Cloning and in silico study of an endoglucanase from a thermophilic bacterium isolated from a hydrothermal vent of West Kawio, Sangihe‐Talaud waters, North Sulawesi, Indonesia

https://doi.org/10.22146/ijbiotech.48272

Edvan Arifsaputra Suherman(1*), Maelita Ramdani Moeis(2), Elvi Restiawaty(3)

(1) School of Life Sciences and Technology, Institut Teknologi Bandung, Jalan Ganesha 10, Bandung 40132, Indonesia
(2) School of Life Sciences and Technology, Institut Teknologi Bandung, Jalan Ganesha 10, Bandung 40132, Indonesia
(3) Department of Chemical Engineering, Institut Teknologi Bandung, Jalan Ganesha 10, Bandung 40132, Indonesia
(*) Corresponding Author

Abstract


Endoglucanase is used in industries that apply high temperatures, such as bioethanol, detergent, paper, and animal feed industries. Most available endoglucanases have very low stability at high temperatures. Therefore, this study aimed to identfy a new thermostable endoglucanase that is able to maintain its actvity at high temperatures. Five isolates of thermophilic bacteria were previously isolated from the hydrothermal vent of West Kawio, Indonesia. Among them, the DSI2 isolate showed the highest endoglucanase actvity, and was identfed and named as Bacillus safensis DSI2. The EgDSI2 gene was cloned from B. safensis DSI2. EgDSI2 is 1851 bp long encoding a protein of 616 amino acids. The encoded protein, EgDSI2, has high sequence identty to other B. safensis endoglucanases and was predicted with the Compute pI/Mw tool to be 69.41 kDa. EgDSI2 was high in hydrophobic amino acids. The enzyme had higher percentage of Ala and
Pro, and lower percentage of Gly compared to thermolabile endoglucanases from two Bacillus species. EgDSI2 harbored a catalytc domain belonging to glycosyl hydrolase family 9 (GH9) and a type 3 cellulose‐binding domain (CBM3). Propertes of endoglucanases with GH9‐CBM3 modular organizaton include actvity over a wide pH range, high optmum temperature, and thermostablity. Therefore, EgDSI2 has potental applicatons in the industries.



Keywords


cloning; endoglucanase; thermostable; hydrothermal vent; in silico

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References

Altschul SF, Gish W,  Miller  W,  Myers  EW, Lipman DJ.  1990. Basic local alignment  search  tool.   J Mol Biol 215(3):403–410. doi:10.1016/S0022­ 2836(05)80360­2.

Bajaj K, Madhusudhan MS, Adkar BV, Chakrabarti P, Ra­ makrishnan C, Sali A, Varadarajan R. 2007. Stereo­ chemical criteria for prediction of the effects of pro­ line mutations on protein stability. PLoS Comput Biol 3(12):e241. doi:10.1371/journal.pcbi.0030241.

Dalhus B, Saarinen M, Sauer UH, Eklund  P,  Johans­ son K, Karlsson A, Ramaswamy S, Bjørk A, Syn­ stad B, Naterstad K, et al. 2002.    Structural ba­    sis for thermophilic protein stability: structures of thermophilic and mesophilic malate dehydrogenases. J Mol Biol 318(3):707–721. doi:10.1016/S0022­ 2836(02)00050­5.

De Castro E, Sigrist CJA, Gattiker A, Bulliard V, Langendijk­Genevaux PS, Gasteiger E, Bairoch A, Hulo N. 2006. ScanProsite: detection of PROSITE signature matches and ProRule­associated functional and structural residues in proteins. Nucleic Acids Res 34(suppl_2):W362–W365. doi:10.1093/nar/gkl124.

De Marco EG, Heck  K,  Martos  ET,  Van  Der  Sand  ST. 2017. Purification and characterization of a thermostable alkaline cellulase produced by Bacillus licheniformis 380 isolated from compost. An Acad Bras Cienc 89(3):2359–2370. doi:10.1590/0001­ 3765201720170408.

Dos Santos YQ, De Veras BO, De Franca AFJ, Gorlach­ Lira K, Velasques J, Migliolo L, Dos Santos EA. 2018. A new salt­tolerant thermostable cellulase from a marine Bacillus sp. strain. J Microbiol Biotechnol 28(7):1078–1085. doi:10.4014/jmb.1802.02037.

