APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Nov. 1994, p.

3981-3988

Vol. 60, No. 11

0099-2240/94/$04.00+0
Copyright X) 1994, American Society for Microbiology

Cloning and Sequencing of a Serine Proteinase Gene from a Thermophilic Bacillus Species and Its Expression in Escherichia coli
BRYCE MACIVER, RONNIE H. McHALE, DAVID J. SAUL, AND PETER L. BERGQUIST* Centre for Gene Technology, University ofAuckland, Auckland, New Zealand
Received 28 March 1994/Accepted 15 August 1994

The gene for a serine proteinase from a thermophilic Bacilus species was identified by PCR amplification, and the complete gene was cloned after identification and isolation of suitably sized restriction fragments from Southern blots by using the PCR product as a probe. Two additional, distinct PCR products, which were shown to have been derived from other serine proteinase genes present in the thermophilic BaciUlus species, were also obtained. Sequence analysis showed an open reading frame of 1,206 bp, coding for a polypeptide of 401 amino acids. The polypeptide was determined to be an extracellular serine proteinase with a signal sequence and prosequence. The mature proteinase possessed homology to the subtilisin-like serine proteinases from a number of Bacillus species and had 61% homology to thermitase, a serine proteinase from Thermoactinomyces vulgaris. The gene was expressed in Escherichia coli in the expression vector pJLA602 and as a fusion with the at-peptide of the lacZ gene in the cloning vector pGEM5. A recombinant proteinase from the lacZ fusion plasmid was used to determine some characteristics of the enzyme, which showed a pH optimum of 8.5, a temperature optimum of 75C, and thermostabilities ranging from a half-life of 12.2 min at 90C to a half-life of 40.3 h at 75C. The enzyme was bound to a bacitracin column, and this method provided a simple, one-step method for producing the proteinase, purified to near homogeneity.
Proteinases are an important group of enzymes both physiologically and commercially. Microbial proteinases dominate commercial applications, with a large market share taken by subtilisin proteinases from Bacillus spp. for laundry detergent applications (43). A major requirement for commercial enzymes is thermal stability, because thermal denaturation is a common cause of enzyme inactivation, and there have been a number of recent efforts to improve the thermostability of some enzymes on the basis of the currently limited knowledge of protein engineering (7, 21, 35, 38). An alternative method of obtaining enzymes with improved thermostability is to isolate enzymes from naturally occurring thermophilic organisms. However, a disadvantage of this approach is that it is often impractical to produce large quantities of enzymes from thermophiles, as yields may be low because of imprecise growth conditions, in addition, high-temperature fermentations may require specialized equipment (34). Therefore, the preferred method is to apply recombinant gene technology to isolate, clone, and express thermophilic genes of interest in mesophilic organisms. Serine proteinases from the subtilisin family that have been sequenced show considerable amino acid homology proximal to the residues involved in the catalytic site (Asp-32, His-64, Ser-221 [subtilisin BPN numbering]). We have designed oligonucleotide primers corresponding to the active-site histidine and serine regions, on the basis of this homology, and we have used them in the PCR to allow isolation of a serine proteinase from Thermus sp. strain Rt41A (22). These primers have been used to screen for serine proteinases in a thermophilic Bacillus strain, designated Akl, in our collection. We report the
* Corresponding author. Mailing address: Molecular Genetics and Microbiology, School of Biological Sciences, University of Auckland, Private Bag 92019, Auckland, New Zealand. Phone: 64-9-3737 599, ext. 7712. Fax: 64-9-3737 414. Electronic mail address: pbergquist@ sbsnovl.auckland.ac.nz.

cloning, sequencing, and initial expression of an extracellular serine proteinase from this thermophilic Bacillus sp.
MATERIALS AND METHODS

Bacteria, plasmids, media, and transformation. The bacterial strains used for all recombination and expression work were Escherichia coli DHSat (9) and JM101 (44). Cultivation of these strains was carried out at 32 or 37C in L broth. The plasmids used for recombinant work were pGEM5Zf+ (Promega Corp., Madison, Wis.) and pBR322, which was used for estimating the transformation frequency. Plasmid pJLA602 (32) was used for expression of the thermophilic proteinase. Ampicillin at 100 j,g/ml was used for the selection of DH5cx containing either pGEM5 recombinants or pBR322, whereas ampicillin at 50 jig/ml was used for pJLA602-based recombinants. Transformation of bacterial strains was performed with bacteria suspended in L broth containing 10% polyethylene glycol 8000, 25 mM MgCl2, and 5% (vol/vol) dimethyl sulfoxide (4). Blue-white colony screening of pGEM5 recombinants was done with 20 ,ug of X-Gal (5-bromo-4-chloro-3-indolyl-PD-galactopyranoside) in agar plates. Isolation and analysis of DNA. Genomic Bacillus strain Akl DNA was isolated by using the ASAP kit and the recommended procedure (Boehringer Mannheim, Sylvia Park, Auckland, New Zealand). Small-scale plasmid DNA was isolated by the alkaline lysis method (3). Larger quantities of plasmid DNA were prepared by a modified LiCl method (2) or by CsCl ethidium bromide density centrifugation (11). DNA manipulation. Digestion of DNA with restriction endonucleases was performed as specified by the manufacturers (Bethesda Research Laboratories, Inc., Gaithersburg, Md.; New England Biolabs, Beverly, Mass.). DNA was ligated with T4 DNA ligase supplied by Boehringer Mannheim and used as specified. Other DNA modification enzymes were used as specified by the manufacturers. DNA fragments were purified
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APPL. ENVIRON. MICROBIOL.

