氨基树脂固定化L-苏氨酸醛缩酶及其应用<sup>*</sup>

doi:10.13523/j.cb.2106029

中国生物工程杂志

2021, Vol. 41

Issue (9): 55-63 DOI: 10.13523/j.cb.2106029

技术与方法

氨基树脂固定化L-苏氨酸醛缩酶及其应用^*

陈开通¹,郑文隆^1,²,杨立荣^1,²,徐刚¹,吴坚平^1,^2,^**(

)

1 浙江大学化学工程与生物工程学院生物工程研究所杭州 310027
2 杭州国际科创中心杭州 311200

Immobilized L-threonine Aldolase by Amino Resin and Its Application

CHEN Kai-tong¹,ZHENG Wen-long^1,²,YANG Li-rong^1,²,XU Gang¹,WU Jian-ping^1,^2,^**(

)

1 Institute of Bioengineering, College of Chemical and Biological Engineering, Zhejiang University, Hangzhou 310027, China
2 Hangzhou Global Scientific and Technological Innovation Center, Zhejiang University,Hangzhou 311200, China

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摘要：

L-苏氨酸醛缩酶(L-Threonine aldolase,L-TA)可以催化甘氨酸和醛合成β-羟基-α-氨基酸。β-羟基-α-氨基酸具有两个手性中心,是多种手性药物的中间体。但是,游离的L-TA难以重复利用,分离纯化困难,严重阻碍了工业化应用。固定化技术可以有效解决这些问题。利用氨基树脂NAA固定化来源于Bacillus nealsonii的L-苏氨酸醛缩酶,采用戊二醛作为交联剂,经过条件优化确定最佳固定化条件为:加酶量13 U、载体量0.6 g、0.4%(V/V)戊二醛、活化时间2 h、pH 8.5、35℃、固定化5 h。在此条件下,固定化酶酶活回收率为85.7%。在30℃下半衰期可达59天,为游离酶的6.5倍。将其应用于合成L-syn-对甲砜基苯丝氨酸,使用460 h后,残余酶活为79.4%。进一步开发了载体再利用策略,将失活固定化酶表面的氨基用戊二醛活化后,再与新的游离酶进行固定化,实现载体的再利用。利用该方法载体可重复利用两次,制备的固定化酶仍能使用460 h。该方法大大降低了固定化成本,为固定化L-TA的工业化应用打下坚实的基础。

关键词： L-苏氨酸醛缩酶; 固定化; 载体再利用; 氨基树脂; L-syn-对甲砜基苯丝氨酸

Abstract:

L-threonine aldolase (L-TA) synthesizes β-hydroxy-α-amino from glycine and aldehyde. β-hydroxy-α-amino acids with two chiral centers is an important pharmaceutical intermediate for the synthesis of many drugs. However, free threonine aldolase is difficult to be reused and separated, which seriously hinders industrial application. Immobilization technology can effectively solve these problems. L-threonine aldolase was immobilized by amino carrier NAA and glutaraldehyde was used as the crosslinking reagent. After optimization of conditions, the optimal immobilization conditions were determined as: enzyme loading 13 U, 0.6 g carrier, glutaraldehyde concentration of 0.4 %(V/V), activation time of 2 h, pH of 8.5 and temperature of 35℃, and immobilization time of 5 h. Under these conditions, the activity recovery of immobilized enzyme was 85.7%. The half-life at 30℃ can reach 59 days, which was 6.5 times that of the free enzyme. The immobilized enzyme was applied in the synthesis of L-syn-3-[4-(methylsulfonyl)phenylserine] (L-syn-MTPS) for 460 h still remained activity of 79.4%. Furthermore, the method to reuse carrier was developed. In this method, the residual amino groups on the surface of the inactivated immobilized enzyme were first activated by glutaraldehyde, and then further combined with the fresh enzyme to realize the reuse of the resin. Using this method, the carrier can be reused twice and the prepared immobilized enzyme can still be used for 460 h. This method greatly reduces the cost of immobilization and lays a solid foundation for the industrial application of immobilized L-TA.

Key words: L-Threonine aldolase Immobilization Reuse carrier Amino resin L-syn-3-[4-(methylsulfonyl)phenylserine]

收稿日期: 2021-06-16 出版日期: 2021-09-30

ZTFLH:

Q814

基金资助: * 国家重点研发计划(2019YFA09005000)

通讯作者: 吴坚平 E-mail: wjp@zju.edu.cn

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引用本文:

陈开通,郑文隆,杨立荣,徐刚,吴坚平. 氨基树脂固定化L-苏氨酸醛缩酶及其应用^*[J]. 中国生物工程杂志, 2021, 41(9): 55-63.