Ebrahimi M, Lakizadeh A, Agha­Golzadeh P, Ebrahimie E, Ebrahimi M. 2011. Prediction of  thermosta­ bility from amino acid  attributes  by  combination of clustering with attribute weighting:  a new vista  in engineering enzymes. PloS ONE 6(8):e23146. doi:10.1371/journal.pone.0023146.

Gasteiger E, Hoogland C, Gattiker A, Wilkins MR, Appel RD, Bairoch A. 2005. Protein identification and anal­ ysis tools on the ExPASy server. In: WJ M, editor, The Proteomics Protocols Handbook, pages 571–607. Totowa: Humana Press.

Grosjean H, Oshima T.  2007.  How  Nucleic  Acids Cope   with   High   Temperature.   In:   C   Ger­ day, N Glansdorff, editors, Physiol Biochem Ex­ tremophiles., pages 39–56. Washington DC: ASM Press. doi:10.1128/9781555815813.ch4.

Gupta A, Verma JP. 2015. Sustainable bio­ethanol production from agro­residues: a review. Re­ newable Sustainable Energy Rev 41:550–567. doi:10.1016/j.rser.2014.08.032.

Hall TA. 1999. BioEdit: a user­friendly biological se­ quence alignment editor and analysis program for Windows 95/98/NT. In: Nucl Acids Symp Series., volume 41, pages 95–98. [London]: Information Re­ trieval Ltd., c1979­c2000.

Henrissat B, Callebaut I, Fabrega S, Lehn P,  Mornon   JP, Davies G. 1995. Conserved catalytic machin­  ery and the prediction of a common fold for several families of glycosyl hydrolases. Proc Natl Acad Sci 92(15):7090–7094. doi:10.1073/pnas.92.15.7090.

Hitomi J, Hatada Y, Kawaminami S, Kawai S, Ito S. 1997. Amino acid sequence and stereoselective hydrolytic reaction of an endo­1, 4­β­glucanase from a Bacil­ lus strain. Biosci, Biotechnol, Biochem 61(12):2004–2009. doi:10.1271/bbb.61.2004.

Hulo N,  Bairoch  A,  Bulliard  V,  Cerutti  L,  Cuche  BA, De Castro E, Lachaize C, Langendijk­ Genevaux PS, Sigrist  CJA. 2007.   The 20  years   of PROSITE. Nucl Acids Res 36(suppl_1):D245– D249. doi:10.1093/nar/gkm977.

Ishak SNH, Masomian M, Kamarudin NHA, Ali MSM, Leow  TC,  Rahman  RNZRA.  2019.     Changes    of thermostability, organic solvent, and pH sta­ bility in Geobacillus zalihae HT1  and  its  mutant by calcium ion. Int J Mol Sci 20(10):2561. doi:10.3390/ijms20102561.

Jang HD, Chen KS. 2003. Production and characteri­ zation of thermostable cellulases from Streptomyces transformant T3­1. World J Microbiol Biotechnol 19(3):263–268. doi:10.1023/A:1023641806194.

Jeong E, Kim H, Lee SW, Han KS. 2003. Discovering the interaction propensities of amino acids and nu­ cleotides from protein­RNA complexes. Mol Cells 16(2):161–167.

Jones P, Binns D, Chang HY, Fraser M, Li W, McAnulla C, McWilliam H, Maslen J, Mitchell A, Nuka G, et al. 2014. InterProScan 5: genome­scale protein func­ tion classification. Bioinformatics 30(9):1236–1240. doi:10.1093/bioinformatics/btu031.

Kajander T,  Kahn PC, Passila SH, Cohen DC, Lehtiö   L, Adolfsen W, Warwicker J, Schell U, Goldman A. 2000. Buried charged surface in proteins. Structure 8(11):1203–1214. doi:10.1016/S0969­ 2126(00)00520­7.

Kanchanadumkerng P, Sakka M, Sakka K, Wiwat C. 2017. Characterization and secretive expression in Bacillus subtilis of endoglucanase from Bacillus safensis iso­ lated from freshwater swamp forest. Walailak J Sci & Tech 14(3):199–213.

