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Applied Microbiology and Biotechnology

, Volume 103, Issue 2, pp 807–817 | Cite as

Study on expression and action mode of recombinant alginate lyases based on conserved domains reconstruction

  • Min Yang
  • Nannan Li
  • Suxiao Yang
  • Yuan Yu
  • Zhenlian Han
  • Li LiEmail author
  • Haijin MouEmail author
Biotechnologically relevant enzymes and proteins
  • 131 Downloads

Abstract

Understanding the effect of conserved domains reconstruction of alginate lyases on action mode is essential for their application and in-depth study. We report the expression and action mode of recombinant alginate lyase (AlyM) and its conserved domain reconstruction forms (AlyMΔCBM, cAlyM, and AlyMΔ58C). The enzymatic activities of AlyM, AlyMΔCBM, cAlyM, and AlyMΔ58C were 61.77, 150.57, 388.97, and 308.21 U/mg, respectively. The transcription level of cAlyM was 49.89-fold of AlyM. cAlyM and AlyMΔ58C showed higher thermal stability than AlyM, indicating that the removal of F5_F8_type_C domain was beneficial for the increase of thermal stability. The enzymes were bifunctional alginate lyases and preferred polyG to polyM. The enzymes degraded alginate to produce unsaturated disaccharide, trisaccharide, and tetrasaccharide as the main end-products. Pentamannuronic acid and pentaguluronic acid were the smallest substrates that could be degraded by AlyM, with unsaturated trisaccharide/tetrasaccharide (40.61%/44.42%) and disaccharide/trisaccharide (10.57%/83.85%) as the main products, respectively. The action modes of enzymes remain unaffected after conserved domain reconstruction, but the affinity of AlyMΔ58C toward polyM increased. This study provides a new strategy for rational modification of alginate lyase based on conserved domain reconstruction.

Keywords

Alginate lyase Action mode Conserved domain reconstruction Transcription level Thermal stability Microbulbifer 

Notes

Funding information

This work was supported by the Shandong Province Key Research and Development Project (2017YYSP003), Natural Science Foundation of Shandong Province (ZR2017MD006), and Public Science and Technology Research Funds Projects of Ocean (201505022).

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.

