33
linker peptide, relative to the parental linker peptide. Modification of cellulases so
as to have an increased net negative charge compared to the parental form has also
been shown to be effective in reducing affinity to lignin and noncellulosic materials,
with improved hydrolytic activity (Cascao-Pereira et al. 2013). Iogen Energy
Corporation patented a modified cellulase with lower affinity for lignin and
increased activity in its presence (Lavigne et al. 2010). The inventors discovered
that substitution of a basic or charge-neutral amino acid at position 129, 322, 363,
or 410 or of the amino acid at position 186 by a threonine results in a decrease in the
extent of deactivation of the modified T. reesei Cel6A cellulase by lignin relative to
that of a parental Cel6A cellulase from which it is derived. There is at least a 15%
reduction in the extent of deactivation by lignin relative to that of a parental form
and this decreased lignin inactivation contributes to increased activity for the hydrolysis of lignocellulose (Lavigne et al. 2010). Inhibition by glucose, the end product
of biomass breakdown, is a major problem in the enzymatic hydrolysis of lignocellulose, and glucose-tolerant enzymes (enzymes that can act at glucose concentrations that are otherwise inhibitory to normal enzymes) are highly desired. Another
patent application from Iogen described cellulase variants with reduced glucose
inhibition (Lavigne et al. 2009). In yet another recent report, Cao et al. (2015)
described engineering a highly glucose-tolerant beta glucosidase for thermostability
and proved its efficacy in enhancing hydrolysis of sugar cane bagasse.
One of the most obvious means of enzyme cost reduction is the reuse of enzymes.
After hydrolysis of lignocellulosic substrates, some amount of enzymes remains
free in solution, whereas some still remain bound to the unhydrolyzed substrate/
residues (Weiss et al. 2013). Ability to recover and reuse these can significantly
reduce the cost of biomass conversion, since enzymes are the most costly reagents
in biomass hydrolysis. There have been a large number of studies ranging from beta
glucosidase immobilization on magnetite (Dekker 1990) to recycling of the entire
unhydrolyzed residues (Visser et al. 2015). The major strategies in use for recycling
of enzyme used for biomass hydrolysis include (1) immobilization of the enzymes,
(2) ultrafiltration to recover free enzymes from hydrolysates, (3) readsorption of
free enzymes from hydrolysates using fresh substrates, and (4) reuse of bound
enzymes through recycling of residual substrates (Gomes et al. 2015; Visser et al.
2015). Immobilization as a recycling strategy can be applied effectively only to beta
glucosidases since the mode of action of endo- and exoglucanases constrains their
use in immobilized form. Apparently, most of the studies on immobilization of
enzymes for biomass hydrolysis have concentrated on beta glucosidases (Dekker
1990; Tu et al. 2006; Song et al. 2016). Recapturing of free cellulase from hydrolysates through use of ultrafiltration has been described by several authors (Qi et al.
2012; Chen et al. 2013). Free enzymes from hydrolysates can also be captured back
by readsorption onto fresh substrates. Here, fresh substrates can be added after completion of hydrolysis and a solid liquid separation step to recover the clear hydrolysates (Tu et al. 2007b; Shang et al. 2014). Alternatively, fresh substrates can be
added into an ongoing hydrolysis process so that the enzyme preferentially adsorbs
on the fresh substrates due to the higher cellulose content (Du et al. 2014; Huang
et al. 2016). In the case of cellulase adsorbed on to the residual solids, recycling of
the enzyme is effected by the recycling of part or all of the residues. The residues
1 Enzymes for Bioenergy
linker peptide, relative to the parental linker peptide. Modification of cellulases so
as to have an increased net negative charge compared to the parental form has also
been shown to be effective in reducing affinity to lignin and noncellulosic materials,
with improved hydrolytic activity (Cascao-Pereira et al. 2013). Iogen Energy
Corporation patented a modified cellulase with lower affinity for lignin and
increased activity in its presence (Lavigne et al. 2010). The inventors discovered
that substitution of a basic or charge-neutral amino acid at position 129, 322, 363,
or 410 or of the amino acid at position 186 by a threonine results in a decrease in the
extent of deactivation of the modified T. reesei Cel6A cellulase by lignin relative to
that of a parental Cel6A cellulase from which it is derived. There is at least a 15%
reduction in the extent of deactivation by lignin relative to that of a parental form
and this decreased lignin inactivation contributes to increased activity for the hydrolysis of lignocellulose (Lavigne et al. 2010). Inhibition by glucose, the end product
of biomass breakdown, is a major problem in the enzymatic hydrolysis of lignocellulose, and glucose-tolerant enzymes (enzymes that can act at glucose concentrations that are otherwise inhibitory to normal enzymes) are highly desired. Another
patent application from Iogen described cellulase variants with reduced glucose
inhibition (Lavigne et al. 2009). In yet another recent report, Cao et al. (2015)
described engineering a highly glucose-tolerant beta glucosidase for thermostability
and proved its efficacy in enhancing hydrolysis of sugar cane bagasse.
One of the most obvious means of enzyme cost reduction is the reuse of enzymes.
After hydrolysis of lignocellulosic substrates, some amount of enzymes remains
free in solution, whereas some still remain bound to the unhydrolyzed substrate/
residues (Weiss et al. 2013). Ability to recover and reuse these can significantly
reduce the cost of biomass conversion, since enzymes are the most costly reagents
in biomass hydrolysis. There have been a large number of studies ranging from beta
glucosidase immobilization on magnetite (Dekker 1990) to recycling of the entire
unhydrolyzed residues (Visser et al. 2015). The major strategies in use for recycling
of enzyme used for biomass hydrolysis include (1) immobilization of the enzymes,
(2) ultrafiltration to recover free enzymes from hydrolysates, (3) readsorption of
free enzymes from hydrolysates using fresh substrates, and (4) reuse of bound
enzymes through recycling of residual substrates (Gomes et al. 2015; Visser et al.
2015). Immobilization as a recycling strategy can be applied effectively only to beta
glucosidases since the mode of action of endo- and exoglucanases constrains their
use in immobilized form. Apparently, most of the studies on immobilization of
enzymes for biomass hydrolysis have concentrated on beta glucosidases (Dekker
1990; Tu et al. 2006; Song et al. 2016). Recapturing of free cellulase from hydrolysates through use of ultrafiltration has been described by several authors (Qi et al.
2012; Chen et al. 2013). Free enzymes from hydrolysates can also be captured back
by readsorption onto fresh substrates. Here, fresh substrates can be added after completion of hydrolysis and a solid liquid separation step to recover the clear hydrolysates (Tu et al. 2007b; Shang et al. 2014). Alternatively, fresh substrates can be
added into an ongoing hydrolysis process so that the enzyme preferentially adsorbs
on the fresh substrates due to the higher cellulose content (Du et al. 2014; Huang
et al. 2016). In the case of cellulase adsorbed on to the residual solids, recycling of
the enzyme is effected by the recycling of part or all of the residues. The residues
1 Enzymes for Bioenergy