Abstract:Enzymatic deglycosylation is an important means to study the structure and function of glycoproteins. Traditional enzymatic deglycosylation mostly uses free enzymes, so that the residual deglycosylase becomes a potential source of contamination signals for subsequent glycoprotein analysis (such as mass spectrometry analysis). To explore new utilization methods of deglycosylation is of great value for the analysis of glycoprotein glycan chain structure. In this study, the recombinant endo-β-N-acetylglucosaminidase (EndoEf) of Enterococcus faecaliswas expressed and purified, using sepharose CL 6B activated by epichlorohydrin as the carrier to immobilize, and the catalytic characteristics of immobilized EndoEf were analyzed. The results showed that EndoEf could be efficiently expressed in Escherichia coli BL21 Star (DE3), and 186.5 mg of enzyme protein could be purified per liter of bacterial solution. The optimal conditions for immobilization of EndoEf were 4.0 mg enzyme per gram of carrier in 50 mmol/L phosphate buffer at pH 8.0, 12 h conjugated at 4 ℃. Immobilized enzymes could remove N-glycan chains of ribonuclease B (RNase B) and ovalbumin (Ova), remaining active when stored at 4 ℃ for 60 d. Immobilized EndoEf had higher activity in the range of 30~40 ℃ and pH 6.0~7.0. The tolerance to DL-dithiothreitol (DTT) and sodium dodecyl sulfate (SDS) was significantly improved than that of the free enzyme, which could tolerate denaturation conditions of 12.0 mmol/L DTT and 2.0% SDS, and tolerate 1.0 mol/L NaCl. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) analysis showed that centrifugation completely removed immobilized EndoEf from the reaction system. The establishment of immobilization method and the analysis of catalytic characteristics could provide a basis for the application of immobilized EndoEf in glycoprotein research.
[1] 李玲聪, 胡少锋, 谷天燕, 等. 2020. 苜蓿链霉菌内切β-N-乙酰氨基葡萄糖苷酶的克隆、表达及酶学性质[J]. 生物工程学报, 36(05): 932-941.
(Li L C, Hu S F, Gu T Y, et al.2020. Cloning, expression and characterization of a new endo-β-N-acetylglucosaminidase from Streptomyces alfalfae[J]. Chinese Journal of Biotechnology, 36(5): 932-941.)
[2] 史清洪, 彭冠英, 孙舒, 等. 2007. 环氧氯丙烷活化琼脂糖凝胶过程强化及性能评价[J]. 过程工程学报, 7(4): 743-746.
(Shi Q H, Peng G Y, Sun S, et al.2007. Process intensification and characterization of agarose gel activation with epichlorohydrin[J]. The Chinese Journal of Process Engineering, 7(4): 743-746.)
[3] 王家红, 童玥, 朱玥, 等. 2011. 蛋白质糖基化的研究进展[J]. 药物生物技术, 18(01): 77-80.
(Wang J H, Tong Y, Zhu Y, et al.2011. The research progress in protein glycosylation[J]. Pharmaceutical Biotechnology, 18(1): 77-80.)
[4] 王薇, 王晓蕾, 张琇. 2011. 酶固定化技术的应用[J]. 畜牧与饲料科学, 32(1): 63-65.
(Wang W, Wang X L, Zhang X.2011. Application on enzyme immobilization technique[J]. Animal Husbandry and Feed Science, 32(1): 63-64.)
[5] 徐云巧, 李婷婷, 吴彩娥, 等. 2017. 糖蛋白的去糖基化方法研究进展[J]. 中国生物工程杂志, 37(5): 97-106.
(Xu Y Q, Li T T, Wu C E, et al.2017. Research progress on the methods of deglycosylation of glycoproteins[J]. China Biotechnology, 37(5): 97-106.)
[6] 杨开伦, 雒秋江. 2002. 胰蛋白酶在环氧氯丙烷活化的2%珠状琼脂糖凝胶上固定化条件的研究[J]. 新疆农业大学学报, 25(1): 35-37.
(Yang K L, Luo Q J.2002. Study on trypsin under immobilizing conditions of epichlorohydrin-activating 2% beaded-agarose[J]. Journal of Xinjiang Agricultural University, 25(1): 35-37.)
[7] 杨清香, 曹军卫, 黄国锦. 1997. 糖基转移酶和去糖基化酶[J]. 氨基酸和生物资源, 19(02): 40-45.
(Yang Q X, Cao J W, Huang G J.1997. Glycosyltransferases and the enzymes of deglycosylation[J]. Amino Acids & Biotic Resources, 19(2): 38-43.)
[8] 张英, 刘影, 刘洋, 等. 2016. 微量生物样品中糖蛋白N-糖链电喷雾电离质谱分析前处理方法的建立[J]. 分析化学, 44(02): 265-272.
(Zhang Y, Liu Y, Liu Y, et al.2016. Pretreatment method for detecting of N-linked glycans of glycoprotein in micro-scale complex biological sample based on electrospray ionization mass spectrometry[J]. Chinese Journal of Analytical Chemistry, 44(02): 265-272.)
[9] 支艳艳, 唱韶红, 巩新, 等. 2014. Endo-H在毕赤酵母中的表达、纯化及其在N-糖基化分析中的应用[J]. 军事医学, 38(03): 193-197.
(Zhi Y Y, Chang S H, Gong X, et al.2014. Expression of endo-beta-N-acetylglucosaminidase H in Pichia pastoris and its application to N-glycosylation analysis[J]. Millitary Medical Sciences, 38(3): 193-197.)
[10] Apweiler R, Hermjakob H, Sharon N.1999. On the frequency of protein glycosylation, as deduced from analysis of the SWISS-PROT database[J]. Biochimica et Biophysica Acta-Bioenergetics, 1473(1): 4-8.
