Subtilisin ALPI is extremely sensitive to highly alkaline conditions, even though the enzyme is produced by alkalophilic Bacillus, whereas sub-tilisin Sendai from alkalophilic Bacillus i
Trang 1The molecular surface of proteolytic enzymes has an important role
in stability of the enzymatic activity in extraordinary environments
Youhei Yamagata1, Hiroshi Maeda1, Tasuku Nakajima1and Eiji Ichishima2
1
Laboratory of Molecular Enzymology, Division of Life Science, Graduate School of Agricultural Science, Tohoku University, Aoba-ku, Sendai, Japan;2Department of Biotechnology, Faculty of Engineering, Soka University, Hachioji, Tokyo, Japan
It is scientifically and industrially important
stabilizing mechanism of proteases in extraordinary
envi-ronments We used subtilisins ALPI and Sendai as models
to study the mechanism Subtilisin ALPI 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 ALPI enzymes by mutating the amino acid
residues specific for subtilisin ALPI 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, are some of the most
investigated enzymes [1,2] Subtilisins are classified into
three groups, the neutral subtilisins, the alkaline subtilisins
and the ALPI-type 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 ALPI (ALPI) from alkalophilic
Bacillus NKS-21 [3] is only member of the ALPI-type
subtilisins ALPI 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 ALPI 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 ALPI from the alkaline subtilisins might depend on the structure of the enzyme In particular, the instability of ALPI in highly alkaline conditions might be caused by the existence of consensus amino acid sequences of ALPI and the neutral subtilisins and/or the peculiar residues in the amino acid sequence of ALPI We selected 12 consensus amino acid residues from the amino acid sequence alignment of ALPI 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 ALPI, 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 ALPI 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: yamagata@biochem.tohoku.ac.jp
Abbreviations: ALPI, subtilisin ALPI; 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 ALPI (EC 3.4.21.64); subtilisin Sendai
(EC 3.4.21.64).
(Received 8 May 2002, revised 23 July 2002,
accepted 26 July 2002)
Trang 2E X P E R I M E N T A L P R O C E D U R E S
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 (tetr)/F¢ (tra36, proAB+, lacIq, lacZDM15) [14], and
BMH71-18 mutS [D(lac-proAB), supE, thi, mutS215:: Tn10
(tetr)/F¢ (tra36, proAB+, lacIq, 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 ALPI
gene (aprQ), Sendai gene (aprS) and NAT gene (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 NdeI site at the initiation site of the ALPI 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 The NdeI–PstI fragment of the mutated pALP3 + Nd was ligated with the mutated plasmid, pTnat3 + Nd, cleaved with NdeI and PstI and treated with calf alkaline phosphatase The plasmid carrying the fusion gene of the promoter region of aprN and the
