Skip to main content
SpringerLink
Log in

Heterologous expression of Thermobifida fusca thermostable alpha-amylase in Yarrowia lipolytica and its application in boiling stable resistant sago starch preparation

  • Original Paper
  • Published:
Journal of Industrial Microbiology & Biotechnology

Abstract

A gene encoding the thermostable α-amylase in Thermobifida fusca NTU22 was amplified by PCR, sequenced, and cloned into Yarrowia lipolytica P01g host strain using the vector pYLSC1 allowing constitutive expression and secretion of the protein. Recombinant expression resulted in high levels of extracellular amylase production, as high as 730 U/l in the Hinton flask culture broth. It is higher than that observed in P. pastoris expression system and E. coli expression system. The purified amylase showed a single band at about 65 kDa by SDS-polyacrylamide gel electrophoresis and this agrees with the predicted size based on the nucleotide sequence. About 70% of the original activity remained after heat treatment at 60°C for 3 h. The optimal pH and temperature of the purified amylase were 7.0 and 60°C, respectively. The purified amylase exhibited a high level of activity with raw sago starch. After 72-h treatment, the DP w of raw sago starch obviously decreased from 830,945 to 237,092. The boiling stable resistant starch content of the sago starch increased from 8.3 to 18.1%. The starch recovery rate was 71%.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6

Similar content being viewed by others

References

  1. Henrissat B (1991) A classification of glycohydrolases based on amino acid sequence similarities. Biochem J 280:309–316

    CAS  PubMed  Google Scholar 

  2. Nguyen QD, Rezessy-Szabo JM, Claeyssens M, Stals I, Hoschke A (2002) Purification and characterization of amylolytic enzymes from thermophilic fungus Thermomyces lanuginosus strain ATCC 34626. Enzyme Microb Technol 31:345–352

    Article  CAS  Google Scholar 

  3. Liu WH, Yang CH (2002) The isolation and identification of a lignocellulolytic and thermophilic actinomycete. Food Sci Agric Chem 4:89–94

    CAS  Google Scholar 

  4. Yang CH, Cheng KC, Liu WH (2003) Optimization of medium composition for production of extracellular amylase by Thermobifida fusca using a response surface methodology. Food Sci Agric Chem 5:35–40

    CAS  Google Scholar 

  5. Yang CH, Liu WH (2004) Purification and properties of a maltotriose-producing α-amylase from Thermobifida fusca. Enzyme Microb Technol 35:254–260

    Article  CAS  Google Scholar 

  6. Yang CH, Liu WH (2007) Cloning and characterization of a maltotriose-producing α-amylase gene from Thermobifida fusca. J Ind Microbiol Biotechnol 34:325–330

    Article  CAS  PubMed  Google Scholar 

  7. Zamost BL, Nielsen HK, Starnes RL (1991) Thermostable enzymes for industrial application. J Ind Microbiol 8:71–82

    Article  CAS  Google Scholar 

  8. Cheng YF, Yang CH, Liu WH (2005) Cloning and expression of Thermobifida xylanase gene in the methylotrophic yeast Pichia pastoris. Enzyme Microb Technol 37:541–546

    Article  CAS  Google Scholar 

  9. Yang CH, Huang YC, Chen CY, Wen CY (2010) Expression of Thermobifida fusca thermostable raw starch digesting alpha-amylase in Pichia pastoris and its application in raw sago starch hydrolysis. J Ind Microbiol Biotechnol. doi:10.1007/s10295-009-0686-9

  10. Fickers P, Benetti PH, Wache Y, Marty A (2005) Hydrophobic substrate utilization by the yeast Yarrowia lipolytica, and its potential applications. FEMS Yeast Res 5:527–543

    Article  CAS  PubMed  Google Scholar 

  11. Madzak C, Treton B, Blanchin-Roland S (2000) Strong hybrid promoters and integrative expression/secretion vectors for quasi-constitutive expression of heterologous proteins in the yeast Yarrowia lipolytica. J Mol Microbiol Biotechnol 2:207–216

    CAS  PubMed  Google Scholar 

  12. Madzak C, Gaillardin C, Beckerich JM (2004) Heterologous protein expression and secretion in the non-conventional yeast Yarrowia lipolytica. J Biotechnol 109:63–81

