PROTEASE VARIANTS AND USES THEREOF

Abstract

Disclosed herein is one or more subtilisin variant useful for cleaning applications and in methods of cleaning, as well as in a variety of industrial applications. One embodiment is directed to one or more subtilisin variant, including one or more Bacillus sp. subtilisin polypeptide variant, and one or more cleaning composition comprising one or more such variant.

Claims

1. An subtilisin variant comprising an amino acid sequence comprising an amino acid substitution at one, two, three, or four or more positions selected from 12, 14, 15, 26, 37, 48, 53, 55, 56, 78, 101, 105, 120, 122, 133, 143, 155, 164, 165, 166, 169, 175, 184, 197, 198, 220, 238, 243, 248, 254, 255, 259, 264, 268, 272, 274, and 275; and wherein the amino acid positions of the variant are numbered by correspondence with the amino acid sequence of SEQ ID NO:1.

2. The subtilisin variant of claim 1, wherein said variant comprises an amino acid sequence comprising one, two, three, or four or more amino acid substitutions selected from (i) Q12X, T14X, D15X, V26X, R37X, H48X, D53X, A55X, N56X, N78X, S101X, S105X, G/N120X, D122X, S133X, I143X, A155X, R164X, G165X, D166X, G169X, D175X, D184X, P/S197X, A198X, T220X, L/W238X, N243X, R248X, N254X, D255X, N259X, N264X, H/N268X, R272X, V274X, and D175X; (ii) Q12X, T14X, D15X, V26X, R37X, H48X, D53X, A55X, N56X, N78X, S101X, S105X, N120X, D122X, S133X, I143X, A155X, R164X, G165X, D166X, G169X, D175X, D184X, P197X, A198X, T220X, L238X, N243X, R248X, N254X, D255X, N259X, N264X, N268X, R272X, V274X, and D175X; or (ii) X12K, X14D, X15K, X26R, X37A, X48R, X53G/R, X55R, X56K, X78D, X101R, X105K, X120R, X122H, X133R, X143R, X155V, X164N, X165R, X166N, X169D, X175N/P/S, X184S, X197K, X198E, X220R, X238K, X243R, X248Q, X254K, X255N, X259R, X264K, X268K, X272N, X274E, and X275S/K; and wherein X is any amino acid.

3. The subtilisin variant of any preceding claim, with the proviso that the (i) amino acid substitution at position 120 is not a G; (ii) amino acid substitution at position 197 is not an S; (iii) amino acid substitution at position 238 is not a W; (iv) amino acid substitution at position 268 is not H; (v) said variant does not consist of the amino acid sequence of BAD02409 or JP2003325186-0001; and/or (vi) said variant does not consist of the amino acid sequence of SWT183_1430046.

4. The subtilisin variant of claim 1 or 2, wherein said variant comprises an amino acid sequence comprising one, two, three, or four or more amino acid substitutions selected from: (i) Q12K, T14D, DISK, V26R, R37A, H48R, D53G/R, A55R, N56K, N78D, S101R, S105K, G/N120R, D122H, S133R, I143R, A155V, R164N, G165R, D166N, G169D, D175N/P/S, D184S, P/S197K, A198E, T220R, L/W238K, N243R, E248Q, N254K, D255N, N259R, N264K, H/N268K, R272N, V274E, and D275S/K; or (ii) Q12K, T14D, DISK, V26R, R37A, H48R, D53G/R, A55R, N56K, N78D, S101R, S105K, N120R, D122H, S133R, I143R, A155V, R164N, G165R, D166N, G169D, D175N/P/S, D184S, P197K, A198E, T220R, L238K, N243R, E248Q, N254K, D255N, N259R, N264K, N268K, R272N, V274E, and D275S/K.

