Tài liệu Báo cáo khoa học: The molecular surface of proteolytic enzymes has an important role in stability of the enzymatic activity in extraordinary environments pptx

The molecular surface of proteolytic enzymes has an important role
in stability of the enzymatic activity in extraordinary environments
Youhei Yamagata
1
, Hiroshi Maeda
1
, Tasuku Nakajima
1
and Eiji Ichishima
2
1
Laboratory of Molecular Enzymology, Division of Life Science, Graduate School of Agricultural Science, Tohoku University,
Aoba-ku, Sendai, Japan;
2
Department of Biotechnology, Faculty of Engineering, Soka University, Hachioji, Tokyo, Japan
It is scientifically and industrially important
1
to clarify the
stabilizing mechanism of proteases in extraordinary envi-
ronments. We used subtilisins ALP I and Sendai as models
to study the mechanism. Subtilisin ALP I is extremely
sensitive to highly alkaline conditions, even though the
enzyme is produced by alkalophilic Bacillus, whereas sub-
tilisin Sendai from alkalophilic Bacillus is stable under
conditions of high alkalinity. We constructed mutant
subtilisin ALP I enzymes by mutating the amino acid
residues specific for subtilisin ALP I to the residues at the
corresponding positions of amino acid sequence alignment
of alkaline subtilisin Sendai. We observed that the two
mutations in the C-terminal region were most effective for
improving stability against surfactants and heat as well as

high alkalinity. We predicted that the mutated residues are
located on the surface of the enzyme structures and, on
the basis of three-dimensional modelling, that they are
involved in stabilizing the conformation of the C-terminal
region. As proteolytic enzymes frequently become inactive
due to autocatalysis, stability of these enzymes in an
extraordinary environment would depend on the confor-
mational stability of the molecular surface concealing
scissile peptide bonds. It appeared that the stabilization of
the molecular surface structure was effective to improve the
stability of the proteolytic enzymes.
Keywords: alkalophilic alkaline resistance; Bacillus;mole-
cular surface structure; serine protease; subtilisin.
There have been several studies of the difference aspects
of proteolytic enzymes and they have been used in various
industrial fields. In particular, subtilisins, serine proteases
from a variety of Bacillus species,aresomeofthemost
investigated enzymes [1,2]. Subtilisins are classified into
three groups, the neutral subtilisins, the alkaline subtilisins
and Ôthe ALP I-typeÕ subtilisin (Fig. 1) [3]. The neutral
subtilisins consist of the subtilisins from neutrophilic
Bacillus such as subtilisin BPN¢ [4], Carlsberg [5], E [6],
and NAT [7]. The alkaline subtilisin group contains the
enzymes from alkalophilic Bacillus such as subtilisin YaB
[8], no. 221 protease [9], Savinase [10], subtilisin Sendai
(Sendai) [11]. Subtilisin ALP I (ALP I) from alkalophilic
Bacillus NKS-21 [3] is only member of the ALP I-type
subtilisins. ALP I is extremely sensitive to high alkaline
conditions, even though the enzyme is produced by an
alkalophilic Bacillus. On the other hand, Sendai from

alkalophilic Bacillus sp. G-825-6, categorized as an
alkaline subtilisin, is very stable under highly alkaline
conditions.
Maeda et al. reported that the inactivation of subtilisin
ALP I at high alkalinity was caused by the instability of
its molecular surface structure and autolysis in the
N-terminal region and/or the C-terminal region [12,13].
We hypothesized that the divergence of the properties of
ALP I from the alkaline subtilisins might depend on the
structure of the enzyme. In particular, the instability of
ALP I in highly alkaline conditions might be caused by
the existence of consensus amino acid sequences of
ALP I and the neutral subtilisins and/or the peculiar
residues in the amino acid sequence of ALP I. We
selected 12 consensus amino acid residues from the
amino acid sequence alignment of ALP I and the neutral
subtilisins. These candidate residues did not occur at the
corresponding positions of the alkaline subtilisins. Fur-
thermore, on the basis of the predicted three-dimensional
structure of ALP I, we believed that the C-terminal
region was located on the molecular surface and was
exposed to the solvent phase; therefore two unique
residues in the C-terminal region were replaced by the
residues at corresponding positions of amino acid
sequence of Sendai. As a result of analysing the mutant
ALP I s, two amino acid residues in the C-terminal
region were found to play important roles in maintaining
stability in highly alkaline conditions. The double muta-
tions prolonged the half-lifetime by more than 120-fold.
The substitutions of the amino acid residues also

improved the stability of the enzyme to detergents and
heat.
Correspondence to: Y. Yamagata, Laboratory of Molecular
Enzymology, Division of Life Science, Graduate School of
Agricultural Science, Tohoku University, 1-1, Tsutsumidori-
Amamiyamachi, Aoba-ku, Sendai, Japan, 981-8555.
Fax: + 81 22717 8778, Tel.: + 81 22717 8776,
E-mail:
Abbreviations: ALP I, subtilisin ALP I; Sendai, subtilisin Sendai;
Suc-Ala-Ala-Pro-Phe-MCA, succinyl-
L
-alanyl-
L
-alanyl-
L
-proryl-
L
-
phenylalanyl-4-methylcoumaryl-7-amide; DSC, differential scanning
calorimetry; LAS, sodium lauryl benzene sulfate.
Enzymes: Subtilisin ALP I (EC 3.4.21.64); subtilisin Sendai
(EC 3.4.21.64).
(Received 8 May 2002, revised 23 July 2002,
accepted 26 July 2002)
Eur. J. Biochem. 269, 4577–4585 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.03153.x
EXPERIMENTAL PROCEDURES
Bacterial strains and plasmids
Escherichia coli DH5a [F–, /80DlacZDM15, D(lacZYA-
argF) U169, deoR, recA1, endA1, hsdR17 (rk
–

