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Applied Microbiology and Biotechnology

, Volume 101, Issue 8, pp 3247–3258 | Cite as

Immobilization of alkaline polygalacturonate lyase from Bacillus subtilis on the surface of bacterial polyhydroxyalkanoate nano-granules

  • GanQiao Ran
  • Dan Tan
  • WeiEr Dai
  • XinLiang Zhu
  • JiPing Zhao
  • Qi MaEmail author
  • XiaoYun LuEmail author
Biotechnologically relevant enzymes and proteins

Abstract

Alkaline polygalacturonate lyase (PGL), one of the pectinolytic enzymes, has been widely used for the bioscouring of cotton fibers, biodegumming, and biopulp production. In our study, PGL from Bacillus subtilis was successfully immobilized on the surface of polyhydroxyalkanoate (PHA) nanogranules by fusing PGL to the N-terminal of PHA synthase from Ralstonia eutropha via a designed linker. The PGL-decorated PHA beads could be simply achieved by recombinant fermentation and consequent centrifugation. The fused PGL occupied 0.985% of the total weight of purified PHA granules, which was identified by mass spectrometer-based quantitative proteomics. The activity of immobilized PGL (184.67 U/mg PGL protein) was a little lower than that of the free PGL (215.93 U/mg PGL protein). The immobilization process did not affect the optimal pH and the optimal temperature of the PGL, but it did enhance the thermostability as well as the pH stability at certain conditions, which will extend the practicability of the immobilized PGL-PHA beads in the alkaline and generally harsh bioscouring process. Furthermore, the immobilized PGL still retained more than 60% of its initial activity after 8 cycles of reuse. Our study provided a novel and promising approach for cost-efficient in vivo PGL immobilization, contributing to wider commercialization of this environmental-friendly biocatalyst.

Keywords

Alkaline polygalacturonate lyase Polyhydroxyalkanoates Immobilization Green chemistry Bioscouring 

Notes

Acknowledgments

This work was financially supported by the National Natural Science Foundation of China (No. 21306110, No. 21602171) and China Postdoctoral Science Foundation (No. 2015M582640). We offer our acknowledgment to both Prof. Alexander Steinbuchel and Prof. Guo-Qiang Chen for kind donation of plasmid pBHR68. Besides, we also thanked Prof. Bernd H. A. Rehm for providing us many suggestions for the granule purification as well as the quantification of immobilized protein.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical statement

This article does not contain any studies with human participants or animals performed by any of the authors.

Supplementary material

253_2016_8085_MOESM1_ESM.xlsx (73 kb)
Table S1 (XLSX 73 kb.)
253_2016_8085_MOESM2_ESM.pdf (377 kb)
Figure S1 (PDF 376 kb.)

