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: yamagata@biochem.tohoku.ac.jp 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. 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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. Ó FEBS 2002 1 The molecular surface in enzymatic stability in extraordinary environments (Eur. J. Biochem. 269) 4585 . The molecular surface of proteolytic enzymes has an important role in stability of the enzymatic activity in extraordinary environments Youhei. 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