5. The subtilisin variant of any preceding claim, wherein said variant comprises an amino acid sequence comprising nine or more amino acid substitutions selected from: R37A-D53G-S101R-N120R-I143R-D166N-D184S-P197K-L238K-E248Q-D255N-R272N; D15K-D53R-N56K-S105K-D122H-D166N-D184S-P197K-E248Q-D255N-V274E; D15K-A55R-S101R-N120R-I143R-D166N-D184S-P197K-L238K-E248Q-N268K-V274E; H48R-A55R-S101R-N120R-S133R-R164N-L238K-E248Q-D255N-N268K; Q12K-D15K-R37A-D122H-R164N-G169D-E248Q-N268K-D275S; D15K-H48R-A55R-S101R-N120R-I143R-G165R-D175N-A198E-N243R-N254K-D275K; Q12K-V26R-R37A-A55R-N78D-S105K-I143R-A155V-D184S-A198E-N243R-N259R-D275K; T14D-D53R-S101R-I143R-R164N-D175S-A198E-N243R-D275K; D15K-D53R-S101R-N120R-I143R-G165R-D175S-N243R-D255N-N264K-D275K; Q12K-V26R-R37A-H48R-A55R-N78D-S101R-D122H-I143R-D166N-D175P-D184S-Y220R-N243R-D255N-R272N; and combinations thereof.

6. The subtilisin variant of any preceding claim, wherein said variant further comprises one or more motif selected from a: i. DTGIXXXHXDLXXXGGXSVFXXXXXXXXXXDXXGH (SEQ ID NO:31) motif, wherein the initial D is the active site Aspartic acid, the terminal H is the active site Histidine, and X is any amino acid; ii. DTGIXXXHXDLXXXGGXSVFXXXXXXDPXXDXXGH (SEQ ID NO:32) motif, wherein the initial D is the active site Aspartic acid, the terminal H is the active site Histidine, and X is any amino acid; iii. DTGIXXXHXDLNVXGGXSVFXXXXXXXXXXDXXGH (SEQ ID NO:33) motif, wherein the initial D is the active site Aspartic acid, the terminal H is the active site Histidine, and X is any amino acid; iv. DTGIXXXHXDLNVXGGXSVFXXXXXXDPXXDXXGH (SEQ ID NO:34) motif, wherein the initial D is the active site Aspartic acid, the terminal H is the active site Histidine, and X is any amino acid; v. DTGIDXXHXDLNVXGGXS VFXXXXXXXXXXDXXGH (SEQ ID NO:35) motif, wherein the initial D is the active site Aspartic acid and the terminal H is the active site Histidine, and X is any amino acid; vi. DTGIDXNHXDL NVRGGXSVFTXXXXX DPXXDXXGH (SEQ ID NO:36) motif, wherein the initial D is the active site Aspartic acid, the terminal H is the active site Histidine, and X is any amino acid; and vii. DTGIDXNHXDLNVRGGXSVFTXXXXX DPYYDXXGH (SEQ ID NO:37) motif, wherein the initial D is the active site Aspartic acid and the terminal H is the active site Histidine, and X is any amino acid.

7. The subtilisin variant of any preceding claim, wherein said variant is from a reference subtilisin comprising an amino acid sequence having 70, 75, 80, 85, 90 or 95% amino acid sequence identity to the amino acid sequence of SEQ ID NO:1, 28, 29, or 34.

8. The subtilisin variant of any preceding claim, wherein said variant further comprises an amino acid sequence having at least 70, 80, 90, or 95% amino acid sequence identity to an amino acid sequence of SEQ ID NO:1 or 34.

9. The subtilisin variant of any preceding claim, wherein said variant has protease activity and/or is isolated.

10. The subtilisin variant of any preceding claim, wherein said variant has one or more improved property when compared to a reference subtilisin, wherein the improved property is selected from improved protease activity, improved cleaning performance in detergent, and improved thermostability in detergent.

11. The subtilisin variant of any preceding claim, wherein the reference subtilisin comprises an amino acid sequence of SEQ ID NO:1 or 34.