PB92 1 AQSVPWGISRVQAPAAHNRGLTGSGVKVAVLDTGI-STHPDLNIRGGASFVPGEPST-QDGNGHGTHVAG
SAVI 1 AQSVPWGISRVQAPAA-NRGLTGSGVKVAVLDTGI-STHTDLNIRGGASFVPVEPST-QDGNGHGTHVAG
YAB 1 QTVPWGINRVQAPIAQSRGFTGTGVRVAVLDTGI-SNHADLRIRGGASFVPGEPN-ISDGNGHGTQVAG
* * * * * * * * * ** ** * ** **
ALP1 1 QTVPWGIPYIYSDVVHRQGYFGNGVKVAVLDTGV-APHPDLHIRGGVSFISTE-NTYVDYNGHGTHVAG
* * * * * * * * * ** ** * ** **
CARL 1 AQTVPYGIPLIKADKVQAQGFKGANVKVAVLDTGIQASHPDLNVVGGASFVAGQAYN-TDGNGHGTHVAG
DY 1 AQTVPYGIPLIKADKVQAQGFKGANVKVGIIDTGIAASHTDLKVVGGASFVSGESYN-TDGNGHGTHVAG
E 1 AQSVPYGISQIKAPALHSQGYTGSNVKVAVIDSGIDSSHPDLNVRGGASFVPSETNPYQDGSSHGTHVAG
AMYL 1 AQSVPYGISQIKAPALHSQGYTGSNVKVAVIDSGIDSSHPDLNVRGGASFVPSETNPYQDGSSHGTHVAQ
MECE 1 AQSVPYGISQIKAPALHSQGYTQSNVKVAVIDSGIDSSHTDLQVRGGASFVPSETNPYQPGSSHGTHVAG
80 90 100 110 120 130 140
221 69 TIAALNNSIGVLGVAPSAELYAVKVLGASGSGSVSSIAQGLEWAGNNGMHVANLSLGSPSPSATLEQAVN
SEND 69 TIAALNNSIGVVGVAPNAELYAVKVLGANGSGSVSSIAQGLQWTAQNNIHVANLSLGSPVGSQTLELAVN
* *** * ***** * ** *** *** * * * * ***
ALP1 68 TVAALNNSYGVLGVAPGAELYAVKVLDRNGSGSHASIAQGIEWAMNNGMDIANMSLGSPSGSTTLQLAAD
* *** * ***** * ** *** *** * * * * ***
CARL 70 TVAALDNTTGVLGVAPSVSLYAVKVLNSSGSGTYSGIVSGIEWATTNGMDVINMSLGGPSGSTAMKQAVD
NAT 71 TIAALNNSIGVLGVAPSASLYAVKVLDSTGSGQYSWIINGIEWAISNNMDVINMSLGGPTGSTALKTVVD
J 71 TIAALNNSIGVLGVSPSASLYAVKVLDSTGSGQYSWIINGIEWAISNNMDVINMSLGGPSGSTALKTVVD
BPN' 71 TVAALNNSIGVLGVAPSASLYAVKVLGADGSGQYSWIINGIEWAIANNMDVINMSLGGPSGAAALKAAVD
150 160 170 180 190 200 210
221 139 SATSRGVLVVAASGNSGA-GSIS -YPARYANAMAVGATDQNNNRASFSQYGAGLDIVAPGVNVQSTYP
SEND 139 QATNAGVLVVAATGNNGS-G -TVSYPARYANALAVGATDQNNNRASFSQYGTGLNIVAPGVGIQSTYP
* * * ** * * * * **** ** ** * * *** **
ALP1 138 RARNAGVLLIGAAGNSGQQGGSNNMGYPARYASVMAVGAVDQNGNRANFSSYGSELEIMAPGVNINSTYL
* * * ** * * * * **** ** ** * * *** **
CARL 140 NAYARGVVVVAAAGNSGSSGNTNTIGYPAKYDSVIAVGAVDSNSNRASFSSVGAELEVMAPGAGVYSTYP
NAT 141 KAVSSGIVVAAAAGNEGSSGSTSTVGYPAKYPSTIAVGAVNSSNQRASFSSVGSELDVMAPGVSIQSTLP
J 141 KAVSSGIVVAAAAGNEGSSGSSSTVGYPAKYPSTIAVGAVNSSNQRASFSSAGSELDVMAPGVSIQSTLP
BPN' 141 KAVASGVVVVAAAGNEGTSGSSSTVGYPGKYPSVIAVGAVDSSNQRASFSSVGPELDVMAPGVSIQSTLP
PB92 205 GSTYASLNGTSMATPHVAGAAALVKQKNPSWSNVQIRNHLKNTATSLGSTNLYGSGLVNAEAATR
SAVI 204 GSTYASLNGTSMATPHVAGAAALVKQKNPSWSNVQIRNHLKNTATSLGSTNLYGSGLVNAEAATR
YAB 204 GNGYASFNGTSMATPHVAGVAALVKQKNPSWSNVQIRNHLKNTATNLGNTTQFGSGLVNAEAATR
* ***** **** *** * * * * * ** * ** *
ALP1 208 NNGYRSLNGTSMASPHVAGVAALVKQKHPHLTAAQIRNRMNQTAIPLGNSTYYGNGLVDAEYAAQ
* ***** **** *** * * * * * ** * ** *
CARL 210 TSTYATLNGTSMASPHVAGAAALILSKHPNLSASQVRNRLSSTATYLGSSFYYGKGLINVEAAAQ
NAT 211 GGTYGAYNGTSMATPHVAGAAALILSKHPTWTNAQVRDRLESTATYLGNSFYYGKGLINVQAAAQ
J 211 GGTYGAYNGTSMATTHVAGAAALILSKHPTWTNAQVRDRLESTATYLGNSFYYGKGLINVQAAAQ
BPN' 211 GNKYGAYNGTSMASPHVAGAAALILSKHPNWTNTQVRSSLENTTTKLGDSFYYGKGLINVQAAAQ
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 ALPI and the neutral subtilisins, and among ALPI and the alkaline subtilisins, respectively s, Unique amino acid sequence in the C-terminal region