from agarose gels with DEAE membranes (Schleicher & Schuell, Dassel, Germany) or by the GeneClean method (Bio 101, La Jolla, Calif.). Oligonucleotide synthesis. Oligonucleotides were synthesized by cyanoethyl phosphoramidite chemistry with an Applied Biosystems 380A oligonucleotide synthesizer. PCR. PCRs were performed in a Perkin-Elmer Cetus (Norwalk, Conn.) thermal cycler according to the recommended procedures. PCR primers RM5 and RM6 were designed to amplify the region between the highly conserved active-site histidine and serine residues from serine proteinases. The primers were RM5 [5' CA(CT) GG(ACGT) ACC AA(CT) GTG GC(CGT) GG 3'] and RM6 [5' (ACG)GG GGT (ACG)GC CAT GGA GGT (CGT)CC 3']. Each primer (100 ng) was used in a standard reaction volume of 50 ,ul in sterile 0.5-ml tubes, with 20 to 50 ,lI of mineral oil added to prevent evaporation. Pharmacia deoxynucleoside triphosphates (0.25 mM) were used for all reactions. The standard' PCR buffer used was 50 mM KCl-20 mM Tris-HCl (pH 8.3)-2 mM MgCl2-1 mg of gelatin per ml. Amplitaq (Cetus) Taq polymerase was used at 0.5 U per reaction. Southern blots. Hybond N+ filters (Amersham Australia Pty. Ltd., Auckland, New Zealand) were used for Southern blots as recommended by the manufacturer. Hybridization with probes labelled with [a-P32]dCTP with a Bresatec random priming kit was carried out for a minimum of 12 h at 60C. A wash procedure with 2x SSC (lx SSC is 0.15 M NaCl plus 0.015 M sodium citrate)-0.1% sodium dodecyl sulfate (SDS) (twice) at room temperature for 10 min each, followed by 1x SSC-0.1% SDS at 60C for 15 min was used for all hybridizations. DNA sequencing. Cloned PCR products were sequenced initially by the dideoxy termination method of Sanger et al. (30). The complete gene sequence was obtained by using universal primers and Sequenase (U.S. Biochemicals, Cleveland, Ohio). Some DNA sequencing was performed with an Applied Biosystems 373A automated sequencer; both strands of the DNA were sequenced. Computer analysis. DNA and protein sequences were analyzed with the Genetics Computer Group (Madison, Wis.) package of Devereux et al. (6), running on a Silicon Graphics 4D/30 workstation. Proteinase assays. Cell extracts were assayed for proteinase activity with 0.2% (wt/vol) azocasein (Sigma Chemical Co., St. Louis, Mo.) in HCT buffers (50 mM HEPES [N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid] at a specified pH, 2 mM CaCl2, 0.01% [vol/vol] Triton X-100) at the required temperature. After a suitable period, the protein was precipitated by the addition of 0.5 ml of 15% (wt/vol) trichloroacetic acid and pelleted by centrifugation. Release of acid-soluble azopeptides was measured by reading the A420. Casein-agar plates consisting of 2% (wt/vol) casein (technical grade; Sigma) and 2.4% milk agar (Oxoid) were used to screen recombinants for proteinase activity (5). Recombinants were grown at 28C until they reached a diameter of approximately 1 mm and then incubated for 1 to 2 h at 42C to induce expression from pJLA602-derived recombinants. Colonies which produced active proteinase were identified by the formation of a halo or precipitation ring around the colony after incubation at 70C for 2 to 5 h. Expression in E. coli. Strains carrying pGEM5-based recombinant plasmids were tested for expression by preparing a cell lysate as follows: cells were grown to late log phase in 15 to 25 ml of L broth at 32C, pelleted for 15 min at 2,000 x g, resuspended in HCT5 buffer (5 mM HEPES [pH 9.1] at 25C,

2 mM CaCl2, 0.01% [vol/vol] Triton X-100) containing 1 mg of lysozyme per ml, and incubated on ice for 30 min. Proteinase activity was detected by adding 25 to 50 ,ul of lysate to the azocasein assay mixture described above. Strains carrying the expression plasmid pNZ1931 or pNZ1932 (see Results) were grown in L broth at 28C until late log phase and then transferred to a water bath incubator at 42C, and incubation was continued for a further 90 min. Cell lysates were prepared as described above. Purification of the Akl proteinase on a bacitracin column. The bacitracin column (40) was prepared by coupling bacitracin (Sigma) to CNBr-activated Sepharose 4B (Sigma), according to the instructions supplied with the matrix. A bacitracin column was prepared in a 1-ml syringe and equilibrated with 5 volumes of HCT5, pH 9.1. Cells (1.5 ml from 10 ml of culture grown overnight) were lysed with lysozyme and detergent as described above. Cell lysates were passed through a Pharmacia PD-10 column, also equilibrated with the same buffer, and the eluate (3 ml) was applied to the bacitracin-Sepharose column. The column was washed with 1.5 ml of HCT5 buffer, and then the proteinase was eluted with 1.5 ml of 0.1 M Tris-HCl (pH 8)-0.5 M NaCl-25% (vol/vol) isopropanol, as described previously (40). All fractions were collected and assayed by azocasein for proteinase activity. Ninety-seven percent of the proteinase activity loaded onto the bacitracin column was recovered in the eluate. Nucleotide sequence accession number. The sequence for the gene shown in Fig. 2 is available from GenBank under the accession number L29506.