References

  1. Botha J, Mizrachi E, Myburg AA, Cowan DA (2017) Carbohydrate active enzyme domains from extreme thermophiles: components of a modular toolbox for lignocellulose degradation. Extremophiles 1:1–12Google Scholar
  2. Cheng Y, Wang D, Gu J, Li J, Liu H, Li F, Han WJ (2017) Biochemical characteristics and variable alginate-degrading modes of a novel bifunctional endolytic alginate lyase. Appl Environ Microbiol 83:1608–1617Google Scholar
  3. Day DF (1998) Alginates. Carbohydr Polym 8:161–182Google Scholar
  4. Dhillon A, Fernandes VO, Dias FM, Prates JA, Ferreira LM, Fontes CM, Centeno MS, Goyal A (2016) A new member of family 11 polysaccharide lyase, rhamnogalacturonan lyase (CtRGlF) from Clostridium thermocellum. Mol Biotechnol 58:232–240CrossRefGoogle Scholar
  5. Dou W, Wei D, Li H, Li H, Rahman MM, Shi J, Xu ZH, Ma YH (2013) Purification and characterisation of a bifunctional alginate lyase from novel Isoptericola halotolerans CGMCC 5336. Carbohydr Polym 98(2):1476–1482CrossRefGoogle Scholar
  6. Duan X, Wu J (2015) Enhancing the secretion efficiency and thermostability of a Bacillus deramificans pullulanase mutant (D437H/D503Y) by N-terminal domain truncation. Appl Environ Microbiol 81:1926–1931CrossRefGoogle Scholar
  7. Elleuche S, Krull A, Lorenz U, Antranikian G (2017) Parallel N- and C-terminal truncations facilitate purification and analysis of a 155-kDa cold-adapted type-I pullulanase. Protein J 36:56–63CrossRefGoogle Scholar
  8. Gimmestad M, Ertesvag H, Heggeset TM, Aarstad O, Svanem BI, Valla S (2009) Characterization of three new Azotobacter vinelandii alginate lyases, one of which is involved in cyst germination. J Bacteriol 191:4845–4853CrossRefGoogle Scholar
  9. Han W, Gu J, Cheng Y, Liu H, Li Y, Li F (2015) Novel alginate lyase (Aly5) from a polysaccharide-degrading marine bacterium Flammeovirga sp. strain MY04: effects of module truncation on biochemical characteristics, alginate degradation patterns, and oligosaccharide-yielding properties. Appl Environ Microbiol 82:864–874Google Scholar
  10. Holtan S, Zhang Q, Strand WI, Skjanbreak G (2006) Characterization of the hydrolysis mechanism of polyalternating alginate in weak acid and assignment of the resulting MG-oligosaccharides by NMR spectroscopy and ESI-mass spectrometry. Biomacromolecules 7:2108–2121CrossRefGoogle Scholar
  11. Imran M, Pant P, Shanbhag YP, Sawant SV, Ghadi SC (2017) Genome sequence of Microbulbifer mangrovi DD-13T, reveals its versatility to degrade multiple polysaccharides. Mar Biotechnol 19:116–124CrossRefGoogle Scholar
  12. Inoue A, Nishiyama R, Ojima T (2016) The alginate lyases FlAlyA, FlAlyB, FlAlyC, and FlAleX from Flavobacterium sp. UMI-01 have distinct roles in the complete degradation of alginate. Algal Res 19:355–362CrossRefGoogle Scholar
  13. Ishikawa K, Kataoka M, Yanamoto T, Nakabayashi M, Watanabe M, Ishihara S, Yamaguchi S (2015) Crystal structure of β-galactosidase from Bacillus circulans ATCC 31382 (BgaD) and the construction of the thermophilic mutants. FEBS J 282:2540–2552CrossRefGoogle Scholar
  14. Jagtap SS, Hehemann JH, Polz MF, Lee JK, Zhao H (2014) Comparative biochemical characterization of three exolytic oligoalginate lyases from Vibrio splendidus reveals complementary substrate scope, temperature, and pH adaptations. Appl Environ Microbiol 80:4207–4214CrossRefGoogle Scholar
  15. Jia X, Qiao W, Tian W, Peng X, Mi S, Su H, Han YJ (2016) Biochemical characterization of extra- and intracellular endoxylanse from thermophilic bacterium Caldicellulosiruptor kronotskyensis. Sci Rep 6:21672–21684CrossRefGoogle Scholar
  16. Kimoto H, Akamatsu M, Fujii Y, Tatsumi H, Kusaoke H, Taketo A (2010) Discoidin domain of chitosanase is required for binding to the fungal cell wall. J Molecul Microb Biotechnol 18:14–23CrossRefGoogle Scholar
  17. Lee KY, Mooney DJ (2012) Alginate: properties and biomedical applications. Prog Polym Sci 37:106–126CrossRefGoogle Scholar
  18. Li S, Yang X, Bao M, Wu Y, Yu W, Han F (2015) Family 13 carbohydrate-binding module of alginate lyase from Agarivorans sp. L11 enhances its catalytic efficiency and thermostability, and alters its substrate preference and product distribution. FEMS Microbiol Lett 362:1–7Google Scholar
  19. Li S, Wang L, Chen X, Zhao W, Sun M, Han Y (2018) Cloning, expression, and biochemical characterization of two new oligoalginate lyases with synergistic degradation capability. Mar Biotechnol 20(1):75–86CrossRefGoogle Scholar
  20. Lundqvist LC, Jam M, Barbeyron T, Czjzek M, Sandstrom C (2012) Substrate specificity of the recombinant alginate lyase from the marine bacteria Pseudomonas alginovora. Carbohydr Res 352:44–50CrossRefGoogle Scholar
  21. Miller G (1959) Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal Chem 31:426–428CrossRefGoogle Scholar
  22. Moh TH, Lau NS, Furusawa G, Amirul AAA (2017) Complete genome sequence of Microbulbifer sp. CCB-MM1, a halophile isolated from Matang Mangrove Forest, Malaysia. Stand Genomic Sci 12:36–45CrossRefGoogle Scholar
  23. Moore CE, Mota SRD, Mikolajek H, Proud CG (2014) A conserved loop in the catalytic domain of eukaryotic elongation factor 2 kinase plays a key role in its substrate specificity. Mol Cell Biol 34(12):2294–2307CrossRefGoogle Scholar
