A fast, sensitive and easy colorimetric assay for chitinase and cellulase activity detection

Biotechnology for Biofuels

Alessandro R Ferrari²  Yasser Gaber¹  Marco W Fraaije² 

[1] Microbiology Department, Faculty of Pharmacy, Beni-Suef University, 62511 Beni-Suef, Egypt

[2] Molecular Enzymology Group, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Nijenborgh 4, 9747 AG, Groningen, The Netherlands

关键词: Colorimetric assay;    Cellulose;    Schales’ procedure;    DNS;    Chitin;    High-throughput screening;    Chito-oligosaccharide oxidase;    Cellulase;    Chitinase;   

Others  :  793217
DOI  :  10.1186/1754-6834-7-37

 received in 2013-11-18, accepted in 2014-02-19,  发布年份 2014

【 摘 要 】

Background

Most of the current colorimetric methods for detection of chitinase or cellulase activities on the insoluble natural polymers chitin and cellulose depend on a chemical redox reaction. The reaction involves the reducing ends of the hydrolytic products. The Schales’ procedure and the 3,5-dinitrosalicylic acid (DNS) method are two examples that are commonly used. However, these methods lack sensitivity and present practical difficulties of usage in high-throughput screening assays as they require boiling or heating steps for color development.

Results

We report a novel method for colorimetric detection of chitinase and cellulase activity. The assay is based on the use of two oxidases: wild-type chito-oligosaccharide oxidase, ChitO, and a mutant thereof, ChitO-Q268R. ChitO was used for chitinase, while ChitO-Q268R was used for cellulase activity detection. These oxidases release hydrogen peroxide upon the oxidation of chitinase- or cellulase-produced hydrolytic products. The hydrogen peroxide produced can be monitored using a second enzyme, horseradish peroxidase (HRP), and a chromogenic peroxidase substrate. The developed ChitO-based assay can detect chitinase activity as low as 10 μU within 15 minutes of assay time. Similarly, cellulase activity can be detected in the range of 6 to 375 mU. A linear response was observed when applying the ChitO-based assay for detecting individual chito-oligosaccharides and cello-oligosaccharides. The detection limits for these compounds ranged from 5 to 25 μM. In contrast to the other commonly used methods, the Schales’ procedure and the DNS method, no boiling or heating is needed in the ChitO-based assays. The method was also evaluated for detecting hydrolytic activity on biomass-derived substrates, that is, wheat straw as a source of cellulose and shrimp shells as a source of chitin.

Conclusion

The ChitO-based assay has clear advantages for the detection of chitinase and cellulase activity over the conventional Schales’ procedure and DNS method. The detection limit is lower and there is no requirement for harsh conditions for the development of the signal. The assay also involves fewer and easier handling steps. There is no need for boiling to develop the color and results are available within 15 minutes. These aforementioned features render this newly developed assay method highly suitable for applications in biorefinery-related research.

【 授权许可】

   
2014 Ferrari et al.; licensee BioMed Central Ltd.

【 图 表 】

Figure 1.

Figure 2.

Figure 3.

Figure 4.

Figure 5.

Figure 6.

Figure 7.

【 参考文献 】

• [1]Chandel AK, Chandrasekhar G, Silva MB, Silvério da Silva S: The realm of cellulases in biorefinery development. Crit Rev Biotechnol 2012, 32:187-202.
• [2]Howard MB, Ekborg NA, Weiner RM, Hutcheson SW: Detection and characterization of chitinases and other chitin-modifying enzymes. J Ind Microbiol Biotechnol 2003, 30:627-635.
• [3]Vaaje-Kolstad G, Horn SJ, Sørlie M, Eijsink VGH: The chitinolytic machinery of Serratia marcescens - a model system for enzymatic degradation of recalcitrant polysaccharides. FEBS J 2013, 280:3028-3049.
• [4]Percival Zhang YH, Himmel ME, Mielenz JR: Outlook for cellulase improvement: screening and selection strategies. Biotechnol Adv 2006, 24:452-481.
• [5]Shih CY, Khan AA, Jia S, Wu J, Shih DS: Purification, characterization, and molecular cloning of a chitinase from the seeds of Benincasa hispida. Biosci Biotechnol Biochem 2001, 65:501-509.
• [6]Eveleigh DE, Mandels M, Andreotti R, Roche C: Measurement of saccharifying cellulase. Biotechnol Biofuels 2009, 2:21. BioMed Central Full Text
• [7]Schales O, Schales SS: Simple method for the determination of glucose in blood. Proc Am Fed Clin Res 1945, 2:78.
• [8]Shen CR, Chen YS, Yang CJ, Chen JK, Liu CL: Colloid chitin azure is a dispersible, low-cost substrate for chitinase measurements in a sensitive, fast, reproducible assay. J Biomol Screen 2010, 15:213-217.
• [9]Horn SJ, Eijsink VGH: A reliable reducing end assay for chito-oligosaccharides. Carbohydr Polym 2004, 56:35-39.
• [10]Heuts DP, Winter RT, Damsma GE, Janssen DB, Fraaije MW: The role of double covalent flavin binding in chito-oligosaccharide oxidase from Fusarium graminearum. Biochem J 2008, 413:175-183.
• [11]Heuts DP, Janssen DB, Fraaije MW: Changing the substrate specificity of a chitooligosaccharide oxidase from Fusarium graminearum by model-inspired site-directed mutagenesis. FEBS Lett 2007, 581:4905-4909.
• [12]Obzansky DM, Rabin BR, Simons DM, Tseng SY, Severino DM, Eggelte H, Fisher M, Harbron S, Stout RW, Di Paolo MJ: Sensitive, colorimetric enzyme amplification cascade for determination of alkaline phosphatase and application of the method to an immunoassay of thyrotropin. Clin Chem 1991, 37:1513-1518.
• [13]Imoto T, Yagishita K: A simple activity measurement of lysozyme. Agric Biol Chem 1971, 35:1514-1516.
• [14]Miller GL: Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal Chem 1959, 31:426-428.
• [15]Dashtban M, Maki M, Leung KT, Mao C, Qin W: Cellulase activities in biomass conversion: measurement methods and comparison. Crit Rev Biotechnol 2010, 30:302-309.
• [16]Coward-Kelly G, Aiello-Mazzari C, Kim S, Granda C, Holtzapple M: Suggested improvements to the standard filter paper assay used to measure cellulase activity. Biotechnol Bioeng 2003, 82:745-749.
• [17]Hildén L, Eng L, Johansson G, Lindqvist SE, Pettersson G: An amperometric cellobiose dehydrogenase-based biosensor can be used for measurement of cellulase activity. Anal Biochem 2001, 290:245-250.
• [18]Nitschke J, Altenbach HJ, Malolepszy T, Mölleken H: A new method for the quantification of chitin and chitosan in edible mushrooms. Carbohydr Res 2011, 346:1307-1310.