RESULTS Phylogeny of Bacillus strain Akl. The 16S rRNA gene was isolated and sequenced by methods described elsewhere (31). The sequence is available from GenBank under the accession number L29507 and is not repeated here. Phylogenetic analysis showed that Akl is a member of the Bacillus rRNA group 5. This group includes Bacillus stearothermophilus and other thermophilic Bacillus spp. The most closely related organism for which 16S rRNA sequence data are available is Bacillus thermoglucosidasius (1). Identification of genes for serine proteinases in Bacillus strain Akl. PCR of genomic DNA from Akl using an annealing temperature of 55C and primers RM5 and RM6 yielded a 500-bp fragment. The 500-bp band was gel purified; treated with T4 polymerase, the Klenow fragment of E. coli DNA polymerase, and T4 polynucleotide kinase; and then ligated into pGEM5, which was cut with EcoRV and treated with alkaline phosphatase. Three recombinants were sequenced with both universal forward and reverse primers to produce sufficient information to construct a continuous sequence of the inserted DNA. A comparison of the sequences was made with the EMBL and GenBank databases, using the FASTA and TFASTA algorithms, and all three sequences showed homology to sequenced serine proteinases. One was highly homologous to an intracellular serine proteinase from Bacillus subtilis, and another had 43% identity with the Bacillus licheniformis subtilisin Carlsberg peptide sequence and 41% identity with the B. subtilis epr gene (reference 10 and data not shown). Further studies with the latter gene from Akl were conducted, and the pGEM5 construct containing the appropriate 500-bp insert was designated pNZ1294. Cloning of the gene. Approximately 2 ,ug of genomic DNA was digested with several restriction endonucleases, the fragments were separated by electrophoresis, and the DNA was transferred to a Hybond N+ filter for Southern hybridization

VOL. 60, 1994

pNZ1915
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Sp

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pNZ1 919
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CLONED BACILLUS SERINE PROTEINASE GENE

3983

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1500

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pNZ1 932

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Expression Constructs

FIG. 1. Diagrammatic representation of insertions containing the alkaline serine proteinase cloned in pGEM5. pNZ1915 carries the 4.5-kb PstI fragment, pNZ1919 contains the SphI fragment from pNZ1915 in pCEM5, and pNZ1917 contains a SmaI-DraI fragment that contains the open reading frame for the alkaline serine proteinase. Abbreviations: P, PstI; E, EcoRI; Sp, SphI; Pv, PvuII; D, DraI; S, SmaI; Nd, NdeI; Sn, SnaBI; Ns, NsiI; B, BamHI; Bs, BstEII; N, NcoI; H, HindlIl.

analysis. This filter was probed with the insert from pNZ1294, and restriction enzyme fragments containing the gene were identified (data not shown). Genomic DNA digested with SnaBI produced a band of 2.6 kb, which hybridized to the probe. A second sample of genomic DNA digested with this enzyme was separated on an agarose gel, and fragments of 2 to 3 kb were isolated and ligated into the EcoRV site of pGEM5 to yield the recombinant plasmid pNZ1910. Sequence analysis of DNA from this recombinant showed that the gene was incomplete and lacked the amino-terminal pre-pro sequence (data not shown). A PstI site mapped on this recombinant suggested that the entire gene would be contained within a 4.5-kb PstI fragment also identified in the Southern blot. DNA fragments of 4 to 5 kb were isolated from a PstI-digested genomic DNA, and the fragments were ligated into the PstI site of pGEM5. Transformants were selected and screened by colony hybridization with a probe made from the 500-bp PCR product described above. Plasmid DNA was isolated from a transformant that hybridized to the PCR probe, and separate samples were digested with PstI plus SnaBI, PvuII, and SspI and were found to contain a 4.5-kb PstI fragment with SnaBI and PvuII sites at the positions predicted from the partial sequence described above. This plasmid was designated pNZ1915. Restriction mapping showed that the gene could be isolated on a 1.7-kb SphI fragment, which was inserted into pGEM5 to form pNZ1919 (Fig. 1). Construction of expression plasmids. Plasmid pNZ1917 was initially constructed for sequencing purposes but was later shown to have significant levels of expression of the proteinase. This plasmid consists of a 1.2-kb fragment from a DraI digest of pNZ1915 ligated into the EcoRV site of pGEM5. Plasmid pNZ1931 was constructed by ligating a 1.3-kb fragment from an SspI digest of plasmid pNZ1915 into pJLA602 at the NcoI site, which had been blunt ended by the use of mung bean nuclease (New England Biolabs). Plasmid pNZ1932 was constructed by PCR amplification of the proteinase gene with primers which incorporated suitable restriction endonuclease

sites: an SphI site at the N terminus and a BglII site at the C terminus. The PCR product was ligated into plasmid pJLA602 at the SphI and BamHI sites. Sequence analysis. All sequencing was carried out by producing further recombinants and deletions from pNZ1915 and pNZ1919 by using restriction sites from the initial maps and from new restriction sites recognized in the sequence as it was generated. The sequence of the gene is shown in Fig. 2, with the putative promoter, ribosome binding site, and transcriptional termination sequences marked. Transcriptional terminators can be identified frequently by the presence of an inverted repeat, which can form a split hairpin structure in the RNA, followed by a run of uracil bases (28). Application of the routine STEMLOOP analysis to the sequence downstream of the assigned terminator identified a number of hairpin structures, with one such structure being a good candidate for a transcriptional terminator (Fig. 2). The sequence has two possible ATG start codons. The preferred start codon was assigned on the basis of peptide sequence homology with other serine proteinases and the positioning of the ATG with respect to the putative promoter and ribosome binding site (20). Translational termination codons TGA, TAG, and TAA were identified at the Cterminal end, clustered in all three frames. The C-terminal end of the proteinase was also identified by comparison with other serine proteinases. The open reading frame codes for a protein of 401 amino acid residues. Homology comparisons show that the putative amino acid sequence has significant similarities (50 to 70%) to five well-characterized subtilisin-like serine proteinases: aqualysin I (16), proteinase K (8), subtilisin BPN (41), subtilisin Carlsberg (12), and thermitase (19). Figure 3 shows that the Akl proteinase has considerable homology to thermitase, an extracellular serine proteinase from Thermoactinomyces vulgaris (19). Serine proteinases are characterized by the catalytic triad of aspartate, histidine, and serine residues (24). The Akl proteinase contains an aspartate