  24. Park HH, Kam N, Lee EY, Kim HS (2012) Cloning and characterization of a novel oligoalginate lyase from a newly isolated bacterium Sphingomonas sp. MJ-3. Mar Biotechnol 14:189–202CrossRefGoogle Scholar
  25. Peng C, Wang Q, Lu D, Han W, Li F (2018) A novel bifunctional endolytic alginate lyase with variable alginate-degrading modes and versatile monosaccharide-producing properties. Front Microbiol 9:167CrossRefGoogle Scholar
  26. Rahman MM, Wang L, Inoue A, Ojima T (2012) cDNA cloning and bacterial expression of a PL-14 alginate lyase from a herbivorous marine snail Littorina brevicula. Carbohydr Res 360:69–77CrossRefGoogle Scholar
  27. Sari-Chmayssem N, Taha S, Mawlawi H, Guégan JP, Jeftić J, Benvegnu T (2016) Extracted and depolymerized alginates from brown algae Sargassum vulgare of Lebanese origin: chemical, rheological, and antioxidant properties. J Appl Phycol 28:1915–1929CrossRefGoogle Scholar
  28. Sen M (2011) Effects of molecular weight and ratio of guluronic acid to mannuronic acid on the antioxidant properties of sodium alginate fractions prepared by radiation-induced degradation. Appl Radiat Isot 69:126–129CrossRefGoogle Scholar
  29. Sim PF, Furusawa G, Teh AH (2017) Functional and structural studies of a multidomain alginate lyase from Persicobacter sp. CCB-QB2. Sci Rep 7:13656–13665CrossRefGoogle Scholar
  30. Stabler C, Wilks K, Sambanis A, Constantinidis I (2001) The effects of alginate composition on encapsulated βTC3 cells. Biomaterial 22:1301–1310CrossRefGoogle Scholar
  31. Sun C, Chen YJ, Zhang XQ, Pan J, Cheng H, Wu M (2014) Draft genome sequence of Microbulbifer elongatus strain HZ11, a brown seaweed-degrading bacterium with potential ability to produce bioethanol from alginate. Mar Genomics 18:83–85CrossRefGoogle Scholar
  32. Thomas F, Lundqvist LC, Jam M, Jeudy A, Barbeyron T, Sandström C, Michel G, Czjzek M (2013) Comparative characterization of two marine alginate lyases from Zobellia galactanivorans reveals distinct modes of action and exquisite adaptation to their natural substrate. J Biol Chem 288:23021–23037CrossRefGoogle Scholar
  33. Tsuji H, Nishimura S, Inui T, Kado Y, Ishikawa K, Nakamura T, Uegaki K (2010) Kinetic and crystallographic analyses of the catalytic domain of chitinase from Pyrococcus furiosus – the role of conserved residues in the active site. Early Child Res Q 277(12):2683–2695Google Scholar
  34. Wakabayashi M, Sakatoku A, Noda F, Noda M, Tanaka D, Nakamura S (2012) Isolation and characterization of Microbulbifer species 6532A degrading seaweed thalli to single cell detritus particles. Biodegradation 23:93–105CrossRefGoogle Scholar
  35. Wang Y, Yuan H, Wang J, Yu Z (2009) Truncation of the cellulose binding domain improved thermal stability of endo-beta-1,4-glucanase from Bacillus subtilis JA18. Bioresour Technol 100:345–349CrossRefGoogle Scholar
  36. Wang J, Zeng D, Liu G, Wang S, Yu S (2014) Truncation of a mannanase from Trichoderma harzianum improves its enzymatic properties and expression efficiency in Trichoderma reesei. J Ind Microbiol Biotechnol 41:125–133CrossRefGoogle Scholar
  37. Wang L, Li S, Yu W, Gong Q (2015) Cloning, overexpression and characterization of a new oligoalginate lyase from a marine bacterium Shewanella sp. Biotechnol Lett 37(3):665–671CrossRefGoogle Scholar
  38. Wang H, He W, Jiang P, Yu Y, Lin L, Sun X, Koffas M, Zhang F, Linhardt RJ (2017) Construction and functional characterization of truncated versions of recombinant keratanase II from Bacillus circulans. Glycoconj J 34:643–649CrossRefGoogle Scholar
  39. Wong TY, Preston LA, Schiller NL (2000) Alginate lyase: review of major sources and enzyme characteristics, structure-function analysis, biological roles, and applications. Annu Rev Microbiol 5:289–340CrossRefGoogle Scholar
  40. Xu F, Dong F, Wang P, Cao HY, Li CY, Li PY, Pang XH, Zhang YZ, Chen XL (2017) Novel molecular insights into the catalytic mechanism of marine bacterial alginate lyase AlyGC from polysaccharide lyase family 6. J Biol Chem 292:4457–4468CrossRefGoogle Scholar
  41. Yang M, Yu Y, Jin TY, Mou HJ, Li L (2018) Genomic analysis of Microbulbifer sp. Q7 exhibiting degradation activity toward seaweed polysaccharides. Mar Genomics 39:7–10CrossRefGoogle Scholar
  42. Yu Y, Liu ZM, Yang M, Chen M, Wei Z, Shi LX, Li L, Mou HJ (2017) Characterization of full-length and truncated recombinant κ-carrageenase expressed in Pichia pastoris. Front Microbiol 8:1544–1556CrossRefGoogle Scholar
  43. Zhou R, Shi XY, Bi DC, Fang WS, Wei GB, Xu X (2015) Alginate-derived oligosaccharide inhibits neuroinflammation and promotes microglial phagocytosis of β-amyloid. Mar Drugs 13:5828–5846CrossRefGoogle Scholar
  44. Zhu B, Chen M, Yin H, Du Y, Ning L (2016) Enzymatic hydrolysis of alginate to produce oligosaccharides by a new purified endo-type alginate lyase. Mar Drugs 14:108–119CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  1. 1.College of Food Science and EngineeringOcean University of ChinaQingdaoChina

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