# Patent

## Title
Recombinant bacillus  proteases and uses thereof

## Abstract

The present invention provides genetically engineered Bacillus strains that can secrete large amount of Bacillus proteases in the extracellular culture medium. More particularly, this invention relates to a process of producing recombinant protease molecules of Bacillus origin in a Bacillus subtilis strain 168, utilizing a strong prophage promoter.

## Background
FIELD OF THE INVENTION
In this invention, a rapid expression system for Bacillus proteases was established. Genetically engineered Bacillus strains that can secrete large amount of Bacillus proteases in the extracellular culture medium were generated. More particularly, this invention relates to a process of producing recombinant protease molecules of Bacillus origin in a Bacillus subtilis strain 168, utilizing a strong prophage promoter.
BACKGROUND OF INVENTION
Subtilisin enzymes usually refer to extracellular serine endopeptidases from related Bacillus species: for example subtilisin Carlsberg from Bacillus licheniformis (Jacobs et al., Nucleic Acids Res 13: 8913–8926, 1985); subtilisin BPN from Bacillus amyloliquifaciens (Wells et al., Nucleic Acids Res 11: 7911–7925, 1983) and alkaline protease PB92 from Bacillus alcalophilus PB92 (Van Der Laan et al., Appl. Environ. Microbiol. 57, 901–909, 1991) etc. Subtilsin enzymes have been studied extensively in last decades because of their usefulness as additives to detergents, esp. to laundry detergents.
There are several advantages about these subtilisins mentioned above. They usually possess high efficiency and little specificity e.g. they can degrade almost all kinds of proteins. They can exhibit activity at high pH (pH 8–12) and in the presence of some surfactants. In addition they are extracellular enzymes secreted by the bacteria into the medium. Thus they can be isolated without breaking the bacterial cells, which makes the purification process easier and less costly.
To be suitable for use in detergents, proteases must exhibit the following properties:

    1. They must possess broad substrate specificity;
    2. They must have activity and stability at alkaline pH range.
    3. They must be stable at high temperature and in the presence of chelating agents, perborates and surfactants.
    4. They must be efficacious at low temperatures (20–40° C.).


