Abstract
A simple optimized microplate-based method to assay endo-1,4-β-mannosidase activity was described as an improved high-throughput screening method. A series of experimental conditions were optimized. It is revealed that the optimum measurement procedure is as follows: adding 50 μL of diluted enzyme sample and 50 μL substrate, incubating at 45 °C for exactly 5 min in micro-plate, mixing with 100 μL 3,5-dinitrosalicylic acid (DNS) reagent, maintaining at boiling point for 15 min, cooling down to room temperature before determining the ABS value at 540 nm using an ELISA micro-plate reader. The reaction volume of the optimized microplate-assay is reduced to 200 μL from 2 500 μL used in the standard β-mannanase macro-assay. The optimized micro-assay is significantly more sensitive in all of the 643 candidates during endo-1,4-β-mannosidase screening. Statistical analyses show that the sensitivity of the optimized micro-method is significantly greater than that of the macro-assay. The optimized method is convenient, fast, and cheap for high throughput enzyme screening.
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DHAWAN S, KAUR J. Microbial mannanases: An overview of production and applications [J]. Crit Rev Biotechnol, 2007, 27(4): 197–216.
GIRIO F M, FONSECA C, CARVALHEIRO F, DUARTE L C, MARQUES S, BOGEL-LUKASIK R. Hemicelluloses for fuel ethanol: A review [J]. Bioresourource Technology, 2010, 101(13): 4775–800.
GONZALEZ C F, FARINA J I, FIGUEROA L I C. A critical assessment of a viscometric assay for measuring Saccharomycopsis fibuligera alpha-amylase activity on gelatinised cassava starch [J]. Enzyme and Microbial Technology, 2002, 30(2): 169–175.
DO B C, DANG T T, BERRIN J G, HALTRICH D, TO K A, SIGOILLOT J C, YAMABHAI M. Cloning, expression in Pichia pastoris, and characterization of a thermostable GH5 mannan endo-1,4-β-mannosidase from Aspergillus niger BK01 [J]. Microbial Cell Factories, 2009 (8): 59–63.
ZHANG Yun-xiong, LIU Zheng-chu, CHEN Xin-bo. Cloning and expression of a mannanase gene from Erwinia carotovora CXJZ95-198 [J]. Annals of Microbiology, 2007, 57(4): 623–628.
MILLER G L. Use of dinitrosalicylic acid reagent for determination of reducing sugar [J]. Analytical Chemistry, 1959, 31(3): 426–428.
ZHAO Wei, ZHENG Jia, ZHOU Hong-bo. A thermotolerant and cold-active β-mannanase from Aspergillus niger CBS 513.88: Constitutive overexpression and high-density fermentation in Pichia pastoris [J]. Bioresour Technol, 2011, 102(12): 7538–7547.
SAMBROOK J, RUSSELL D W. Molecular cloning: A laboratory manual [M]. New York: Cold Spring Harbor Laboratory Press, 2001: 155–157.
JOHN G T, GOELLING D, KLIMAN T I, SCHNEIDER H, HEINZLE E. Integrated optical sensing of dissolved O2 in microtiter plates: A novel tool for microbial cultivation [J]. Biotechnology and Bioengineering, 2003, 81(7): 829–836.
LUO H, WANG Y, WANG H, YANG J, YANG Y, HUANG H, YANG P, BAI Y, SHI P, FAN Y, YAO B. A novel highly acidic beta-mannanase from the acidophilic fungus Bispora sp. MEY-1: Gene cloning and overexpression in Pichia pastoris [J]. Appllied Microbiology Biotechnology, 2009, 82(3): 453–461.
RAMADA M H, LOPES F A, ULHOA C J, SILVA RDO N. Optimized microplate beta-1,3-glucanase assay system for Trichoderma spp. screening [J]. Journal Microbiological Methods, 2010, 81(1): 6–10.
KING B C, DONNELLY M K, BERGSTROM G C, WALKER L P, GIBSON D M. An optimized microplate assay system for quantitative evaluation of plant cell wall-degrading enzyme activity of fungal culture extracts [J]. Biotechnology and Bioengineering, 2009, 102(4): 1033–1044.
LÁSZLÓ FÜLÖP, TAMÁS PONYI. Rapid screening for endo-[beta]-1,4-glucanase and endo-[beta]-1,4-mannanase activities and specific measurement using soluble dye-labelled substrates [J]. Journal of Microbiological Methods, 1997, 29(1): 15–21.
BOETTNER M, PRINZ B, HOLZ C, STAHL U, LANG C. High-throughput screening for expression of heterologous proteins in the yeast Pichia pastoris [J]. Journal of Biotechnology, 2002, 99(1): 51–62.
BOURGAULT R, BEWLEY J D. Gel diffusion assays for endo-beta-mannanase and pectin methylesterase can underestimate enzyme activity due to proteolytic degradation: A remedy [J]. Analytical Biochemistry, 2002, 300(1): 87–93.
MANSUR M, CABELLO C, HERNANDEZ L, PAIS J, VARAS L, VALDES J, TERRERO Y, HIDALGO A, PLANA L, BESADA V, GARCIA L, LAMAZARES E, CATELLANOS L, MARTINEZ E. Multiple gene copy number enhances insulin precursor secretion in the yeast Pichia pastoris [J]. Biotechnology Letters, 2005, 27(5): 339–345.
VASSILEVA A, CHUGH D A, SWAMINATHAN S, KHANNA N. Effect of copy number on the expression levels of hepatitis B surface antigen in the methylotrophic yeast Pichia pastoris [J]. Protein Expression and Purification, 2001, 21(1): 71–80.
ZHU T C, GUO M J, SUN C, QIAN J C, ZHUANG Y P, CHU J, ZHANG S. A systematical investigation on the genetic stability of multi-copy Pichia pastoris strains [J]. Biotechnology Letters, 2009, 31(5): 679–684.
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Foundation item: Project(31000350) supported by the National Natural Science Foundation of China
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Shen, L., Wang, Xl., Zheng, J. et al. An optimized micro-plate assay for high-throughput screening of recombinant Pichia pastoris strains. J. Cent. South Univ. 19, 3046–3054 (2012). https://doi.org/10.1007/s11771-012-1377-7
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DOI: https://doi.org/10.1007/s11771-012-1377-7