12. The subtilisin variant of claim 10 or 11, wherein the improved property is (i) improved protease activity, wherein said variant has a PI>1 on N-suc-AAPF-pNA or dimethyl casein substrate; (ii) improved cleaning performance, wherein said variant has a BMI, POM, and/or egg yolk with pigment swatch cleaning PI>1; and/or (iii) improved thermostability, wherein said variant has a stability PI>1.

13. The subtilisin variant of claim 12, wherein said (i) protease activity is measured in accordance with the protease activity assay of Example 1; (ii) cleaning performance in detergent is measured in accordance the cleaning performance in detergent assay of Example 1; and/or (iii) thermostability in detergent is measured in accordance with the thermostability in detergent assay of Example 1.

14. A composition comprising one or more subtilisin variant of any preceding claim.

15. The composition of claim 14, wherein said composition is a detergent composition.

16. The composition of claim 15, wherein the detergent composition is selected from the group consisting of a laundry detergent, a fabric softening detergent, a dishwashing detergent, and a hard-surface cleaning detergent.

17. The composition of any one of claims 14-16, wherein said composition further comprises at least one ion selected from calcium and zinc; at least one stabilizer; from about 0.001% to about 1.0 weight % of said variant; at least one bleaching agent; at least one adjunct ingredient; and/or one or more additional enzymes or enzyme derivatives selected from the group consisting of acyl transferases, alpha-amylases, beta-amylases, alpha-galactosidases, arabinosidases, aryl esterases, beta-galactosidases, carrageenases, catalases, cellobiohydrolases, cellulases, chondroitinases, cutinases, endo-beta-1,4-glucanases, endo-beta-mannanases, esterases, exo-beta-mannanases, galactanases, glucoamylases, hemicellulases, hyaluronidases, keratinases, laccases, lactases, ligninases, lipases, lipoxygenases, mannanases, oxidases, pectate lyases, pectin acetyl esterases, pectinases, pentosanases, perhydrolases, peroxidases, phenoloxidases, phosphatases, phospholipases, phytases, polygalacturonases, additional proteases, pullulanases, reductases, rhamnogalacturonases, beta-glucanases, tannases, transglutaminases, xylan acetyl-esterases, xylanases, xyloglucanases, xylosidases, metalloproteases, and combinations thereof.

18. The composition of any one of claims 14-17, wherein said composition contains phosphate or is phosphate-free and/or contains borate or is borate-free.

19. The composition of any one of claims 14-18, wherein said composition is a granular, powder, solid, bar, liquid, tablet, gel, paste or unit dose composition.

20. A method of cleaning, comprising contacting a surface or an item in need of cleaning with the variant of any one of claims 1-13 or the composition of any one of claims 14-19; and optionally further comprising the step of rinsing said surface or item after contacting said surface or item with said variant or composition.

21. The method of claim 20, wherein said item is dishware or fabric.

22. A polynucleotide comprising a nucleic acid sequence that encodes the variant of any one of claims 1-13, wherein said polynucleotide is optionally isolated.

23. An expression vector or cassette comprising the polynucleotide of claim 22.

24. The expression vector or cassette of claim 23, wherein the nucleic acid sequence is operably linked to a promoter.

25. A recombinant host cell comprising the vector or cassette of claim 23 or the polynucleotide of claim 22.

26. A composition comprising the variant of any one of claims 1-13, wherein said composition is an animal feed, contact lens cleaning, wound cleaning, or textile, leather or feather processing composition.

Description

EXAMPLE 1

Assays

Performance Index

[0153] The performance index (PI) of an enzyme compares the performance of the variant (measured value) with the parent enzyme (theoretical value or measured value) at the same protein concentration. Theoretical concentrations for the parent enzyme can be calculated using the parameters extracted from a Langmuir fit of a standard curve of the parent enzyme. A PI that is greater than 1 (PI>1) indicates improved performance by a variant as compared to a parent or benchmark protease (defined for each analyzed data set), while a PI of 1 (PI=1) identifies a variant that performs the same as the parent, and a PI that is less than 1 (PI<1) identifies a variant that performs worse than the parent.

