2003 Progress Report: Engineering Design of Stable Immobilized Enzymes for the Hydrolysis and Transesterification of Triglycerides

EPA Grant Number: R829479C008
Subproject: this is subproject number 008 , established and managed by the Center Director under grant R829479
(EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).

Center: The Consortium for Plant Biotechnology Research, Inc., Environmental Research and Technology Transfer Program
Center Director: Schumacher, Dorin
Title: Engineering Design of Stable Immobilized Enzymes for the Hydrolysis and Transesterification of Triglycerides
Investigators: Noureddini, Hossein , Larsen, Gustavo
Institution: University of Nebraska at Lincoln
EPA Project Officer: Lasat, Mitch
Project Period: July 1, 2002 through June 30, 2004
Project Period Covered by this Report: July 1, 2002 through June 30, 2003
RFA: The Consortium for Plant Biotechnology Research, Inc., Environmental Research and Technology Transfer Program (2001) RFA Text |  Recipients Lists
Research Category: Targeted Research , Hazardous Waste/Remediation

Objective:

The objectives of this research project are as follows:

• Catalyst Improvement Strategies—to increase the specific surface area of the immobilized matrix within a specific range of macropore size.

• Materials Characterization—to provide feedback to the catalyst improvement studies regarding pore size distributions, specific surface areas, and enzyme distribution within the alcogel.

• Reactor Studies—to systematically feed back to the catalyst improvement studies on the reactivity, reusability, and stability of the immobilized enzyme.

Progress Summary:

Enzyme Immobilization

Our investigation of the immobilization of lipase PS from Pseudomonas cepacia by entrapment within a chemically inert hydrophobic sol-gel support is complete. The gel-entrapped lipase was prepared by the hydrolysis of tetramethoxysilane (TMOS) with methyltrimethoxysilane (MTMS), isobutyltrimethoxysilane (iso-BTMS), and n-butyltrimethoxysilane (n-BTMS). The immobilized lipase subsequently was used in the hydrolysis of soybean oil to determine its activity, recyclability, and thermostability. The biocatalyst thus prepared was equally effective or was better in its hydrolytic activity relative to the free enzyme. The catalytic activity of the entrapped lipase strongly depended on the type of precursor that was used in its preparation. The entrapped lipase within TMOS/iso-BTMS showed the highest activity. The catalytic activity of the immobilized lipase was more pronounced during the earlier stages of the reaction. Thermostability of the lipase significantly was improved in the immobilized form. The immobilized lipase was stable up to 70°C, whereas, for the free enzyme, moderate to severe loss of activity was observed beyond 40°C. The immobilized lipase was consistently more active and stable, than the free enzyme. The immobilized lipase also proved to be very stable, as it retained more than 95 percent of its initial activity after 12 1-hour reactions.

Enzyme Immobilization—Structure Modification

The structure modification work involved the use of glucose and vacuum. Glucose was introduced with the monomers into the immobilization process, while vacuum was applied during the sol-gel aging step. Lipase AY from Candida rugosa was used in these experiments. The immobilized lipase subsequently was used in the hydrolysis of soybean oil to determine its stability within the support structure, as well as its thermostability. To examine this, the immobilized enzyme was subjected to a period of preincubation at the reaction temperature prior to the experiments. The hydrolysis reaction then was carried out for 1 hour. Results showed that the modified, immobilized enzyme initially equaled in its hydrolytic activity relative to free enzyme and retained more than 95 percent of its activity after 120 hours of incubation at 40°C, whereas the free enzyme lost 67 percent of its activity after 24 hours of incubation and lost almost all of its activity after 96 hours of incubation at 40°C. Compared to our initial structure, the activity of the modified structure was higher by more than fourfold. At this point, our immobilization work for the hydrolysis reaction is complete. Immobilization studies now will concentrate on developing a practical, immobilized enzyme for the transesterification reactions.

Future Activities:

Enzyme Immobilization. Work will continue in the area of enzymatic transesterification reaction (biodiesel). Both methyl and ethyl esters will be used in this study. Unlike the chemical reaction where methanol has a clear advantage over ethanol, ethanol can be used as easily as methanol in the enzymatic reaction.

Sol/Gel Structure Modification. Work will concentrate on the effect of the vacuum procedure on pore size and distribution for the transesterification reaction. Additives such as glucose have been very effective in the hydrolysis reaction and will be explored further in the transesterification reaction.

