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. 2020 Dec 10:8:622325.
doi: 10.3389/fchem.2020.622325. eCollection 2020.

Biocatalytic Synthesis of D-Allulose Using Novel D-Tagatose 3-Epimerase From Christensenella minuta

Yang Wang  1 , Yuvaraj Ravikumar  2 , Guoyan Zhang  2 , Junhua Yun  2 , Yufei Zhang  2 , Amreesh Parvez  2 , Xianghui Qi  1   2 , Wenjing Sun  2
Affiliations

Biocatalytic Synthesis of D-Allulose Using Novel D-Tagatose 3-Epimerase From Christensenella minuta

Yang Wang et al. Front Chem. .

Abstract

D-allulose, which is one of the important rare sugars, has gained significant attention in the food and pharmaceutical industries as a potential alternative to sucrose and fructose. Enzymes belonging to the D-tagatose 3-epimerase (DTEase) family can reversibly catalyze the epimerization of D-fructose at the C3 position and convert it into D-allulose by a good number of naturally occurring microorganisms. However, microbial synthesis of D-allulose is still at its immature stage in the industrial arena, mostly due to the preference of slightly acidic conditions for Izumoring reactions. Discovery of novel DTEase that works at acidic conditions is highly preferred for industrial applications. In this study, a novel DTEase, DTE-CM, capable of catalyzing D-fructose into D-allulose was applications. In this study, a novel DTEase, DTE-CM, capable of catalyzing D-fructose into D-allulose was DTE-CM on D-fructose was found to be remarkably influenced and modulated by the type of metal ions (co-factors). The DTE-CM on D-fructose was found to be remarkably influenced and modulated by the type of metal ions (co-factors). The 50°C from 0.5 to 3.5 h at a concentration of 0.1 mM. The enzyme exhibited its maximum catalytic activity on D-fructose at pH 6.0 and 50°C from 0.5 to 3.5 h at a concentration of 0.1 mM. The enzyme exhibited its maximum catalytic activity on -fructose at pH 6.0 and 50°C with a K cat /K m value of 45 mM-1min-1. The 500 g/L D-fructose, which corresponded to 30% conversion rate. With these interesting catalytic properties, this enzyme could be a promising candidate for industrial biocatalytic applications.

Keywords: Christensenella minuta; D-allulose; D-tagatose 3-epimerase; biocatalysis; biochemical characterization.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
The epimerization reactions of D-fructose and D-tagatose catalyzed by DTEase family.
Figure 2
Figure 2
Alignment of the amino acid sequences of DTEase family. The origins of DTEase family and their GenBank Nos are as follows: P. cichorii (BAA24429.1); R. sphaeroides (ACO59490.1); C. fortuita (WP_061137998.1); Sinorhizobium sp. (WP_069063284.1); A. tumefaciens (AAK88700.1); C. cellulolyticum H10 (ACL75304); Ruminococcus sp. (ZP_04858451); Clostridium sp. (WP_014314767.1);C. scindens (B0NGC5); Desmospora sp. (F5SL39); C. bolteae (A8RG82); Dorea sp. (CDD07088.1); T. primitia (WP_010256447.1); F. plautii (EHM40452.1); A. globiformis (BAW27657.1); Agrobacterium sp. (EGL65884.1); P. senegalensis (WP_010270828.1); S. aureus (SQA09501.1); DaeM (MN337631); C. minuta WP_066519968.1. The residues involved in the metal coordinating site (▴) are symbolized according to the crystal structures of A. tumefaciens DAEase (Kim et al., 2006b), C. cellulolyticum DAEase (Chan et al., 2012) and P. cichorii DTEase (Yoshida et al., 2007), The alignment was prepared using the program ESPript.
Figure 3
Figure 3
The phylogenetic tree of DTEase family, the microorganism origins of DTEase family with GenBank No.s as follows: P. cichorii (BAA24429.1); R. sphaeroides (ACO59490.1); C. fortuita (WP_061137998.1); Sinorhizobium sp. (WP_069063284.1); A. tumefaciens (AAK88700.1); C. cellulolyticum H10 (ACL75304); Ruminococcus sp. (ZP_04858451); Clostridium sp. (WP_014314767.1); C. scindens (B0NGC5); Desmospora sp. (F5SL39); C. bolteae (A8RG82); Dorea sp. (CDD07088.1); T. primitia (WP_010256447.1); F. plautii (EHM40452.1); A. globiformis (BAW27657.1); Agrobacterium sp. (EGL65884.1); P. senegalensis (WP_010270828.1); S. aureus (SQA09501.1); DaeM (MN337631); C. minuta (This study) AQ.
Figure 4
Figure 4
SDS-PAGE analysis of the DTE-CM and HPLC analysis of the isomerization products by the purified DTE-CM. (A) SDS-PAGE analysis. Lane M, protein marker; Lane 1, the control E. coli Rosetta (DE3) cells harboring plasmid pANY1; Lane 2, E. coli Rosetta (DE3) harboring pANY-Cm-dte; Lane 3, purified recombinant DTEase. (B) The HPLC map of DTE biotransformation sample compared with standard D-allulose. Peak 1 is D-fructose; peak 2 is D-allulose; peak 3 is the isomerization products.
Figure 5
Figure 5
Effect of metal ions (A) and metal ion concentration (B) on DTE-CM activity.
Figure 6
Figure 6
Effect of pH and temperature on activity of DTE-CM: (A) temperature dependence; (B) pH dependence; (C) pH stability; (D) thermostability analysis.
Figure 7
Figure 7
Bioconversion of D-fructose to D-allulose by DTE-CM: 100 (formula image); 300 (formula image); 500 g/L (formula image).
See this image and copyright information in PMC

References

    1. Afach G., Kawanami Y., Cheetangdee N., Fukada K., Izumori K. (2008). Lipase-catalyzed synthesis of D-psicose satty acid diesters and their emulsification activities. J. Am. Oil Chem. Soc. 85, 755–760. 10.1007/s11746-008-1242-x - DOI
    1. Bhosale S. H., Rao M. B., Deshpande V. V. (1996). Molecular and industrial aspects of glucose isomerase. Microbiol. Rev. 60, 280–300. 10.1128/MMBR.60.2.280-300.1996 - DOI - PMC - PubMed
    1. Bilik V., Tihlarik K. (1973). Reaction of saccharides catalyzed by molybdate ions. IX. epimerization of ketohexoses. Chem. Zvesti. 28, 106–109.
    1. Bradford M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248–254. 10.1016/0003-2697(76)90527-3 - DOI - PubMed
    1. Chan H. C., Zhu Y., Hu Y., Ko T. P., Huang C. H., Ren F., et al. . (2012). Crystal structures of D-psicose 3-epimerase from Clostridium cellulolyticum H10 and its complex with ketohexose sugars. Protein Cell 3, 123–131. 10.1007/s13238-012-2026-5 - DOI - PMC - PubMed

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