mk
+
), phoA,
supE44, k-, thi-1, gyrA96, relA1] was used for cloning with
M13 derivatives mp18 and mp19. E. coli MV1184 [ara,
D(lac-proAB), rpsL, thi (F80 lacZDM15) D(srl-recA)306::
Tn10 (tet
r
)/F¢ (tra36, proAB
+
, lacI
q
, lacZDM15) [14], and
BMH71-18 mutS [D(lac-proAB), supE, thi, mutS215:: Tn10
(tet
r
)/F¢ (tra36, proAB
+
, lacI
q
, lacZDM15] was used for site-
directed mutagenesis. B. subtilis KN2 (phe-I, lys-I, nprR2,
nprE18, aprE3, ispA) [15] was used for protein expression.
Plasmids pUC119 and pUC118 [14] were used as the vectors
for construction of the mutant enzymes and for site-directed
mutagenesis. Plasmids pALP3 [3], pALP1 [11] and pTnat3
[7] were the recombinant plasmids containing intact ALP I
gene (aprQ), Sendai gene (aprS)andNATgene(aprN),
respectively. Plasmid pUB110 [16] was used for transfor-
mation of B. subtilis.

Expression of ALP I
Site-directed mutagenesis was carried out by the modified
method of Carter et al. [17]. Construction of the expression
plasmids is summarized in Fig. 2. A 27-mer synthetic
oligonucleotide, 5¢-CGCTCACATATGAAGGTTAAGC
AATCG-3¢, was used to introduce a unique NdeIsiteat
the initiation site of the ALP I gene, aprQ, and to change the
initiation codon from TTG to ATG. A 26-mer oligonu-
cleotide, 5¢-TTTGCTTCTCATATGTTACCCTCTCC-3¢,
was used to introduce a new NdeI site at the initiation site of
the NAT gene, aprN.TheNdeI–PstIfragmentofthe
mutated pALP3 + Nd was ligated with the mutated
plasmid, pTnat3 + Nd, cleaved with NdeIandPstIand
treated with calf alkaline phosphatase. The plasmid carrying
the fusion gene of the promoter region of aprN and the
10 20 30 ∆ 40 50 60 ∆ 70
PB92 1 AQSVPWGISRVQAPAAHNRGLTGSGVKVAVLDTGI-STHPDLNIRGGASFVPGEPST-QDGNGHGTHVAG
221 1 AQSVPWGISRVQAPAAHNRGLTGSGVKVAVLDTGI-STHPDLNIRGGASFVPGEPST-QDGNGHGTHVAG
SAVI 1 AQSVPWGISRVQAPAA-NRGLTGSGVKVAVLDTGI-STHTDLNIRGGASFVPVEPST-QDGNGHGTHVAG
SEND 1 NQVTPWGITRVQAPTAWTRGYTGTGVRVAVLDTGI-STHPDLNIRGGVSFVPGEPS-YQDGNGHGTHVAG
YAB 1 QTVPWGINRVQAPIAQSRGFTGTGVRVAVLDTGI-SNHADLRIRGGASFVPGEPN-ISDGNGHGTQVAG
* * * * * * * * * ** ** * ** **
ALP1 1 QTVPWGIPYIYSDVVHRQGYFGNGVKVAVLDTGV-APHPDLHIRGGVSFISTE-NTYVDYNGHGTHVAG
* * * * * * * * * ** ** * ** **
CARL 1 AQTVPYGIPLIKADKVQAQGFKGANVKVAVLDTGIQASHPDLNVVGGASFVAGQAYN-TDGNGHGTHVAG
DY 1 AQTVPYGIPLIKADKVQAQGFKGANVKVGIIDTGIAASHTDLKVVGGASFVSGESYN-TDGNGHGTHVAG
NAT 1 AQSVPYGISQIKAPALHSQGYTGSNVKVAVIDSGIDSSHPDLNVRGGASFVPSETNPYQDGSSHGTHVAG
E 1 AQSVPYGISQIKAPALHSQGYTGSNVKVAVIDSGIDSSHPDLNVRGGASFVPSETNPYQDGSSHGTHVAG
J 1 AQSVPYGISQIKAPALHSQGYTGSNVKVAVIDSGIDSSHPDLNVRGGASFVPSETNPYQDGSSHGPHVAG
AMYL 1 AQSVPYGISQIKAPALHSQGYTGSNVKVAVIDSGIDSSHPDLNVRGGASFVPSETNPYQDGSSHGTHVAQ