3984

MACIVER ET AL.

APPL. ENVIRON. MICROBIOL.

100

1 GCATGCTACGATTAAATATCTAAGAATCCCCAATTAAATACCATTATITTIT|ACAAAATTATATTATATTAAAATAAATGTCGAGATTTTT |GAGAAAAAATT
- -35 -10 . r.b.s. . S1t1

101 TTCATTCCTCCCCC MCCTCAACGTAAAATTAATACCGCAATTCCA I iAAAGGGGGAATAI I CCATGAAGTTTAAAGCGATTGTAAGI I ATCCCTC

200 300 400 500 600 700 800

M K F K A I V S L S L
201 GCTGTTTCCATGTCAC ZCCATTCCTTGTGGAAGCAGCCTCTAATGATGGGGTAGAATCTCCAAAAACCGTTCCGAAATTAACGTGTCTCATGAAA A V S M S L F P F L V E A A S N D G V E S P K T V S E I N V S H E K

301 AAGGAGCATATGTCCAGGGAGAAGTCATTGTCCAATTCAAAGAACAAGTAAATGCTGAAGAAAAGGCAAAGGCATTAAAAGAAGTTGGGGCAACGGCGGT G A Y V Q G E V I V Q F K E Q V N A E E K A K A L K E V G A T A V
401 TCCAGATAATGATCGAGTTAAATCTAAATTAATGTACTAAAAGTAGGAAATGTGGAAGCTGTTGTGAAAGCATTAAATAATAACCCGTTIAGTAGAATAC P D N D R V K S K F N V L K V G N V E A V V K A L N N N P L V E Y 501 GCAGAGCCAAACTATTATTTAATGCAGCTTGGACTCCAAATGATACGTACTATCAGGGTTATCAATATGGTCCACAAAATACGTATACCGACTATGCTT A E P N Y L F N A A W T P N D T Y Y Q G Y Q Y G P Q N T Y T D Y A W

601 GGGATGTT IACAAAAGGCAGTAGCGGTCAAGAGATTGCTGTTATTGATACAGGTGTAGATTATACACATCCTGATTAGATGGAAAAGTCATCAAAGGATA D V T K G S S G Q E I A V I D T G V D Y T H P D L D G K V I K G Y

701 TGATTTCGTAGATAATGATTACGACCCAATGGAs M GAATAATCATGGTACGCACGTAGCTGGAATAGCAGCTGCGGAAACAAACAATGCTACAGGTATT D F V D N D Y D P M D L N N H G T H V A G I A A A E T N N A T G I

801 GCCGGCATGGCCCCAAACACAAGAATTTTI FGGCTGTGCGCGCTT|TAGATCGAAATGGCAGTGGTACTCTAAGTGATATTGCCGATGCGATCATTT |ATGCTG A G M A P N T R I L A V R A L D R N G S G T L S D I A D A I I Y A A

901 CTGATTCAGGCGCTGAAGTCATTAACCTGTCACTTGGTTGTGATTGTCATACAACCACATTGGAGAATGCTGTAAACTATGCATGGAATAAGGGTT |CTGT D S G A E V I N L S L G C D C H T T T L E N A V N Y A W N K G S V
1001

900
1000
1100

AGTAGTTGCCGCAGCCGGAAATAATGGATCCTCTACAACATTGAACCGGCTTCTTATGAAAATGTAATTGCAGTTGGCGCAGTAGATCAATATGATCGG V V A A A G N N G S S T T F E P A S Y E N V I A V G A V D Q Y D R

1101 TTAGCATCATTCTCGAACTATGGAACATGGGTAGATGTCGTAGCTCCAGGTGTAGACATTGTCTCAACTATTACTGGTAACCGATATGCCTATATGTCGG L A S F S N Y G T W V D V V A P G V D I V S T I T G N R Y A Y M S G 1201 GCACTTCCATGGCATCCCCTCATGTAGCCGGTCTTGCAGCCTTACTAGCGAGTCAAGGACGGAATAACATAGAAATTCGTCAAGCCATCGAGCAAACAGC T S M A S P H V A G L A A L L A S Q G R N N I E I R Q A I E Q T A

1200

1300 1400

1301 AGACAAAATCTCCGGAACTGGAACATA TTCAAATATGGAAGAATCAATTCTTATAATGCTGTAACATATTAAATAGATTTAAATAACGACACCGTGCCA D K I S G T G T Y F K Y G R I N S Y N A V T Y *Inverted 1401 ACGCCAAGGGCACGGTGTCTTAATTCAAGCTTTTT ZGGAAAATCACGACATACAAATTATGTAAGAAAGCTTATTCTCCCATAGGAATTGAAGAGGAAAAC