[11] Atsushi M.2015. Improvement of endo-β-N-acetylglucosaminidase H production using silkworm-baculovirus protein expression system[J]. Journal of Asia Pacific Entomology, 18(2): 175-180.
[12] Chen C C, Su W C, Huang B Y, et al.2014. Interaction modes and approaches to glycopeptide and glycoprotein enrichment[J]. Analyst, 139(4): 688-704.
[13] Collin M, Olsén A.2001. EndoS, a novel secreted protein from Streptococcus pyogenes with endoglycosidase activity on human IgG[J]. EMBO Journal, 20(12): 3046-3055.
[14] Collin M, Fischetti V A.2004. A novel secreted endoglycosidase from Enterococcus faecalis with activity on human immunoglobulin G and ribonuclease B[J]. Journal of Biological Chemistry, 279(21): 22558-22570.
[15] Dall'Olio F.1996. Protein glycosylation in cancer biology: An overview[J]. Journal of Clinical Pathologu-Molecula Pathology, 49(3): M126-M135.
[16] Damm J B, Kamerling J P, van Dedem G W, et al.1987. A general strategy for the isolation of carbohydrate chains from N-,O-glycoproteins and its application to human chorionic gonadotrophin[J]. Glycoconjugate Journal, 4(2): 129-144.
[17] Datta S, Christena L R, Rajaram Y R S.2013. Enzyme immobilization: An overview on techniques and support materials[J]. 3 Biotech, 3(1): 1-9.
[18] Dicosimo R, Mcauliffe J, Poulose A J, et al.2013. Industrial use of immobilized enzymes[J]. Chemical Society Reviews, 42(15): 6437-6474.
[19] Frisch E, Schwedler C, Kaup M, et al.2013. Endo-β-N-acetylglucosaminidase H de-N-glycosylation in a domestic microwave oven: Application to biomarker discovery[J]. Analytical Biochemistry, 433: 65-69.
[20] Farsang R, Kovács N, Szigeti M, et al.2022. Immobilized exoglycosidase matrix mediated solid phase glycan sequencing[J]. Analytica Chimica Acta, 1215: 339906.
[21] Filho D G, Silva A G, Guidini C Z.2019. Lipases: Sources, immobilization methods, and industrial applications[J]. Applied Microbiology and Biotechnology, 103(18): 7399-7423.
[22] Karav S, Cohen J L, Barile D, et al.2017. Recent advances in immobilization strategies for glycosidases[J]. Biotechnology Progress, 33(1): 104-112.
[23] Kim M I, Ham H O, Oh S D, et al.2006. Immobilization of Mucor javanicus lipase on effectively functionalized silica nanoparticles[J]. Journal of Molecular Catalysis B-Enzymatic, 39(1-4): 62-68.
[24] Krenkova J, Szekrenyes A, Keresztessy Z, et al.2013. Oriented immobilization of peptide-N-glycosidase F on a monolithic support for glycosylation analysis[J]. Journal of Chromatography A, 1322: 54-61.
[25] Krištić J, Lauc G.2017. Ubiquitous importance of protein glycosylation[J]. Methods in Molecular Biology, 1503: 1-12.
[26] Kwan E M, Boraston A B, Mclean B W, et al.2005. N-glycosidase-carbohydrate-binding module fusion proteins as immobilized enzymes for protein deglycosylation[J]. Protein Engineering Design & Selection Peds, 18(10): 497-501.
[27] Masahara-Negishi Y, Hosomi A, Mea M D, et al.2012. A plant peptide: N-glycanase orthologue facilitates glycoprotein ER-associated degradation in yeast[J]. Biochimica et Biophysica Acta-bioenergetics, 1820: 1457-1462.
[28] Nuck R, Zimmermann M, Sauvageot D, et al.1990. Optimized deglycosylation of glycoproteins by peptide-N4-(N-acetyl-beta-glucosaminyl)-asparagine amidase from Flavobacterium meningosepticum[J]. Glycoconjugate Journal, 7(4): 279-286.
[29] Ohtsubo K, Marth J D.2006. Glycosylation in cellular mechanisms of health and disease[J]. Cell, 8(19): 855-867.
[30] Ongay S, Boichenko A, Govorukhina N, et al.2012. Glycopeptide enrichment and separation for protein glycosylation analysis[J]. Journal of Separation Science, 35(18): 2341-2372.
[31] Parc A L, Karav S, Bell, et al.2015. A novel endo-β-N-acetylglucosaminidase releases specific N-glycans depending on different reaction conditions[J]. Biotechnology Progress, 31(5): 1323-1330.
[32] Szabo Z, Guttman A, Karger B L.2010. Rapid release of N-linked glycans from glycoproteins by pressure-cycling technology[J]. Analytical Chemistry, 82(6): 2588-2593.
[33] Takashima S, Kurogochi M, Osumi K, et al.2020. Novel endo-β-N-acetylglucosaminidases from Tannerella species hydrolyze multi-branched complex-type N-glycans with different specificities[J]. Glycobiology, 30(11): 923-934.
[34] Wang L X.2011. The amazing transglycosylation activity of endo-β-N-acetylglucosaminidases[J]. Trends in Glycoscience & Glycotechnology, 23(129): 33-52.
[35] Yamagami M, Matsui Y, Hayakawa T, et al.2017. Plug-plug kinetic capillary electrophoresis for in-capillary exoglycosidase digestion as a profiling tool for the analysis of glycoprotein glycans[J]. Journal of Chromatography A, 1496: 157-162.
[36] Zacchi L F, Schulz B L.2016. N-glycoprotein macroheterogeneity: Biological implications and proteomic characterization[J]. Glycoconjugate Journal, 33(3): 359-376.