Page 37 - Advances in Textile Biotechnology

16 Advances in textile biotechnology

recently, for the treatment of wool. Most subtilisin protein engineering
continues to involve enhancement of catalytic activity and thermostability,

as well as, substrate specificity and oxidation resistance.

The first improvement of a detergent protease was performed in B. amylo-
liquefaciens subtilisin (subtilisin BPN′), to achieve oxidative stability. Met
in position 222, adjacent to the Ser221 in the enzyme active site was susbti-
tuted by non-oxidizable amino acids preserving the three-dimensional con-
figuration of the protein (Estell et al., 1985). The mutant form of the enzyme

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became a commercial detergent protease under the tradename Maxapem .
A similar variant with the substitution Met222Ala was later introduced in
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the market under the name Durazyme .
The industrial use of proteases in detergents or for leather processing
also requires an enzyme that is stable at higher temperatures. Several
approaches have been taken to increase the thermostability of detergent
proteases in order to achieve improved storage stability. Bryan et al. (1986)
reported the thermal stabilization of subtilisin BPN′ as a result of the single
substitution of Ser for Asn at position 218. The results of x-ray analysis
indicated that slight improvements in the hydrogen-bond parameters
enhanced the enzyme’s thermal stability. Potential salt bridges have been
introduced into subtilisin BPN′ by protein engineering to improve the
thermal stability (Erwin et al., 1990). Takagi et al. (1990) succeeded in sta-

bilizing subtilisin E by introduction of a disulfide bond between residues
Cys61 and Cys98. The two cysteines were introduced by protein engineering
based on structural comparison with a thermophilic serine protease. The
structure of subtilisin BPN′ was compared with that of subtilisin Carlsberg
and the presence of a leucine at position 217 in subtilisin Carlsberg was

identified as responsible for the high specific activity of this enzyme for

synthetic substrates (Wells et al., 1987). By replacing the tyrosine that was
at position 217 in subtilisin BPN′ by leucine, the resulting mutant enzyme

(BPN′ Tyr217Leu) exhibited a ten-fold increase in efficiency, compared with
the wild-type subtilisin BPN′, for the hydrolysis of synthetic substrates.
When tested for performance in neutral pH liquid detergents, the Tyr-
217Leu variant was found to be twice as efficient as subtilisin BPN′ and

also more stable (Wolff et al., 1996). Thus, a single amino acid change in

subtilisin BPN′ yielded an enzyme that is significantly better for laundry
applications than the parent enzyme. In a similar study, the structure of the
subtilisin produced by Bacillus lentus (subtilisin BL) was compared with
the structure of subtilisin BPN′ and it was found that both enzymes differ
in 103 out of 275 positions. One of the sequence differences between the
two enzymes is that subtilisin BL has valine at position 104 whereas sub-
tilisin BPN′ has tyrosine at this position. The amino acid at position 104 in
the sequence of subtilisin BPN′ was previously identified and reported as

important for performance of the enzyme and that a tyrosine was preferred

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