

Section: Sources and Biochemical Properties Of Amylomaltasementioning

confidence: 97%

Production of Large-Ring Cyclodextrins by Amylomaltases

et al. 2022

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Amylomaltase is a well-known glucan transferase that can produce large ring cyclodextrins (LR-CDs) or so-called cycloamyloses via cyclization reaction. Amylomaltases have been found in several microorganisms and their optimum temperatures are generally around 60–70 °C for thermostable amylomaltases and 30–45 °C for the enzymes from mesophilic bacteria and plants. The optimum pHs for mesophilic amylomaltases are around pH 6.0–7.0, while the thermostable amylomaltases are generally active at more acidic conditions. Size of LR-CDs depends on the source of amylomaltases and the reaction conditions including pH, temperature, incubation time, and substrate. For example, in the case of amylomaltase from Corynebacterium glutamicum, LR-CD productions at alkaline pH or at a long incubation time favored products with a low degree of polymerization. In this review, we explore the synthesis of LR-CDs by amylomaltases, structural information of amylomaltases, as well as current applications of LR-CDs and amylomaltases.

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“…An error-prone PCR assay was also carried out to isolate enzymes with higher thermostability. It allowed for the isolation of the A406V mutant, which showed a higher thermostability at 50 °C than the wild type [ 68 ]. The catalytic efficiency values k cat / K m of the mutant was 2.9 times higher than that of the wild type.…”

Section: Mutational Analysesmentioning

confidence: 99%

Amylomaltases in Extremophilic Microorganisms

et al. 2021

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Amylomaltases (4-α-glucanotransferases, E.C. 2.4.1.25) are enzymes which can perform a double-step catalytic process, resulting in a transglycosylation reaction. They hydrolyse glucosidic bonds of α-1,4′-D-glucans and transfer the glucan portion with the newly available anomeric carbon to the 4′-position of an α-1,4′-d-glucan acceptor. The intramolecular reaction produces a cyclic α-1,4′-glucan. Amylomaltases can be found only in prokaryotes, where they are involved in glycogen degradation and maltose metabolism. These enzymes are being studied for possible biotechnological applications, such as the production of (i) sugar substitutes; (ii) cycloamyloses (molecules larger than cyclodextrins), which could potentially be useful as carriers and encapsulating agents for hydrophobic molecules and also as effective protein chaperons; and (iii) thermoreversible starch gels, which could be used as non-animal gelatin substitutes. Extremophilic prokaryotes have been investigated for the identification of amylomaltases to be used in the starch modifying processes, which require high temperatures or extreme conditions. The aim of this article is to present an updated overview of studies on amylomaltases from extremophilic Bacteria and Archaea, including data about their distribution, activity, potential industrial application and structure.

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Mutagenesis for improvement of activity and thermostability of amylomaltase from Corynebacterium glutamicum

Cited by 24 publications

References 43 publications

Amylomaltase fromThermus filiformis: expression inSaccharomyces cerevisiaeand its use in starch modification

Amylomaltase fromThermus filiformis: expression inSaccharomyces cerevisiaeand its use in starch modification

Production of Large-Ring Cyclodextrins by Amylomaltases

Amylomaltases in Extremophilic Microorganisms

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