However, the yield of subtilisin naturally secreted by Bacillus species is usually low and could not meet the requirement of industry. Fortunately, the application of genetic engineering has greatly enhanced its production yield (Jacobs et al., Gene 152: 69–74, 1995; Zaghloul et al., Enzyme Microb Technol 16: 534–537, 1994). Now subtilisins can be industrially produced. In this patent an expression system based on Bacillus subtilis was successfully used to produce subtilisins with high yield in a short period of time.
Enzyme Production by the Phase φ105 Overexpression System
In a previously established φ105 system (Thornewell et al., Gene 133:47–53, 1993), a defective prophage vector, φ105MU331 was derived for high-level protein over-expression expression in B. subtilis (Leung & Errington, Gene 154(1):1–6, 1995). In this derived system, not only efficient inducible (by heat) transcription of the gene is provided, but also, it prevented the lysis of the host cell. Thus the enzyme produced can be collected easily in the culture media without disruption of the cells, which means the purification steps can be greatly diminished. In addition to this, unlike E. coli, Bacilli are GRAS bacteria, the genes encoding their proteins are also GRAS to animals and thus, human.
SUMMARY OF THE INVENTION
The present invention provides genetically engineered Bacillus strains that can secrete large amount of Bacillus proteases in the extracellular culture medium. More particularly, this invention relates to a process of producing recombinant protease molecules of Bacillus origin in a Bacillus subtilis strain 168, utilizing a strong prophage promoter and a signal peptide from subtilisin E of the Bacillus subtilis. 
Preferred molecules of the present invention include protease genens subtilisin Carlsberg (SEQ ID NO:9), and subtilisin BPN′ (SEQ ID NO:7) from Bacillus licheniformis (ATCC 10716) and Bacillus amyloliquifaciens (ATCC 23844), respectively.
It is another object of the present invention to provide a rapid process for producing large quantity of protease enzyme.
In accordance with one aspect of the present invention, there are provided protease enzymes for applications in commercial processes, such as, detergent applications.
In a further aspect of the present invention, there is provided a process for producing related proteases by recombinant technology comprising a Bacillus host and the strong prophage promoter described in this invention.
In this invention, an integration vector pSGE containine the DNA of the signal peptide (SEQ ID NO:15) of the subtilisin E gene from a Bacillus subtilis strain was constructed. Subtilisin E is a natural secretion protein of Bacillus subtilis. In our studies, the expression yields of the proteases subtilisin Carlsberg and subtilisin BPN′ cloned from Bacillus licheniformis;(ATCC 10716) and Bacillus amyloliquifaciens (ATCC 23844) were greatly enhanced by replacing the native signal peptides of these proteases with that of the Subtilisin E. Since both target proteases were heterologous proteins to Bacillus subtilis, their signal peptides could not be properly processed by the secretory machinery of B. subtilis. By exchanging their signal peptides with that of the subtilisin E, this allowed proper secretion of these proteins.
The signal peptide DNA sequence from subtilisin E gene was first amplified by PCR and then cloned into the plasmid pSGt, which contains a terminator DNA from the α-amylase gene of Bacillus licheniformis. The plasmid containing signal peptide sequence of subtilisin E gene and terminator sequence of α-amylase gene was designated pSGE (FIG. 1).
Two alkaline protease genes, including subtilisin Carlsberg (SEQ ID NO:9) from Bacilus licheniformis (ATCC 10716) and subtilisin BPN′ (SEQ ID NO:7) from Bacillus amyloliquifaciens (ATCC 23844), were amplified and cloned into the integration vector pSGE, to create pSG-C and pSG-B, respectively. Plasmids containing these protease genes were then transformed into B. subtilis. Then transformants were screened on milk plate. Some transformants that showed larger clear zones than negative control on milk plate were used to overproduce the target proteases.
The invention features nucleic acid molecules which are at least 45% (or 55%, 65%, 75%, 85%, 95%, 98% or 99%) identical to the nueleotide sequence of any of SEQ ID NOS:7, 9, 14, or a complement thereof.
The invention features nucleic acid molecules which are at least 45% (or 55%, 65%, 75%, 85%, 95%, or 98%) identical to the nucleotide sequence of any of SEQ ID 7, 9, or a complement thereof, wherein such nucleic acid molecules encode polypeptides or proteins that exhibit at least one structural and/or functional feature of a polypeptide of the invention.
The invention features nucleic acid molecules of at least 200, 250, 300, 350, 400, 450, 500, 550, 575, 600, 625, 650, 675, 700, 725, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1125 or 1149 nucleotides of the nucleotide sequence of SEQ ID NO: 7.
The invention features nucleic acid molecules which include a fragment of at least least 200, 250, 300, 350, 400, 450, 500, 550, 575, 600, 625, 650, 675, 700, 725, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1125 or 1140 nucleotides of the nucleotide sequence of SEQ ID NO:9, or a complement thereof.
The invention features nucleic acid molecules of at least 250, 275, 300, 325, 350, 375, 400, 425, 450, or 479 nucleotides of the nucleotide sequence of SEQ ID NO:14, or a complement thereof.
The invention also features nucleic acid molecules which include a nucleotide sequence encoding a protein having an amino acid sequence that is at least 45% (or 55%, 65%, 75%, 85%, 95%, 98%, or 99%) identical to the amino acid sequence of any of SEQ ID Nos:8, 10, 11, or 12.
The invention also features nucleic acid molecules which include a nucleotide sequence encoding a protein having an amino acid sequence that is at least 45% (or 55%, 65%, 75%, 85%, 95%, 98%, or 99%) identical to the amino acid sequence of any of SEQ ID Nos:8, 10, 11, or 12, wherein the protein encoded by the nucleotide sequence also exhibits at least one structural and/or functional feature of a polypeptide of the invention.
Also within the invention are nucleic acid molecules which encode a fragment of a polypeptide having the amino acid sequence of any of SEQ ID Nos: 8, 10, 11, or 12, the fragment including at least 15 (20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 381 or 382) contiguous amino acids of any of SEQ ID NOs:8, 10, 11, or 12.
Also within the invention are nucleic acid molecules which encode a fragment of a polypeptide having the amino acid sequence of any of SEQ ID Nos: 8, 10, 11, or 12, the fragment including at least 15 (20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 381 or 382) contiguous amino acids of any of SEQ ID NOs:8, 10, 11, or 12, wherein the fragment exhibits at least one structural and/or functional feature of a polypeptide of the invention.
The invention includes nucleic acid molecules which encode a naturally occurring allelic variant of a polypeptide comprising the amino acid sequence of any of SEQ ID Nos: 8, 10, 11, or 12, wherein the nucleic acid molecule hybridizes under stringent conditions to a nucleic acid molecule having a nucleic acid sequence encoding any of SEQ ID Nos:8, 10, 11, or 12, or a complement thereof.
The invention includes nucleic acid molecules which encode a naturally occurring allelic variant of a polypeptide comprising the amino acid sequence of any of SEQ ID Nos:8, 10, 11, or 12, wherein the nucleic acid molecule hybridizes under stringent conditions to a nucleic acid molecule having a nucleic acid sequence encoding any of SEQ ID Nos:8, 10, 11, or 12, or a complement thereof, wherein such nucleic acid molecules encode polypeptides or proteins that exhibit at least one structural and/or functional feature of a polypeptide of the invention.
Also within the invention are isolated polypeptides or proteins having an amino acid sequence that is at least about 65%, preferably 75%, 85%, 95%, 98%, or 99% identical to the amino acid sequence of any of SEQ ID Nos:8, 10, 11, or 12.
Also within the invention are isolated polypeptides or proteins having an amino acid sequence that is at least about 65%, preferably 75%, 85%, 95%, or 98% identical to the amino acid sequence of any of SEQ ID Nos:8, 10, 11, or 12, wherein the polypeptides or proteins also exhibit at least one structural and/or functional feature of a polypeptide of the invention.
Also within the invention are polypeptides which are naturally occurring allelic variants of a polypeptide that includes the amino acid sequence of any of SEQ ID Nos:8, 10, 11, or 12, wherein the polypeptide is encoded by a nucleic acid molecule which hybridizes under stringent conditions to a nucleic acid molecule having the sequence of any of SEQ ID Nos:7, 9, or a complement thereof.
Also within the invention are polypeptides which are naturally occurring allelic variants of a polypeptide that includes the amino acid sequence of any of SEQ ID Nos:8, 10, 11, or 12, wherein the polypeptide is encoded by a nucleic acid molecule which hybridizes under stringent conditions to a nucleic acid molecule having the sequence of any of SEQ ID Nos:7 or 9, or a complement thereof, wherein such nucleic acid molecules encode polypeptides or proteins that exhibit at least one structural and/or functional feature of a polypeptide of the invention.
In yet another embodiment, a method is provided for producing a polypeptide, comprising:

    
    
        (a) cultivating a Bacillus cell in a medium conducive for the production of a polypeptide, wherein the Bacillus cell comprises a nucleic acid construct comprising a Φ105MU331 promoter in which the Φ105MU331 promoter sequence is operably linked to a nucleic acid sequence encoding the polypeptide; and
        (b) isolating the polypeptide from the cultivation medium.
    
    


In yet another embodiment of the above recited method, the nucleic acid sequence encodes a protease subtilisin gene product, Carlsberg (SEQ ID NO:10), cloned from a Bacillus licheniformis strain (ATCC No. 10716), the protease subtilisin BPN′ gene product (SEQ ID NO:8) from Bacillus amyloliquifaciens (ATCC No. 23844), or a combination thereof.
In yet another embodiment of the above recited method, the nucleic acid sequence encodes a protease subtilisin gene product coding sequence, Carlsberg (SEQ ID NO:9), cloned from a Bacillus licheniformis strain (ATCC No. 10716), the protease subtilisin BPN′ gene (SEQ ID NO:7) from Bacillus amyloliquifaciens (ATCC No. 23844), or a combination thereof.
In yet another embodiment of the above recited method, the nucleic acid sequence encodes a protease subtilisin gene product coding sequence, Carlsberg (SEQ ID NO:9), cloned from a Bacillus licheniformis strain (ATCC No. 10716), or the protease subtilisin BPN′ gene (SEQ ID NO:7) from Bacillus amyloliquifaciens (ATCC No. 23844) operably linked to the 30 amino acid signal peptide (SEQ ID NO:15) of Bacillus subtilis 168 subtilisin E.
In yet another embodiment of the above recited method, the nucleic acid construct further comprises a selectable marker gene.
In yet another embodiment of the above recited method, the selectable marker is the CAT gene.
In yet another embodiment of the above recited method, the Bacillus cell contains no selectable marker gene.
In yet another embodiment of the above recited method, the nucleic acid sequence encodes a polypeptide heterologous to the Bacillus cell.
In yet another embodiment of the above recited method, the polypeptide is a hormone or variant thereof, enzyme, receptor or portion thereof, antibody or portion thereof, or reporter.
In yet another embodiment of the above recited method, the enzyme is an aminopeptidase, amylase, carbohydrase, carboxypeptidase, catalase, cellulase, chitinase, cutinase, cyclodextrin glycosyltransferase, deoxyribonuclease, esterase, alpha-galactosidase, beta-galactosidase, glucoamylase, alpha-glucosidase, beta-glucosidase, invertase, laccase, lipase, mannosidase, mutanase, oxidase, pectinolytic enzyme, peroxidase, phytase, polyphenoloxidase, proteolytic enzyme, ribonuclease, transglutaminase, or xylanase.
In yet another embodiment of the above recited method, the Bacillus host cell is a Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillus firmus, Bacillus lautus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus pumilus, Bacillus stearothermophilus, Bacillus subtilis, Bacillus thuringiensis, Bacillus thermoleovorans strain (ATCC No. 43506), Bacillus subtilis strain 168, or Bacillus subtilis strain MU331.
In yet another embodiment of the above recited method, the nucleic acid sequence encodes a polypeptide homologous to the Bacillus cell.
In yet another embodiment of the above recited method, the polypeptide is a protease.
In yet another embodiment of the above recited method, the Bacillus cell is a Bacillus amyloliquefaciens cell.
In yet another embodiment of the above recited method, the Bacillus cell is a Bacillus licheniformis cell.