of ALPI; *, consensus sequence in the sub-tilisins; n, catalytic triad ALPI, subtilisin ALPI (accession number; BAA06158); PB92, serine protease from B alcalophilus PB92 (A49778); 221, no 221 protease from alkalo-philic Bacillus sp no 221 (S27501); SAVI, SavinaseTM(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).
Trang 3coding 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 ALPI 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 BigDyeTM
Terminator Cycle Sequencing Kit and ABI PRISMTM377
DNA sequencer (Applied Biosystems)
Production and purification of recombinant subtilisins
B subtilis KN2 carrying each recombinant plasmid was
grown aerobically at 37C 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 mM Mes buffer pH 6.5 containing
2 mMCaCl2 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 0–1.0MNaCl linear gradient and pooled The active fraction was dialysed with 10 mM Mes at pH 6.5 containing 2 mMCaCl2 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 0–0.5M 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 30C 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, 70C) Differential scanning calorimetry (DSC)
For determination of unfolding temperature (Tm), 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]
R E S U L T S
Expression of ALP I ALPI 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 subtilisKN2 Therefore the promoter region of subtilisin NAT (NAT) from B subtilis (natto) was used for expres-sion The open reading frame of the ALPI gene, aprQ, was ligated of the downstream of the promoter region of the
pALP1
Ampr
aprQ P A
Pst I EcoRI pTnat3
Ampr
aprN P N PstI
Nde I pALP1+Nd
Ampr
aprQ P A PstI
EcoRI
Nde I pTnat3+Nd Ampr
aprN P N PstI
Nde I pNALP3 Ampr
aprQ P N Pst I EcoRI
pUB110 Neor
EcoRI
pNALP3B aprQ
P N
Ampr
Neor
EcoRI
EcoRI
Nde I Pst I
Introduction of a new NdeI site
NdeI and Pst I
EcoRI
EcoRI NdeI and Pst I Introduction of a new NdeI 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.
Trang 4NAT gene, aprN The E 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)1ALPI 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 ALPI 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 ALPI
We attempted to express all of the mutant enzymes using
the same method, but Q18R-, I108L-, D137N-, A150T- and
S170N-ALPI 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 ALPI (data not shown)
Activities of the enzymes
The specific activities of ALPI, 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
ALPI lost its enzymatic activity after only a few minutes’
incubation in 0.1M Na2HPO4/NaOH buffer pH 12
(Fig 3A) After 2 min, ALPI 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-ALPI, were most stable retaining 60% of the original activity after 1 h, and 30% after 6 h
D266N-ALPI 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-ALPI in alkaline
Table 2 Specific activities of the mutant subtilisins.