CHEN Kai-tong,ZHENG Wen-long,YANG Li-rong,XU Gang,WU Jian-ping. Immobilized L-threonine Aldolase by Amino Resin and Its Application. China Biotechnology, 2021, 41(9): 55-63.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.2106029 或 https://manu60.magtech.com.cn/biotech/CN/Y2021/V41/I9/55

图1 固定化条件的优化

图2 固定化酶和游离酶酶学性质表征

图3 L-TA@NAA的操作稳定性

图4 载体再利用方案设计

图5 再利用树脂特性

图6 NAA、L-TA@NAA、L-TA@NAA-1的透射电镜图

表S1 不同方法固定化L-TA的活性回收率和蛋白质回收率

[1]	Chen Q J, Chen X, Feng J H, et al. Improving and inverting cβ-stereoselectivity of threonine aldolase via substrate-binding-guided mutagenesis and a stepwise visual screening. ACS Catalysis, 2019, 9(5):4462-4469. doi: 10.1021/acscatal.9b00859
[2]	Liu Z C, Chen X, Chen Q J, et al. Engineering of l-threonine aldolase for the preparation of 4-(methylsulfonyl)phenylserine, an important intermediate for the synthesis of florfenicol and thiamphenicol. Enzyme and Microbial Technology, 2020, 137:109551. doi: 10.1016/j.enzmictec.2020.109551
[3]	Wang L C, Xu L, Xu X Q, et al. An L-threonine aldolase for asymmetric synthesis of β-hydroxy-α-amino acids. Chemical Engineering Science, 2020, 226:115812. doi: 10.1016/j.ces.2020.115812
[4]	Xu L, Wang L C, Xu X Q, et al. Characteristics of l-threonine transaldolase for asymmetric synthesis of β-hydroxy-α-amino acids. Catalysis Science & Technology, 2019, 9(21):5943-5952.
[5]	Zheng W L, Chen K T, Wang Z, et al. Construction of a highly diastereoselective aldol reaction system with l-threonine aldolase by computer-assisted rational molecular modification and medium engineering. Organic Letters, 2020, 22(15):5763-5767. doi: 10.1021/acs.orglett.0c01792
[6]	López C, Ríos S D, López-Santín J, et al. Immobilization of PLP-dependent enzymes with cofactor retention and enhanced stability. Biochemical Engineering Journal, 2010, 49(3):414-421. doi: 10.1016/j.bej.2010.02.004
[7]	Mateo C, Palomo J M, Fernandez-Lorente G, et al. Improvement of enzyme activity, stability and selectivity via immobilization techniques. Enzyme and Microbial Technology, 2007, 40(6):1451-1463. doi: 10.1016/j.enzmictec.2007.01.018
[8]	Rodrigues R C, Ortiz C, Berenguer-Murcia Á, et al. Modifying enzyme activity and selectivity by immobilization. Chemical Society Reviews, 2013, 42(15):6290-6307. doi: 10.1039/c2cs35231a pmid: 23059445
[9]	Zhang Y F, Ge J, Liu Z. Enhanced activity of immobilized or chemically modified enzymes. ACS Catalysis, 2015, 5(8):4503-4513. doi: 10.1021/acscatal.5b00996
[10]	Pal A, Khanum F. Covalent immobilization of xylanase on glutaraldehyde activated alginate beads using response surface methodology: Characterization of immobilized enzyme. Process Biochemistry, 2011, 46(6):1315-1322. doi: 10.1016/j.procbio.2011.02.024
[11]	Fu H, Dencic I, Tibhe J, et al. Threonine aldolase immobilization on different supports for engineering of productive, cost-efficient enzymatic microreactors. Chemical Engineering Journal, 2012, 207-208:564-576. doi: 10.1016/j.cej.2012.07.017
[12]	Tibhe J D, Fu H, Noël T, et al. Flow synthesis of phenylserine using threonine aldolase immobilized on eupergit support. Beilstein Journal of Organic Chemistry, 2013, 9:2168-2179. doi: 10.3762/bjoc.9.254
[13]	Zheng W L, Chen K T, Fang S, et al. Construction and application of PLP self-sufficient biocatalysis system for threonine aldolase. Enzyme and Microbial Technology, 2020, 141:109667. doi: 10.1016/j.enzmictec.2020.109667