BPN' 1 AQSVPYGVSQIKAPALHSQGYTGSNVKVAVIDSGIDSSHPDLKVAGGASMVPSETNPFQDNNSHGTHVAG
MECE 1 AQSVPYGISQIKAPALHSQGYTQSNVKVAVIDSGIDSSHTDLQVRGGASFVPSETNPYQPGSSHGTHVAG
80 90 100 110 120 130 140
PB92 69 TIAALNNSIGVLGVAPNAELYAVKVLGASGSGSVSSIAQGLEWAGNNGMHVANLSLGSPSPSATLEQAVN
221 69 TIAALNNSIGVLGVAPSAELYAVKVLGASGSGSVSSIAQGLEWAGNNGMHVANLSLGSPSPSATLEQAVN
SAVI 68 TIAALNNSIGVLGVAPSAELYAVKVLGASGSGSVSSIAQGLEWAGNNGMHVANLSLGSPSPSATLEQAVN
SEND 69 TIAALNNSIGVVGVAPNAELYAVKVLGANGSGSVSSIAQGLQWTAQNNIHVANLSLGSPVGSQTLELAVN
YAB 68 TIAALNNSIGVLGVAPNVDLYGVKVLGASGSGSISGIAQGLQWAANNGMHIANMSLGSSAGSATMEQAVN
* *** * ***** * ** *** *** * * * * ***
ALP1 68 TVAALNNSYGVLGVAPGAELYAVKVLDRNGSGSHASIAQGIEWAMNNGMDIANMSLGSPSGSTTLQLAAD
* *** * ***** * ** *** *** * * * * ***
CARL 70 TVAALDNTTGVLGVAPSVSLYAVKVLNSSGSGTYSGIVSGIEWATTNGMDVINMSLGGPSGSTAMKQAVD
DY 70 TVAALDNTTGVLGVAPNVSLYAIKVLNSSGSGTYSAIVSGIEWATQNGLDVINMSLGGPSGSTALKQAVD
NAT 71 TIAALNNSIGVLGVAPSASLYAVKVLDSTGSGQYSWIINGIEWAISNNMDVINMSLGGPTGSTALKTVVD
E 71 TIAALNNSIGVLGVSPSASLYAVKVLDSTGSGQYSWIINGIEWAISNNMDVINMSLGGPTGSTALKTVVD
J 71 TIAALNNSIGVLGVSPSASLYAVKVLDSTGSGQYSWIINGIEWAISNNMDVINMSLGGPSGSTALKTVVD
AMYL 71 TIAALNNSIGVLGVSPSASLYAVKVLDSTGSGQYSWIINGIEWAISNNMDVINMSLGGPSGSTALKTVVD
BPN' 71 TVAALNNSIGVLGVAPSASLYAVKVLGADGSGQYSWIINGIEWAIANNMDVINMSLGGPSGAAALKAAVD
MECE 71 TIAALNNSIGVLGVAPSSALYAVKVLDSTGSGQYSWIINGIEWAISNNMDVINMSLGGPTGSTALKTVVD
150 160 170 180 190 200 210
PB92 139 SATSRGVLVVAASGNSGA-GSIS YPARYANAMAVGATDQNNNRASFSQYGAGLDIVAPGVNVQSTYP
221 139 SATSRGVLVVAASGNSGA-GSIS YPARYANAMAVGATDQNNNRASFSQYGAGLDIVAPGVNVQSTYP
SAVI 138 SATSRGVLVVAASGNSGA-GSIS YPARYANAMAVGATDQNNNRASFSQYGAGLDIVAPGVNVQSTYP
SEND 139 QATNAGVLVVAATGNNGS-G TVSYPARYANALAVGATDQNNNRASFSQYGTGLNIVAPGVGIQSTYP
YAB 138 QATASGVLVVAASGNSGA-G N-VGFPARYANAMAVGATDQNNNRATFSQYGAGLDIVAPGVGVQSTVP
* * * ** * * * * **** ** ** * * *** **
ALP1 138 RARNAGVLLIGAAGNSGQQGGSNNMGYPARYASVMAVGAVDQNGNRANFSSYGSELEIMAPGVNINSTYL
* * * ** * * * * **** ** ** * * *** **
CARL 140 NAYARGVVVVAAAGNSGSSGNTNTIGYPAKYDSVIAVGAVDSNSNRASFSSVGAELEVMAPGAGVYSTYP
DY 140 KAYASGIVVVAAAGNSGSSGSQNTIGYPAKYDSVIAVGAVDSNKNRASFSSVGAELEVMAPGVSVYSTYP

NAT 141 KAVSSGIVVAAAAGNEGSSGSTSTVGYPAKYPSTIAVGAVNSSNQRASFSSVGSELDVMAPGVSIQSTLP
E 141 KAVSSGIVVAAAAGNEGSSGSTSTVGYPAKYPSTIAVGAVNSSNQRASFSSAGSELDVMAPGVSIQSTLP
J 141 KAVSSGIVVAAAAGNEGSSGSSSTVGYPAKYPSTIAVGAVNSSNQRASFSSAGSELDVMAPGVSIQSTLP
AMYL 141 KAVSSGIVVAAAAGNEGSSGSSSTVGYPAKYPSTIAVGAVNSSVQRASFSSAGSELDVMAPGVSIQSTLP
BPN' 141 KAVASGVVVVAAAGNEGTSGSSSTVGYPGKYPSVIAVGAVDSSNQRASFSSVGPELDVMAPGVSIQSTLP
MECE 141 KAYSSGIVVAAAAGNEGSSGSTSTVGYPAKYPSTIAVGAVNSANQRASFSSAGSELDVMAPGVSIQSTLP
220∆ 230 240 250 260 270
PB92 205 GSTYASLNGTSMATPHVAGAAALVKQKNPSWSNVQIRNHLKNTATSLGSTNLYGSGLVNAEAATR
221 205 GSTYASLNGTSMATPHVAGAAALVKQKNPSWSNVQIRNHLKNTATSLGSTNLYGSGLVNAEAATR
SAVI 204 GSTYASLNGTSMATPHVAGAAALVKQKNPSWSNVQIRNHLKNTATSLGSTNLYGSGLVNAEAATR
SEND 205 GNRYASLSGTSMATPHVAGVAALVKQKNPSWSNTQIRQHLTSTATSLGNSNQFGSGLVNAEAATR
YAB 204 GNGYASFNGTSMATPHVAGVAALVKQKNPSWSNVQIRNHLKNTATNLGNTTQFGSGLVNAEAATR
* ***** **** *** * * * * * ** * ** *
ALP1 208 NNGYRSLNGTSMASPHVAGVAALVKQKHPHLTAAQIRNRMNQTAIPLGNSTYYGNGLVDAEYAAQ
* ***** **** *** * * * * * ** * ** *
CARL 210 TSTYATLNGTSMASPHVAGAAALILSKHPNLSASQVRNRLSSTATYLGSSFYYGKGLINVEAAAQ
DY 210 SNTYTSLNGTSMASPHVAGAAALILSKYPTLSASQVRNRISSTATNLGDSFYYGKGLINVEAAAQ
NAT 211 GGTYGAYNGTSMATPHVAGAAALILSKHPTWTNAQVRDRLESTATYLGNSFYYGKGLINVQAAAQ
E 211 GGTYGAYNGTSMATPHVAGAAALILSKHPTWTNAQVRDRLESTATYLGNSFYYGKGLINVQAAAQ
J 211 GGTYGAYNGTSMATTHVAGAAALILSKHPTWTNAQVRDRLESTATYLGNSFYYGKGLINVQAAAQ
AMYL 211 GGTYGAYNGTSMATPHVAGAAALILSKHPTWTNAQVRDRLESTATYLGNSFYYGKGLINVQAAAQ
BPN' 211 GNKYGAYNGTSMASPHVAGAAALILSKHPNWTNTQVRSSLENTTTKLGDSFYYGKGLINVQAAAQ
MECE 211 GGTYGAYNGTSMATPHVAGAAALILSKIPTWTNAQVRDRLESTATYLGSSFYYGKGLINVQAAAQ
Fig. 1. Alignments of the amino acid sequence
of the subtilisins. The amino acid sequence
enclosed in dark boxes and in open boxes are
common sequences among ALP I and the
neutral subtilisins, and among ALP I and the
alkaline subtilisins, respectively. s, Unique
amino acid sequence in the C-terminal region

