Identification and Enzymatic Characterization of a Sugar Phosphatase

doi:10.13560/j.cnki.biotech.bull.1985.2024-0095

Abstract

Abstract:

【Objective】 High value-added monosaccharides can be synthesized through multi-enzyme cascade reactions using starch or dextrin as substrates. In the reaction systems, sugar phosphatase catalyzes the irreversible dephosphorylation reaction of the phosphorylated sugar intermediates to generate the corresponding sugars. This work is aimed to mine the new sugar phosphatase and to characterize this enzyme. 【Method】 In this study, a gene with unknown function, named as TsPase derived from the thermophilic bacterium Thermoproteus sp.CIS_19, was obtained from gene mining and screening. TsPase was found to have sugar phosphatase activity after its heterologous soluble expression with Escherichia coli BL21（DE3）, purification and enzymatic characterization. 【Result】 TsPase has a T_m value of（80.3 ± 1.3）℃, while the optimal reaction temperature of 70℃ and pH of 4. Our study indicated that metal ion Mg²⁺ has a strong promoting effect on TsPase. With the substrate tagatose 6 phosphate kinetic constant K_m is（2.40 ± 0.98）mmol/L, and the catalytic constant k_cat is（102.50 ± 8.60）min^-1. TsPase was integrated into an in vitro synthetic enzymatic biosystem for the one-pot production of D-tagatose from maltodextrin. The production of D-tagatose in the reaction equilibrium system was（4.26 ± 0.03）g/L and the conversion rate reach 42.6% ± 0.3%. 【Conclusion】 Overall, TsPase possesses fine thermal stability and activity, as well as high conversation rate in vitro synthetic enzymatic biosystem. These characteristics make it great value in future theoretical research and industrial production.

Key words: sugar phosphatase, enzymatic characterization, thermal stability, gene mining

QIAO Ye, ZHANG Nan, YANG Jian-hua, ZHANG Cui-ying, ZHU Lei-lei. Identification and Enzymatic Characterization of a Sugar Phosphatase[J]. Biotechnology Bulletin, 2024, 40(7): 299-306.

Figures/Tables 5

Fig. 1 Phylogenetic tree constructed using AfPase as a template

Fig. 2 SDS-PAGE analysis of expression（A）and purification（B）of TsPase （A）1: The crude supernatant of pET28a（+）; 2: the crude supernatant of TsPase; 3: protein marker.（B）1: The purified enzyme of TsPase; 2: protein marker

Fig. 3 Determination of catalytic activity of TsPase with different substrates

Fig. 4 Enzymatic properties of TsPase A: Catalytic activity curve of TsPase with different temperature. B: pH Curve of TsPase. C: Effect of different metal ions on the activity of TsPase. D: The catalytic activity of TsPase with different concentration of Mg2+

Fig. 5 D-tagatose bioproduction from maltodextrin by an in vitro synthetic enzymatic biosystem

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