Improving the Thermal Stability of Lactate Oxidase by ETSS

doi:10.13560/j.cnki.biotech.bull.1985.2018-0177

Abstract

Abstract: Lactate oxidase can catalyze the oxidation of lactic acid to pyruvate,thus it has important industrial application value. However,lactate oxidases from different sources share the common problem of poor thermal stability,which impacts its application in industry. The researches on lactate oxidase are less reported,and there was no report on the improvement of its thermal stability,therefore improving the thermal stability of lactate oxidase is of great significance. In this study,we cloned and expressed highly specific lactate oxidase EgLOD,and used bioinformatics and surface charge analysis software ETSS to analyze the protein structure of lactate oxidase. We designed 9 mutants for the experiment of improving their thermal stability based on the distribution and optimization of protein surface char,2 mutants D250N and D281N presented efficient enhanced thermal stability after screening. After incubated under 60℃ for 30 min,they remained more than 50% of the enzyme activity,while the wild type EgLOD remained only 20%.

Key words: lactate oxidase, thermal stability, molecular modification, ETSS

HUA Chen, LI Xin-xin, TU Tao, YANG Hong, LUO Hui-ying, CHEN Jia-ming, YAO Bin, BAI Ying-guo, PENG Shu-chuan. Improving the Thermal Stability of Lactate Oxidase by ETSS[J]. Biotechnology Bulletin, 2018, 34(8): 144-150.

References

[1] 谷劲松, 曲音波. 丙酮酸的酶法转化及乳酸氧化酶的研究进展[J]. 微生物学通报, 2003, 30(1):86-90.
[2] Park HS, Lee JY, Kim HS.Production of L-DOPA(3, 4-dihydroxyphenyl-L-alanine)from benzene by using a hybrid pathway[J]. Biotechnology & Bioengineering, 1998, 58(2-3):339.
[3] Gavalas VG, Chaniotakis NA.Lactate biosensor based on the adsorption of polyelectrolyte stabilized lactate oxidase into porous conductive carbon[J]. Microchimica Acta, 2001, 136(3-4):211-215.
[4] Xu JJ, Zhao W, Luo XL, et al.A sensitive biosensor for lactate based on layer-by-layer assembling MnO₂ nanoparticles and lactate oxidase on ion-sensitive field-effect transistors[J]. Chemical Communications, 2005, 6(6):792-794.
[5] Unterweger B, Stoisser T, Leitgeb S, et al.Engineering of Aerococcus viridans L-lactate oxidase for site-specific PEGylation:characterization and selective bioorthogonal modification of a S218C mutant[J]. Bioconjugate Chemistry, 2012, 23(7):1406.
[6] Toda A, Nishiya Y.Gene cloning, purification, and characterization of a lactate oxidase from Lactococcus lactis, subsp. cremoris, IFO3427[J]. Journal of Fermentation & Bioengineering, 1998, 85(5):507-510.
[7] Sztajer H, Wang W, Lünsdorf H, et al.Purification and some properties of a novel microbial lactate oxidase[J]. Applied Microbiology & Biotechnology, 1996, 45(5):600.
[8] Lillis B, Grogan C, Berney H, et al.Investigation into immobilisation of lactate oxidase to improve stability[J]. Sensors & Actuators B Chemical, 2000, 68(1-3):109-114.
[9] Hamamatsu N, Nomiya Y, Aita T, et al.Directed evolution by accumulating tailored mutations:thermostabilization of lactate oxidase with less trade-off with catalytic activity[J]. Protein Engineering Design & Selection, 2006, 19(11):483-489.
[10] 刘真, 常雁红, 刘仁霖, 等. 磁性壳聚糖微球的制备及对乳酸氧化酶的固定化研究[J]. 现代化工, 2007(s2):310-313.
[11] 王艳, 周林, 姚丽丽. 耻垢分枝杆菌产乳酸氧化酶条件的研究[J]. 食品与发酵工业, 2006, 32(8):28-31.
[12] Umena Y, Yorita K, Matsuoka T, et al.The crystal structure of L-lactate oxidase from Aerococcus viridans at 2. 1A resolution reveals the mechanism of strict substrate recognition[J]. Biochemical & Biophysical Research Communications, 2006, 350(2):249.
[13] Demirjian A.Crystallographic study on the interaction of L-lactate
oxidase with pyruvate at 1. 9 Angstrom resolution at 1. 9 Angstrom resolution[J]. Biochemical & Biophysical Research Communications, 2007, 358(4):1002-1007.
[14] Zhang L, Tang X, Cui D, et al.A method to rationally increase protein stability based on the charge-charge interaction, with application to lipase LipK107[J]. Protein Science A Publication of the Protein Society, 2014, 23(1):110-116.
[15] Tu T, Luo H, Meng K, et al.Improvement in Thermostability of an Achaetomium sp. strain Xz8 endopolygalacturonase via the optimization of charge-charge interactions[J]. Applied & Environmental Microbiology, 2015, 81(19):6938-6944.
[16] 谷劲松. 乳酸氧化酶转化DL-乳酸生成丙酮酸[D]. 济南:山东大学, 2002.
[17] 周林. 乳酸氧化酶的制备及其固定化催化DL-乳酸生产丙酮酸研究[D]. 长沙:中南大学, 2007.
[18] Minagawa H, Shimada J, Kaneko H.Effect of mutations at Glu160 and Val198 on the thermostability of lactate oxidase[J]. Febs Journal, 2003, 270(17):3628-3633.
[19] Strickler SS, Gribenko AV, Gribenko AV, et al.Protein stability and surface electrostatics:a charged relationship[J]. Biochemistry, 2006, 45(9):2761.
[20] Schweiker KL, Zarrine-Afsar A, Davidson AR, et al.Computational design of the Fyn SH3 domain with increased stability through optimization of surface charge charge interactions[J]. Protein Science, 2007, 16(12):2694-2702.