Enzyme
Specific activity (katÆkg)1) a
Casein
Suc-Ala-Ala-Pro-Phe-MCA
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 s- were used for the mutation of Sendai + Nd primers were used for the construction of expression plasmids The other primers were used for each mutation of ALPI Restriction enzyme recognition sequences are shown in italics.
Trang 5conditions 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 ALPI
Resistance to surfactants
Residual activities of the mutant enzymes were measured
after incubation with 0.1% SDS in 0.1MH3BO3/Na2CO3/
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) ALPI
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-ALPI 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-ALPI 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
6 5 4 3 2 1 0 0 20 40 60 80 100 120
A
6 5 4 3 2 1 0 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 ALPI-derived mutant enzymes are shown s, ALP I; d, D266N-ALPI; n, Y269A-ALPI; m, D266N/Y269A-ALPI; h, Y269A/A271T/Q272R-ALPI;
j, D266N/Y269A/A271T/Q272R-ALPI In (B) the stability of Sendai-derived mutant enzymes are shown d, Sendai; m, N263-Sendai; j, N263D/A266Y-Sendai.
12 10 8 6 4
Time (h)
2 0 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 ALPI; d, D266N-ALPI; n, Y269A-D266N-ALPI; m, D266N/Y269A-D266N-ALPI; h, Y269A/ A271T/Q272R-ALPI; j, D266N/Y269A/A271T/Q272R-ALPI; e, D117H-ALPI.
Trang 6mutations were most effective (Fig 6) Thermostability (Tm:
mid point in the thermally induced transition from the
folded to the unfolded state) of wild-type ALPI, Sendai and
D266N/Y269A-ALPI were estimated by differential
scan-ning calorimetry (DSC) The Tmof D266N/Y269A-ALPI
was 74.4C It was higher than that of the wild-type ALPI,
70.2C, and almost the same as that of wild-type Sendai,
73.6C
Stability of mutant Sendai in alkaline conditions
The substitutions of Asp266Asn and Tyr269Ala were most
effective in improving the stability of ALPI 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
D I S C U S S I O N
Based on our previous results we hypothesized that the
sensitivity of ALPI to high alkalinity depends on structural
divergence from the alkaline subtilisins and that the altered
residues causing the sensitivity of ALPI interacted with the
molecular surface region, or were located on the surface of
the molecule [12,13] Twelve consensus amino acid residues
of ALPI 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 ALPI were constructed
We have confirmed that the C-terminal region is very important for enzymatic stability under conditions of high alkalinity In particular, 266Asp is responsible for the instability of ALPI 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-ALPI 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 ALPI includes the peptide bonds that are digestible by the other ALPIs [12] The molecular surface structures of ALPI 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 ALPI 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 ALPI 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.
70 60
50 40
30
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 ALPI; d, D266N-ALPI; n, Y269A-ALPI; m, D266N/
Y269A; h, wild-type Sendai.
Trang 7penetrated 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-ALPI were more sensitive to the surfactant
than ALPI, we conclude that the mutations decreased the
conformational stability of ALPI
Thermostability of D266N-, D266N/Y269A and
Y269A-ALPI were also improved In particular the inactivation
temperatures of D266N and D266N/Y269A-ALPI were
10C higher than that of wild-type ALPI On the other
hand, the Tmof D266N/Y269A-ALPI increased by only
4C The denaturation temperature indicates the stability
of the structure of the protein The difference of the
increments between the inactive temperature and the Tm
should indicate that the mutations improved the stability of
surface region ALPI was not denatured at 55C, but the
enzymatic activity was lost This indicates degradation of
the ALPI 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 Tmdid 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 ALPI, 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 ALPI 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 ALPI independently As the side chain of Asn266 would be able to bind to the main chain of 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 ALPI These facts would be consistent with
a scenario in which the first cleavage site of ALPI 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 ALPI, 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 ALPI 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
17C 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 consensus 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 ALPI, but replacement by consensus amino acid residues
of all the subtilisins other than ALPI were effective We thought that the effectiveness of the approach would
Trang 8depend 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
A C K N O W L E D G E M E N T S
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.