FIGURE LEGENDS
The following figures illustrate the embodiments of the invention and are not meant to limit the scope of the invention encompassed by the claims.
FIG. 1. (A) Schematic map of the expression vector pSGt. Heterologous gene(s) is inserted into the multicloning sites (MCS) of the vector. The vector carries a pBR 322 replication origin ORI for E. coli, the promoter and the ribosome binding site of the ORF 19 of the bacteriophage phi-105 (Φ-105), the terminator of B. licheniformis α-amylase (term), a cat gene for chloramphenical resistance and a bla gene for ampicillin resistance. (B) Schematic maps of the expression vectors pSG-E, pSG-C and pSG-B.
FIG. 2. The amino acid sequences of the subtilisin E-protease fusion proteins. Amino acid residues representing the signal peptide of B. subtilis subtilisin E were shown in Italics. The DNA sequences encoding the mature proteases subtilisin Carlsberg and subtilisin BPN′ were cloned in frame with the signal peptide of B. subtilis subtilisin E. The amino acid residues determined by N-terminal protein sequencing were underlined.
FIG. 3. Overexpression of the recombinant proteases in shaking flasks using various media. (A) Overexpression of protease BPN′. (B) Overexpression of protease Carlsberg. At 4 hours after heat induction, 10 ul culture supernatants were run into each lane. Protein markers were loaded on the first lane on the left. CP: 100 ug commercial enzyme powder concentrate from NovoNordis (Savinase 4.0T).
FIG. 4. Overexpression of the recombinant proteases in a 2 L fermentor. (A) Overexpression of protease BPN′. (B) Overexpression of protease Carlsberg. Protein markers were loaded on the first lane on the left. CP: 100 ug commercial enzyme powder concentrate from NovoNordis (Savinase 4.0T).
FIG. 5. Thermal stability test.
FIG. 6. Overexpression of protease BPN′ using a medium rich in soybean meal (SBM). 10 μl supernatants were run into each lane. CP: 100 ug commercial enzyme powder concentrate from NovoNordis (Savinase 4.0T).



EXAMPLES
1. Amplication of Protease Genes from B. subtilis Strain 168, Bacillus amyloliquifaciens and Bacillus licheniformis 
Bacillus subtilis 168, Bacillus amyloliquifaciens (ATCC 23844) and Bacillus licheniformis (ATCC 10716) were used as the source of chromosomal DNA. PCR was performed with three pairs of primer (Table 1). The first pair of primer, SubES (SEQ. ID No. 1) and SubEA (SEQ. ID No. 2) was used to amplify the signal peptide of the subtilisin E gene from Bacillus subtilis 168. The other two pairs of primers, BPN′-S (SEQ. ID No. 3)/BPN′-A (SEQ. ID No. 4) and CAR-S (SEQ. ID No. 5)/CAR-A(SEQ. ID No. 6) were used to amplify the protease genes BPN′ and Carlsberg from B. licheniformis and B. amyloliquifaciens, respectively. PCR was carried out for 30 cycles with each cycle composed of 4 min at 94° C. (denaturation), 40 second at 53° C. (annealing), 3 min at 75° C. (extension), Pfu polymerase was employed to safeguard the fidelity of the reaction. The DNA and its deduced amino acid sequences of protease BPN′ are shown in SEQ. ID No. 7 and SEQ. ID No. 8, whereas those of the protease Carlsberg are shown in SEQ. ID No. 9 and SEQ. ID No. 10. It was expected that the signal peptides of the proteases from B. licheniformis and B. amyloliquifaciens might not be correctly processed in the expression host B. subtilis, therefore only the gene fragment encoding for the mature enzyme was amplified.