1501 CAATATTATTCTTTTI |ATCTCAGCAAAAATAAAAAAACTGCCAAATGGCAGACTCTAACTAACTA s| aa iGCTTCCTTCTTTCTGCTGCAG 1591
< ItRepeat

~~~~~~~~~Run of Uracils in mRNA

1500

FIG. 2. Nucleotide sequence of the gene encoding the alkaline serine proteinase and the deduced protein sequence. Putative -35 and -10 sequences and ribosome binding sites (r.b.s.) are labeled. A possible termination sequence is identified by an inverted repeat and a run of uracil bases. The asterisk denotes the stop codon.

residue at position 160 of the complete amino acid sequence, which corresponds to the correct position for this residue to be the third catalytic residue which is conserved throughout the serine proteinases in the alignment (the histidine and serine residues were part of the product of the PCR oligonucleotide primers). The amino acid sequence of the Akl proteinase contains a region of 97 residues between the putative signal peptide and the mature proteinase which was designated a prosequence. A number of other extracellular proteinases have been shown to contain a prosequence; studies with the a-lytic proteinase from Lysobacter enzymogenes (33) and subtilisin E (46) support the view that the role of the prosequence is to aid in the correct folding of the proteinases. The exact mechanisms have yet to be elucidated, but it is apparent from those prosequences shown in the homology lineup of proteinases in Fig. 3 that there are no obvious common or conserved motifs. Signal peptide. Signal peptides from Bacillus spp. tend to be longer than those from E. coli and have on average more positive charges (29). The deduced peptide sequence of the Akl serine proteinase has most of the characteristics of a signal peptide in the first 25 amino acid residues. (Fig. 4). At the N terminus are two basic lysine residues, followed by a sequence containing a high proportion of nonpolar (hydropho-

bic) residues. A helix-breaking proline occurs at position 19, and the -3 and -1 rules (42) predict a cleavage site between residues 24 and 25, which are both alanines. Verification of the leader sequence and propeptide cleavage sites was obtained from amino-terminal sequencing of both the proproteinase and the mature proteinase from the recombinant proteinase expressed in E. coli (27). Expression of the Akl proteinase in E. coli. Preliminary experiments using the azocasein assay (see Materials and Methods) demonstrated that the Akl proteinase was being produced by pNZ1915 (Fig. 1), presumably from its own promoter sequence, and also by pNZ1917, a plasmid carrying a 1.2-kb Dral fragment containing a truncated proteinase gene which produces a protein fused with the first 38 residues of the cx-peptide of the lacZ gene. This fusion protein lacks the first three amino acids of the signal peptide. SDS-polyacrylamide gel electrophoresis (PAGE) examination of native and heattreated cell extracts showed that strains carrying either of these plasmids produced the Akl proteinase as the proenzyme and the mature enzyme. The amounts were increased substantially for pNZ1917 by the addition of the inducer IPTG (isopropyl,B-D-thiogalactopyranoside) (data not shown). Early experiments concentrated on the intracellular levels of Akl proteinase activity by using cell extracts as described

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CLONED BACILLUS SERINE PROTEINASE GENE

Akl . MKFK AIVSLSLAVS Thermitase AQlprepro MRKTYWLMAL FAVLVLGGCQ Protk ..MRLSVLLS LLPLALGAPA M Carlsberg .......... SubtDY BPN ..........
..........
..........

3985

MSLFPFLVEA ASNDGVESPK TVSEINVSHE KGAYVQGEVI VQFKEQVNAE EKAKALKEVG ATAVPDNDRV

..........

..........

..........

..........

..........

..........

..........

.........

MASRSDPTPT LAEAFWPKEA PVYGLDDPEA IPGRYIWFK KGKGQSLLQG GITTLQARLA PQGVVVTQAY ARGEM VANKYIVKFK EGSALSALDA AMEKISGK ..... PDHVY VEQRSEAAPL IE ........ MRKKSFWLGM LTAFMLVFTM AFSDSASAAQ PAK.NVEKDY IVGFKSGVKT ASV ..KKDII KESGGKVDKQ
.....
..

..........

..........

..........

..........

..........

..........

..........

..........

..........

..........

Amylosacc
Subtmesen BSUISPI

..........
..........

..........

MRGKKVWISL LFALALIFTM AFGSTSSAQA AGKSNGEKKY IVGFKQTMST MSAAKKKDVI SEKGGKVQKQ VRSKKLWISL LFALTLIFTM AF.SNMSAQA AGKSSTEKKY IVGFKQTMSA MSSAKKKDVI SEKGGKVQKQ
.......... .......... ..........
..........

..........

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81 Akl KSKFNVLKVG NVEAWKALN NNPLVEYAEP NYLFNAAWTP NDTYYQGYQY YTP NDPYFSSRQY Thermitase .......... AQlprepro TGALQGFAAE MAPQALEAFR QSPDVEFIEA DKVVRAWATQ SPAPWGLDRI Protk KNVFSGFAAT LDENMVRVLR AHPDVEYIEQ DAWTINAAQ TNAPWGLARI Carlsberg FRIINAAKAK LDKEALKEVK NDPDVAYVEE DHV ....... AHALAQTVPY SubtDY ........................................... AQTVPY BPN FKYVDAASAT LNEKAVKELK KDPSVAYVEE DHV....... AHAYAQSVPY Amylosacc FKYVNAAAAT LDEKAVKELK KDPSVAYVEE DHI ....... AHEYAQSVPY Subtmesen ............................................ AQSVPY BSUISPI .... MNG EIRLIPYVTN EQI ....... MD. .VNELPE
