A novel d-allulose 3-epimerase from Staphylococcus aureus (SaDAE) has been screened as a d-allulose 3-epimerase family enzyme based on its high specificity for d-allulose. It usually converts both d-fructose and d-tagatose to respectively d-allulose and d-sorbose. We targeted potential biocatalysts for the large-scale industrial production of rare sugars. SaDAE showed a high activity on d-allulose with an affinity of 41.5 mM and catalytic efficiency of 1.1 s−1 mM−1. Four residues, Glu146, Asp179, Gln205, and Glu240, constitute the catalytic tetrad of SaDAE. Glu146 and Glu240 formed unique interactions with substrates based on the structural model analysis. The redesigned SaDAE_V105A showed an improvement of relative activity toward d-fructose of 68%. The conversion rate of SaDAE_V105A reached 38.9% after 6 h. The triple mutant S191D/M193E/S213C showed higher thermostability than the wild-type enzyme, exhibiting a 50% loss of activity after incubation for 60 min at 74.2 °C compared with 67 °C for the wild type. We redesigned SaDAE for thermostability and biocatalytic production of d-allulose. The research will aid the development of industrial biocatalysts for d-allulose.

Ulvans, complex polysaccharides found in the ulvales (green seaweed) cell wall, contain predominantly 3-sulfated rhamnose (Rha3S) linked to either D-glucuronic acid, L-iduronic acid or D-xylose. The ulvan lyase endolytically cleaves the glycoside bond between Rha3S and uronic acid via a β-elimination mechanism. Ulvan lyase has been identified as belonging to the polysaccharide lyase family PL24 or PL25 in the carbohydrate active enzymes database, in which fewer members have been characterized. We present the cloning and characterization of a novel ulvan lyase from Pseudoalteromonas sp. strain PLSV (PsPL). The enzymes were heterologously expressed in Escherichia coli BL21 (DE3) and purified as the His-tag fusion protein using affinity chromatography, ion-exchange chromatography and size-exclusion chromatography. The degradation products were determined by thin-layer chromatography (TLC), liquid chromatography-mass spectrometry (LC-MS) to be mainly disaccharides and tetrasaccharides. Ulvan lyase provides an example of degrading ulvales into oligosaccharides. Arg265, His152 and Tyr249 were considered to serve as catalytic residues based on PsPL structural model analysis.