Fusion and secretory expression of an exo‐inulinase and a d‐allulose 3‐epimerase to produce d‐allulose syrup from inulin. (21st August 2020)

• Record Type: Journal Article
• Authors: Huang, Yueyuan; Li, Liangfei; Chi, Yaowei; Sha, Yuanyuan; Wang, Rui; Xu, Zheng; Xu, Xiaoqi; Li, Sha; Gao, Zhen; Xu, Hong
• Is Part Of: Journal of the science of food and agriculture. Volume 101:Number 2(2021)
• Page Start: 693
• Page End: 702
• Publication Date: 2020-08-21
• Subjects: inulin -- fusion enzyme -- d‐Allulose -- Exo‐inulinase -- immobilized enzyme; Food -- Periodicals; Agriculture -- Periodicals; 664
• DOI: 10.1002/jsfa.10682
• Languages: English
• ISSNs: 0022-5142

Abstract

Abstract: BACKGROUND: This study developed a feasible catalytic method for d ‐allulose syrup production using a fusion enzyme, either in free or immobilized form, through hydrolysis of inulin extracted from Jerusalem artichoke tubers. RESULTS: d ‐Allulose 3‐epimerase (DAE) was actively expressed in secretory form by fusing with the extracellular exo‐inulinase CSCA in Escherichia coli BL21 (DE3). The best linker ligating the two enzymes was a flexible peptide containing 12 residues (GSAGSAAGSGEF). At 55 °C and pH 8.0, and as with the addition of 1 mmol L −1 Mn 2+, the CSCA‐linkerE‐DAE fusion enzyme obtained through high cell‐density cultivation displayed a maximal exo‐inulinase activity of 21.8 U mg −1 and resulted in a yield of 6.3 g L −1 d ‐allulose and 39.2 g L −1 d ‐fructose using 60 g L −1 inulin as the raw material. Catechol‐modified alginate with titanium ions (Alg(Ti)PDA) was found to be a promising immobilization material for the fusion enzyme. After conversion for 8 days, the Alg(Ti)PDA‐immobilized CSCA‐linkerE‐DAE (8 U g −1 ) completed 24 reaction cycles and retained over 80% of its original activity. Each reaction obtained an average of 19.8 g L −1 d ‐allulose and 32.7 g L −1 D‐fructose from 60 g L −1 inulin. CONCLUSION: This study shed light on a feasible and cost‐effective approach for the production of syrup containing d ‐allulose and D‐fructose with inulin as the raw material via the use of a CSCA and DAE fusion enzyme. This syrup is of added value as a functional sweetener. © 2020 Society of Chemical Industry