.......... .......... .......

GPQNTYTDYA WDVTKGSSGQ GPQKIQAPQA WDIAEG.SGA DQRDLPLSNS YTYTATGRGV SSTSPGTSTY YYDESAGQGS GIPLIKADKV QAQGFKGANV GIPLIKADKV QAQGYKGANV GVSQIKAPAL HSQGYTGSNV

EIAVIDTGVD YTHPDLDGKV NVYV IR TTHREFGGRA

CVYVIDTGIE KVAVLUVGIQ KVGIITGIA KVAVIQSGID

KIAIflTGVQ SNHPDLAGKV

ASHPEFEGRA ASHPDLN ..V ASiTDLK ..V SSHPDLK ..V GISQIKAPAL HSQGYTGSNV KVAVIPqGID SSHPDLN ..V GISQIKAPAL HSQGYTGSNV KVAVIISGID SSHPDLN ..V GIKVIKAPEM WAKGVKGKNI KVAVLDTG,CD TSHPDLKNQI
.......

161

Akl IKGYDFVDND YDP MDL NNHGVAGI AAAETNNATG IAGMAPNTRI LAVRALDRN. GTLSDIAD AIIYAADSGA E VI Thermitase VGGWDFVDND STP QNG NGHGTCGI AAAVTNNSTG IAGTAPKASI LAVRVLDNS. GSGTWTAVAN GITYAADQGA K VI QDC NGHGV IGGVT..... . YGVAKAVNL YAVRVLDCN. GSGSTSGVIA GVDWVT.... RNHRRPAVA AQlprepro RVGYDAL .. G NG Protk QMVKTYYY..S RDG NG HHCA VGSRT S YGVAKKTQL FGVKVLDDN. GSGQYSTIIA GMDFVASDKN NRNCPKGWA Carlsberg VGGASFVAGE A YN.TDG NGHGV VAAL.DNTTG VLGVAPSVSL YAVKVLNSS. GSGTYSGIVS GIEWATTNGM D VI SubtDY VGGASFVSGE S YN.TDG NGHV VAAL.DNTTG VLGVAPNVSL YAIKVLNSS. tS' TYSAIVS GIEWATQNGL D VI BPN AGGASMVPSE T NPFQDN N VAAL.NNSIG VLGVAPSASL YAVKVLGAD. wGYSWIIN GIEWAIANNM D VI Amylosacc RGGASFVPSE T NPYQDG SS IAAL.NNSIG VLGVSPSASL YAVKVLDST. GSGQYSWIIN GIEWAISNNM D VI Subtmesen RGGASFVPSE T NPYQDG SSHVA IAAL.NNSIG VLGVAPSSAL YAVKVLDST. -GGQYSWIIN GIEWAISNNM D VI BSUISPI IGGKNFSDDD GGKEDAISDY NGHGVAG IAAN.DSNGG IAGVAPEASL LIVKVAGGEN SQYEWIIN GINYAVEQKV D II
.... ....

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241

Akl Thermitase

NL$LCDCHT
NM

TTLENAVNYA

WNKGSVVVAA

AiNNGSS

..TTFEPASY

ENVIAVGAVD QYDRLASFSN YGTWVDWAP SSDARASFSN YGSCVDLFAP

GVDIVSTITG

AGNAGNT ....APNYPAYY SNAIAVASTD QNDNKSSFST YGSWDVAAP GSWIYSTYPT

G.GVS
TALDNAVKNS SSVNSAAARL

AQlprepro
Protk Carlsberg SubtDY BPN Amylosacc Subtmesen BSUISPI

IAA6VVYAV
QSSGVMVAVA

AGNDNANACN
NNNADARN

YS

.PARV

AEALTGATT

GASIPSAWYT

SLSG.GYS

YS

PASE

PSVCTVGASD

RYDRRSSFSN YGSVLDIFGP GTSILSTWIG

NM GPSGS TAMKQAVDNA YARGVVVVAA NM LGPSGS TALKQAVDKA YAS4IVVVAA

ASGSSGN. TNTIGYPAKY DSVIAVGAVD SNSNRASPSS VGAELEVMAP iNSGSSGS. QNTIGYPAKY DSVIAVGAVD SNKNRASFS VGAELEVMAP

AAGVYSTYPT G'VSVYSTYPS

NMSLtGPSGS AALKAAVDKA VASGVVVVA

NM3~GPSGS

TALKTVVDKA

VSSGIVVAAA, AGEGSSGS.

NMSGPTGS TALKTVVDKA VSSGIWAAA SMSLGPSDV PELEEAVKNA VKNGVLWCA

321

A,GNEGSSGS.

EGTSGS. SSTVGYPGKY SSTVGYPAKY TSTVGYPAKY AGDGDER TEELSYPAAY

PSVIAVGAVD SSNQRASFSS VGPELDVMAP VSIQSTLPG PSTIAVGN SSNQRASFSS AGSELDVMAP GVSIQSTLPG PSTIAVGAVN SANQRASFSS AGSELDVMAP GVSIQSTLPG NEVIAVGSVS VARELSEFSN ANKEIDLVAP GENILSTLPN

Akl NRYAY ..MSG AMAS LAALLAS QGRNNI EIRQAIEQTA DKISGTGT YFKYGR INSYNAVTY. Thermitase STYAS LSG AG VAGLLAS QGRSAS NIRAAIENTA DKISGTGT YWAKGR VNAYKAVQY. AQlprepro SDTATQTLNG STAG VAALYLEQNP SATPASVASA ILNGATTGRL SGIGSGSPNR LLYSLLSSGS ............... Protk GS..TRSISG' fAVAG LAA.YLMTLG KTTAASACRY IADTANKGDL SNIPFGTVNL LAYNNYQA ................. Carlsberg STYAT LNO RLSSTATYL GSSFYYGKGL INVEAAAQ. AAALILSK.....HPNLSAS QVRN SubtDY NTYTS LNG RLSSTATNL GDSFYYGKGL INVEAAAQ. SAg AAALILSK.....YPTLSAS QVRN BPN NKYGA ..YNG SLENTTTKL GDSFYYGKGL INVQAAAQ AAALILSK.....HPNWTNT QVRS Amylosacc GTYGA ..YNGS AG AAALILSK.....HPTWTNA QVRD RLESTATYL GNSFYYGKGL INVQAAAQ. Subtmesen GTYGA YNG AAALILSK.....HPTWTNA QVRD RLESTATYL GSSFYYGKGL INVQAAAQ. BSUISPI KKYGK..LTG TSNAAPNSG ALALIKSYEE ESFQRKLSES EVFAQLIRRT LPLDIAKTLA GNGFLYLTAP DELAEKAEQS HLLTL
... .... ...... .. ... .... ...... ..

......

..

......

......

.......

......

..

......

FIG. 3. Homology lineup of alkaline serine proteinases. Identical amino acid residues are shaded. Akl, extracellular serine proteinase from Akl (this study); thermitase, extracellular proteinase from T. vulgaris (19); AQlprepro, extracellular proteinase from Thermus aquaticus YT-1 (16); Protk, extracellular proteinase from Tritirachium album Limber (8); Carlsberg, extracellular proteinase from B. licheniformis (12); SubtDY, extracellular proteinase from B. subtilis DY (23); BPN, extracellular proteinase from Bacillus amyloliquefaciens (41); Amylosacc, extracellular proteinase from Bacillus amylosacchariticus (45); Subtmesen, extracellular proteinase from Bacillus mesentericus (36); BSU-ISPI, major intracellular serine proteinase from B. subtilis (14).

above. However, a significant level of proteinase (18 to 25%) was detectable in the cell-free culture supernatant (data not shown). These results suggest that either cell lysis had occurred (although it was not apparent on inspection of the cultures) or

10

15
A
A

20

K A

I V

S
A

S
A

L A V S M S L F P F L V E A
-_

25 A

FIG. 4. Signal peptide sequence of alkaline serine proteinase from Bacillus strain Akl. +, basic charged residue; A, polar residue; acidic charged residue. The cleavage point is indicated by the arrow.
-,