R E F E R E N C E S
1 Ottesen, M & Svendsen, I (1970) The subtilisins In Methods in
Enzymology 19 (Perlmann, G.E & Lorand, L., eds), pp 199–215.
Academic press, New York, London.
2 Bryan, P.N (2000) Protein engineering of subtilisin Biochim.
Biophys Acta 1543, 203–222.
3 Yamagata, Y., Sato, T., Hanzawa, S & Ichishima, E (1995) The
structure of subtilisin ALPI from alkalophilic Bacillus sp
NKS-21 Curr Microbiol 30, 201–209.
4 Wells, J.A., Ferrari, E., Henner, D.J., Estell, D.A & Chen, E.Y.
(1983) Cloning, sequencing, and secretion of Bacillus
amylolique-faciens subtilisin in Bacillus subtilis Nucleic Acids Res 11, 7911– 7925.
5 Jacobs, M., Eliasson, M., Uhlen, M & Flock, J.I (1985) Cloning, sequencing and expression of subtilisin Carlsberg from Bacillus licheniformis Nucleic Acids Res 13, 8913–8926.
6 Stahl, M.L & Ferrari, E (1984) Replacement of the Bacillus subtilis subtilisin structural gene with an in vitro-derived deletion mutation J Bacteriol 158, 411–418.
7 Nakamura, T., Yamagata, Y & Ichishima, E (1992) Nucleotide sequence of the subtilisin NAT gene, aprN, of Bacillus subtilis (natto) Biosci Biotechnol Biochem 56, 1869–1871.
8 Kaneko, R., Koyama, N., Tsai, Y.C., Juang, R.Y., Yoda, K & Yamasaki, M (1989) Molecular cloning of the structural gene for alkaline elastase YaB, a new subtilisin produced by an alkalophilic Bacillus strain J Bacteriol 171, 5232–5236.
9 Takami, H., Kobayashi, T., Kobayashi, M., Yamamoto, M., Nakamura, S., Aono, R & Horikoshi, K (1992) Molecular cloning, nucleotide sequence, and expression of the structural gene for alkaline serine protease from alkaliphilic Bacillus sp 221 Biosci Biotechnol Biochem 56, 1455–1460.
10 Betzel, C., Klupsch, S., Papendorf, G., Hastrup, S., Branner, S & Wilson, K.S (1992) Crystal structure of the alkaline proteinase Savinase from Bacillus lentus at 1.4 A˚ resolution J Mol Biol 223, 427–445.
11 Yamagata, Y., Isshiki, K & Ichishima, E (1995) Subtilisin Sendai from alkalophilic Bacillus sp molecular and enzymatic properties Fig 7 The putative substituted model of the C-terminal region and its surroundings White, red and blue indicate C, O and N, respectively The green dotted lines indicate calculated hydrogen bonds (A) Wild-type ALPI (B) D266N-ALPI (C) Y269A-ALPI (D) D266N/Y269A-ALPI.
Trang 9of the enzyme and molecular cloning and characterization of the
gene Aprs Enzyme Microb Technol 17, 653–663.
12 Maeda, H., Mizutani, O., Yamagata, Y., Ichishima, E &
Naka-jima, T (2001) Alkaline-resistance model of subtilisin ALPI, a
novel alkaline subtilisin J Biochem (Tokyo) 129, 675–682.
13 Maeda, H., Yamagata, Y., Ichishima, E & Nakajima, T (2001)
Identification of N-terminal autodigestion target site in subtilisin
ALPI Biosci Biotechnol Biochem 65, 1255–1257.
14 Vieira, J & Messing, J (1987) Production of single-stranded
plasmid DNA In Methods in Enzymoogyl (Wu, R., ed.), pp 3–11.
Academic Press, Orland, San Diego, New York, Austin, Boston,
London, Sydney, Tokyo, Toronto.
