Immobilization of N-acetylneuraminic Acid Aldolaseand Properties of the Immobilized Enzyme

doi:10.13560/j.cnki.biotech.bull.1985.2019-1027

Abstract

Abstract: N-acetylneuraminic acid aldolase（NAL）was immobilized using LX-1000HFA amino resin. The immobilized conditions were optimized,and the enzymatic properties and stabilitiesbetweenfree and immobilized enzymes were compared. The optimized conditions of immobilization were asthat the quantity of carrier was 5.0 g,the immobilization time was 12 h,the buffer concentration was 1.0 mol/L,the pH was 7.5,and the temperature was 25℃. The prepared immobilized NAL had the highest viability and the enzyme activity,reached 200 U/g wet resin. Compared with the free enzyme,the optimal reaction temperature increased by 5℃,the optimal reaction pH remainedunchanged,and thermal- and pH-tolerancewere significantly improved. Meanwhile,the storage stability and operational stability of the immobilized enzyme were also greatly enhanced. Only 6% of the enzyme activity was lost after storage for 10 d at 4℃,and the immobilized enzyme retained 80% itsactivity after repeated usage for 10 times.Therefore,the immobilized enzyme hasfine thermal stability,pH stability,storage stability,and operational stability,which provides a theoretical basis for the industrial production of N-acetylneuraminic acid via enzymatic method.

Key words: N-acetylneuraminic acid aldolase, amino resinLX-1000HFA, immobilization, enzymatic property, N-acetylneuraminic acid

WANG Hui-lan, WU Jin-yong, CHEN Xiang-song, YUAN Li-xia, ZHU Wei-wei, YAO Jian-ming. Immobilization of N-acetylneuraminic Acid Aldolaseand Properties of the Immobilized Enzyme[J]. Biotechnology Bulletin, 2020, 36(6): 165-173.

