Page 35 - Advances in Textile Biotechnology

14 Advances in textile biotechnology

optimum pH. They designed the mutations based on the sequence com-
parisons of family 7 cellulases with different pH behaviors. The comparison
studies revealed that a histidine residue adjacent to the acid/base catalyst
could account for the higher pH optimum of the Humicola insolens Cel7B
endoglucanase and therefore a mutation Ala224His was designed.
Modeling studies further suggested that four additional amino acid changes
(Glu223Ser/Leu225Val/ Thr226Ala/Asp262Gly) would be required in order

to fit the bulkier histidine side-chain. As the optimum pH of the Cel7A
mutant enzyme became more alkaline, however, the overall activity on both
soluble and insoluble substrates decreased (Becker et al., 2001). Wang et al.
(2005) demonstrated that a single substitution of Asn342 by Thr in T. reesei
endoglucanase Cel5A causes a pH optimum shift from 4.8 to 5.4. More
recently, the same enzyme was successfully engineered not only to a more
alkaline pH optimum but also to a higher catalytic effi ciency (Qin et al.,
2008). Residue 342 was subjected to saturation mutagenesis, and the enzyme
was further changed by random mutagenesis and two rounds of DNA shuf-

fling. A mutant form Asn342Val, with an optimal activity at pH 5.8, was
obtained. This corresponds to a basic shift of one pH unit compared with
the wild-type enzyme. The same mutant exhibited also improved catalytic

efficiency for the main substrates at pH 6.2. Other variants obtained, namely
Leu218His, Gln139Arg/Asn342Thr and Gln139Arg/Leu218His/Trp276Arg/
Asn342Thr, presented more than a 4.5-fold higher activity in reactions
compared with the wild-type at pH 7.0. The increase in activities and pH
optima of the variants was attributed to the existence of more stable helixes
and to the changed electrostatic interactions between the catalytic residues
and substrates (Qin et al., 2008).
Voutilainen et al. (2007) recently reported the successful expression of
the single-module cellobiohydrolase Cel7B from the thermophilic fungus
Melanocarpus albomyces (Ma Cel7B) in Saccharomyces cerevisiae (Sc
Cel7B) and used random mutagenesis to improve thermostability of the
enzyme. Three interesting mutants (Ala30Thr, Gly184Asp and Ser290Thr)
with increases in thermostability from 1–3 °C were obtained. Two different
strategies were used to improve the hydrolytic activity of this enzyme
towards crystalline cellulose at elevated temperatures: structure-guided
protein engineering was used to introduce an additional tenth disulfi de
bridge to the Ma Cel7B catalytic module, and a fusion protein was also
constructed by linking a cellulose-binding module (CBM) and a linker from
the T. reesei Cel7A to the C terminus of Ma Cel7B. Both approaches proved
to be successful (Voutilainen et al., 2009). Moreover, the disulfi de bridge
mutation Gly4Cys/Met70Cys was added to the most thermostable mutant
(Ser209Thr) created previously and the double mutant Gly4Cys/Met70Cys/
Ser209Thr appears to be a promising candidate for textile applications
requiring high temperatures (Voutilainen et al., 2009).

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