of ALP I; *, consensus sequence in the sub-
tilisins; n, catalytic triad. ALP I, subtilisin
ALP I (accession number; BAA06158); PB92,
serine protease from B. alcalophilus PB92
(A49778); 221, no. 221 protease from alkalo-
philic Bacillus sp. no. 221 (S27501); SAVI,
Savinase
TM
(P29600); SEND, subtilisin Sen-
dai (BAA06157), YAB, alkaline esterase Ya-B
(P20724); CARL, subtilisin Carlsberg
(P00780); DY, subtilisin DY (P00781): NAT,
subtilisin NAT (JH0778); E, subtilisin E
(P04189); J, subtilisin J (P29142); AMYL,
subtilisin amylosacchariticus (P00783); BPN¢,
subtilisin BPN¢ (P00782); MECE, mecente-
ricopeptidase (P07518).
4578 Y. Yamagata et al. (Eur. J. Biochem. 269) Ó FEBS 2002
coding region of aprQ was designated pNALP3. Plasmid
pNALP3 digested with EcoRI was ligated with pUB110
digested with EcoRI. The shuttle vector carrying aprQ was
designated pNALP3B. It was used in the protoplast
transformation of B. subtilis KN2 [18]. Plasmid pALP1,
carrying the Sendai gene, aprS [11], was digested with EcoRI
and ligated with pUB110. The constructed plasmid was
named pSen6B. It was also introduced into B. subtilis KN2.
Construction of mutant enzymes
Oligonucleotides for introducing the mutation to the
enzymes are shown in Table 1. The oligonucleotides were
used to replace the amino acid residues of ALP I with the

amino acid residues at the corresponding position in Sendai
using the method described above.
DNA sequencing
To confirm the nucleotide sequences of the mutated genes,
DNA sequencing was carried out by using a BigDye
TM
Terminator Cycle Sequencing Kit and ABI PRISM
TM
377
DNA sequencer (Applied Biosystems).
Production and purification of recombinant subtilisins
B. subtilis KN2 carrying each recombinant plasmid was
grown aerobically at 37 °C in 1000 mL 2% beef extract, 2%
polypeptone, 0.2% casein, 0.7% NaCl (w/v) until the cells
entered the late log phase of growth. The culture broth was
centrifuged at 12 000 g for 30 min with a Hitachi
SCR20BA centrifuge. A crude enzyme solution was
dialysed against 10 m
M
Mes buffer pH 6.5 containing
2m
M
CaCl
2
. The solution was applied to a cation exchange
column of SP-TOYOPEARL 650M (3.8 · 25 cm) equili-
brated with the same buffer. The enzyme active fraction was
eluted with a 0–1.0
M
NaCl linear gradient and pooled. The

active fraction was dialysed with 10 m
M
Mes at pH 6.5
containing 2 m
M
CaCl
2
. The enzyme solution was loaded to
an FPLC-Hitrap SP (Amersham Pharmacia Biotech)
equilibrated with the same buffer. The enzyme active
fraction was eluted with a 0–0.5
M
NaCl linear gradient.
The purified enzymes were monitored by SDS/PAGE [19]
and immunoblot analysis [20]. Purified enzyme was also
blotted onto poly(vinylidene difluoride) (PVDF) membrane
[21]. Amino-terminal amino acid sequence analysis of each
enzyme blotted onto PVDF membrane was performed with
an ABI protein sequencer Model 491 (Applied Biosystems).
Assay of enzymatic activities
Protease activities towards milk casein were examined as
described in an according to a previous report [22]. Fluoro-
metric assays were conducted as described previously [23].
Protein was measured by Lowry’s method using BSA
fraction V (Seikagaku ko-gyo, Tokyo, Japan) as the
standard. The alkaline stability was measured at 30 °C
and pH 10.0 using succinyl-
L
-alanyl-
L