References

  1. Boersema PJ, Raijmakers R, Lemeer S, Mohammed S, Heck AJ (2009) Multiplex peptide stable isotope dimethyl labeling for quantitative proteomics. Nat Protoc 4(4):484–494. doi: 10.1038/nprot.2009.21 CrossRefPubMedGoogle Scholar
  2. Blatchford PA, Scott C, French N, Rehm BH (2012) Immobilization of organophosphohydrolase OpdA from Agrobacterium radiobacter by overproduction at the surface of polyester inclusions inside engineered Escherichia coli. Biotechnol and Bioeng 109:1101–1108. doi: 10.1002/bit.24402 CrossRefGoogle Scholar
  3. Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254CrossRefPubMedGoogle Scholar
  4. Brockelbank JA, Peters V, Rehm BH (2006) Recombinant Escherichia coli strain produces a ZZ domain displaying biopolyester granules suitable for immunoglobulin G purification. Appl Environ Microbiol 72(11):7394–7397. doi: 10.1128/AEM.01014-06 CrossRefPubMedPubMedCentralGoogle Scholar
  5. Bustamante-Vargas CE, de Oliveira D, Valduga E, Venquiaruto LD, Paroul N, Backes GT, Dallago RM (2016) Biomimetic mineralization of the alginate/gelatin/calcium oxalate matrix for immobilization of pectinase: influence of matrix on the pectinolytic activity. Appl Biochem Biotech 179(6):1060–1072. doi: 10.1007/s12010-016-2050-7 CrossRefGoogle Scholar
  6. Chen SY, Chien YW, Chao YP (2014) In vivo immobcion of D-hydantoinase in Escherichia coli. J Biosci Bioeng 118(1):78–81. doi: 10.1016/j.jbiosc.2013.12.020 CrossRefPubMedGoogle Scholar
  7. Chen GQ, Hajnal I, Wu H, Lv L, Ye J (2015) Engineering biosynthesis mechanisms for diversifying polyhydroxyalkanoates. Trends Biotechnol 33:565–574. doi: 10.1016/j.tibtech.2015.07.007 CrossRefPubMedGoogle Scholar
  8. Chiliveri SR, Linga VR (2014) A novel thermostable, alkaline pectate lyase from Bacillus tequilensis SV11 with potential in textile industry. Carbohyd Polym 111:264–272. doi: 10.1016/j.carbpol.2014.04.065 CrossRefGoogle Scholar
  9. Denner EBM, Mcgenity TJ, Busse HJ, Grant WD, Wanner G, Stan-Lotter H (1994) Halococcus salifodinae sp. nov., an archaeal isolate from an Austrian salt mine. Int J Syst Bacteriol 44:774–780CrossRefGoogle Scholar
  10. Fang S, Li J, Liu L, Du G, Chen J (2011) Overproduction of alkaline polygalacturonate lyase in recombinant Escherichia coli by a two-stage glycerol feeding approach. Bioresource Technol 102:10671–10678. doi: 10.1016/j.biortech.2011.09.020 CrossRefGoogle Scholar
  11. Hay ID, Du J, Reyes PR, Rehm BH (2015) In vivo polyester immobilized sortase for tagless protein purification. Microb Cell Factories 14:190. doi: 10.1186/s12934-015-0385-3 CrossRefGoogle Scholar
  12. Hooks DO, Rehm BH (2015) Surface display of highly-stable Desulfovibrio vulgaris carbonic anhydrase on polyester beads for CO2 capture. Biotechnol Lett 37(7):1415–1420. doi: 10.1007/s10529-015-1803-7 CrossRefPubMedGoogle Scholar
  13. Hooks DO, Blatchford PA, Rehm BH (2013) Bioengineering of bacterial polymer inclusions catalyzing the synthesis of N-acetylneuraminic acid. Appl Environ Microbiol 79:3116–3121. doi: 10.1128/AEM.03947-12 CrossRefPubMedPubMedCentralGoogle Scholar
  14. Hooks DO, Venning-Slater M, Du J, Rehm BH (2014) Polyhydroyxalkanoate synthase fusions as a strategy for oriented enzyme immobilisation. Molecules 19:8629–8643. doi: 10.3390/molecules19068629 CrossRefPubMedGoogle Scholar