References

[1] Schauer R.Achievements and challenges of sialic acid research[J]. Glycoconjugate Journal, 2000, 17(7-9):485-499.
[2] Saleem T.Soluble CD14, Sialic Acid and L-Fucose in breast milk and their role in increasing the immunity of breast-fed infants[J]. American Journal of Biochemistry and Biotechnology, 2011, 7(1):S35.
[3] Schnaar R L, Gerardy-Schahn R, Hildebrandt H.Sialic acids in the brain:Gangliosides and polysialic acid in nervous system development, stability, disease, and regeneration[J]. Physiological Reviews, 2014, 94(2):461-518.
[4] Kawai N, Ikematsu H, Iwaki N, et al.Comparison of the effectiveness of zanamivir and oseltamivir against influenza A/H1N1, A/H3N2, and B[J]. Clinical Infectious Diseases, 2009, 48(7):996-997.
[5] Wang B.Molecular Mechanism underlying sialic acid as an essential nutrient for brain development and cognition[J]. Advances in Nutrition:An International Review Journal, 2012, 3(3):465S-472S.
[6] Koketsu M, Juneja LR, Kawanami H, et al.Preparation of N-acetyln-eurammic acid from delipidated egg yolk[J]. Glycoconjugate Jou-rnal, 1992, 9(2):70-74.
[7] KimioFuruhata. Chemistry of N-Acetylneuraminic Acid(Neu5Ac)
[J]. Trends in Glycoscience and Glycotechnology, 2004, 16(89):143-169.
[8] Ishikawa M, Koizumi S.Microbial production of N-acetylneuraminic acid by genetically engineered Escherichia coli[J]. Carbohydrate Research, 2010, 345(18):2605-2609.
[9] 朱德强. 产N-乙酰神经氨酸重组大肠杆菌的构建及其生物转化合成[D]. 无锡:江南大学, 2017.
[10] 李宏越. 微生物发酵法生产N-乙酰神经氨酸的研究[D]. 大连:大连工业大学, 2014.
[11] Maru I, Ohnishi J, Ohta Y, et al.Simple and large-scale production of N-acetylneuraminic acid from N-acetyl-D-glucosamine and pyruvate using N-acyl-D-glucosamine 2-epi-merase and N-acetylneuraminatelyase[J]. Carbohydrate Research, 1998, 306:575-578.
[12] Mahmoudian M, Noble D, Drake C S, et al.An efficient process for production of N-acetylneuraminic acid using N-acetylneuraminic acid aldolase[J]. Enzyme MicrobTechnol, 1997, 20(5):393-400.
[13] Lin BX, Zhang ZJ, Liu WF, et al.Enhanced production of N-acetyl-D-neuraminic acid by multi-approachwhole-cell biocatalyst[J]. Applied Microbiology and Biotechnology, 2013, 97(11):4775-4784.
[14] Sun W, Ji W, Li N, et al.Construction and expression of a polycistronic plasmid encoding N-acetylglucosamine 2-epimerase and N-acetylneuraminic acid lyase simultaneously for production of N-acetylneuraminic acid[J]. Bioresource Technology, 2013, 130:23-29.
[15] Krajewska B.Application of chitin- and chitosan-based materials for enzyme immobilizations:A review[J]. Enzyme and Microbial Technology, 2004, 35(2-3):126-139.
[16] Mateo C, Palomo J M, Fernandez-Lorente G, et al.Improvement of enzyme activity, stability and selectivity via immobilization techniques[J]. Enzyme and Microbial Technology, 2007, 40(6):1451-1463.
[17] Wang M, Hua X, Yang R, et al.Immobilization of cellobiose 2-epimerase from Caldicellulosiruptorsaccharolyticus on commercial resin DuoliteA568[J]. Food Bioscience, 2016, 14:47-53.
[18] 钟汕. 碱性蛋白酶的固定化及其应用[D]. 杭州:浙江大学, 2018.
[19] Cheng J, Zhuang W, Tang C, et al.Efficient immobilization of AGE and NAL enzymes onto functional amino resin as recyclable and high-performance biocatalyst[J]. Bioprocess and Biosystems Engineering, 2017, 40(3):331-340.
[20] 尹春丽, 曹珊珊, 许乐, 等. 氨基树脂固定化S-腺苷甲硫氨酸合成酶的研究[J]. 化学与生物工程, 2014(9):17-20.
[21] Mendes AA, Giordano RC, Giordano RDLC, et al.Immobilization and stabilization of microbial lipases by multipoint covalent attachment on aldehyde-resin affinity:Application of the biocatalysts in biodiesel synthesis[J]. Journal of Molecular Catalysis B:Enzymatic, 2011, 68(1):109-115.
[22] He H, Wei Y, Luo H, et al.Immobilization and stabilization of cephalosporin C acylase on aminated support by crosslinking with glutaraldehyde and further modifying with aminated macromolecules[J]. Biotechnology Progress, 2015, 31:387-395.
[23] 许敬亮, 袁振宏, 张宇, 等. 氨基功能载体固定化酶研究进展[J]. 化工进展, 2010, 29(3):494-497.
[24] 杨茂区. 固定化N-乙酰-D神经氨酸醛缩酶的制备及生化性质研究[D]. 杭州:浙江大学, 2006.
[25] Hu S, Chen J, Yang Z, et al.Coupled bioconversion for preparation of N-acetyl-d-neuraminic acid using immobilized N-acetyl-d-glucosamine-2-epimerase and N-acetyl-d-neuraminic acid lyase[J]. Applied Microbiology Biotechnology, 2010, 85(5):1383-1391.
[26] 顾恺, 邹树平, 王志才, 等. 环氧树脂固定化卤醇脱卤酶的研究[J]. 现代化工, 2016(11):69-72.
[27] 嵇东情, 郑仁朝, 郑裕国, 等. 环氧树脂固定化拟南芥腈水解酶NIT的研究[J]. 精细与专用化学品, 2015, 23(5):12-16.
[28] 徐珊, 李任强, 张继福, 等. 使用国产环氧树脂LXEP-120固定化脂肪酶研究[J]. 广西师范大学学报:自然科学版, 2018, 36(4):113-123.
[29] 刘天龙. 异丙醇脱氢酶固定化工艺研究[D]. 杭州:浙江大学, 2018.
[30] Mendes AA, Freitas L, De Carvalho AKF, et al.Immobilization of a commercial lipase from Penicilliumcamembertii(Lipase G)by different strategies[J]. Enzyme Research, 2011, 2011:1-8.