the E. coli export functions were able to recognize the Bacillus signal sequence. Expression in the temperature-inducible vector, pJLA602. E. coli strains carrying the pJLA602 constructs (see above) were grown at 28C until mid-log phase and induced at 42C to inactivate the X repressor. Preliminary experiments showed that maximal proteinase activity was present after 45 min at 42C. Assays showed that about 27% of the total enzyme activity was secreted into the medium and that the remainder was present in the whole-cell extract. This experiment demonstrated that similar amounts of enzyme activity were produced with IPTG induction of pNZ1917 and heat induction of pNZ1931. Hence, the additional amino acids from the lacZ

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l
67 43

30

FIG. 5. SDS-polyacrylamide gel of material eluted from a bacitracin column (40) and concentrated with a Centricon 30 apparatus (Amicon, Danvers, Mass.). Lane 1, molecular mass markers (in kilodaltons); lane 2, eluate from the bacitracin column. The arrow indicates the proteinase band.

fusion had no effect on the yield of the enzyme and, of course, the prosequence was cleaved off in each case following heat treatment, to give rise to the active mature enzyme (27). Purification of the Akl proteinase on a bacitracin column. A recent publication by van Noort et al. (40) demonstrated that the Bacillus antibiotic bacitracin could be used to affinity purify a proteinase from Aspergillus niger. Bacitracin has been shown to bind all four major mechanistic classes of proteinase, that is, the metalloproteinases and the serine, cysteine, and aspartate proteinases (40). The bacitracin column prepared as described in Materials and Methods successfully bound the Akl serine proteinase and provided a simple and effective method for producing proteinase purified to near homogeneity. Fractions were checked for purity by SDS-PAGE with the Pharmacia PhastSystem. Gels were silver stained, and two major bands are visible from the bacitracin-purified material, migrating at approximately 36 and 25 kDa (Fig. 5). Peek et al. (27) have shown that the proteinase corresponds to the 36-kDa band. Characterization of the Akl proteinase produced from the two expression vectors. The detailed characterization of the recombinant Akl proteinase has been published previously (27). The enzyme requires thermal activation (100 min at 70C) and has a pH optimum of 8.5, a broad temperature optimum of 70 to 90C, and a half-life at 80C of 120 min (27). Thermostability curves for the proteinase were obtained at temperatures of 75, 80, 85, and 90C with proteinase purified by the bacitracin affinity method. These data were used to determine the half-lives of the proteinase at these temperatures, which are 12.2 min at 90C, 1.7 h at 850C, 12.4 h at 800C, and 40.3 h at 75C, all data collected in the absence of substrate.

DISCUSSION
We have used PCR to identify an internal fragment from a serine proteinase gene from a thermophilic Bacillus sp. The primers used were originally designed to amplify a similar region from a Thermus species, an organism with a high G+C ratio (22). However, the redundancy designed into these primers has allowed their use in amplifying the fragment from the Bacillus species, even though the region sequenced has a much lower G+C ratio. The success in amplifying this region is explained by the high degree of homology which exists

between all subtilisin-like serine proteinases around the activesite histidine and serine residues. Examination of the sequence showed that putative control sequences could be identified (Fig. 2). A number of promoters and ribosome binding sites have been reported in the literature for B. subtilis genes (23). In addition to these, several neutral metalloproteinase genes have been cloned from strains of B. stearothermophilus (25), for which the promoters and ribosome binding sites have been identified. Takagi et al. (37) compared the promoter of the nprT gene by Si nuclease mapping with other cloned metalloproteinase genes, using this information to identify their promoter regions (14, 26, 39). From this information, it has been possible to assign a putative promoter region (-10 and -35 sequences) and a ribosome binding site to the gene for the Akl serine proteinase (Fig. 2). Another feature of the deduced sequence of the mature proteinase is the occurrence of two cysteine residues at positions 258 and 260. The formation of two disulfide bonds has been demonstrated for aqualysin 1 (18) and proteinase K (13). The cysteine residues in the Akl proteinase are separated by only one amino acid, and it seems unlikely that an effective disulfide bond would be formed between them, although this suggestion has not been tested experimentally. It should be noted that thermitase contains only one cysteine residue, while the subtilisins (Carlsberg, BPN, amylosacchariticus, etc.) contain no cysteine residues. Limited expression of the proteinase in E. coli appears to come from its own promoter system, as evidenced by the results from recombinant