Continuous Spectrophotometric Assay for High-Throughput Screening of Predominant d-Allulose 3-Epimerases

Abstract

d-Allulose is an attractive noncaloric sugar substitute with numerous health benefits, which can be biosynthesized by d-allulose 3-epimerases (DAEases). However, enzyme instability under harsh industrial reaction conditions hampered its practical applications. Here, we developed a continuous spectrophotometric assay (CSA) for the efficient analysis of d-allulose in a mixture. Furthermore, a high-throughput screening strategy for DAEases was developed using CSA by coupling DAEase with a NADH-dependent ribitol dehydrogenase, enabling high-throughput screening of DAEase variants with desired properties. The variant M15S/P40N/S209N exhibited a half-life of 22 h at 60 °C and an 8.7 °C increase of the T 50 60 value, with a 1.2-fold increase of activity. Structural modeling and molecular dynamics simulations indicated that the improvement of thermostability and activity was due to some new hydrogen bonds between chains at the dimer interface and between the residue and the substrate d-fructose. This work offers a robust tool and theoretical basis for the improvement of DAEases, which will benefit the enzymatic biosynthesis of d-allulose and promote its industrial application.

Computer-Aided Targeted Mutagenesis of Thermoclostridium caenicola d-Allulose 3-Epimerase for Improved Thermostability

“…To date, about 20 microorganisms have been reported as the sources of DAEase, and nearly half were identified in the last 3 years, such as Paenibacillus senegalensis, 13 Staphylococcus aureus, 14 Rhodopirellula baltica, 15 DaeM, 16 Thermoclostridium caenicola, 17 Bacillus sp., 18 Pirellula sp., 19 Novibacillus thermophilus, 20 Halanaerobium congolense, 21 and Sinorhizobium fredii. 22 Thermostability is an important indicator of enzyme performance. Thermostable enzymes exhibit higher kinetic stability and operational stability at elevated temperatures.…”

“…d -Allulose 3-epimerase (DAEase, EC 5.1.3.30) is one of the most important members of the KEase family and has a pivotal role in the biosynthesis of d -allulose due to the high catalytic activity toward d -fructose relative to other KEase family members. To date, about 20 microorganisms have been reported as the sources of DAEase, and nearly half were identified in the last 3 years, such as Paenibacillus senegalensis, Staphylococcus aureus, Rhodopirellula baltica, DaeM, Thermoclostridium caenicola, Bacillus sp., Pirellula sp., Novibacillus thermophilus, Halanaerobium congolense, and Sinorhizobium fredii …”

“…Recently, a heat-tolerant DAEase (DaeM) from a thermal spring metagenome in India has been reported with half-life values of 165 and 54 h at 60 and 70 °C, respectively; however, its specific activity for catalyzing the epimerization of d -fructose into d -allulose is only 7 U/mg . On the other hand, several methods of molecular modification have been used for engineering thermostable versions of DAEase, such as gene fusion, , directed evolution, ,, and rational/semirational design. − Identifying potential mutation hotspots that affect thermostability plays a critical role in the rational/semirational design. Extensive research has shown that the difference in the free energy of folding between a wild-type protein and its mutant (ΔΔ G fold ) indicates the mutational effect .…”

Fine-Tuning of Carbon Flux and Artificial Promoters in Bacillus subtilis Enables High-Level Biosynthesis of d-Allulose

“…The health-promoting properties have inspired great interest in the development of the large-scale production process for d -allulose. ,, Conventional chemical synthesis is not suitable for industrial production due to harsh reaction conditions, low yield of the product, and environmental concerns. , By contrast, a highly specific and environmentally friendly biosynthetic pathway for d -allulose production by enzymatically epimerizing the C-3 position of d -fructose has tremendous potential as an industrial biocatalyst. , At present, the identified DAEases have high substrate specificity and mild reaction conditions, but low thermostability precludes their application under harsh industrial conditions. Li et al screened the thermostable DAEase variant M15S/P40N/S209N with a half-life of 22 h at 60 °C . Patel et al mined the thermostable DaeM with a half-life of 165 h at 60 °C and DaeB with about 600 h at 50 °C, respectively. , The enhanced thermostability and reusability of these enzymes indicate that they have the potential for the industrial production of d -allulose.…”

“…Li et al screened the thermostable DAEase variant M15S/ P40N/S209N with a half-life of 22 h at 60 °C. 11 Patel et al mined the thermostable DaeM with a half-life of 165 h at 60 °C and DaeB with about 600 h at 50 °C, respectively. 12,13 The enhanced thermostability and reusability of these enzymes indicate that they have the potential for the industrial production of D-allulose.…”