Journal of Microbiology and Biotechnology

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
권호 표지
DOI QR코드

DOI QR Code

Effective Microwell Plate-Based Screening Method for Microbes Producing Cellulase and Xylanase and Its Application

  • Kim, Jennifer Jooyoun (School of Marine and Atmospheric Science, Stony Brook University) ;
  • Kwon, Young-Kyung (Korea Institute of Ocean Science and Technology) ;
  • Kim, Ji Hyung (Korea Institute of Ocean Science and Technology) ;
  • Heo, Soo-Jin (Korea Institute of Ocean Science and Technology) ;
  • Lee, Youngdeuk (Korea Institute of Ocean Science and Technology) ;
  • Lee, Su-Jin (Korea Institute of Ocean Science and Technology) ;
  • Shim, Won-Bo (Department of Plant Pathology and Microbiology, Texas A&M University) ;
  • Jung, Won-Kyo (Department of Biomedical Engineering, Pukyong National University) ;
  • Hyun, Jung-Ho (Department of Environmental Marine Sciences, Hanyang University) ;
  • Kwon, Kae Kyoung (Korea Institute of Ocean Science and Technology) ;
  • Kang, Do-Hyung (Korea Institute of Ocean Science and Technology) ;
  • Oh, Chulhong (Korea Institute of Ocean Science and Technology)
  • Received : 2014.05.21
  • Accepted : 2014.07.24
  • Published : 2014.11.28
Download PDF
⟨ Previous Next ⟩

Abstract

Cellulase and xylanase are main hydrolysis enzymes for the degradation of cellulosic and hemicellulosic biomass, respectively. In this study, our aim was to develop and test the efficacy of a rapid, high-throughput method to screen hydrolytic-enzyme-producing microbes. To accomplish this, we modified the 3,5-dinitrosalicylic acid (DNS) method for microwell plate-based screening. Targeted microbial samples were initially cultured on agar plates with both cellulose and xylan as substrates. Then, isolated colonies were subcultured in broth media containing yeast extract and either cellulose or xylan. The supernatants of the culture broth were tested with our modified DNS screening method in a 96-microwell plate, with a $200{\mu}l$ total reaction volume. In addition, the stability and reliability of glucose and xylose standards, which were used to determine the enzymatic activity, were studied at $100^{\circ}C$ for different time intervals in a dry oven. It was concluded that the minimum incubation time required for stable color development of the standard solution is 20 min. With this technique, we successfully screened 21 and 31 cellulase- and xylanase-producing strains, respectively, in a single experimental trial. Among the identified strains, 19 showed both cellulose and xylan hydrolyzing activities. These microbes can be applied to bioethanol production from cellulosic and hemicellulosic biomass.

