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首页> 外文期刊>BMC Biotechnology >Characterization of purified and Xerogel immobilized Novel Lignin Peroxidase produced from Trametes versicolor IBL-04 using solid state medium of Corncobs
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Characterization of purified and Xerogel immobilized Novel Lignin Peroxidase produced from Trametes versicolor IBL-04 using solid state medium of Corncobs

机译：使用玉米芯固体培养基从云芝Trametes IBL-04生产的纯化和干凝胶固定的新型木质素过氧化物酶的表征

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Background Cost-effective production of industrially important enzymes is a key for their successful exploitation on industrial scale. Keeping in view the extensive industrial applications of lignin peroxidase (LiP), this study was performed to purify and characterize the LiP from an indigenous strain of Trametes versicolor IBL-04. Xerogel matrix enzyme immobilization technique was applied to improve the kinetic and thermo-stability characteristics of LiP to fulfil the requirements of the modern enzyme consumer sector of biotechnology. Results A novel LiP was isolated from an indigenous T. versicolor IBL-04 strain. T. versicolor IBL-04 was cultured in solid state fermentation (SSF) medium of corn cobs and maximum LiP activity of 592?±?6 U/mL was recorded after five days of incubation under optimum culture conditions. The crude LiP was 3.3-fold purified with specific activity of 553 U/mg after passing through the DEAE-cellulose and Sephadex -G-100 chromatography columns. The purified LiP exhibited a relatively low molecular weight (30?kDa) homogenous single band on native and SDS-PAGE . The LiP was immobilized by entrapping in xerogel matrix of trimethoxysilane (TMOS) and proplytetramethoxysilane (PTMS) and maximum immobilization efficiency of 88.6% was achieved. The free and immobilized LiPs were characterized and the results showed that the free and immobilized LiPs had optimum pH 6 and 5 while optimum temperatures were 60°C and 80°C, respectively. Immobilization was found to enhance the activity and thermo-stability potential of LiP significantly and immobilized LiP remained stable over broad pH and temperature range as compare to free enzyme. Kinetic constants K m and V max were 70 and 56?μM and 588 and 417 U/mg for the free and immobilized LiPs, respectively. Activity of this novel extra thermo-stable LiP was stimulated to variable extents by Cu2+, Mn2+ and Fe2+ whereas, Cystein, EDTA and Ag+ showed inhibitory effects. Conclusions The indigenously isolated white rot fungal strain T. versicolor IBL-04 showed tremendous potential for LiP synthesis in SSF of corncobs in high titters (592 U/mL) than other reported Trametes ( Coriolus , Polyporus ) species. The results obtained after dual phase characterization suggested xerogel matrix entrapment a promising tool for enzyme immobilization, hyper-activation and stabilization against high temperature and inactivating agents. The pH and temperature optima, extra thermo-stability features and kinetic characteristics of this novel LiP of T. versicolor IBL-04 make it a versatile enzyme for various industrial and biotechnological applications.
机译：背景技术具有成本效益的工业重要酶的生产是其在工业规模上成功开发的关键。考虑到木质素过氧化物酶（LiP）在工业上的广泛应用，本研究旨在从本地Trametes versicolor IBL-04菌株中纯化和表征LiP。应用干凝胶基质酶固定化技术改善LiP的动力学和热稳定性，以满足现代生物技术酶消费领域的需求。结果从本地云芝TBL IBL-04菌株中分离到了新的LiP。在玉米芯的固态发酵（SSF）培养基中培养云芝TBL IBL-04，在最佳培养条件下培养5天后，其最大LiP活性为592？±？6 U / mL。在通过DEAE-纤维素和Sephadex -G-100色谱柱后，将粗制的LiP纯化3.3倍，比活度为553 U / mg。纯化的LiP在天然和SDS-PAGE上显示出相对较低的分子量（30？kDa）均质单条带。通过捕获三甲氧基硅烷（TMOS）和丙四甲氧基硅烷（PTMS）的干凝胶基质来固定LiP，并实现了88.6％的最大固定效率。对游离和固定化的LiPs进行了表征，结果表明，游离和固定化的LiPs的最佳pH值为6和5，而最佳温度分别为60°C和80°C。与游离酶相比，发现固定化显着增强了LiP的活性和热稳定性，并且固定化LiP在宽的pH和温度范围内保持稳定。动力学常数K m 和V max 分别为70和56？μM以及588和417 U / mg分别表示游离和固定的LiP。 Cu 2 + ，Mn 2+ 和Fe 2 + ，而半胱氨酸，EDTA和Ag + 具有抑制作用效果。结论本地分离的白腐真菌杂色曲霉IBL-04在高滴度（592 U / mL）的玉米芯SSF中显示出LiP合成的巨大潜力，远高于其他报道的Trametes（Coriolus，Polyporus）种类。在双相表征后获得的结果表明，干凝胶基质的截留是酶固定，超活化以及针对高温和灭活剂的稳定化的有前途的工具。这种新颖的杂色丁香IBL-04的LiP的最适pH和温度，超强的热稳定性和动力学特性使其成为多种工业和生物技术应用的通用酶。

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