TABLE 1







Primers employed in this study












Primer name
Sequence
Orientation
Template





SubES
gcgatcgCATATGAGAAGCAAAAAATTGTGGATCAGC
sense
Signal peptide for subtilisin E



(SEQ ID


NO:1)


 


SubEA
gcggatccGCGGCCGCCTGCGCAGACATGTTGC
antisense
Signal peptide for subtilisin E


(SEQ ID


NO:2)


 


BPN-S
gcgatatcGCGGCCGCAGGGAAATCAAACGGGGAA
sense
Mature BPN gene


(SEQ ID


NO:3)


 


BPN-A
gcGGATCCACTTGGCCGTTACGGGACT
antisense
Mature BPN gene


(SEQ ID


NO:4)


 


CAL-S
gcgatatcGCGGCCGCTCAACCGGCGAAAAATGTT
sense
Mature Calsberg gene


(SEQ ID


NO:5)


 


CAL-A
gcGGATCCTTATTGAGCGGCAGCTTCGAC
antisense
Mature Calsberg gene


(SEQ ID


NO:6)


 


phi-1-5
ATAGACAATCGGCGGTTAAC
sense
phi-105 ORF19 promoter


SEQ ID


NO:13)









2. Construction of pSGt Expression Plasmids Carrying the Protease Genes

The PCR fragments were purified by phenol/chloroform extraction and ethanol precipitation. The DNA fragment obtained by the SubE primers, the BPN′ primers and the CAR primers were subjected to Nde I/Bam H1, Not I/Bam HI and Not I/Bam HI restriction enzyme digestion, respectively. Since Subtilisin E is of B. subtilis origin, its native signal peptide was compatible to the expression host strain B. subtilis. Therefore, the coding sequence of its signal peptide was subcloned into the Nde I and Bam HI sites of the cloning vector pSGt (FIG. 1) to create pSG-E, in which a Not I site was engineered. On the other hand, since the signal peptides of the protease from B. licheniformis and B. amyloliquifaciens were not compatible to B. subtilis, only the coding sequences of the mature enzymes were cloned into the NotI/BamHI sites of the pSGE to create pSG-B and pSG-C. The map of these vectors is shown in FIG. 1B. The amino acid sequences of the resultant fusion proteins (SEQ. ID Nos. 11 and 12) were aligned in FIG. 2. After ligation overnight, the plasmids were ethanol precipitated before transformation into E. coli. Competent E. coli cells (Top 10) were prepared and transformed by electroporation with the Bio Rad pulser under the conditions recommended by the supplier. The electroporated cells were transferred to 2×YT and incubated at 37° C. for 40 minutes before spreading onto LB-agar plates with ampicillin (100 μg/ml). After overnight incubation, colonies were picked from the LA-agar plates and screened by PCR. Positive clones were selected and grown in LB broth with ampicillin (100 μg/ml) overnight. The plasmids were extracted from the cells by using the Bio Rad Quantum prep Plasmid Miniprep kit, under the recommended conditions.
3. Transformation of Bacillus subtilis 
A Bacillus recipient strain (B. subtilis MU331) was streaked onto a LB-agar plate with erythromycin (5 μg/ml) and incubated at 37° C. overnight. Multiple colonies were inoculated into 5 ml pretransformation medium [(PTM)—2.2% (v/v) of 40% w/v glucose; 1% (v/v) solution P (0.5 ml of 0.1M CaCl2.2H2O, 2.5 ml of 1.0M MgSO4.7H2O, 0.01 ml of 1.0M MnSO4.4H2O and 7.0 ml ddH2O); 1.8% (v/v) Casamino (2 g/L); 1% (v/v) Tryptophan (2 mg/ml); 1% (v/v) Isoleucine (20 mg/ml); 1% (v/v) Valine (20 mg/ml); 1% (v/v) Leucine (20 mg/ml); 1% (v/v) Methionine (5 mg/ml); 90% (v/v) of Spizizen minimal medium (0.2% (w/v) ammonium sulphate, 1.4% (w/v) dipotassium phosphate, 0.6% (w/v) potassium dihydrogen phosphate, 0.1% (w/v) sodium citrate dihydrate, 0.02% (w/v) magnesium sulphate)], and incubated at 37° C. with shaking at 280 rpm. Cell growth was monitored, until OD600 reached 3.0 ̃3.3.
One-hundred (100) μl of competent cells were mixed with about 2 μg DNA and transferred into 1 ml pre-warmed (37° C.) transformation medium[(TM)—1.4% (v/v) of 40% (w/v) glucose; 0.47% (v/v) solution F (1.0M MgSO4.7H2O); 0.05% (v/v) Casamino (2 g/l); 0.93% (v/v) Tryptophan (2 mg/ml); 0.93% (v/v) Isoleucine (20 mg/ml); 0.93% (v/v) Valine (20 mg/ml); 0.93% (v/v) Leucine (20 mg/ml); 0.93% (v/v) Methionine (5 mg/ml); 93.4% (v/v) of Spizizen minimal medium (0.2% (w/v) ammonium sulphate, 1.4% (w/v) dipotassium phosphate, 0.6% (w/v) potassium dihydrogen phosphate, 0.1% (w/v) sodium citrate dihydrate, 0.02% (w/v)magnesium sulphate)] and incubated at 37° C. with shaking at 280 rpm for 1.5 hour. The culture was centrifuged (13,200 rpm, 2 min) and 150 μl was spread onto LB-agar plates with chloramphenicol (5 μg/ml). The plates were incubated at 37° C. overnight and each single colony was transferred onto a LB-agar plate with erythromycin (5 μg/ml) and one with chloramphenicol (5 μg/ml) on the next day. PCR screening was performed on the colonies by using a promoter specific primer, phi-105, (SEQ ID NO:13) and one of the protease specific antisense primers.
