Characterization of a F280N variant of l-arabinose isomerase from Geobacillus thermodenitrificans identified as a d-galactose isomerase

Kim, Baek-Joong; Hong, Sunwook; Shin, Kyung‐Chul; Jo, Ye-Seul; Oh, Deok‐Kun

doi:10.1007/s00253-014-5827-z

Cited by 28 publications

(23 citation statements)

References 40 publications

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“…These limitations of LAI for tagatose synthesis have been realized previously, and each issue has been addressed individually. While some successes have been reported, especially by selecting or engineering enzymes with more desirable properties 4,11,35–38 , this is the first study to address and overcome all three issues simultaneously.…”

Section: Discussionmentioning

confidence: 99%

Surpassing thermodynamic, kinetic, and stability barriers to isomerization catalysis for tagatose biosynthesis

Bober

Nair

2019

Preprint

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3There are many enzymes that are relevant for making rare and valuable chemicals that 4 while active, are severely limited by thermodynamic, kinetic, or stability issues (e.g. 5 isomerases, lyases, transglycosidase etc.). In this work, we study an enzymatic reaction 6 system − Lactobacillus sakei L-arabinose isomerase (LsLAI) for D-galactose to D-tagatose 7 isomerizationthat is limited by all three reaction parameters. The enzyme has a low 8 catalytic efficiency for non-natural substrate galactose, has low thermal stability at 9 temperatures > 40 °C, and equilibrium conversion < 50%. After exploring several strategies 10 to overcome these limitations, we finally show that encapsulating the enzyme in a gram-11 positive bacterium (Lactobacillus plantarum) that is chemically permeabilized can enable 12 reactions at high rates, high conversion, and at high temperatures. The modified whole cell 13 system stabilizes the enzyme, differentially partitions substrate and product across the 14 membrane to shift the equilibrium toward product formation enables rapid transport of 15 substrate and product for fast kinetics. In a batch process, this system enables 16 approximately 50 % conversion in 4 h starting with 300 mM galactose (an average 17 productivity of 37 mM/h), and 85 % conversion in 48 h, which are the highest reported for 18 food-safe mesophilic tagatose synthesis. We suggest that such an approach may be 19 invaluable for other enzymatic processes that are similarly kinetically-, thermodynamically-20 , and/or stability-limited. 65We use the food-safe engineered probiotic bacterium Lactobacillus plantarum as the 66 expression host due to its increasing relevance to biochemical and biomedical research 30,31 . 67This approach enabled ~ 50 % conversion of galactose to tagatose in 4 h (productivity of ~ 68 38 mmol tagatose L -1 h -1 ) ultimately reaching ~ 85% conversion after 48 h at high galactose 69 loading (300 mM) in batch culture. This is among the highest conversions and 70 productivities reported to date for tagatose production using a mesophilic enzyme. Such an 71 approach is expected to be applicable to other biocatalytic systems where similar trade-offs 72 between kinetics, thermodynamics, and/or stability pose hurdles to process development. 73 74 Results: 75Characterizing L. sakei LAI (LsLAI) limitations. 76 At high substrate loading (400mM galactose) and 37 °C, the reported optimal temperature 77 for this enzyme 32 , LsLAI purified from L. plantarum exhibited an initial forward reaction 78 (turnover) rate (kinitial) of 9.3 ± 0.3 s -1 , which is lower than the maximum reaction rate 79 possible by this enzyme of 17.0 ± 1.3 s -1 min -1 with its preferred substrate, arabinose, at 400 80 mM. Increasing the reaction temperature to 50 °C increased the initial reaction rate to 11.2 81 s -1 ( Fig. 1a) but was accompanied by rapid enzyme inactivation, which is consistent with 82 previous reports of thermal instability of this enzyme 32 . The enzyme exhibited first-order 83 degradation with a half-life (t½) ...

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Section: Discussionmentioning

confidence: 99%

Surpassing thermodynamic, kinetic, and stability barriers to isomerization catalysis for tagatose biosynthesis

Bober

Nair

2019

Preprint

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“…Different strategies of protein engineering have been employed for improving the properties of l‐ AI (Figure ). Site directed mutagenesis of l‐ AI produced by Geobacillus thermodenitrificans , Bacillus stearothermophilus , and Lactobacillus fermentum , resulted into increase of the specific activity, catalytic efficiency, substrate affinity and change of pH optima (Kim, Hong, Shin, Jo, & Oh, ; Li, Xu, Li, & Xu, ; Rhimi et al., ). Site‐directed mutagenesis study shows that the amino acid residue Lys‐269 of Alicyclobacillus acidocaldarius l‐ arabinose isomerase plays a crucial role to modify the pH optima (Lee et al., ).…”

