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Transgenic Research

, Volume 22, Issue 3, pp 477–488 | Cite as

Heterologous expression of cellobiohydrolase II (Cel6A) in maize endosperm

  • Shivakumar Pattada Devaiah
  • Deborah Vicuna Requesens
  • Yeun-Kyung Chang
  • Kendall R. Hood
  • Ashley Flory
  • John A. Howard
  • Elizabeth E. Hood
Original Paper

Abstract

The technology of converting lignocellulose to biofuels has advanced swiftly over the past few years, and enzymes are a significant constituent of this technology. In this regard, cost effective production of cellulases has been the focus of research for many years. One approach to reach cost targets of these enzymes involves the use of plants as bio-factories. The application of this technology to plant biomass conversion for biofuels and biobased products has the potential for significantly lowering the cost of these products due to lower enzyme production costs. Cel6A, one of the two cellobiohydrolases (CBH II) produced by Hypocrea jecorina, is an exoglucanase that cleaves primarily cellobiose units from the non-reducing end of cellulose microfibrils. In this work we describe the expression of Cel6A in maize endosperm as part of the process to lower the cost of this dominant enzyme for the bioconversion process. The enzyme is active on microcrystalline cellulose as exponential microbial growth was observed in the mixture of cellulose, cellulases, yeast and Cel6A, Cel7A (endoglucanase), and Cel5A (cellobiohydrolase I) expressed in maize seeds. We quantify the amount accumulated and the activity of the enzyme. Cel6A expressed in maize endosperm was purified to homogeneity and verified using peptide mass finger printing.

Keywords

Zea mays Endosperm Hypocrea jecorina Cellobiohydrolase II Cel6A Biomass 

Notes

Acknowledgments

This work was supported by a grant from the US Department of Energy (DE FG36 GO88025) with cost share from the Wal-Mart Foundation, the Walton Family Foundation, and Arkansas State University. The authors would like to thank Dr. Brett Savary and Dr. Prasanna Vasu, Arkansas Biosciences Institute, Arkansas State University, for the use of the HPLC and MALDI–TOF.

Supplementary material

11248_2012_9659_MOESM1_ESM.pptx (152 kb)
Fig. S1: Vector NTI map of transformation vector. Vector CDN (Os Glutelin Promoter and Cel6A, or CBH II). BAASS: barley alpha amylase signal sequence, CAMV35S: cauliflower mosaic virus 35S promoter and terminator, COLE1: E. coli replication origin, LB: left border, moPAT: maize-optimized phosphinothricin acetyl transferase gene, OsGlu: Oryza sativa (Rice) Glutelin Promoter, PIN II: potato protease inhibitor II terminator, RB: right border, COS: cointegration sequence for pSB1, SpecR: spectinomycin resistance. Supplementary material 1 (PPTX 151 kb)
11248_2012_9659_MOESM2_ESM.pptx (107 kb)
Fig. S2: Purification of Cel6A. Coomassie blue stained acrylamide gel of purified Cel6A. M: molecular weight markers. Supplementary material 2 (PPTX 106 kb)
11248_2012_9659_MOESM3_ESM.doc (42 kb)
Supplementary material 3 (DOC 42 kb)

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Copyright information

© Springer Science+Business Media Dordrecht 2012

Authors and Affiliations

  • Shivakumar Pattada Devaiah
    • 1
  • Deborah Vicuna Requesens
    • 1
  • Yeun-Kyung Chang
    • 1
  • Kendall R. Hood
    • 2
  • Ashley Flory
    • 1
  • John A. Howard
    • 3
  • Elizabeth E. Hood
    • 1
    • 2
  1. 1.Arkansas Biosciences InstituteArkansas State UniversityState UniversityUSA
  2. 2.Infinite Enzymes, LLCState UniversityUSA
  3. 3.Applied Biotechnology InstituteSan Luis ObispoUSA

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