4. Overexpression of Proteases in B. subtilis MU331
Bacterial cells from frozen stock was streaked onto LB-agar plate with chloramphenicol (5 μg/ml) and incubated at 37° C. overnight. A single colony was inoculated into 15 ml BHY medium (3.7% (w/v) brain-heart infusion broth, 0.5% (w/v) yeast extract) with chloramphenicol (5 μg/ml) and incubated at 37° C. with shaking at 270 rpm overnight. 12 ml of the overnight culture was transferred into 100 ml BHY medium without chloramphenicol, and incubated at 37° C. with shaking at 280 rpm. Cell growth was monitored by reading OD600 until it reached 5.3–5.9. Heat shock was then performed by placing the sample in a 50° C. water-bath for 8 minutes with vigorous shaking and then re-incubated at 37° C., with shaking at 280 rpm. At 4 hours post-induction, all samples were collected and centrifuged to remove cell pellets.
5. N-terminal Amino Acid Sequence Analysis
N-terminal amino acid sequence analysis was performed after protein separation by SDS/PAGE and transferred to PVDF membrane. The band of interest was cut out and subjected to automated Edman degradation with the Hewlett Packard protein sequencer (model G1000A), in accordance with the manufacturer's instruction. Analysis of the N-terminal sequence of the mature protease A shows that the signal peptides and the propeptides of the proteases were correctly cleaved. The first seven amino acid residues of the mature protease Carlsberg and BPN′ were AQTVPYG and AQSVPYG, respectively. As a result, both mature proteases had 275 amino acid residues.
6. Protease Activity Assay Using succinyl-Ala-Ala-Pro-Phe-p-nitroanilide (SucAAPF-pNA) Mitchinson and Wells, Biochemistry 28: 4807–4815, 1989)
A synthetic substrate succinyl-Ala-Ala-Pro-Phe-p-nitroanilide (SucAAPF-pNA) was employed for protease activity enzyme. The colourless substrate can be converted into a yellowish product, p-nitroanilide (pNA), by protease activity. A 0.5 mM stock of SucAAPF-pNA was prepared in a 0.1 M Tris-HCl buffer, pH 8.6 and the enzyme-containing culture supernatants were diluted in the same buffer. 10 ul diluted enzyme was then mixed with 190 ul substrate in triplicate into the wells of a 96 well ELISA plate and incubated at 22° C. A blank was prepared by mixing 10 ul Tris-HCl buffer with 190 ul substrate. OD reading at 405 nm was taken at 20 mins and 30 mins, respectively. To prepare a standard curve, a 1 mM p-nitroaniline (pNA) stock was serially diluted and OD405 readings were measured. One enzyme unit (EU) is defined as the amount of enzyme that releases 1 micromole of pNA per minute at 22° C.
7. Protease Activity Assay Using Azocoll as Substrates (Chavira, et al., Anal Biochem 136: 446–450 1984)
50 mM Tris-HCl containing 5 mM calcium chloride at pH 8.0 was used as the assay buffer. 150 mg Azocoll was first stirred in 30 ml of assay buffer for 2 hours before filtered through a Whatmnan No. 1 filter paper to remove the filtrate. The undissolved pellet was resuspended in 30 ml assay buffer and 1 ml aliquots were made. Protease in the culture supernatant was diluted 50 times in the assay buffer and 20 ul diluted enzyme is added to 1 ml Azocoll suspension which has been preheated at 37° C. After incubating the mixture at 37° C. for 1 hour, the reaction was stopped on ice bath and unhydrolysed azocoll was removed by centrifugation at 10000 g for 5 min. The color of the supernatant was measured at OD520nm. One enzyme unit (EU) is defined as the amount of enzyme that produces an OD520 nm of 0.5.
8. Overexpression of the Proteases in Shake Flasks Using Industrial Media
Five media were formulated by industrial grade chemicals and their ability to support protease overexpression was examined. The formulations are shown in Table 2. A single colony was inoculated into 15 ml BHY medium (3.7% (w/v) brain-heart infision broth, 0.5% (w/v) yeast extract) with chloramphenicol (5 μg/ml) and incubated at 37° C. with shaking at 270 rpm overnight. 1 ml of the overnight culture was transferred into 20 ml formulated media without chloramphenicol, and incubated at 37° C. with shaking at 280 rpm. Heat shock was then performed at 4 hours post-inoculation by placing the flasks in a 50° C. water-bath for 5 minutes with vigorous shaking and then re-incubated at 37° C., with shaking at 280 rpm. At 4 hours post-induction, all samples were collected and centrifuged to remove cell pellets and 20 μl supernatant collected was subjected to SDS/PAGE. The overexpression of protease Carlsberg and BPN′ in various media were shown in FIGS. 3A and 3B, respectively. The enzyme activities were shown in Table 3.







