title: Efficient immobilization of AGE and NAL enzymes onto functional amino resin as recyclable and high-performance biocatalyst.
abstract: N-Acetylglucosamine-2-epimerase (AGE) and N-acetylneuraminic acid lyase (NAL) were immobilized for synthesis of N-acetylneuraminic acid (Neu5Ac) on three resins: Amberzyme oxirane resin (AOR), poly (styrene-co-DVB)-Br resin (PBR) and amino resin (AR). The loading capacity and immobilized enzyme activity showed that AR was the best carrier. Three methods of glutaraldehyde cross-linking were tested and simultaneous cross-linking and immobilization was demonstrated to be the best method. The functional properties of immobilized AGE and NAL were studied and compared to those of the free enzyme. The highest enzyme activities of free and immobilized AGE were obtained in 0.1 M potassium phosphate buffer at pH 7.5 and a temperature of 37 °C. Comparatively, the highest NAL activities were at pH 8.5. Meanwhile, an increase in Km(from 1.14 to 1.31 mg·mL-1for AGE and from 1.05 to 1.25 mg·mL-1for NAL) and a decrease in Vmax(from 177.53 to 106.37 µg·min-1mL-1for AGE and from 126.41 to 95.96 µg·min-1mL-1for NAL) were recorded after immobilization. The AR-glutaraldehyde-enzyme system exhibited better thermal stability than the free enzyme, and retained 72% of its initial activity even after eight repeated runs. The apparent activation energy (Ea) of the free and immobilized AGE (NAL) was 117.14 kJ·mol-1(124.21 kJ·mol-1) and 78.45 kJ·mol-1(66.64 kJ·mol-1), respectively, implying that the catalytic efficiency of the immobilized enzyme was restricted by mass-transfer rather than kinetic limit. Subsequently, Neu5Ac production from GlcNAc using immobilized enzymes in one reactor was carried out resulting 101.45 g·L-1of Neu5Ac and the highest conversion ratio of 82%. This method of enzyme immobilization may have a promising future for Neu5Ac production in industry.
authors.affiliation: State Key Laboratory of Materials-Oriented Chemical Engineering, Nanjing Tech University, No. 5, Xinmofan Road, Nanjing, 210009, People's Republic of China.|College of Biotechnology and Pharmaceutical Engineering, National Engineering Technique Research Center for Biotechnology, Nanjing Tech University, No. 30, Puzhu South Road, Nanjing, 211816, People's Republic of China.|Synergetic Innovation Center for Advanced Materials, Nanjing Tech University, No. 30, Puzhu South Road, Nanjing, 211816, People's Republic of China.
authors.forename: Jian
authors.initials: J
authors.lastname: Cheng
authors.affiliation: State Key Laboratory of Materials-Oriented Chemical Engineering, Nanjing Tech University, No. 5, Xinmofan Road, Nanjing, 210009, People's Republic of China.|College of Biotechnology and Pharmaceutical Engineering, National Engineering Technique Research Center for Biotechnology, Nanjing Tech University, No. 30, Puzhu South Road, Nanjing, 211816, People's Republic of China.|Synergetic Innovation Center for Advanced Materials, Nanjing Tech University, No. 30, Puzhu South Road, Nanjing, 211816, People's Republic of China.
authors.forename: Wei
authors.initials: W
authors.lastname: Zhuang
authors.affiliation: State Key Laboratory of Materials-Oriented Chemical Engineering, Nanjing Tech University, No. 5, Xinmofan Road, Nanjing, 210009, People's Republic of China.|College of Biotechnology and Pharmaceutical Engineering, National Engineering Technique Research Center for Biotechnology, Nanjing Tech University, No. 30, Puzhu South Road, Nanjing, 211816, People's Republic of China.|Synergetic Innovation Center for Advanced Materials, Nanjing Tech University, No. 30, Puzhu South Road, Nanjing, 211816, People's Republic of China.
