Development of chitin cross-linked enzyme aggregates of L-methioninase for upgraded activity, permanence and application as efficient therapeutic formulations.
Development of chitin cross-linked enzyme aggregates of L-methioninase for upgraded activity, permanence and application as efficient therapeutic formulations.
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DOI：
10.1016/j.ijbiomac.2019.08.246
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发表时间：
2019-12
影响因子：
8.2
通讯作者：
S. Kannan;M. Marudhamuthu
中科院分区：
文献类型：
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作者：
S. Kannan;M. Marudhamuthu
本研究从恶臭假单胞菌MTCC 9782中沉淀出L-蛋氨酸酶（MET），并用交联剂戊二醛交联，得到具有催化活性的不溶性酶。在所测试的各种沉淀剂中，硫酸铵显示出最高的沉淀效率（80%）。采用双响应统计概念（提供20次不同运行的软件）来评估沉淀剂的作用、交联剂的浓度和交联的持续时间。在进行的20次不同运行中，在运行6中观察到最高的酶活性（88.17U）：聚集体尺寸为11.57 μm，饱和硫酸铵浓度为80%，戊二醛浓度为2 mM，孵育时间为12 h。得到的R2值为0.9754（酶活性）和0.9203（聚集体大小），这表明增强的关联之间的实验和预测值的酶活性。与甲壳素珠共价交联的酶分子与游离酶和与戊二醛交联的酶相比显示出增加的活性。FTIR光谱证实了CLEA-MET和几丁质交联的CLEA-MET之间的二级结构改变。热稳定性测定表明，几丁质交联的CLEA-MET和CLEA-MET分别在55 °C和60 °C的温度下保持95%和80%的最大酶活性。储存稳定性测定表明，CLEA-MET保留了其初始活性的65%，几丁质固定化CLEA保留了其活性的88%。扫描电子显微镜、透射电子显微镜和高容量筛选成像技术表明，壳聚糖固定化CLEA-MET微球具有良好的单分散性和介孔结构，且CLEA为无定形团簇，孔隙较少。细胞毒性分析表明，几丁质固定化CLEA-MET显着抑制A549细胞的增殖高达96.66%相比，游离酶（72%）和CLEA-MET（76%）。
In this study, L-methioninase (METs) was precipitated fromPseudomonas putidaMTCC 9782 and was cross-linked with a cross-linking agent glutaraldehyde to obtain a catalytically active insoluble enzyme. Among the various precipitants tested, ammonium sulfate displayed the highest precipitating (80%) efficiency. A double-response statistical concept, software that provides 20 different runs, was employed to assess the role of precipitant, concentration of cross-linking agent, and duration of cross linking. From the different 20 runs performed, the highest enzyme activity was observed in run 6 (88.17 U): the aggregate size was 11.57 μm, the concentration of saturated ammonium sulfate was 80% and glutaraldehyde 2 mM, and the incubation period was 12 h. R2values of 0.9754 (enzyme activity) and 0.9203 (aggregate size) were obtained, which showed an enhanced association between the experimental and predicted values of enzyme activity. Enzyme molecules covalently cross-linked with chitin beads showed increased activities compared to free enzymes and enzymes cross-linked with glutaraldehyde. FTIR spectra confirmed the secondary structural alterations between CLEA-METs and chitin-cross-linked CLEA-METs. Thermal stability assays showed that chitin cross-linked CLEA-METs and CLEA-METs retained maximum enzyme activities of 95% and 80% at temperatures 55 °C and 60 °C, respectively. Storage stability assays showed that CLEA-METs retained 65% of their initial activity and chitin-immobilized CLEAs retained 88% of their activity. Moreover, scanning electron microscopy, transmission electron microscopy, and high content screening imaging technique revealed that chitin-immobilized CLEA-MET microspheres showed good monodispersity and mesoporous structure with the amorphous clusters of CLEA with few pores. Cytotoxicity analysis demonstrated that chitin-immobilized CLEA-MET significantly inhibited the proliferation of A549 cells up to 96.66% compared to free enzyme (72%) and CLEA-METs (76%).