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Bioprocess Biosyst Eng. 2017 Mar;40(3):331-340. doi: 10.1007/s00449-016-1700-z. Epub 2016 Nov 15.

Efficient immobilization of AGE and NAL enzymes onto functional amino resin as recyclable and high-performance biocatalyst.

Cheng J1,2,3, Zhuang W1,2,3, Tang C1,2,3, Chen Y1,2,3, Wu J1,2, Guo T1,2, Ying H4,5,6.

Author information

1
State Key Laboratory of Materials-Oriented Chemical Engineering, Nanjing Tech University, No. 5, Xinmofan Road, Nanjing, 210009, People's Republic of China.
2
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.
3
Synergetic Innovation Center for Advanced Materials, Nanjing Tech University, No. 30, Puzhu South Road, Nanjing, 211816, People's Republic of China.
4
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.
5
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.
6
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.

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 K m (from 1.14 to 1.31 mg·mL-1 for AGE and from 1.05 to 1.25 mg·mL-1 for NAL) and a decrease in V max (from 177.53 to 106.37 µg·min-1 mL-1 for AGE and from 126.41 to 95.96 µg·min-1 mL-1 for 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 (E a) 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-1 of Neu5Ac and the highest conversion ratio of 82%. This method of enzyme immobilization may have a promising future for Neu5Ac production in industry.

KEYWORDS:

Amino resin; Immobilization; N-Acetylglucosamine 2-epimerase; N-Acetylneuraminic acid; N-Acetylneuraminic acid lyase

PMID:
27848098
DOI:
10.1007/s00449-016-1700-z
[Indexed for MEDLINE]

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