TABLE 2





Formulations of various media




















Medium A3
g/liter
Medium B2
g/liter







Malto-dextrin
50
Malto-dextrin
50



NH4NO3
 4
NH4NO3
 4



Hydrolyzated
 6
Yeast extract
10



soybean protein







Medium E1
g/liter
Medium E2
g/liter







Hydrolyzated
25
Hydrolyzated
40



soybean protein

soybean protein



CaCO3
 5
CaCO3
 5



Potato Starch
 5
Potato Starch
 5







Medium N5
g/liter







Glucose
50



NaHCO3
 5



(NH4)2HPO4
 2.5



Yeast extract
10

















TABLE 3







Enzyme activities of proteases Carlsberg and BPN′


expressed in various media.















Pro-










teases
Substrates
BHY
A3
B2
E1
E2
N5
CP





Carls-
sAAPF-pNA
50.95
18.39
28.12
43.25
44.77
17.77
0.98


berg


BPN′
sAAPF-pNA
5.70
3.23
5.26
2.32
1.41
3.27
0.98


Carls-
Azocoll
1176
404
604
950
985
391
180


berg


BPN′
Azocoll
866
508
804
324
200
520
180









The enzyme activities are expressed in U/ml for the media BHY, A3, B2, E1, E2 and N5. For CP, a protease powder concentrate (Savinase 4.0T) from NovoNordis, the enzyme unit is expressed in U/mg.

9. Overexpression of Proteases BPN′ and Carlsberg in Fermentor

A fresh bacillus colony was inoculated into 80 ml BHY medium with chloramphenicol and the seed culture was shaken at 37° C. at 280 rpm. After overnight incubation, 80 ml seed culture was inoculated into 1.6 L BHY medium without chloramphenicol in a 2 litre fermentor (Biostat B, B. Bruan International). The temperature was controlled at 37° C. and the pH was controlled at 7.0 by addition of acid and base. Dissolved oxygen was maintained at 30% by a control loop that varied the stir speed. Heat induction was carried out when the OD reached 5.0 by increasing the temperature to 50° C. and then decreased to 37° C. immediately. Usually this heat induction process could be completed in 30 mins. Culture supernatants were recovered at 4 hours and 16 hours post-induction for enzyme activity assay and SDS-PAGE analysis. The results of SDS-PAGE were shown in FIG. 4. In general, higher yields of proteases were obtained from fermentor than from shaking flasks.







