subtilisin are protease which come under serine like protease

1. Engineering Subtilisin Protease toward
Increased Oxidative Resistance

2. Engineering Subtilisin Protease toward Increased
Oxidative Resistance
Introduction
Oxygen-based bleaching agents are a major
component of powder detergent formulations as
active compounds for stain removal. Currently, the
source of active oxygen is hydrogen peroxide
produced at high local concentrations by spontaneous
decomposition of percarbonate and perborate
combined with tetraacetylethylenediamine.

3. chemical generation of hydrogen peroxide in
high local concentration can damage textile and
surfaces.
In addition, bleaching agents can inactivate
enzymes present in detergent formulations.
Therefore, gentle bleaching under mild pH
conditions is more attractive for cosmetics,
therapeutics, laundry, and disinfection
applications.

4. Mild bleaching can be achieved through in situ
production of low concentrations of peroxycarboxylic
acids (R–COOOH), which are strong oxidizing agents
with superior performance compared with hydrogen
peroxide . Thus, when mild bleaching conditions are
required, enzymatic in situ production by ester
hydrolysis in the presence of hydrogen peroxide is
preferred .
On the other hand, the enzymatic generation of
chemical oxidants is often limited by enzyme sensitivity
to oxidative agents. Amino acid residues that are most
prone to oxidation are methionine, cysteine, and
tryptophan.

5. Different enzyme classes were tested regarding resistance
against oxidative agents such as hydrogen peroxide.
Systematic studies proposed several mechanisms for
understanding reduced oxidative resistance of the
enzymes:
(i) sulfoxidation of Cys or Met residues in proximity to or
in the active site that can cause changes in the electronic
environment;
(ii) formation of tyrosine dimers ; and
(iii) reducing the side chain hydrophobicity that can
cause conformational changes in the protein structure.
The conformational changes reduce structural flexibility
and consequently the enzyme activity and can block the
access of the substrate through the substrate-binding
pocket .

6. As attractive enzymes for laundry applications,
subtilisins have been studied and reengineered toward
increased resistance against oxidative bleaching agents.
Subtilisin from B. amyloliquefaciens was stabilized by
substituting all methionine and cysteine residues with
alanine. The obtained subtilisin variant showed a 24-
fold increased half-life in 0.1 M hydrogen peroxide
compared with wild type .

7. A study by Stauffer and Etson showed that out of five
Met residues present in subtilisin Carlsberg, only the
one located in close proximity to the active site was
oxidized and decreased oxidative resistance. The
generation of peroxycarboxylic acids from the
corresponding esters in the presence of hydrogen
peroxide is an attractive side activity discovered in
subtilisin Carlsberg aside from its proteolytic activity.

8. Enzyme engineering to promote perhydrolysis by
subtilisin Carlsberg resulted in a variant containing the
substitutions Thr58Ala, Leu216Trp, and Met221 as in
wild type. This subtilisin Carlsberg variant showed
increased perhydrolytic (∼threefold) and decreased
proteolytic activity (∼eightfold)
Subtilisin Carlsberg variant (Thr58Ala, Leu216Trp, and
Met221) was further reengineered in order to increase the
production of peroxycarboxylic acids. Amino acid
substitutions at position Gly165 resulted in a variant with
increased catalytic constant for perhydrolysis of methyl-
propionate, methylbutyrate, and methylpentanoate up to
3.5-fold (Gly165Tyr), 5.4-fold (Gly165Leu), and 5.5-fold
(Gly165Leu and Gly165Ile), respectively showing that a
single amino acid exchange in the substrate-binding pocket
can promote perhydrolysis.

9. In parallel, the oxidative resistance of subtilisin
Carlsberg variant (Thr58Ala, Leu216Trp, and Met221 as
in wild type) against perhydrolysis products
(peroxycarboxylic acids) was engineered.
Amino acids Trp216 and Met221 were identified as key
residues governing peroxycarboxylic acid formation and
oxidative resistance.

10. Identified variants with increased oxidative resistance
harbor an amino acid exchange from Trp and Met to
Leu and Ser, which are less prone to oxidation.
Substitution of Met221 with Ser in variant E1 and with
Cys in variant E6 causes a dramatic increase in
oxidative resistance suggesting that Met221 is a key
residue for oxidative resistance in subtilisins. In
addition, it was observed that solvent accessibility of
the targeted amino acids could also play a role on
oxidative resistance. Solvent accessibility of Cys221 in
variant E6 was less than 10% and, therefore, it could be
protected and slowly oxidized.

11. On the other hand, variant E4 shows the highest
oxidative resistance (increased 2.6-fold compared with
subtilisin Carlsberg) despite having two Met residues
susceptible to oxidation (Met216 and Met221).
Met216 could act as a “suicide antioxidant” protecting
the key residue Met221.
A subtilisin-like alkaline protease from Bacillus KSM
shows naturally high resistance toward hydrogen
peroxide and harbors two Met residues, Met251 and
Met256. Structural overlay shows the structural identity
of Met216 and Met221 in variant E4 with Met251 and
Met256 in subtilisin-like alkaline protease with high
oxidation tolerance.

12. Thank you