Biochemical and kinetic characterization of laccase and manganese peroxidase from novel Klebsiella pneumoniae strains and their application in bioethanol production.
Biochemical and kinetic characterization of laccase and manganese peroxidase from novel Klebsiella pneumoniae strains and their application in bioethanol production.
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DOI：
10.1039/c8ra01204k
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发表时间：
2018-04-18
期刊：
影响因子：
3.9
通讯作者：
Pydisetty, Y.
中科院分区：
文献类型：
--
作者：
Gaur, Nisha;Narasimhulu, Korrapati;Pydisetty, Y.
来自新型肺炎克雷伯菌分离株的漆酶 (lac) 和锰过氧化物酶 (MnP) 在木质素基础培养基 (LBM) 上生长，通过 80% 硫酸铵分级、透析和 DEAE-琼脂糖柱层析纯化。 NITW715076_2、NITW715076_1 和 NITW715076 菌株生产漆酶的最佳温度分别为 60 °C、50 °C 和 50 °C，生产 MnP 的最佳温度分别为 50 °C、70 °C 和 60 °C。发现从所有分离物中生产这两种酶的最佳生产 pH 值为 5。实现了 2.8–3.5 倍的酶纯化，保留了约 60–70% 的初始活性。 SDS-PAGE显示漆酶和MnP的分子量分别为66 kDa和48 kDa。与 NITW715076_1 (lac: Km = 3.97) 相比，底物 ABTS 和 MnSO4 对 NITW715075_2 衍生的漆酶和 MnP (lac: Km = 0.38 mM, Vmax = 71.42 U ml−1; MnP: Km = 0.17 mM, Vmax = 106.38 U ml−1) 表现出更高的特异性mM，Vmax = 148.8 U ml−1；MnP：Km = 0.90 mM，Vmax = 114.67 U ml−1）和 NITW715076（lac：Km = 0.46 mM，Vmax = 23.42 U ml−1；MnP：Km = 0.19 mM，Vmax = 108.10 U ml−1）。 L-半胱氨酸和叠氮化钠对这两种酶的活性有强烈的抑制作用。 EDTA 在较高浓度下抑制漆酶和 MnP 活性。 SDS 强烈抑制活性，而对于 MnP 则表现出较小的抑制作用。这些酶用于从稻米和麦麸生物质生产乙醇，与对照相比，产量提高了 39.29%。在评估这些酶的适用性后，可以表明肺炎克雷伯菌分离株的木质素分解酶可以有效地用于增强乙醇生产，并且可以探索其他假定的应用。从新型克雷伯氏菌分离株中纯化高达 3 倍的漆酶和 MnP，介导从稻米和麦麸底物生产乙醇，使产量提高近 40%。
Laccase (lac) and manganese peroxidase (MnP) enzymes from the novel Klebsiella pneumoniae isolates, grown on lignin basic media (LBM) were purified by 80% ammonium sulphate fractionation, dialysis and DEAE-sepharose column chromatography. The optimum temperatures for laccase production were 60 °C, 50 °C and 50 °C and for MnP production were 50 °C, 70 °C and 60 °C from NITW715076_2, NITW715076_1 and NITW715076 isolates, respectively. The optimal pH for production was found to be 5 for production of both the enzymes from all the isolates. 2.8–3.5 fold enzyme purification was achieved retaining around 60–70% of the initial activity. SDS-PAGE revealed the molecular mass of laccase and MnP to be 66 kDa and 48 kDa, respectively. The substrate ABTS and MnSO4 exhibited more specificity towards NITW715075_2 derived laccase and MnP (lac: Km = 0.38 mM, Vmax = 71.42 U ml−1; MnP: Km = 0.17 mM, Vmax = 106.38 U ml−1) compared to NITW715076_1 (lac: Km = 3.97 mM, Vmax = 148.8 U ml−1; MnP: Km = 0.90 mM, Vmax = 114.67 U ml−1) and NITW715076 (lac: Km = 0.46 mM, Vmax = 23.42 U ml−1; MnP: Km = 0.19 mM, Vmax = 108.10 U ml−1) derived. l-Cysteine and sodium azide imposed a strong inhibitory effect on the activities of both the enzymes. EDTA inhibited laccase and MnP activity at higher concentration. SDS strongly inhibited activity while for MnP it showed less inhibitory effect. The enzymes were employed for ethanol production from rice and wheat bran biomass which showed 39.29% improved production compared to control. After evaluating the applicability of these enzymes it can be suggested that the ligninolytic enzyme of Klebsiella pneumoniae isolates could be effectively employed in enhanced ethanol production and could be explored for other putative applications. Upto 3 fold purified laccase and MnP from novel Klebsiella isolates, mediated ethanol production from rice and wheat bran substrates lead to almost 40% improvement in production profile.
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