[14]	Kurjatschij S, Katzberg M, Bertau M. Production and properties of threonine aldolase immobilisates. Journal of Molecular Catalysis B: Enzymatic, 2014, 103:3-9. doi: 10.1016/j.molcatb.2014.01.021
[15]	Filho M, Pessela B C, Mateo C, et al. Reversible immobilization of a hexameric α-galactosidase from Thermus sp. strain T2 on polymeric ionic exchangers. Process Biochemistry, 2008, 43(10):1142-1146. doi: 10.1016/j.procbio.2008.05.016
[16]	Yakup Arica M, Soydogan H, Bayramoglu G. Reversible immobilization of Candida rugosa lipase on fibrous polymer grafted and sulfonated p(HEMA/EGDMA) beads. Bioprocess and Biosystems Engineering, 2010, 33(2):227-236. doi: 10.1007/s00449-009-0316-y pmid: 19350276
[17]	Zhao G H, Wang J Z, Li Y F, et al. Reversible immobilization of glucoamylase onto metal-ligand functionalized magnetic FeSBA-15. Biochemical Engineering Journal, 2012, 68:159-166. doi: 10.1016/j.bej.2012.04.009
[18]	Aslan Y, Tanriseven A. Immobilization of pectinex ultra SP-L to produce galactooligosaccharides. Journal of Molecular Catalysis B: Enzymatic, 2007, 45(3-4):73-77. doi: 10.1016/j.molcatb.2006.12.005
[19]	Sui Y, Cui Y, Xia G M, et al. A facile route to preparation of immobilized cellulase on polyurea microspheres for improving catalytic activity and stability. Process Biochemistry, 2019, 87:73-82. doi: 10.1016/j.procbio.2019.09.002
[20]	朱衡, 林海蛟, 张继福, 等. 氨基载体共价结合固定化海洋假丝酵母脂肪酶. 中国生物工程杂志, 2019, 39(7):71-78.
	Zhu H, Lin H J, Zhang J F, et al. Covalent immobilization of marine Candida rugosa lipase using amino carrier. China Biotechnology, 2019, 39(7):71-78.
[21]	Lyu J B, Li Z D, Men J C, et al. Covalent immobilization of Bacillus subtilis lipase A on Fe3O4 nanoparticles by aldehyde tag: an ideal immobilization with minimal chemical modification. Process Biochemistry, 2019, 81:63-69. doi: 10.1016/j.procbio.2019.03.017
[22]	Cherian E, Dharmendirakumar M, Baskar G. Immobilization of cellulase onto MnO2 nanoparticles for bioethanol production by enhanced hydrolysis of agricultural waste. Chinese Journal of Catalysis, 2015, 36(8):1223-1229. doi: 10.1016/S1872-2067(15)60906-8
[23]	Nagar S, Mittal A, Kumar D, et al. Immobilization of xylanase on glutaraldehyde activated aluminum oxide pellets for increasing digestibility of poultry feed. Process Biochemistry, 2012, 47(9):1402-1410. doi: 10.1016/j.procbio.2012.05.013
[24]	Ranjbakhsh E, Bordbar A K, Abbasi M, et al. Enhancement of stability and catalytic activity of immobilized lipase on silica-coated modified magnetite nanoparticles. Chemical Engineering Journal, 2012, 179:272-276. doi: 10.1016/j.cej.2011.10.097
[25]	Keerti, Gupta A, Kumar V, et al. Kinetic characterization and effect of immobilized thermostable β-glucosidase in alginate gel beads on sugarcane juice. ISRN Biochemistry, 2014, 2014:1-8.
[26]	Ozyilmaz G, Gezer E. Production of aroma esters by immobilized Candida rugosa and porcine pancreatic lipase into calcium alginate gel. Journal of Molecular Catalysis B: Enzymatic, 2010, 64(3-4):140-145. doi: 10.1016/j.molcatb.2009.04.013
[27]	Migneault I, Dartiguenave C, Bertrand M J, et al. Glutaraldehyde: behavior in aqueous solution, reaction with proteins, and application to enzyme crosslinking. BioTechniques, 2004, 37(5):790-802. pmid: 15560135
[28]	Wang S M, Su P, Huang J, et al. Magnetic nanoparticles coated with immobilized alkaline phosphatase for enzymolysis and enzyme inhibition assays. Journal of Materials Chemistry B, 2013, 1(12):1749. doi: 10.1039/c3tb00562c

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