  15. Hoondal GS, Tiwari RP, Tewari R, Dahiya N, Beg QK (2002) Microbial alkaline pectinases and their industrial applications: a review. Appl Microbiol Biotechnol 59:409–418. doi: 10.1007/s00253-002-1061-1 CrossRefPubMedGoogle Scholar
  16. Jahns AC, Rehm BH (2015) Immobilization of active lipase B from Candida antarctica on the surface of polyhydroxyalkanoate inclusions. Biotechnol Lett 37(4):831–835. doi: 10.1007/s10529-014-1735-7 CrossRefPubMedGoogle Scholar
  17. Jahns AC, Haverkamp RG, Rehm BH (2008) Multifunctional inorganic-binding beads self-assembled inside engineered bacteria. Bioconjug Chem 19:2072–2080. doi: 10.1021/bc8001979 CrossRefPubMedGoogle Scholar
  18. Kim YJ, Choi SY, Kim J, Jin KS, Lee SY, Kim KJ (2016a) Structure and function of the N-terminal domain of Ralstonia eutropha polyhydroxyalkanoate synthase, and the proposed structure and mechanisms of the whole enzyme. Biotechnol J [Epub ahead of print]Google Scholar
  19. Kim J, Kim YJ, Choi SY, Lee SY, Kim KJ (2016b) Crystal structure of Ralstonia eutropha polyhydroxyalkanoate synthase C-terminal domain and reaction mechanisms. Biotechnol J [Epub ahead of print]Google Scholar
  20. Klug-Santner BG, Schnitzhofer W, Vrsanska M, Weber J, Agrawal PB, Nierstrasz VA, Guebitz GM (2006) Purification and characterization of a new bioscouring pectate lyase from Bacillus pumilus BK2. J Biotechnol 121:390–401. doi: 10.1016/j.jbiotec.2005.07.019 CrossRefPubMedGoogle Scholar
  21. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685. doi: 10.1038/227680a0 CrossRefPubMedGoogle Scholar
  22. Li Z, Bai Z, Zhang B, Li B, Jin B, Zhang M, Lin F, Zhang H (2012) Purification and characterization of alkaline pectin lyase from a newly isolated Bacillus clausii and its application in elicitation of plant disease resistance. Appl Biochem Biotech 167:2241–2256. doi: 10.1007/s12010-012-9758-9 CrossRefGoogle Scholar
  23. Liu K, Zhao G, He B, Chen L, Huang L (2012) Immobilization of pectinase and lipase on macroporous resin coated with chitosan for treatment of whitewater from papermaking. Bioresource Technol 123:616–619. doi: 10.1016/j.biortech.2012.07.074 CrossRefGoogle Scholar
  24. Lopez NI, Pettinari MJ, Nikel PI, Mendez BS (2015) Polyhydroxyalkanoates: much more than biodegradable plastics. Adv Appl Microbiol 93:73–106. doi: 10.1016/bs.aambs.2015.06.001 CrossRefPubMedGoogle Scholar
  25. Parlane NA, Grage K, Lee JW, Buddle BM, Denis M, Rehm BH (2011) Production of a particulate hepatitis C vaccine candidate by an engineered Lactococcus lactis strain. Appl Environ Microbiol 77(24):8516–8522. doi: 10.1128/AEM.06420-11 CrossRefPubMedPubMedCentralGoogle Scholar
  26. Peters V, Rehm BH (2006) In vivo enzyme immobilization by use of engineered polyhydroxyalkanoate synthase. Appl Environ Microbiol 72:1777–1783. doi: 10.1128/AEM.72.3.1777-1783.2006 CrossRefPubMedPubMedCentralGoogle Scholar
  27. Pohlmann A, Fricke WF, Reinecke F, Kusian B, Liesegang H, Cramm R, Eitinger T, Ewering C, Potter M, Schwartz E, Strittmatter A, Voss I, Gottschalk G, Steinbuchel A, Friedrich B, Bowien B (2006) Genome sequence of the bioplastic-producing ‘Knallgas’ bacterium Ralstonia eutropha H16. Nat Biotechnol 24(10):1257–1262. doi: 10.1038/nbt 1244 CrossRefPubMedGoogle Scholar
