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Recovery of bovine lysozyme from transgenic sugarcane stalks: extraction, membrane filtration, and purification

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Abstract

Increased industrial use of sugarcane (Saccharum spp. hybrid) for food and bioenergy has led to considerable improvements in its genetic transformation, which allowed the development of not only pest- and herbicide-resistant lines but also lines expressing high-value bioproducts and biopolymers. However, the economic benefits of using inexpensive transgenic plant systems for the production of industrial proteins could be offset by high downstream processing costs. In this work, transgenic sugarcane expressing recombinant bovine lysozyme (BvLz) was used to evaluate the feasibility of extraction and fractionation of recombinant proteins expressed in sugarcane stalks. Three pH levels (4.5, 6.0 and 7.5) and three salt concentrations (0, 50, and 150 mM NaCl) were tested to determine BvLz and total protein extractability. Two extraction conditions were selected to prepare BvLz extracts for further processing by cross-flow filtration, a suitable method for concentration and conditioning of extracts for direct applications or prior to chromatography. Partial removal of native proteins was achieved using a 100 kDa membrane but 20–30 % of the extracted BvLz was lost. Concentration of clarified extracts using a 3 kDa membrane resulted in twofold purification and 65 % recovery of BvLz. Loading of concentrated sugarcane extract on hydrophobic interaction chromatography (HIC) resulted in 50 % BvLz purity and 69 % recovery of BvLz.

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Acknowledgments

The authors thank BioCane, Inc., a subsidiary of Growers Research Group, LLC. for the financial support.

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Author notes

  1. G. O. F. Barros

    Present address: Embrapa Agricultural Instrumentation, Rua XV de Novembro 1452, São Carlos, SP, 13560-970, Brazil

  2. M. A. T. Ballen

    Present address: Leibniz-Institute for Natural Product Research and Infection Biology, Hans-Knöll-Institute, Beutenbergstr. 11a, 07745, Jena, Germany

  3. S. L. Woodard

    Present address: Kalon Biotherapeutics, LLC, 100 Discovery Drive, Suite 200, College Station, TX, 77845, USA

  4. L. R. Wilken

    Present address: Biological and Agricultural Engineering, Kansas State University, 129 Seaton Hall, Manhattan, KS, 66506-2906, USA

Authors and Affiliations

  1. Department of Biological and Agricultural Engineering, Texas A&M University, 2117 TAMU, College Station, TX, 77843-2117, USA

    G. O. F. Barros, M. A. T. Ballen, S. L. Woodard, L. R. Wilken, S. G. White & Z. L. Nikolov

  2. Department of Plant Pathology and Microbiology, Texas A&M AgriLife Research and Extension Center, Weslaco, TX, 78596, USA

    M. B. Damaj & T. E. Mirkov

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  1. G. O. F. Barros
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  2. M. A. T. Ballen
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  8. Z. L. Nikolov
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Correspondence to Z. L. Nikolov.

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Barros, G.O.F., Ballen, M.A.T., Woodard, S.L. et al. Recovery of bovine lysozyme from transgenic sugarcane stalks: extraction, membrane filtration, and purification. Bioprocess Biosyst Eng 36, 1407–1416 (2013). https://doi.org/10.1007/s00449-012-0878-y

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  • DOI: https://doi.org/10.1007/s00449-012-0878-y

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