15 Nakamura, A., Koide, Y., Kawamura, F., Horinouchi, S.,
Uoz-umi, T & Beppu, T (1990) Construction of a Bacillus subtilis
strain deficient in three proteases Agric Biol Chem 54, 1307–
1309.
16 Keggins, K.M., Lovett, P.S & Duvall, E.J (1978) Molecular
cloning of genetically active fragments of Bacillus DNA in Bacillus
subtilis and properties of the vector plasmid pUB110 Proc Natl
Acad Sci USA 75, 1423–1427.
17 Carter, P , Bedouelle, H & Winter, G (1985) Improved
oligo-nucleotide site-directed mutagenesis using M13 vectors Nucleic
Acids Res 13, 4431–4443.
18 Chang, S & Cohen, S.N (1979) High frequency transformation of
Bacillus subtilis protoplasts by plasmid DNA Mol Gen Genet
168, 111–115.
19 Laemmli, U.K (1970) Cleavage of structural proteins during the
assembly of the head of bacteriophage T4 Nature 227, 680–685.
20 Burnette, W.N (1981) Western blotting: electrophoretic transfer
of proteins from sodium dodecyl sulfate–polyacrylamide gels to
unmodified nitrocellulose and radiographic detection with
antibody and radioiodinated protein A Anal Biochem 112,
195–203.
21 LeGendra, N & Matsudaira, P (1989) Purification of proteins
and peptides by SDS-PAGE In A Practical Guide to Protein and
Peptide Purification for Microsequencing (Matsudaira, T.P., ed.),
pp 49–69 Academic Press, San Diego, New York, Berkley, Boston, London, Sydney, Tokyo, Toronto.
22 Tsuchida, O., Yamagata, Y., Ishizuka, T., Arai, T., Yamada, J., Takeuchi, M & Ichishima, E (1986) An alkaline proteinase of an alkalophilic Bacillus sp Curr Microbiol 14, 7–12.
23 Yamagata, Y & Ichishima, E (1989) A new alkaline proteinase with pI 2.8 from alkalophilic Bacillus sp Curr Microbiol 19, 259– 264.
24 Takagi, H., Takahashi, T., Momose, H., Inouye, M., Maeda, Y., Matsuzawa, H & Ohta, T (1990) Enhancement of the thermo-stability of subtilisin E by introduction of a disulfide bond engineered on the basis of structural comparison with a thermo-philic serine protease J Biol Chem 265, 6874–6878.
25 Vogt, G., Woell, S & Argos, P (1997) Protein thermal stability, hydrogenbonds and ion pair J Mol Biol 269, 631–643.
26 Jaenicke, R & Bohm, G (1998) The stability of proteins in extreme environments Curr Opin Struct Biol 8, 738–748.
27 Facchiano, A.M., Colonna, G & Ragone, R (1998) Helix stabi-lizing factors and stabilization of thermophilic proteins: an X-ray based study Protein Eng 11, 753–760.
28 Zavodszky, P., Kardos, J., Svingor, A., Petsko, A & G (1998) Adjustment of conformation al flexibility is a key event in the thermal adaptation of proteins Proc Natl Acad Sci USA 95, 7406–7411.
29 Spiller, B., Gershenson, A., Arnold, F.H., Stevens, R & C (1999)
A structural view of evolutionary divergence Proc Natl Acad Sci USA 96, 12305–12310.
30 Steipe, B., Schiller, B., Pluckthun, A & Steinbacher, S (1994) Sequence statistics reliably predict stabilizing mutations in a pro-tein domain J Mol Biol 240, 188–192.
31 Lehmann, M., Loch, C., Middendorf, A., Studer, D., Lassen, S.F., Pasamontes, L., van Loon, A.P.G.M & Wyss, M (2002) The consensus concept for thermostability engineering of proteins: further proof of concept Protein Eng 15, 403–411.