Keywords

References

  1. Agrawal M, Pradeep S, Chandraraj K, Gummadi SN. 2005. Hydrolysis of starch by amylase from Bacillus sp. KCA102: a statistical approach. Process. Biochem. 40: 2499-2507. https://doi.org/10.1016/j.procbio.2004.10.006
  2. Bayer EA, Chanzy H, Lamed R, Shoham Y. 1998. Cellulose, cellulases and cellulosomes. Curr. Opin. Struct. Biol. 8: 548-557. https://doi.org/10.1016/S0959-440X(98)80143-7
  3. Beauchemin KA, Rode LM, Sewalt VJH. 1995. Fibrolytic enzymes increase fiber digestibility and growth rate of steers fed dry forages. Can. J. Anim. Sci. 75: 641-644. https://doi.org/10.4141/cjas95-096
  4. Camacho NA, Aguilar OG. 2003. Production, purification, and characterization of a low-molecular-mass xylanase from Aspergillus sp. and its application in baking. Appl. Biochem. Biotechnol. 104: 159-171. https://doi.org/10.1385/ABAB:104:3:159
  5. Chang MCY. 2007. Harnessing energy from plant biomass. Curr. Opin. Chem. Biol. 11: 677-684. https://doi.org/10.1016/j.cbpa.2007.08.039
  6. Chundawat SPS, Balan V, Dale BE. 2008. High-throughput microplate technique for enzymatic hydrolysis of lignocellulosic biomass. Biotechnol. Bioeng. 99: 1281-1294. https://doi.org/10.1002/bit.21805
  7. Collins T, Gerday C, Feller G. 2005. Xylanases, xylanase families and extremophilic xylanases. FEMS Microbiol. Rev. 29: 3-23. https://doi.org/10.1016/j.femsre.2004.06.005
  8. Feng ZH, Wang YS, Zheng YG. 2011. A new microtiter plate-based screening method for microorganisms producing alpha-amylase inhibitors. Biotechnol. Bioprocess Eng. 16: 894-900. https://doi.org/10.1007/s12257-011-0033-7
  9. Fujita Y, Takahashi S, Ueda M, Tanaka A, Okada H, Morikawa Y, et al. 2002. Direct and efficient production of ethanol from cellulosic material with a yeast strain displaying cellulolytic enzymes. Appl. Environ. Microbiol. 68: 5136-5141. https://doi.org/10.1128/AEM.68.10.5136-5141.2002
  10. Goncalves C, Rodriguez-Jasso RM, Gomes N, Teixeira JA, Belo I. 2010. Adaptation of dinitrosalicylic acid method to microtiter plates. Anal. Methods 2: 2046-2048. https://doi.org/10.1039/c0ay00525h
  11. Harbak L, Thygesen HV. 2002. Safety evaluation of a xylanase expressed in Bacillus subtilis. Food Chem. Toxicol. 40: 1-8. https://doi.org/10.1016/S0278-6915(01)00092-8
  12. Hendricks CW, Doyle JD, Hugley B. 1995. A new solid medium for enumerating cellulose-utilizing bacteria in soil. Appl. Environ. Microbiol. 61: 2016-2019.
  13. Henrissat B. 1994. Cellulases and their interaction with cellulose. Cellulose 1: 169-196. https://doi.org/10.1007/BF00813506
  14. Kasana R, Salwan R, Dhar H, Dutt S, Gulati A. 2008. A rapid and easy method for the detection of microbial cellulases on agar plates using Gram's iodine. Curr. Microbiol. 57: 503-507. https://doi.org/10.1007/s00284-008-9276-8
  15. Kirk O, Borchert TV, Fuglsang CC. 2002. Industrial enzyme applications. Curr. Opin. Biotechnol. 13: 345-351. https://doi.org/10.1016/S0958-1669(02)00328-2
  16. Kurakake M, Komaki T. 2001. Production of $\beta$-mannanase and $\beta$-mannosidase from Aspergillus awamori K4 and their properties. Curr. Microbiol. 42: 377-380. https://doi.org/10.1007/s002840010233
  17. Lewis GE, Hunt CW, Sanchez WK, Treacher R, Pritchard GT, Feng P. 1996. Effect of direct-fed fibrolytic enzymes on the digestive characteristics of a forage-based diet fed to beef steers. Anim. Sci. 74: 3020-3028. https://doi.org/10.2527/1996.74123020x
  18. Menon V, Prakash G, Prabhune A, Rao M. 2010. Biocatalytic approach for the utilization of hemicellulose for ethanol production from agricultural residue using thermostable xylanase and thermotolerant yeast. Bioresour. Technol. 101: 5366-5373. https://doi.org/10.1016/j.biortech.2010.01.150
  19. Miyazaki K, Takenouchi M, Kondo H, Noro N, Suzuki M, Tsuda S. 2006. Thermal stabilization of Bacillus subtilis family-11 xylanase by directed evolution. J. Biol. Chem. 281: 10236-10242. https://doi.org/10.1074/jbc.M511948200
  20. Nichols EJ, Beckman JM, Hadwiger LA. 1980. Glycosidic enzyme activity in pea tissue and pea-Fusarium solani interactions. Plant Physiol. 66: 199-204. https://doi.org/10.1104/pp.66.2.199