    Article  CAS  PubMed  Google Scholar 

  13. Barth G, Gaillardin C (1997) Physiology and genetics of the dimorphic fungus Yarrowia lipolytica. FEMS Microbiol Rev 19:219–237

    Article  CAS  PubMed  Google Scholar 

  14. Englyst HN, Kingman SM, Cummings JH (1992) Classification and measurement of nutritionally important starch fractions. Eur J Clin Nutr 46:533–550

    Google Scholar 

  15. Mortensen PB, Clausen MR (1996) Short-chain fatty acids in the human colon: relation to gastrointestinal health and disease. Scand J Gastroenterol 31:32–148

    Google Scholar 

  16. Raben A, Tagliabue A, Christensen NJ, Madsen J, Holst JJ, Astrup A (1994) Resistant starch: the effect on postprandial glycemia, hormonal response, and satiety. Am J Clin Nutr 60:544–551

    CAS  PubMed  Google Scholar 

  17. Philip J, Muir JG, Birkett A, Lu ZX, Jones GP, O’Dea K, Young GP (1995) Effect of resistant starch on fecal bulk and fermentation-dependent events in humans. Am J Clin Nutr 62:121–130

    Google Scholar 

  18. Lin JH, Wang SW, Chang YH (2009) Impacts of acid-methanol treatment and annealing on the enzymatic resistance of corn starches. Food Hydrocoll 23:1465–1472

    Article  CAS  Google Scholar 

  19. Eerlingen RC, Deceuninck M, Delcour JA (1993) Enzyme-resistant starch. II. Influence of amylose chain length on resistant starch formation. Cereal Chem 70:345–350

    CAS  Google Scholar 

  20. Leong YH, Karim AA, Norziah MH (2007) Effect of pullulanase debranching of sago (Metroxylon sagu) starch at subgelatinization temperature on the yield of resistant starch. Starch 59:21–32

    Article  CAS  Google Scholar 

  21. Brumovsky JO, Thompson DB (2001) Production of boiling-stable granular resistant starch by partial acid hydrolysis and hydrothermal treatment of high-amylose maize starch. Cereal Chem 78:680–689

    Article  CAS  Google Scholar 

  22. Xuan JW, Fournier P, Gaillardin C (1988) Cloning of the LYS5 gene encoding saccharopine dehydrogenase from the yeast Yarrowia lipolytica by target integration. Curr Genet 14:15–21

    Article  CAS  Google Scholar 

  23. Lin JH, Lee SY, Chang YH (2003) Effect of acid-alcohol treatment on the molecular structure and physiochemical properties of maize and potato starches. Carbohydr Polym 53:475–482

    Article  CAS  Google Scholar 

  24. Lin JH, Chang YH, Hsu YH (2009) Degradation of cotton cellulose treated with hydrochloric acid either in water or in ethanol. Food Hydrocoll 23:1548–1553

    Article  CAS  Google Scholar 

  25. Bordes F, Fudalej F, Dossat V, Nicaud JM, Marty A (2007) A new recombinant protein expression system for high-throughput screening in the yeast Yarrowia lipolytica. J Microbiol Methods 70:393–502

    Article  Google Scholar 

  26. Madzak C, Otterbein L, Chamkha M, Moukha S, Asther M, Gaillardin C, Beckerich JM (2005) Heterologous production of a laccase from the basidiomycete Pycnoporus cinnabarinus in the dimorphic yeast Yarrowia lipolytica. FEMS Yeast Res 5:635–646

    Article  CAS  PubMed  Google Scholar 

  27. Haralampu SG (2000) Resistant starch—a review of the physical properties and biological impact of RS3. Carbohydr Polym 41:285–292

    Article  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Cosmetic Science, Providence University, Taichung, 43301, Taiwan

    Chao-Hsun Yang, Yu-Chun Huang, Cheng-Yu Chen & Chia-Ying Wen

Authors
  1. Chao-Hsun Yang
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Yu-Chun Huang
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Cheng-Yu Chen
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Chia-Ying Wen
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Chao-Hsun Yang.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Yang, CH., Huang, YC., Chen, CY. et al. Heterologous expression of Thermobifida fusca thermostable alpha-amylase in Yarrowia lipolytica and its application in boiling stable resistant sago starch preparation. J Ind Microbiol Biotechnol 37, 953–960 (2010). https://doi.org/10.1007/s10295-010-0745-2

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10295-010-0745-2

Keywords

Search

Navigation