TABLE 4







Enzyme activities of proteases Carlsberg


and BPN′ produced in fermentor.














Shake
4 hr post-
16 hr post-



Proteases
Substrates
flask
induction
induction
CP















Carlsberg
sAAPF-pNA
40.1
81.4
124.39
0.98


Carlsberg
Azocoll
856
1756
2728
180


BPN′
sAAPF-pNA
2.62
5.32
6.46
0.98


BPN′
Azocoll
514
954
1358
180









The enzyme activities are expressed in U/ml of culture supernatant. For CP, a protease powder concentrate (Savinase 4.0T) from NovoNordis, the enzyme unit is expressed in U/mg.

10. Thermal Stability of the Proteases

The thermal stability of the expressed proteases Carlsberg and BPN′ was compared with a commercial protease (Savinase 4.0T). The protease solutions were first incubated at room temperature, 40° C., 50° C., 60° C., 70° C and 80° C. for 20 min down on ice. Afterwards, the protease solutions were equilibrated to room temperature and a protease activity assay using SucAAPF-pNA as substrate was carried out. As shown in FIG. 5, the protease BPN′ and the commercial protease have better thermal stability than the protease Carlsberg.
11. Overexpression of Protease BPN′ Using a Medium Rich in Soybean Meal
The ability of soybean meal to support the expression of protease BPN′ was investigated by the procedures described in Example 7. Media were prepared with increasing amount of soybean meal, from 25 g to 100 g per litre, and their ability to support protease overexpression were examined in shake flask. At 4 hours post-induction, all samples were collected and 10 μl supernatant was analyzed by SDS/PAGE (FIG. 6). The enzyme activities are shown in table 5.










Media ingredients:










Medium SBM
g/liter







Soybean Meal
 25–100



CaCO3
5



Potato Starch
5

















TABLE 5







Enzyme activities of proteases BPN′ expressed in various media.















Proteases
Substrates
BHY
B2
E1
SBM25 g
SBM50 g
SBM100 g
CP


















BPN′
sAAPF-pNA
9.51
5.29
5.22
2.80
8.42
32.09
0.82









The enzyme activities are expressed in U/ml of culture supernatant. For CP, a protease powder concentrate (Savinase 4.0T), the enzyme unit is expressed in U/mg.

EQUIVALENTS
It will be appreciated that the various features described herein may be used singly or in any combination thereof. Therefore, the present invention is not limited to only the embodiments specifically described herein. While the foregoing description and drawings represent a preferred embodiment of the present invention, it will be understood that various additions, modifications, and substitutions may be made therein without departing from the spirit and scope of the present invention as defined in the accompanying claims. In particular, it will be clear to those skilled in the art that the present invention may be embodied in other specific forms, structures, arrangements, proportions, and with other elements, materials, and components, without departing from the spirit or essential characteristics thereof. One skilled in the art will appreciate that the invention may be used with many modifications of structure, arrangement, proportions, materials, and components and otherwise, used in the practice of the invention, which are particularly adapted to specific environments and operative requirements without departing from the principles of the present invention. The presently disclosed embodiment is therefore to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims, and not limited to the foregoing description.

## Claims

1. A process for production of a protease comprising cultivating a recombinant Bacillus strain containing an expression vector comprising:
a. a promoter region of the ORF19 of the bacteriophage phi-105 consisting of SEQ ID NO:14; and
b. a DNA molecule, encoding the protease, wherein said molecule has over 95% homology to SEQ ID NO:7 or 9.

2. The process according to claim 1, wherein said DNA molecule is SEQ ID NO: 7 or 9.

3. The process according to claim 1, in which the promoter is induced by heat.

4. A process according to claim 1, further comprising the steps of separating or purifying the said protease.

5. A process for production of a protease comprising cultivating a recombinant Bacillus strain containing an expression vector comprising:
a. a promoter region of the ORF19 of the bacteriophage phi-105 consisting of SEQ ID NO: 14; and
b. a DNA molecule encoding the protease wherein said protease is from any prokaryotic or eukaryotic organism and wherein said DNA molecule has over 95% homology with SEQ ID NO: 7 or 9.

6. An expression vector comprising:
a. a promoter region of the OFR19 of the bacteriophage phi-105 consisting of SEQ ID NO:14; and
b. a nucleic acid molecule encoding an amino acid sequence of SEQ ID NO: 8 or 10.