Environmental Engineering Reference
In-Depth Information
2.3.2. Lipase engineering: activity
. Another important challenge faced by lipase use in
biodiesel production has been the diminished efficiency of lipases in transesterifications with
methanol and ethanol in comparison to longer-chain alcohols. Lipases typically accomplish
most rapid catalysis when the substrates are freely soluble in one another; methanol and
ethanol, however, were found to be only soluble in vegetable (soybean and rapeseed) oils at
molar ratios of 1:2 and 2:3 (alcohol : triglyceride fatty acid), respectively. Moreover,
insoluble methanol caused rapid inactivation of the lipases. These realizations led to the
development of a system in which alcohols were added in discrete doses, below their
solubility limits, to batch reaction mixtures, improving the reaction yield to near-completion
(>97 percent) as well as extending the lifetime of the lipases to greater than 100 days [1, 18].
Improving the tolerance of lipases to methanol or ethanol is also possible: certain species
of Fusarium, Pseudomonas, and Bacillus produce solvent-tolerant lipases [1], and directed
evolution has the potential to effect further desired changes in solvent tolerance, catalytic rate,
and substrate specificity in these or other enzymes [24], reviewed in [28]. Immobilization of
lipases within gels may extend lipase activity, as well; work of Noureddini and colleagues in
screening a number of extracellular lipases for methanoloysis and ethanolysis of soybean oil
revealed that several of the enzymes were more active, and retained their activities for longer
periods of time, when subjected to such immobilization [29], building on previous work
investigating lipase immobilization for transesterifiction [30, 31].
2.3.3. Whole-cell systems
. To diminish the time and energy requirements of enzyme
purification, even of a relatively simple purification such as that required with lipases, as well
as problems presented by instability of extracellular enzymes, significant effort has addressed
the direct use of whole cells as biocatalysts [32-34]. For example, lipases can be
overexpressed within biotechnologically tractable hosts in which they are not secreted,
followed by permeabilization of the host to allow catalysis to occur within the (compromised)
cytoplasm. Cytoplasmic overexpression of the Rhizopus oryzae lipase in Saccharomyces
cerevisiae, followed by freeze-thawing and air drying of the yeast cells, resulted in a whole-
cell biocatalyst that effectively catalyzed triglyceride methanolysis [35]; optimization of the
membrane fatty acid composition further improved lipase activity and stability [36].
In addition, cells producing their native lipases have been recruited. In work directed
toward whole-cell lipase optimization by Ban and colleagues, cells were immobilized using
porous biomass support particles (BSPs) made of polyurethane foam by introducing the BSPs
during batch cultivation and allowing the cells to colonize them spontaneously. Cells were
then cross-linked onto the support with glutaraldehyde, a procedure that greatly extended the
enzymatic activity longevity. Once immobilized in this form, the particles could be treated
much as conventional solid-phase catalysts: aseptic handling of the particles was unnecessary,
the particles could withstand mechanical shear, and they could be reused for up to 6 batches.
Mass transfer rates were also sufficiently rapid within the BSPs that conversion rates
approached those obtained with extracellular lipases: methanolysis of soybean oil by
immobilized Rhizopus oryzae cells, in the presence of 10-20 percent water, reached 80-90
percent without any organic solvent pretreatment [37, 1, 38]; additional improvements in
catalytic rate and durability were reported recently with this system in an air-lift reactor
configuration [39]. Further development of whole-cell biocatalysts is thus positioned to make
important contributions to biodiesel production.
