Lei Wang1,2, Meirong Hu3, Yong Tao3,4. 1. Chinese Academy of Sciences Key Laboratory of Microbial Physiological and Metabolic Engineering, Institute of Microbiology, Chinese Academy of Sciences, Beijing, 100101, China. wl8893@163.com. 2. College of Life Science, University of Chinese Academy of Sciences, Beijing, 100049, China. wl8893@163.com. 3. Chinese Academy of Sciences Key Laboratory of Microbial Physiological and Metabolic Engineering, Institute of Microbiology, Chinese Academy of Sciences, Beijing, 100101, China. 4. College of Life Science, University of Chinese Academy of Sciences, Beijing, 100049, China.
Abstract
OBJECTIVE: D-Glucosamine (GlcN) is an important amino sugar with various applications in medicine, food & beverages, nutritional supplements, and dairy products. This study aimed to produce GlcN from N-acetyl-D-glucosamine (GlcNAc) with an efficient deacetylase, and apply different strategies to enhance GlcN production. RESULTS: We screened a series of deacetylases that involved in the deacetylation of GlcNAc to form GlcN. A diacetylchitobiose deacetylase (TKDac) from Thermococcus kodakarensis exhibited high-efficient deacetylation activity for GlcNAc, yet mostly in the form of inclusion bodies. The soluble expression of TKDac was improved by a co-expressing molecular chaperone (groEL) and TKDac, and insertion of rare codon ATA encoding isoleucine. As such, the recombinant strain TKEL4 was constructed to express TKDac, and 48 g/L GlcN was achieved by TKDac-catalyzed deacetylation. To overcome the inhibition of byproduct (acetate), immobilized TKDac was carried out to produce GlcN from GlcNAc. The immobilized TKDac was conveniently re-used for several batches (above 8) with a 90% conversion rate. CONCLUSIONS: TKDac from T. kodakarensis was found to be an efficient deacetylase to produce GlcN. Co-expression of molecular chaperone and target protein, and insertion of rare codons were effective to improve the soluble expression of TKDac. The immobilized TKDac represents a promising method for future GlcN production.
OBJECTIVE: D-Glucosamine (GlcN) is an important amino sugar with various applications in medicine, food & beverages, nutritional supplements, and dairy products. This study aimed to produce GlcN from N-acetyl-D-glucosamine (GlcNAc) with an efficient deacetylase, and apply different strategies to enhance GlcN production. RESULTS: We screened a series of deacetylases that involved in the deacetylation of GlcNAc to form GlcN. A diacetylchitobiose deacetylase (TKDac) from Thermococcus kodakarensis exhibited high-efficient deacetylation activity for GlcNAc, yet mostly in the form of inclusion bodies. The soluble expression of TKDac was improved by a co-expressing molecular chaperone (groEL) and TKDac, and insertion of rare codon ATA encoding isoleucine. As such, the recombinant strain TKEL4 was constructed to express TKDac, and 48 g/L GlcN was achieved by TKDac-catalyzed deacetylation. To overcome the inhibition of byproduct (acetate), immobilized TKDac was carried out to produce GlcN from GlcNAc. The immobilized TKDac was conveniently re-used for several batches (above 8) with a 90% conversion rate. CONCLUSIONS: TKDac from T. kodakarensis was found to be an efficient deacetylase to produce GlcN. Co-expression of molecular chaperone and target protein, and insertion of rare codons were effective to improve the soluble expression of TKDac. The immobilized TKDac represents a promising method for future GlcN production.
Authors: Daniel G Gibson; Lei Young; Ray-Yuan Chuang; J Craig Venter; Clyde A Hutchison; Hamilton O Smith Journal: Nat Methods Date: 2009-04-12 Impact factor: 28.547
Authors: Laura Fernandez-Lopez; Sara G Pedrero; Nerea Lopez-Carrobles; Beatriz C Gorines; Jose J Virgen-Ortíz; Roberto Fernandez-Lafuente Journal: Enzyme Microb Technol Date: 2016-12-16 Impact factor: 3.493
Authors: Ming-De Deng; David K Severson; Alan D Grund; Sarah L Wassink; Richard P Burlingame; Alan Berry; Jeffrey A Running; Candice A Kunesh; Linsheng Song; Thomas A Jerrell; Reinhardt A Rosson Journal: Metab Eng Date: 2005-05 Impact factor: 9.783