plasmids pNZ1915 and pNZ1919. The formation of an in-frame fusion with the o-peptide of the lacZ gene in recombinant plasmid pNZ1917 or with the lambda promoters PL and PR in plasmids pNZ1931 and pNZ1932 allowed much higher levels of expression from the E. coli lacZ promoter. Crude cell lysates from strain PB5517 (carrying plasmid pNZ1917) were used to determine some characteristics of the proteinase, which has a pH optimum of 8.5, a temperature optimum of 75C, and sensitivity to the calcium ion chelator EGTA [ethylene glycol-bis (3-aminoethyl ether)-N,N,N',N'-tetraacetic acid] (27). The Bacillus antibiotic bacitracin was shown to have a high degree of affinity for the proteinase and was used to obtain a good degree of purification, although not to homogeneity. Higher levels of secretion into the culture supernatant were observed for plasmid pNZ1931 than for pNZ1932. This observation may be due to the construction of pNZ1932. Ligation of the PCR-amplified gene into the SphI site has resulted in a fusion of the Akl proteinase with the first four amino acid residues of the atpE leader sequence and also in the mutation of the first lysine (K) in the Akl sequence to a glutamine (Q). The incorporation of the atpE leader sequence in plasmid pJLA602 was designed to provide high levels of translation from this vector (32). The translational start point for proteinase expression from plasmid pNZ1932 should be the atpE start codon, yet the results show that higher levels of expression were achieved with pNZ1931, in which the start codon is effectively 5 bp further from the ribosome binding site than in pNZ1932. Further work which needs to be conducted with these recombinants to provide insights into these observations includes sequencing (to confirm the gene positioning), analysis by SDS-PAGE (to estimate levels of expression as a percentage of total E. coli protein), and determination of the possibility of the formation of inclusion bodies. A number of neutral metalloproteinases from thermophilic Bacillus spp. have been reported, but this is the first example of a serine proteinase from this group of organisms. The closest homolog to the Akl proteinase is thermitase from T. vulgaris.

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This bacterium is included in the order Actinomycetales (17), whereas Bacillus is not. However, members of the genus Thermoactinomyces exhibit many characteristics common to the genus Bacillus, notably the production of a true endospore and lower G+C ratios; they also exhibit 16S rRNA homology to the bacilli. Lacey and Cross (17) comment that members of the genus Thermoactinomyces will probably be reclassified as members of the genus Bacillus. In light of this information, it is not surprising that the Akl proteinase showed the highest degree of homology to thermitase. The information available on serine proteinases from thermophilic bacilli is limited. However, a large quantity of data is available for the mesophile B. subtilis, which produces several proteinases, including both serine proteinases and neutral metalloproteinases. There are at least five distinct extracellular proteinases produced by B. subtilis (9). These are the serine proteinases: subtilisin (gene aprE), a minor serine proteinase (gene epr), bacillopeptidase F (gene bpf), and two neutral metalloproteinases (genes nprE and mpr). Therefore, the finding of three PCR products representing fragments from three distinct serine proteinase genes in this work was not considered unusual. One PCR product has been used to identify an extracellular serine proteinase gene, which forms the subject of this report. A second PCR product showed a very high level of homology to the major intracellular serine proteinase gene of B. subtilis. A third PCR product obtained was also considered a fragment from a serine proteinase gene, but identification of the type of serine proteinase gene from which it was derived will have to await complete cloning and sequencing. A considerable amount of the proteinase activity expressed in E. coli remains in the cell (70 to 80%). This result is undesirable for possible commercial use, since full recovery of proteinase in the presence of E. coli proteins requires complex and expensive purification. However, the fact that proteinase is thermophilic offers some advantage, as cell extracts can be heated to precipitate and digest E. coli proteins (26). Alternatively, further studies could focus on the expression and secretion of the proteinase in a mesophilic Bacillus species, since cloning and expression of the thermophilic neutral metalloproteinase gene nprM from B. stearothermophilus and secretion of its product have been successfully carried out in B. subtilis (15).
ACKNOWLEDGMENTS This work was supported in part by a grant from Pacific Enzymes Ltd. and the University of Auckland Research Committee.
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