Kar T, Scheiner S. 2004. Comparison of cooperativity in CH–O and OH–O hydrogen bonds. J Phys Chem A 108(42):9161–9168. doi:10.1021/jp048546l.

Khianngam S, Pootaeng­on Y, Techakriengkrai T, Tana­ supawat   S.   2014.   Screening   and   identifica­ tion of cellulase producing bacteria  isolated  from oil palm meal. J Appl Pharm Sci 4(4):90. doi:10.7324/JAPS.2014.40416.

Koenig JJ, Neudörfl  JM,  Hansen  A,  Breugst  M.  2018. Redetermination  of  the  solvent­free  crys­ tal   structure   of   l­proline.   Acta   Crystallogr, Sect E: Crystallogr Commun 74(8):1067–1070. doi:10.1107/S2056989018009490.

Kumar S, Arya S, Nussinov R. 2007. Temperature­ dependent molecular adaptation features in pro­ teins. In: C Gerday, N Glansdorff,  editors,  Phys­  iol Biochem Extremophiles, pages 75–85. Wash­ ington DC: American Society of Microbiology. doi:10.1128/9781555815813.ch6.

Kumar S, Stecher G, Li M, Knyaz C, Tamura K. 2018. MEGA X: molecular evolutionary genetics analy­ sis across computing platforms. Mol Biology Evol 35(6):1547–1549. doi:10.1093/molbev/msy096.

Kumwenda B,  Litthauer  D,  Bishop  OT,  Reva  O. 2013. Analysis of protein thermostability enhanc­ ing factors in industrially important Thermus bac­ teria species. Evol Bioinf Online 9:EBO–S12539. doi:10.4137/EBO.S12539.

Ladenstein R, Antranikian G. 1998. Proteins from hy­ perthermophiles: stability and enzymatic catalysis close to the boiling point of water. In: Biotechnol­ ogy of Extremophiles, pages 37–85. Berlin: Springer. doi:10.1007/BFb0102289.

Lane CE, Hulgan D, O’Quinn K, Benton MG. 2015. CE­ MAsuite: open source degenerate PCR primer design.       Bioinformatics 31(22):3688–3690. doi:10.1093/bioinformatics/btv420.

Larkin  MA,  Blackshields  G,   Brown   NP,   Chenna   R,  McGettigan  PA, McWilliam H,  McWilliam H,  Valentin   F,   Wallace   IM,   Wilm   A,   Lopez R, et al.  2007.  Clustal  W  and  Clustal  X  ver­  sion   2.0.  Bioinformatics 23(21):2947–2948. doi:10.1093/bioinformatics/btm404.

Lateef A, Adelere IA, Gueguim­Kana EB. 2015. The biology and potential biotechnological applications of Bacillus safensis. Biologia 70(4):411–419. doi:10.1515/biolog­2015­0062.

Li W, Zhang WW, Yang MM, Chen YL. 2008. Cloning of the thermostable cellulase gene from newly iso­ lated Bacillus subtilis and its expression in Es­ cherichia coli. Mol Biotechnol 40(2):195–201. doi:10.1007/s12033­008­9079­y.

Li YH, Ding M, Wang J, Xu GJ,  Zhao  F.  2006.  A novel thermoacidophilic endoglucanase, Ba­EGA, from a new cellulose­degrading bacterium, Bacillus sp. AC­1. Appl Microbiol Biotechnol 70(4):430–436. doi:10.1007/s00253­005­0075­x.

Lima AOS, Quecine MC,  Fungaro  MHP,  Andreote  FD, Maccheroni W, Araújo WL, Silva­Filho MC, Pizzirani­Kleiner AA, Azevedo J. 2005. Molecular characterization of a β­1, 4­endoglucanase from an endophytic Bacillus pumilus strain. Appl Microbiol Biotechnol 68(1):57–65. doi:10.1007/s00253­004­1740­1.

Lynd LR, Weimer PJ, Van Zyl WH, Pretorius IS. 2002. Microbial cellulose utilization: fundamentals and biotechnology. Microbiol Mol Biol Rev 66(3):506– 577. doi:10.1128/MMBR.66.3.506­577.2002.