Section: Approaches For Improvement Of Tagatose Productionmentioning

confidence: 99%

“…Protein engineering of l‐ arabinose isomerase ( l‐ AI) from different sources: GTAI‐1 from Geobacillu sthermodinitrificans (Oh, Kim, & Oh, ); GTAI‐2 from Geobacillus thermodinitrificans (Kim et al., ); GSAI‐1 from Geobacillus stearothermophilus (Kim, Yoon, Seo, Oh, & Choi, ); GSAI‐2 from Geobacillus stearothermophilus (Kim et al., ); AHAI from Alicyclobacillus hesperidum (Fan et al., ); SHEWAI from Shewanella sp. (Rhimi et al., ); BSAI from Bacillus stearothermophilus (Rhimi et al., ); LFAI‐1 from Lactobacillus fermentum (Xu, Li, Feng, Zhan, & Xu, ); LFAI‐2 from Lactobacillus fermentum (Li, Xu, Li, & Xu,).…”

Section: Approaches For Improvement Of Tagatose Productionmentioning

confidence: 99%

“…d-Tagatose is a potential therapeutic molecule that can blunt the blood glucose levels in both healthy and diabetic patient (Donner, Wilber, & Ostrowski, 1996). d-Tagatose has several advantages over the conventional oral antidiabetic medicines ; GTAI-2 from Geobacillus thermodinitrificans (Kim et al, 2014a); GSAI-1 from Geobacillus stearothermophilus (Kim, Yoon, Seo, Oh, & Choi, 2001b); GSAI-2 from Geobacillus stearothermophilus ; AHAI from Alicyclobacillus hesperidum (Fan et al, 2015); SHEWAI from Shewanella sp. (Rhimi et al, 2011a); BSAI from Bacillus stearothermophilus (Rhimi et al, 2009); LFAI-1 from Lactobacillus fermentum (Xu, Li, Feng, Zhan, & Xu, 2014b); LFAI-2 from Lactobacillus fermentum (Li, Xu, Li, & Xu,2012).…”

Section: Antidiabeticmentioning

confidence: 99%

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Tagatose as a Potential Nutraceutical: Production, Properties, Biological Roles, and Applications

Roy

Chikkerur

Roy

et al. 2018

Journal of Food Science

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Nutraceuticals are gaining importance owing to their potential applications in numerous sectors including food and feed industries. Among the emerging nutraceuticals, d‐tagatose occupies a significant niche because of its low calorific value, antidiabetic property and growth promoting effects on beneficial gut bacteria. As d‐tagatose is present in minute quantities in naturally occurring food substances, it is produced mainly by chemical or biological means. Recently, attempts were made for bio‐production of d‐tagatose using l‐arabinose isomerase enzyme to overcome the challenges of chemical process of production. Applications of d‐tagatose for maintaining health and wellbeing are increasing due to growing consumer awareness and apprehension against modern therapeutic agents. This review outlines the current status on d‐tagatose, particularly its production, properties, biological role, applications, and the future perspectives.

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“… This process occurs in the pentose phosphate pathway of some microorganisms, , such as Bacillus stearothermophilus , Bacillus coagulans , and Thermotoga maritime, among others. To date, AIs from more than 30 different sources have been identified. − Oh D-K and co-workers have applied AI from Geobacillus thermodenitrificans (gtAI) to produce l -ribulose with a conversion rate of 19%, and further converted the product to l -ribose in combination with mannose-6-phosphate isomerase . Moreover, these two enzymes have also been expressed in Escherichia coli cells and used to directly produce l -ribose from l -arabinose .…”

Section: Introductionmentioning

confidence: 99%

Production of l-Ribulose Using an Encapsulated l-Arabinose Isomerase in Yeast Spores

Liu

Zhou

et al. 2019

J. Agric. Food Chem.

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The rare sugar L-ribulose is produced from the abundant sugar L-arabinose by enzymatic conversion. An Larabinose isomerase (AI) from Geobacillus thermodenitrif icans was efficiently expressed and encapsulated in Saccharomyces cerevisiae spores. Deletion of the yeast OSW2 gene, which causes a mild defect in the integrity of the spore wall, substantially improved the activity of encapsulated AI, without damaging its superior enzymatic properties of thermostability, pH tolerance,and resistance toward SDS and proteinase treatments. In a 10 mL reaction, 100 mg of dry AI encapsulated in spores produced 250 mg of L-ribulose from 1 g of L-arabinose, indicating a 25% conversion rate. Notably, the product of L-ribulose was directly purified from the reaction solution with an approximately 91% recovery using a Ca 2+ ion exchange column. Our results describe not only a facile approach for the production of L-ribulose but also a useful strategy for the enzymatic conversion of rare sugars in "Izumoring".

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Characterization of a F280N variant of l-arabinose isomerase from Geobacillus thermodenitrificans identified as a d-galactose isomerase

Cited by 28 publications

References 40 publications

Surpassing thermodynamic, kinetic, and stability barriers to isomerization catalysis for tagatose biosynthesis

Surpassing thermodynamic, kinetic, and stability barriers to isomerization catalysis for tagatose biosynthesis

Tagatose as a Potential Nutraceutical: Production, Properties, Biological Roles, and Applications

Production of l-Ribulose Using an Encapsulated l-Arabinose Isomerase in Yeast Spores

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