d-allulose, a rare sugar that is scarce in nature, exerts several beneficial effects and has commercial potential. d-allulose 3-epimerase (DAEase) plays a vital role in catalyzing the isomerization from d-fructose to d-allulose. However, the industrial application of DAEase for d-allulose production is hindered by its poor long-term thermostability. In the present research, we introduced a proline residue (i) to restrict its spatial conformation and (ii) to reduce the entropy of the unfolded state of DAEase. The t value of the double-site Clostridium bolteae DAEase mutant Cb-51P/89P was prolonged to 58 min at 55 °C, a 2.32-fold increase compared with wild-type DAEase. The manipulation did not cause obvious changes in the enzymatic properties, including optimum pH, optimal temperature, optimum metal ion, and enzymatic activity. As the accumulation of multiple small effects through proline substitution could dramatically improve the thermostability of the mutant protein, our method to improve the thermostability while roughly retaining the original enzymatic properties is promising.
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http://dx.doi.org/10.1016/j.pep.2022.106145 | DOI Listing |
J Agric Food Chem
January 2025
School of Food and Biological Engineering, Jiangsu University, 301 Xuefu Road, Zhenjiang, Jiangsu 212013, PR China.
D-Allose, a rare sugar, has gained significant attention not only as a low-calorie sweetener but also for its anticancer, antitumor, anti-inflammatory, antioxidant, and other pharmaceutical properties. Despite its potential, achieving high-level biosynthesis of D-allose remains challenging due to inefficient biocatalysts, low conversion rates, and the high cost of substrates. Here, we explored the food-grade coexpression of D-allulose 3-epimerase (Bp-DAE) and L-rhamnose isomerase (BsL-RI) within a single cell using WB800N as the host.
View Article and Find Full Text PDFSci Rep
December 2024
State Key Laboratory of Biobased Material and Green Papermaking, Qilu University of Technology, Shandong Academy of Science, Jinan, 250353, People's Republic of China.
D-allulose/D-psicose is a significant rare sugar with broad applications in the pharmaceutical, food, and other industries. In this study, we cloned the D-allulose 3-epimerase (DPEase) gene from Arthrobacter globiformis M30, using pET22b as the vector. The recombinant E.
View Article and Find Full Text PDFJ Agric Food Chem
January 2025
College of Light Industry and Food Engineering, Guangxi University, 100 Daxue Road, Nanning 530004, Guangxi, China.
d-Allulose 3-epimerase (DAEase) derived from has excellent properties in the catalytic production of d-allulose, a rare sugar with unique biological functions. However, the industrial application of DAEase (Cb-DAEase) for d-allulose production is hindered by its low enzyme activity, poor long-term thermostability, and pH tolerance. In this study, we identified potential noncatalytic residues in Cb-DAEase using methods such as proline substitution, surface charge engineering, and surface residue prediction.
View Article and Find Full Text PDFJ Appl Glycosci (1999)
November 2024
1 Matsutani Chemical Industry Co., Ltd.
D-Allulose 3-epimerase catalyzes C-3 epimerization between D-fructose and D-allulose was found in strain M30. The enzyme gene was cloned, and its recombinant enzyme and the mutant variants were expressed in Using the information of the sequence and model structure, we succeed in the improvement of melting temperature for the enzyme without significant loss of the enzyme activity by protein engineering method. The melting temperatures were increased by 2.
View Article and Find Full Text PDFbioRxiv
November 2024
College of Computer, Mathematical and Natural Sciences, University of Maryland, College Park, Maryland, USA.
D-allulose, a rare sugar with emerging potential as a low-calorie sweetener, has garnered attention as an alternative to other commercially available alternative sweeteners, such as sugar alcohols, which often cause severe gastrointestinal discomfort. D-allulose-6-phosphate 3-epimerase (AlsE) is a prokaryotic enzyme that converts D-allulose-6-phosphate into D-fructose-6-phopshate, enabling its use as a carbon source. However, the taxonomic breadth of AlsE across gut bacteria remains poorly understood, hindering insights into the utilization of D-allulose by microbial communities.
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