  21. Oh C, Nikapitiya C, Lee Y, Whang I, Kim SJ, Kang DH, Lee J. 2010. Cloning, purification and biochemical characterization of beta agarase from the marine bacterium Pseudoalteromonas sp. AG4. J. Ind. Microbiol. Biotechnol. 37: 483-494. https://doi.org/10.1007/s10295-010-0694-9
  22. Prade RA. 1996. Xylanases: from biology to biotechnology. Biotechnol. Genet. Eng. 13: 101-131. https://doi.org/10.1080/02648725.1996.10647925
  23. Rahkamo L, Siika-Aho M, Vehvilainen M, Dolk M, Viikari L, Nousiainen P, Buchert J. 1996. Modification of hardwood dissolving pulp with purified Trichoderma reesei cellulases. Cellulose 3: 153-163. https://doi.org/10.1007/BF02228798
  24. Reczey K, Szengyel Zs, Eklund R, Zacchi G. 1996. Cellulase production by T. reesei. Bioresour. Technol. 57: 25-30. https://doi.org/10.1016/0960-8524(96)00038-7
  25. Rubin EM. 2008. Genomics of cellulosic biofuels. Nature 454: 841-845. https://doi.org/10.1038/nature07190
  26. Schwald W, Chan M, Breuil C, Saddler JN. 1988. Comparison of HPLC and colorimetric methods for measuring cellulolytic activity. Appl. Microbiol. Biotechnol. 28: 398-403. https://doi.org/10.1007/BF00268203
  27. Shankar M, Priyadharshini R, Gunasekaran P. 2009. Quantitative digital image analysis of chromogenic assays for high throughput screening of $\alpha$-amylase mutant libraries. Biotechnol. Lett. 31: 1197-1201. https://doi.org/10.1007/s10529-009-9999-z
  28. Sumner JB. 1921. Dinitrosalicyclic acid: a reagent for the estimation of sugar in normal and diabetic urine. J. Biol. Chem. 47: 5-9.
  29. Sumner JB. 1924. The estimation of sugar in diabetic urine, using dinitrosalicylic acid. J. Biol. Chem. 62: 287-290.
  30. Teather RM, Wood PJ. 1982. Use of Congo red-polysaccharide interactions in enumeration and characterization of cellulolytic bacteria from the bovine rumen. Appl. Environ. Microbiol. 43: 777-780.
  31. Ten LN, Im WT, Kim MK, Kang MS, Lee ST. 2004. Development of a plate technique for screening of polysaccharidedegrading microorganisms by using a mixture of insoluble chromogenic substrates. J. Microbiol. Methods 56: 375-382. https://doi.org/10.1016/j.mimet.2003.11.008
  32. Twomey LN, Pluske JR, Rowe JB, Choct M, Brown W, McConnell MF, Pethick DW. 2003. The effects of increasing levels of soluble non-starch polysaccharides and inclusion of feed enzymes in dog diets on faecal quality and digestibility. Anim. Feed Sci. Tech. 108: 71-82. https://doi.org/10.1016/S0377-8401(03)00161-5
  33. Wilson DB. 2009. Cellulases and biofuels. Curr. Opin. Biotechnol. 20: 295-299. https://doi.org/10.1016/j.copbio.2009.05.007
  34. Wood IP, Elliston A, Ryden P, Bancroft I, Roberts IN, Waldron KW. 2012. Rapid quantification of reducing sugars in biomass hydrolysates: improving the speed and precision of the dinitrosalicylic acid assay. Biomass Bioenergy 44: 117-121. https://doi.org/10.1016/j.biombioe.2012.05.003
  35. Xiao Z, Storms R, Tsang A. 2006. A quantitative starchiodine method for measuring alpha-amylase and glucoamylase activities. Anal. Biochem. 351: 146-148. https://doi.org/10.1016/j.ab.2006.01.036

Cited by

  1. Analysis of the Biotechnological Potential of a Lentinus crinitus Isolate in the Light of Its Secretome vol.15, pp.12, 2014, https://doi.org/10.1021/acs.jproteome.6b00636
  2. Dosagem de açúcares redutores com o reativo DNS em microplaca vol.20, pp.None, 2014, https://doi.org/10.1590/1981-6723.11315
  3. Identification and Characterization of Pathogenic and Endophytic Fungal Species Associated with Pokkah Boeng Disease of Sugarcane vol.33, pp.3, 2017, https://doi.org/10.5423/ppj.oa.02.2017.0029
  4. Deep Hypersaline Anoxic Basins as Untapped Reservoir of Polyextremophilic Prokaryotes of Biotechnological Interest vol.18, pp.2, 2014, https://doi.org/10.3390/md18020091
  5. Characterization of glycoside hydrolase family 11 xylanase from Streptomyces sp. strain J103; its synergetic effect with acetyl xylan esterase and enhancement of enzymatic hydrolysis of lignocellulosi vol.20, pp.1, 2014, https://doi.org/10.1186/s12934-021-01619-x