Mc Gowan J. 2015. Bioinformatics Tools. URL https://jamiemcg.github.io/bioinf/gc.html.

Milburn D, Laskowski RA, Thornton JM. 1998. Se­ quences annotated by structure: a tool  to  fa­  cilitate the use of structural information in se­ quence analysis. Protein Eng 11(10):855–859. doi:10.1093/protein/11.10.855.

Moeis MR, Natalia D, Ningrum RW, Dwijayanti A. 2014. Cloning and expression of endoglucanase gene from thermophilic bacteria Bacillus sp. RP1. Microbiology Indonesia 8(4):4. doi:10.5454/mi.8.4.4.

Nakashima H, Fukuchi S, Nishikawa K. 2003. Compo­ sitional changes in RNA, DNA and proteins for bacterial adaptation to higher and lower temperatures. J Biochem 133(4):507–513. doi:10.1093/jb/mvg067.

Patel K, Patel H, Shah G. 2014. Production and optimiza­ tion of cellulase enzymes from newly isolated fungi. J Pure Appl Microbiol 8(5):4163–4169.

Ragone R. 2001. Hydrogen­bonding classes in proteins and their contribution to the unfolding reaction. Pro­ tein Sci 10(10):2075–2082. doi:10.1110/ps.09201.

Rice P, Longden I, Bleasby A. 2000. EMBOSS: the European molecular biology open software suite. Trends Genet 16(6):276–277. doi:10.1016/S0168­ 9525(00)02024­2.

Sadeghi M, Naderi­Manesh H, Zarrabi M, Ranjbar B. 2006. Effective factors in thermostability of ther­ mophilic proteins. Biophys Chem 119(3):256–270. doi:10.1016/j.bpc.2005.09.018.

Satomi M, La Duc M, Venkateswaran K. 2006. Bacil­ lus safensis sp. nov., isolated from spacecraft and assembly­facility surfaces. Int J Syst Evol Microbiol 56(8):1735–1740. doi:10.1099/ijs.0.64189­0.

Sheng P, Huang S, Wang Q, Wang A, Zhang H. 2012. Isolation, screening, and optimization of the fer­ mentation conditions of highly  cellulolytic  bacte­ ria from the hindgut of Holotrichia parallela larvae (Coleoptera: Scarabaeidae). Appl Biochem Biotech­ nol 167(2):270–284. doi:10.1007/s12010­012­9670­

3.

Shimon L, Belaich A, Belaich JP, Bayer E, Lamed R, Shoham Y, Frolow F, et al. 2000. Structure of a family IIIa scaffoldin CBD from the cellulosome of Clostrid­ ium cellulolyticum at 2.2 Å resolution. Acta Crys­ tallogr, Sect D: Biol Crystallogr 56(12):1560–1568. doi:10.1107/S0907444900012889.

Sievers F, Wilm A, Dineen D, Gibson T, Karplus K, Li W, Lopez R, McWilliam H, Remmert M, Söding J, Thompson J, Higgins D. 2011. Fast, scalable gener­ ation of high­quality protein multiple sequence align­ ments using Clustal Omega. Mol Syst Biol 7(1). doi:10.1038/msb.2011.75.

Slepecky R, Hemphill H. 2006. The Genus Bacillus— Nonmedical, pages 530–562. New York: Springer US. doi:10.1007/0­387­30744­3_16.

Srinivas R, Panda T. 1998. pH and thermal stability studies of carboxymethyl cellulase from intergeneric fusants of Trichoderma reesei/Saccharomyces cere­ visiae. J Ind Microbiol Biotechnol 21(4­5):178–183. doi:10.1038/sj.jim.2900556.

Sukumaran R, Singhania RR, Pandey A. 2005. Microbial cellulases­production, applications and challenges. J Sci Ind Res 64:832–844.

Teather R, Wood P. 1982. Use of Congo red­ polysaccharide interactions in enumeration and char­ acterization of cellulolytic bacteria from the bovine rumen. Appl Environ Microbiol 43(4):777–780.