-alanyl-
L
-proryl-
L
-
phenylalanyl-4-methylcoumaryl-7-amide (Suc-Ala-Ala-
Pro-Phe-MCA) as a substrate after incubating the enzyme
for various length of time (2, 4, 6, 8, 10, 20, 30, 60, 120, 180,
240, 360 min) at pH 12. The resistance to surfactants was
measured after incubating the enzyme with 0.1% surfactant
at pH 10. Thermostability of the enzymes was measured
after incubating the enzyme for 10 min at a range of
temperature (30, 40, 50, 55, 57.5, 60, 62.5, 65, 70 °C).
Differential scanning calorimetry (DSC)
For determination of unfolding temperature (T
m
), calori-
metric measurements were carried out using a heater flux-
type SSC 560 U instrument (Seiko Instrument & Electronics
Ltd., Tokyo) [24].
Construction of the putative three-dimensional model
The putative three-dimensional structure and the putative
mutation models were constructed by the methods reported
previously [12].
RESULTS
Expression of ALP I
ALP I was not expressed by using the original promoter in
B. subtilis KN2 as a host, possibly because the promoter
sequence is not be suitable for the expression system in
B. subtilis KN2. Therefore the promoter region of subtilisin

NAT (NAT) from B. subtilis (natto) was used for expres-
sion. The open reading frame of the ALP I gene, aprQ,was
ligated of the downstream of the promoter region of the
pALP1
Amp
r
aprQ
PA
Pst
I
Eco
RI
pTnat3
Amp
r
aprN
PN
Pst
I
Nde
I
pALP1+Nd
Amp
r
aprQ
PA
Pst
I
Eco
RI

Nde
I
pTnat3+Nd
Amp
r
aprN
PN
Pst
I
Nde
I
pNALP3
Amp
r
aprQ
PN
Pst
I
Eco
RI
pUB110
Neo
r
Eco
RI
pNALP3B
aprQ
PN
A
mp

r
Neo
r
Eco
RI
Eco
RI
Nde
I
Pst
I
Introduction of a new
Nde
I site
Nde
I and
Pst
I
Eco
RI
Eco
RI
Nde
I and
Pst
I
Introduction of a new
Nde
I site
Fig. 2. Construction of the expression plasmid for ALP I. Thick arrows

indicate subtilisin genes. Dark grey and light grey thick lines show the
promoter region of aprQ and aprN, respectively.
Ó FEBS 2002
1
The molecular surface in enzymatic stability in extraordinary environments (Eur. J. Biochem. 269) 4579
NAT gene, aprN.TheE. coli–B. subtilis shuttle vector
containing the chimeric gene was named pNALP3B.
B. subtilis KN2 was transformed with pNALP3B. The
transformant expressed  20–30 mgÆL
)1
ALP I in culture
broth. The amount of the expressed enzyme was equal to
those of NAT and Sendai using the original promoters from
culture broth of the transformed B. subtilis KN2. The
expressed ALP I was purified up to a single band by SDS/
PAGE and confirmed with immunoblot analysis (data not
shown). The N-terminal amino acid sequence was identified
as that of native ALP I.
We attempted to express all of the mutant enzymes using
the same method, but Q18R-, I108L-, D137N-, A150T- and
S170N-ALP I were not expressed. The other mutant
enzymes were expressed in almost same quantities as the
wild-type enzymes. The expressed mutant enzymes were
purified, and all of the enzymes were confirmed by SDS/
PAGE, immunoblot analysis and N-terminal sequencing to
be derivatives of ALP I (data not shown).
Activities of the enzymes
The specific activities of ALP I, Sendai and the mutant
enzymes were measured with casein and Suc-Ala-Ala-Pro-
Phe-MCA as substrates. The values of the mutant enzymes

were consistent with those of the wild-type enzymes
(Table 2).
Stability under alkaline conditions
ALP I lost its enzymatic activity after only a few minutes’
incubation in 0.1
M
Na
2
HPO
4
/NaOH buffer pH 12
(Fig. 3A). After 2 min, ALP I showed only 27% of the
original activity, and after 10 min the enzyme showed
just 1% of its original activity. On the other hand, Sendai
was stable under these conditions and held 63% of the
original activity after 6 h at pH 12 (Fig. 3B). Two mutant
enzymes, D266N/Y269A- and D266N/Y269A/A271T/
Q272R-ALP I, were most stable retaining 60% of the
original activity after 1 h, and 30% after 6 h.
2
The D266N-
ALP I showed 40 and 20% of the original activity after
1 h and 6 h of incubation, respectively. The stability of
Y269A/A271T/Q272R- and Y269A-ALP I in alkaline
Table 2. Specific activities of the mutant subtilisins.
Enzyme
Specific activity (katÆkg
)1
)
a

Casein
Suc-Ala-Ala-
Pro-Phe-MCA
Wild-type ALP I 0.290 26.2
D117H 0.237 21.2
V177T 0.282 21.1
E192G 0.310 25.0
M196V 0.262 19.9
Y259Q 0.277 23.3
D266N 0.300 24.0
Y269A 0.265 29.9
Q272R 0.265 29.9
D266N/Y269A 0.360 21.9
A271T/Q272R 0.265 21.9
Y269A/A271T/Q272R 0.300 23.8
D266N/Y269A/A271T/Q272R 0.270 20.0
Wild-type Sendai 0.330 148
N263D 0.327 152
N263D/A266Y 0.319 147
a
Enzymatic activities were measured at 30 °C.
Table 1. Sequences of primers used for mutagensis. Small letters show substituted nucleotides. Primers with an attached Ôs-Õ were used for the
mutation of Sendai. Ô+NdÕ primers were used for the construction of expression plasmids. The other primers were used for each mutation of
ALP I. Restriction enzyme recognition sequences are shown in italics.
Primer Sequence Restriction enzyme
ALP + Nd
5¢-TTAACCTTCAtatgTGAGGGTATTTTTTG-3¢ NdeI
NAT + Nd 5¢-TTTGCTTCTCAtatgTTACCCTCTCC-6¢ NdeI
I7V 5¢-AATATAAGGGAcTCCCCAtGGAACAGTCTG-3¢ NcoI
Q18R 5¢-CCCAAAGTAAGGTcGACGgTGcACAACATCCGA-3¢ ApaLI