  28. Rehman HU, Aman A, Silipo A, Qader SA, Molinaro A, Ansari A (2013) Degradation of complex carbohydrate: immobilization of pectinase from Bacillus licheniformis KIBGE-IB21 using calcium alginate as a support. Food Chem 139:1081–1086. doi: 10.1016/j.foodchem.2013.01.069 CrossRefPubMedGoogle Scholar
  29. Rehm FB, Chen S, Rehm BH (2016) Enzyme engineering for in situ immobilization. Molecules 21(10):1370. doi: 10.3390/molecules21101370 CrossRefGoogle Scholar
  30. Seyedarabi A, To TT, Ali S, Hussain S, Fries M, Madsen R, Clausen MH, Teixteira S, Brocklehurst K, Pickersgill RW (2010) Structural insights into substrate specificity and the anti beta-elimination mechanism of pectate lyase. Biochemistry 49:539–546. doi: 10.1021/bi901503g CrossRefPubMedGoogle Scholar
  31. Solbak AI, Richardson TH, McCann RT, Kline KA, Bartnek F, Tomlinson G, Tan X, Parra-Gessert L, Frey GJ, Podar M, Luginbuhl P, Gray KA, Mathur EJ, Robertson DE, Burk MJ, Hazlewood GP, Short JM, Kerovuo J (2005) Discovery of pectin-degrading enzymes and directed evolution of a novel pectate lyase for processing cotton fabric. J Biol Chem 280:9431–9438. doi: 10.1074/jbc.M411838200 CrossRefPubMedGoogle Scholar
  32. Spiekermann P, Rehm BH, Kalscheuer R, Baumeister D, Steinbuchel A (1999) A sensitive, viable-colony staining method using Nile red for direct screening of bacteria that accumulate polyhydroxyalkanoic acids and other lipid storage compounds. Arch Microbiol 171:73–80CrossRefPubMedGoogle Scholar
  33. Saleem Z, Rennebaum H, Pudel F, Grimm E (2008) Treating bast fibres with pectinase improves mechanical characteristics of reinforced thermoplastic composites. Compos Sci Technol 68:471–476. doi: 10.1016/j.compscitech.2007.06.005 CrossRefGoogle Scholar
  34. Tan D, Xue YS, Aibaidula G, Chen GQ (2011) Unsterile and continuous production of polyhydroxybutyrate by Halomonas TD01. Bioresource Technol 102:8130–8136. doi: 10.1016/j.biortech.2011.05.068 CrossRefGoogle Scholar
  35. Thakur J, Gupta R (2012) Improvement of tea leaves fermentation through pectinases. Acta Microbiol Imm H 59:321–334. doi: 10.1556/AMicr.59.2012.3.3 CrossRefGoogle Scholar
  36. Tzanova T, Calafell M, Guebitz GM, Cavaco-Paulo A (2001) Bio-preparation of cotton fabrics. Enzyme Microb Tech 29:357–362. doi: 10.1016/S0141-0229(01)00388-X CrossRefGoogle Scholar
  37. Yao YC, Zhan XY, Zhang J, Zou XH, Wang ZH, Xiong YC, Chen J, Chen GQ (2008) A specific drug targeting system based on polyhydroxyalkanoate granule binding protein PhaP fused with targeted cell ligands. Biomaterials 29(36):4823–4830. doi: 10.1016/j.biomaterials.2008.09.008 CrossRefPubMedGoogle Scholar
  38. Yuan P, Meng K, Luo HY, Shi PJ, Huang HQ, Bai YG, Yang PL, Yao B (2011) A novel low-temperature active alkaline pectate lyase from Klebsiella sp. Y1 with potential in textile industry. Process Biochem 46:1921–1926. doi: 10.1016/j.procbio.2011.06.023 CrossRefGoogle Scholar
  39. Yadav S, Yadav PK, Yadav D, Yadav KDS (2009) Pectin lyase: a review. Process Biochem 44:1–10. doi: 10.1016/j.procbio.2008.09.012 CrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2017

Authors and Affiliations

  1. 1.Key Laboratory of Biomedical Information Engineering of Ministry of Education, School of Life Science and TechnologyXi’an Jiaotong UniversityXi’anPeople’s Republic of China
  2. 2.Institute of Enzyme EngineeringShannxi Academy of ScienceXi’anPeople’s Republic of China

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