Page 38 - Advances in Textile Biotechnology

Design and engineering of novel enzymes for textile applications 17

over valine at this position (Estell and Wells, 1988). In separate mutagen-
esis experiments on the subtilisin BL enzyme, it was found that mutations
at positions 123 and 274 were also important for performance of this enzyme
(Caldwell et al., 1991). A variant of subtilisin BL was therefore constructed
in which tyrosine replaced valine at position 104, serine replaced asparagine
at position 123, and alanine replaced threonine at position 274. The result-

ing enzyme was more than twice as efficient as the parent subtilisin BL in
the cleaning of enzyme sensitive stains in a granular alkaline detergent
matrix (Caldwell et al., 1991).
SDM was also used to introduce a Asn218Ser mutation that increased
the thermostability of the enzyme (Wang et al., 1993). SDM was also used
by Yang et al. (2000a) to generate a Ser236Cys mutant subtilisin E with a
half-life, at 60 °C, four-fold longer than that of native subtilisin E. Using this
mutant, thermostability could also be increased, by forming a disulfi de
bridge between two molecules of Ser236Cys subtilisin E. Yang et al. (2000b)
also used random mutagenesis PCR technique to develop a thermally stable
and oxidation-resistant mutant. The new Met222Ala/Asn118Ser subtilisin

E was five-fold more thermally stable than native enzyme. In another
report, the thermal stability of subtilisin E was increased using directed
evolution to convert B. subtilis subtilisin E into an enzyme functionally
equivalent to its thermophilic homolog thermitase from Thermoactinomy-
ces vulgaris (Zhao and Arnold, 1999).
Mainly for ecological reasons, proteases of the subtilisin type are also
being studied as an alternative for chemical pre-treatment of wool. However,
the increase in subtilisin molecular weight is crucial for its successful appli-

cation in wool finishing because, owing to its small size, the enzyme is able
to penetrate into the fi bre cortex causing the destruction of the inner parts
of wool structure (Shen et al., 1999). Araújo et al., (2008a) attempted to
create recombinant subtilisin E with high molecular weights using two
novel approaches: the construction of two polysubtilisins, (fusing two and
four subtilisin E genes in frame), and the formation of a subtilisin trimer
by fusion of native prosubtilisin E gene with the gene coding for SP-D
neckdomain. Both approaches resulted in the expression of three modifi ed
subtilisins although no activity was recovered for these enzymes. They also
reported a different approach to increase the subtilisin E molecular weight
based on the fusion of the DNA sequences coding for B. subtilis proSub-
tilisin E and for an elastin-like polymer (ELP) (Araújo et al., 2009). The
ELP gene used is based on 220 repetitions of the monomeric sequence Val
Pro Ala Val Gly (VPAVG), and it was constructed in the same laboratory.
The recombinant protein, exhibiting a molecular weight above 116 kDa (an
increase of more than four-fold in the weight of the native enzyme), was

biologically produced in E. coli, purified and used for wool-fi nishing assays.
Both yarns and fabrics treated with genetically engineered enzyme

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