I108L 5¢-ATTCATCGCCCAcTCgAgTCCTTGAGCAAT-3¢ XhoI
D117H 5¢-GTTGGCAATATgCATCCCATTATT-3¢ EcoT221
D137N 5¢-CTAGCGCGGTtTGCTGCcAgcTGCAGGGTTGT-3¢ PvuII
A150T 5¢-TTGTCCTGAGTTaCCgGtCGCCCCAATTAA-3¢ AgeI
S170N 5¢-TCCAACAGCCATaACgttTGCATAGCGCHC-3¢ AclI
V177T 5¢-TCCATTTTGGTC CgtCGCTCCAACAGC-3¢
E192G
5¢-AATCTCAAGTcCgGATCCATAGCT-3¢ BamHI
M196V 5¢-TAATATTGACcCCgGGCGCCAcAATCTCAAG-3¢ SmaI
Y259Q 5¢-GCCATTTCCATAtTgaGTaCTGTTACCAAG-3¢ ScaI
D266N 5¢-CATACTCAGCgTtaACTAAGCCATTTC-3¢ HpaI
Y269A 5¢-TTGAGCCGCAgcCTCAGCgTCgAC TAAGCCATTTC-3¢ SalI
Q272R 5¢-CTTAGGGATTAacGAGCCGCATACTCAGCgTCgAC TAAGCCATTTC-3¢ HpaI
D266N/Y269A 5¢-ATTGAGCCGCAgcCTCAGCgTtaACTAAGCCATTTC-3¢ HpaI
A271T/Q272R 5¢-CTTAGGGATTAacGcGtCGCATACTCAG-3¢ MluI
Y269A/A271T/ Q272R 5¢-CTTAGGGATTAacGcGtCGCAgcCTCAGCATCC-3¢ MluI
D266N/Y269A/ 5¢-CTTAGGGATTAacGcGtCGCAgcCTCAGCATtCACTAAGCCA-3¢ MluI
A271T/Q272R s-N263D 5¢-TGCAGCTTCTGCgTcgACAAGTCCACTGCC-3¢ SalI
s-N263D/A266Y 5¢-AATATAAGCTTAaCgcGTTGCAtaTTCTGCgTcgAcAAGTCCACTGCC-3 ¢ MluI/SalI
4580 Y. Yamagata et al. (Eur. J. Biochem. 269) Ó FEBS 2002
conditions was also improved. We did not observe
improved stability under alkaline conditions in the other
mutant enzymes. They lost the activity within 10 min as did
wild-type ALP I.
Resistance to surfactants
Residual activities of the mutant enzymes were measured
after incubation with 0.1% SDS in 0.1
M
H
3

BO
3
/Na
2
CO
3
/
KCl buffer at pH 10.0. The results were different from those
obtained by treating the enzymes with high alkalinity.
Several mutant enzymes showed drastically improved
stability in solutions containing SDS (Fig. 4). ALP I
maintained 60% of enzymatic activity after 1 h, and 20%
of activity after 4 h whereas mutant enzymes D266N/
Y269A/A271T/Q272R-, Q272R/D266N/Y269A-, Y269A/
A271T/Q272R-, D266N- and Y269A-ALP I showed the
highest stability retaining > 90% of the original activities
after 4 h, and  60% after 24 h (data not shown). Mutant
enzyme Asp177His also showed improved resistance to the
surfactants, but the mutant enzyme lost about 90% of its
enzymatic activity during 12 h of incubation. The mutant
enzymes E192G- and M196V-ALP I showed almost the
same stability as the wild-type enzyme. The other mutations,
Val177Thr, Tyr259Gln, Gln272Arg and Ala271Thr/
Gln272Arg, make the enzyme sensitive to SDS. The same
results were obtained from the investigation using 0.1%
sodium lauryl benzene sulfate (LAS) instead of SDS (data
not shown).
Thermostability of the enzymatic activity
The residual activities of the mutant enzymes with improved
resistibility against alkalinity and surfactants were measured

after incubation for 10 min at pH 10.0 and at a variety of
temperatures (Fig. 5). The substitution of Asp266Asn and
Asp266Asn/Tyr269Ala improved the thermostability by
 10 °C, and Tyr269Ala substitution improved the thermo-
stability by  5 °C.
Protein denaturation by thermal treatment
Our investigation of enzymatic stability against alkalinity
and surfactants showed that the Asp266Asn and Tyr269Ala
6543210
0
20
40
60
80
100
120
Time (h
)
Time (h
)
A
6543210
0
20
40
60
80
100
120
B

Fig. 3. Alkaline resistance of mutant-ALP Is (A) and mutant-Sendai (B). The enzymes (0.1 mgÆmL
)1
) were incubated for each time at 30 °C, pH 12,
and then the residual activities were measured with Suc-Ala-Ala Pro-Phe MCA as a substrate at 30 °C and pH 10.0. In (A) the results of ALP I-
derived mutant enzymes are shown. s,ALP I;d, D266N-ALP I; n, Y269A-ALP I; m, D266N/Y269A-ALP I; h, Y269A/A271T/Q272R-ALP I;
j, D266N/Y269A/A271T/Q272R-ALP I. In (B) the stability of Sendai-derived mutant enzymes are shown. d, Sendai; m, N263-Sendai; j,
N263D/A266Y-Sendai.
1210864
Time (h)
20
0
20
40
60
80
100
120
Fig. 4. Stability of mutant-ALP Is against SDS. The enzymes
(0.1 mgÆmL
)1
) were incubated with 0.1% SDS solution for 10, 20, 30,
60, 120, 180, 240, 360, 480, and 720 min at pH 10.0, and then the
residual activities were measured with Suc-Ala-Ala Pro-Phe MCA as a
substrate at 30 °C and pH 10.0. s, wild-type ALP I; d, D266N-
ALP I; n, Y269A-ALP I; m, D266N/Y269A-ALP I; h, Y269A/
A271T/Q272R-ALP I; j, D266N/Y269A/A271T/Q272R-ALP I; e,
D117H-ALP I.
Ó FEBS 2002
1
The molecular surface in enzymatic stability in extraordinary environments (Eur. J. Biochem. 269) 4581