authors.forename: Chenglun
authors.initials: C
authors.lastname: Tang
authors.affiliation: State Key Laboratory of Materials-Oriented Chemical Engineering, Nanjing Tech University, No. 5, Xinmofan Road, Nanjing, 210009, People's Republic of China.|College of Biotechnology and Pharmaceutical Engineering, National Engineering Technique Research Center for Biotechnology, Nanjing Tech University, No. 30, Puzhu South Road, Nanjing, 211816, People's Republic of China.|Synergetic Innovation Center for Advanced Materials, Nanjing Tech University, No. 30, Puzhu South Road, Nanjing, 211816, People's Republic of China.
authors.forename: Yong
authors.initials: Y
authors.lastname: Chen
authors.affiliation: State Key Laboratory of Materials-Oriented Chemical Engineering, Nanjing Tech University, No. 5, Xinmofan Road, Nanjing, 210009, People's Republic of China.|College of Biotechnology and Pharmaceutical Engineering, National Engineering Technique Research Center for Biotechnology, Nanjing Tech University, No. 30, Puzhu South Road, Nanjing, 211816, People's Republic of China.
authors.forename: Jinglan
authors.initials: J
authors.lastname: Wu
authors.affiliation: State Key Laboratory of Materials-Oriented Chemical Engineering, Nanjing Tech University, No. 5, Xinmofan Road, Nanjing, 210009, People's Republic of China.|College of Biotechnology and Pharmaceutical Engineering, National Engineering Technique Research Center for Biotechnology, Nanjing Tech University, No. 30, Puzhu South Road, Nanjing, 211816, People's Republic of China.
authors.forename: Ting
authors.initials: T
authors.lastname: Guo
authors.affiliation: State Key Laboratory of Materials-Oriented Chemical Engineering, Nanjing Tech University, No. 5, Xinmofan Road, Nanjing, 210009, People's Republic of China. yinghanjie@njtech.edu.cn.|College of Biotechnology and Pharmaceutical Engineering, National Engineering Technique Research Center for Biotechnology, Nanjing Tech University, No. 30, Puzhu South Road, Nanjing, 211816, People's Republic of China. yinghanjie@njtech.edu.cn.|Synergetic Innovation Center for Advanced Materials, Nanjing Tech University, No. 30, Puzhu South Road, Nanjing, 211816, People's Republic of China. yinghanjie@njtech.edu.cn.
authors.forename: Hanjie
authors.initials: H
authors.lastname: Ying
mesh_terms: D000117:Acetylglucosamine; D002021:Buffers; D002238:Carbohydrate Epimerases / Q000737:chemistry*; D002384:Catalysis; D003432:Cross-Linking Reagents / Q000737:chemistry; D004800:Enzymes, Immobilized / Q000737:chemistry*; D004852:Epoxy Compounds / Q000737:chemistry; D004926:Escherichia coli / Q000201:enzymology; D005976:Glutaral / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008190:Lyases / Q000737:chemistry*; D019158:N-Acetylneuraminic Acid / Q000737:chemistry*; D009438:Neuraminic Acids / Q000737:chemistry; D007652:Oxo-Acid-Lyases / Q000737:chemistry*; D010710:Phosphates / Q000737:chemistry; D017680:Potassium Compounds / Q000737:chemistry; D019998:Racemases and Epimerases / Q000737:chemistry*; D015203:Reproducibility of Results; D012117:Resins, Synthetic; D013696:Temperature
chemical_list: D002021:Buffers; D003432:Cross-Linking Reagents; D004800:Enzymes, Immobilized; D004852:Epoxy Compounds; D009438:Neuraminic Acids; D010710:Phosphates; D017680:Potassium Compounds; D012117:Resins, Synthetic; C013216:potassium phosphate; D008190:Lyases; D007652:Oxo-Acid-Lyases; C018728:N-acetylneuraminate lyase; D019998:Racemases and Epimerases; D002238:Carbohydrate Epimerases; D019158:N-Acetylneuraminic Acid; D005976:Glutaral; D000117:Acetylglucosamine
keywords: Amino resin; Immobilization; N-Acetylglucosamine 2-epimerase; N-Acetylneuraminic acid; N-Acetylneuraminic acid lyase