Characterization. The developed material will be characterized for the pore size and distribution by electron microscopy and Brunauer-Emmett-Teller (BET) procedures. We also will use scanning electron microscopy and transition electron microscopy methods to investigate the distribution of the immobilized lipases within the supporting matrix.


Journal Articles on this Report : 1 Displayed | Download in RIS Format

Other subproject views: All 2 publications 2 publications in selected types All 2 journal articles
Other center views: All 211 publications 48 publications in selected types All 44 journal articles
Type Citation Sub Project Document Sources
Journal Article Noureddini H, Gao X, Philkana RS. Immobilized Pseudomonas cepacia lipase for biodiesel fuel production from soybean oil. Bioresource Technology 2005;96(7):769-777. R829479 (2006)
R829479C008 (2003)
R829479C008 (Final)
  • Abstract from PubMed
  • Full-text: ScienceDirect-PDF
    Exit
  • Abstract: ScienceDirect-Abstract
    Exit
  • Other: University of Nebraska-Lincoln - PDF
    Exit
  • Supplemental Keywords:

    entrapment, immobilization, lipase, sol-gel, hydrolysis, triglycerides, transesterification, soybean oil., Scientific Discipline, TREATMENT/CONTROL, Sustainable Industry/Business, Genetics, Technology, New/Innovative technologies, Environmental Engineering, agricultural oils, hydrolysis, reactor studies, transesterification, bioengineering, triglycerides, plant genes, catalytic studies, biotechnology, plant biotechnology, bioenergy

    Relevant Websites:

    http://www.cpbr.org Exit

    Progress and Final Reports:

    Original Abstract
  • Final Report

  • Main Center Abstract and Reports:

    R829479    The Consortium for Plant Biotechnology Research, Inc., Environmental Research and Technology Transfer Program

    Subprojects under this Center: (EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).
    R829479C001 Plant Genes and Agrobacterium T-DNA Integration
    R829479C002 Designing Promoters for Precision Targeting of Gene Expression
    R829479C003 aka R829479C011 Biological Effects of Epoxy Fatty Acids
    R829479C004 Negative Sense Viral Vectors for Improved Expression of Foreign Genes in Insects and Plants
    R829479C005 Development of Novel Plastics From Agricultural Oils
    R829479C006 Conversion of Paper Sludge to Ethanol
    R829479C007 Enhanced Production of Biodegradable Plastics in Plants
    R829479C008 Engineering Design of Stable Immobilized Enzymes for the Hydrolysis and Transesterification of Triglycerides
    R829479C009 Discovery and Evaluation of SNP Variation in Resistance-Gene Analogs and Other Candidate Genes in Cotton
    R829479C010 Woody Biomass Crops for Bioremediating Hydrocarbons and Metals
    R829479C011 Biological Effects of Epoxy Fatty Acids
    R829479C012 High Strength Degradable Plastics From Starch and Poly(lactic acid)
    R829479C013 Development of Herbicide-Tolerant Energy and Biomass Crops
    R829479C014 Identification of Receptors of Bacillus Thuringiensis Toxins in Midguts of the European Corn Borer
    R829479C015 Coordinated Expression of Multiple Anti-Pest Proteins
    R829479C016 A Novel Fermentation Process for Butyric Acid and Butanol Production from Plant Biomass
    R829479C017 Molecular Improvement of an Environmentally Friendly Turfgrass
    R829479C018 Woody Biomass Crops for Bioremediating Hydrocarbons and Metals. II.
    R829479C019 Transgenic Plants for Bioremediation of Atrazine and Related Herbicides
    R829479C020 Root Exudate Biostimulation for Polyaromatic Hydrocarbon Phytoremediation
    R829479C021 Phytoremediation of Heavy Metal Contamination by Metallohistins, a New Class of Plant Metal-Binding Proteins
    R829479C022 Development of Herbicide-Tolerant Energy and Biomass Crops
    R829479C023 A Novel Fermentation Process for Butyric Acid and Butanol Production from Plant Biomass
    R829479C024 Development of Vectors for the Stoichiometric Accumulation of Multiple Proteins in Transgenic Crops
    R829479C025 Chemical Induction of Disease Resistance in Trees
    R829479C026 Development of Herbicide-Tolerant Hardwoods
    R829479C027 Environmentally Superior Soybean Genome Development
    R829479C028 Development of Efficient Methods for the Genetic Transformation of Willow and Cottonwood for Increased Remediation of Pollutants
    R829479C029 Development of Tightly Regulated Ecdysone Receptor-Based Gene Switches for Use in Agriculture
    R829479C030 Engineered Plant Virus Proteins for Biotechnology