mutations were most effective (Fig. 6). Thermostability (T
m
:
mid point in the thermally induced transition from the
folded to the unfolded state) of wild-type ALP I, Sendai and
D266N/Y269A-ALP I were estimated by differential scan-
ning calorimetry (DSC). The T
m
of D266N/Y269A-ALP I
was 74.4 °C. It was higher than that of the wild-type ALP I,
70.2 °C, and almost the same as that of wild-type Sendai,
73.6 °C.
Stability of mutant Sendai in alkaline conditions
The substitutions of Asp266Asn and Tyr269Ala were most
effective in improving the stability of ALP I. To estimate the
effects of the corresponding amino acid residues in Sendai,
N266D- and N266D/Y269A-Sendai were constructed by
using primers s-N263D and s-N263D/A266Y (Table 1).
Compared with the stability of wild-type Sendai, both
mutant enzymes, N266D- and N266D/Y269A-Sendai
showed decreased stability under alkaline conditions
(Fig. 3B). Wild-type Sendai was stable at pH 12 and it
maintained 80% of the original activity after 6 h. The
activity of N263D-Sendai decreased to 45% and 10% of the
original after 2 and 6 h of incubation at pH 12, respectively.
N263D/A266Y-Sendai showed only 10% of the original
activity after 2 h, and little enzymatic activity was observed
after 4 h.
DISCUSSION
Based on our previous results we hypothesized that the

sensitivity of ALP I to high alkalinity depends on structural
divergence from the alkaline subtilisins and that the altered
residues causing the sensitivity of ALP I interacted with the
molecular surface region, or were located on the surface of
the molecule [12,13]. Twelve consensus amino acid residues
of ALP I and the neutral subtilisins and two specific
residues were selected as targets on the basis of amino acid
sequence alignment and predicted three-dimensional struc-
ture. Seventeen mutants of ALP I were constructed.
We have confirmed that the C-terminal region is very
important for enzymatic stability under conditions of high
alkalinity. In particular,
266
Asp is responsible for the
instability of ALP I at pH 12. The mutation Asp266Asn
caused only 40% of the original activity to be retained after
1 h. The mutation Tyr269Ala improved enzymatic stability
of D266N-ALP I cumulatively. The double mutant enzyme
showed 63% of the original activity after 1 h of incubation.
However, the single mutation Tyr269Ala showed only 1%
of the original activity after alkaline treatment for 1 h. The
other substituted residues did not improve the stability in
conditions of high alkalinity. The molecular surface region
of ALP I includes the peptide bonds that are digestible by
the other ALP Is [12]. The molecular surface structures of
ALP I are perturbed by alkaline, and the covered scissile
peptide bonds appear at the molecular surface and become
exposed to the solvent; the exposed peptide bonds are
digested by one another and the enzyme becomes inactive.
We hypothesize that substitutions of the amino acid residues

in ALP I restrain the conformational changes of the
molecular surface responsible for degradation.
The substitution of Asp266Asn, Tyr269Ala and
Asp266Asn/Tyr269Ala were also effective in increasing
resistance of ALP I to anionic surfactants. The unfolding
caused by surfactants occurred in a moderate manner in
comparison with denaturation by high alkalinity, and the
structural change of the molecular surface proceeded slowly.
In conditions of high alkalinity, a hydroxyl ion probably
Fig. 6. Thermostability of the enzyme structure. The denaturing
temperatures of the enzymes (3.3 nmol) were measured by DSC. The
arrowheads indicate the midpoints of the thermally induced phase
transitions.
7060504030
0
20
40
60
80
100
120
Tem p. ( C
)
Fig. 5. Thermostability of the mutant-ALP Is. The enzymes
(0.1 mgÆmL
)1
) were incubated for 10 min at 30, 40, 50, 55, 57, 60, 63,
65, 70 °C and pH 10.0, and then the residual activities were measured
with Suc-Ala-Ala-Pro-Phe-MCA as a substrate at 30 °C and pH 10.0.
s, wild-type ALP I; d, D266N-ALP I; n, Y269A-ALP I; m, D266N/

Y269A; h, wild-type Sendai.
4582 Y. Yamagata et al. (Eur. J. Biochem. 269) Ó FEBS 2002
penetrated from the molecular surface into the inside. Many
functional groups would then be deprotonated and it would
become difficult to maintain intra-molecular hydrogen and
ionic bond networks, causing rapid conformational changes
on the surface. On the other hand, anionic surfactants such
as SDS and LAS bind to the surface regions of protein, and
the surface regions would be unfolded and removed from
the core structure of the enzyme slowly. Then the scissile
bonds concealed by the surface regions would be exposed to
solvent and digested by one another. The mutation
Asp117His improved stability against SDS, but the sensi-
tivity of the mutant enzyme to alkalinity was not improved.
The mutation would essentially contribute to the improve-
ment of enzymatic stability, but it was not an indication of
the improvement in alkaline conditions, as the denaturation
by alkaline was very fast. As V177T-, Y259A-, Q272R- and
A271T/Q272R-ALP I were more sensitive to the surfactant
than ALP I, we conclude that the mutations decreased the
conformational stability of ALP I.
Thermostability of D266N-, D266N/Y269A and Y269A-
ALP I were also improved. In particular the inactivation
temperatures of D266N and D266N/Y269A-ALP I were
10 °C higher than that of wild-type ALP I. On the other
hand, the T
m
of D266N/Y269A-ALP I increased by only
4 °C. The denaturation temperature indicates the stability
of the structure of the protein. The difference of the