Tormo J, Lamed R, Chirino A, Morag E, Bayer E, Shoham   Y,   Steitz   T.   1996.    Crystal   structure of a bacterial family­III cellulose­binding domain:   a general mechanism for attachment to cellulose. EMBO J 15(21):5739–5751. doi:10.1002/j.1460­ 2075.1996.tb00960.x.

Turner P, Mamo G, Karlsson E. 2007. Potential and utilization of thermophiles and thermostable en­ zymes in biorefining. Microb Cell Fact 6(1):9. doi:10.1186/1475­2859­6­9.

Van den Burg B, Vriend G, Veltman OR, Venema G, Ei­ jsink VGH. 1998.  Engineering an enzyme to re­   sist boiling. Proc Natl Acad Sci 95(5):2056–2060. doi:10.1073/pnas.95.5.2056.

Veltman OR, Vriend G, Middelhoven PJ, van den Burg B, Venema G, Eijsink VGH. 1996. Analysis of struc­ tural determinants of the stability of thermolysinlike proteases by molecular modelling and site­directed mutagenesis. Protein Eng, Des Sel 9(12):1181–1189. doi:10.1093/protein/9.12.1181.

Watanabe K, Chishiro K, Kitamura K, Suzuki Y. 1991. Proline residues responsible for thermostability oc­ cur with high frequency in the loop regions of an extremely thermostable oligo­1, 6­glucosidase from Bacillus thermoglucosidasius KP1006. J Biol Chem 266(36):24287–24294.

Waterhouse A, Bertoni M, Bienert S, Studer G, Tauriello G, Gumienny R, Heer FT,  de Beer TAP,  Rempfer  C, Bordoli L, et al. 2018. SWISS­MODEL: ho­ mology modelling of protein structures and com­ plexes. Nucl Acids Res 46(W1):W296–W303. doi:10.1093/nar/gky427.

Wood TM, Bhat KM. 1988. Biomass, Part A: Cellulose and hemicellulose. In: WA Wood, ST Kellog, editors, Methods in Enzymology, volume 160, pages 87–91. New York: Academic Press.

Yachdav G, Kloppmann E, Kajan L, Hecht M, Goldberg T, Hamp T, Hönigschmid P, Schafferhans A, Roos M, Bernhofer M, et al. 2014. PredictProtein—an open resource for online prediction of protein structural and functional features. Nucl Acids Res 42(W1):W337– W343. doi:10.1093/nar/gku366.

Yan BX, Sun YQ. 1997. Glycine residues provide flexibil­ ity for enzyme active sites. J Biol Chem 272(6):3190– 3194. doi:10.1074/jbc.272.6.3190.

Yang D, Weng H, Wang M, Xu W, Li Y, Yang H. 2010. Cloning and expression of a novel thermostable cel­ lulase from newly isolated Bacillus subtilis strain I15. Mol Biol Rep 37(4):1923–1929. doi:10.1007/s11033­ 009­9635­y.

Ye J, Coulouris G, Zaretskaya I, Cutcutache I, Rozen S, Madden TL. 2012. Primer­BLAST: a tool to design target­specific primers for polymerase chain reaction. BMC Bioinf 13(1):134. doi:10.1186/1471­2105­13­ 134.

Yu H,  Zhao  Y,  Guo  C,  Gan  Y,   Huang  H.  2015.   The role of proline substitutions within flexible re­ gions on thermostability of luciferase. Biochim Biophys Acta, Proteins Proteomics 1854(1):65–72. doi:10.1016/j.bbapap.2014.10.017.

Zarafeta D, Kissas D, Sayer C, Gudbergsdottir SR, Ladoukakis E, Isupov MN, Chatziioannou A, Peng X, Littlechild JA, Skretas G, et al. 2016.  Dis­ covery and characterization of a thermostable and highly halotolerant GH5 cellulase from an icelandic hot spring isolate. PLoS One 11(1):e0146454. doi:10.1371/journal.pone.0146454.

Zhang YHP, Himmel ME,  Mielenz  JR.  2006.  Out­ look for cellulase improvement: screening and se­ lection strategies. Biotechnol Adv 24(5):452–481. doi:10.1016/j.biotechadv.2006.03.003.

 



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