increments between the inactive temperature and the T
m
should indicate that the mutations improved the stability of
surface region. ALP I was not denatured at 55 °C, but the
enzymatic activity was lost. This indicates degradation of
the ALP I molecular begins as soon as the conformational
change occurs on the surface region. As improvement of
structural stability at the molecule surface would repress
autolysis, the inactivation temperature increases. However,
the effect of the mutation should not extent the whole
protein and so the T
m
did not increase likewise.
Stability of N263D- and N263D/A266Y-Sendai were
observed in alkaline conditions. The mutated residues of
Asn263 and Ala266 in Sendai correspond to Asp266 and
Ala269 in ALP I, respectively. The mutant Sendai became
sensitive to high alkalinity. The double-mutated Sendai was
additively more sensitive to alkalinity than N263D-Sendai.
The mutations at these positions in Sendai should promote
instability of the surface region. As the C region of Sendai
also would play an important role in restraining the
conformational change of the surface regions, wild-type
Sendai could be resistant to highly alkaline conditions.
The putative three-dimensional models of the enzymes
were constructed to clarify the location of substituted
residues and their interactions with surrounding residues.
The effective mutation sites of Asp266 and Tyr269 in the
C-terminal region were located on the back surface of a
catalytic triad, and it was understandable that the substi-

tutions did not influence the activities or specificity of the
mutant enzymes. The N-terminal region of ALP I was also
on the surface. The side chains of Ile10 and Tyr11 were close
to the residue Asp266 (Fig. 7A). The residues Thr250,
Ala251 and Tyr252 that lead to the C-terminal region were
located near the C-terminal region on the opposite side of
the N-terminal region on the molecular surface. In the wild-
type enzyme, the side chain of Asp266 interacted with the
amino group of the main chain at Glu268. The oxygen of
the main chain at Asp266 was bound to amino groups of
main chain of Tyr269 and Ala270 by hydrogen bonds. The
interactions should maintain the structure of the C-terminal
region. In the wild-type enzyme, no bonds were observed
between the N-terminal region and the C-terminal region on
the molecular surface, and the two regions would interact
with the core structure of ALP I independently. As the side
chainofAsn266wouldbeabletobindtothemainchainof
Ile10 with a hydrogen bond by substitution of Asp266Asn,
the mobility of the two regions on the surface would be
reduced by the interaction (Fig. 7B and D). We thought
that structural change of the molecular surface would be
unlikely to occur. The mutation of Tyr269 to Ala caused the
disappearance of a large aromatic polarized side chain
projecting to solvent, and the surface structure of the
C-terminal region would become highly dense (Fig. 7C and
D). The structure of the region would not be influenced by
environmental stress. These results indicate that the C-ter-
minal region and enforcement of the interaction between the
C- and N-terminal regions could be very important for the
stabilization of ALP I. These facts would be consistent with

a scenario in which the first cleavage site of ALP I occurs at
Glu18–Gly19 in the N-terminal region, and the next is
located in the C-terminal region [13]. The mutation
Asp117His contributed to the resistance to surfactants.
Aspartic acid at position 117 was located in the bottom of
the depression on the surface of ALP I, and it was adjacent
to Lys26, which was the last residue of N-terminal region on
the molecular surface. As a result of substitution of Asp117
to His, the side chain is larger. The mutation seemed to fill in
the gap between surrounding residues of the depression, and
the side chain of the mutated residue might restrain the
mobility of the N-terminal region on the surface by
interaction with the main chain of Lys26 by van der Waals’
forces (data not shown).
Altering core packing, helix stabilization, introduction of
surface salt bridges and reduction of flexibility in surface
loops are proposed mechanisms for the thermostability of
proteins [25–29]. The stability of ALP I under alkaline
conditions was caused by the stabilization of the surface
structure. Similar results are obtained from the structural
studies of shuffled p-nitrobenzyl esterases with improved
solvent stability and thermostability. The enzyme obtains a
17 °C increase in thermostability with 13 amino acid
residues replacements out of 484 residues with the eight
times reiterative random mutations [29]. Some of the
mutations decrease the conformational freedom. The
mutations fix disordered loops of esterase.
We selected the amino acid residues to mutate on the
basis of the predicted three-dimensional protein structure
and the alignment of amino acid sequences of the subtilisins.

Steipe et al. showed that the frequently occurring amino
acids at a given position in an amino acid sequence
alignment have a lager stabilizing effect than less frequently
occurring amino acids [30]. According to this concept,
Lehmann et al. presented a new semi-rational ÔconsensusÕ
approach for increasing the thermostability of proteins [31].
In the consensus phytase, four out of 32 replaced residues
increase thermostability and 10 decrease it. In our results,
replacement with the consensus amino acid residues of the
alkaline subtilisins did not improve the alkaline stability of
ALP I, but replacement by consensus amino acid residues
of all the subtilisins other than ALP I were effective. We
thought that the effectiveness of the approach would
Ó FEBS 2002
1
The molecular surface in enzymatic stability in extraordinary environments (Eur. J. Biochem. 269) 4583
depend on the population of the amino acid alignment
sequences of the proteins. To improve stability according to
this concept, it might not be enough to use information
from stable enzymes, but from many counterparts contain-
ing unstable ones.
3
ACKNOWLEDGEMENTS
We thank Prof Takeshi Uozumi of the Department of Biotechnology,
Tokyo University, for the permission to use B. subtilis KN2, and Prof
Koji Takahashi of the Department of Applied Biochemical Science,
Tokyo University of Agriculture and Technology, for the DSC
measurements.
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