Protein Determination Assay

[0154] Protein concentration determination for BspE04637-T1 parent protease (SEQ ID NO:1) and BspE04637 variants obtained from culture supernatant was performed with an Agilent U-HPLC system. A calibration curve (0-500 ppm) using purified BspE04637-T1 protein was generated and used to generate a standard curve. Aliquots of clarified culture supernatants were diluted 10 fold in dilution buffer (Tris 25 mM, pH 7.4, 5 mM CaCl.sub.2) and then mixed on a 1:1 ratio with an Acetonitrile containing buffer (22.5 mM Tris, pH 7.4, 4.5 mM CaCl.sub.2, 9% acetonitrile). These samples were filtered on 45 m filter plates and filtrate was loaded via an auto-sampler onto a reverse phase column (Zorbax 300 SB-C3 column). A linear elution gradient of Buffer A (0.1% Trifluoroacatic acid) and Buffer B (0.07% Acetonitrile was used. At a flow rate of 1 mL/min with a 4 min run time and a 1 min post-run column equilibration. Absorbance was measured at 220 nm, and peaks were integrated using ChemStation software (Agilent Technologies). The protein concentration of the samples was calculated based on the standard curve of the purified BspE04637-T1 parent enzyme.

Protease Activity Assays

[0155] The protease activity of BspE04637-T1 parent protease (SEQ ID NO:1) and variants thereof was tested by measuring hydrolysis of N-suc-AAPF-pNA, dimethyl casein (DMC).

[0156] For the AAPF assay, the reagent solutions used were: 100 mM Tris pH 8.6, 10 mM CalCl.sub.2, 0.005% Tween-80 (Tris/Ca buffer) and 160 mM suc-AAPF-pNA in DMSO (suc-AAPF-pNA stock solution) (Sigma: S-7388). To prepare a working solution, 1 mL suc-AAPF-pNA stock solution was added to 100 mL Tris/Ca buffer and mixed. An enzyme sample was added to a microtiter plate (MTP) containing 1 mg/mL suc-AAPF-pNA working solution and assayed for activity at 405 nm over 3 minutes using a SpectraMax plate reader in kinetic mode at RT. The protease activity was expressed as mOD/min.

[0157] For the DMC assay, the reagent solutions used were: 2.5% Dimethylcasein (DMC, Sigma) in 100 mM Sodium Carbonate pH 9.5, 0.075% TNBSA (2,4,6-trinitrobenzene sulfonic acid, Thermo Scientific) in Reagent A. Reagent A: 45.4 g Na.sub.2B.sub.4O.sub.7.10H.sub.2O (Merck) in 15 mL 4N NaOH to reach a final volume of 1000 mL in MQ water, Dilution Solution: 10 mM NaCl, 0.1 mM CaCl.sub.2, 0.005% Tween-80, 0.02% Na-azide. MTPs were filled with 47.5 uL DMC substrate following the addition of 2.5 uL of 20 ppm protease supernatant. 50 uL of TNBSA in Reagent A was then added with slow mixing. Activity was measured at 405 nm over 5 minutes using a SpectraMax plate reader in kinetic mode at RT. As stated above for the AAPF assay, activity was expressed as mOD/min.

Detergents Compositions Evaluated

[0158] Proteases were tested for cleaning performance and/or stability in custom-made liquid detergent formulations (Test HDL1 and Test HDL2) having the composition set forth in Tables 1.1 and 1.2. The HDL detergents, Test HDL1 and Test HDL 2 are considered boron-free since they contained 5 mg/Kg of boron, when tested for elemental boron content.

TABLE-US-00002 TABLE 1.1 Composition of Test HDL1 Detergent Formula Component Ingredient Trade name Wt. % Solvent Water (total) 64.89 Surfactant C12-C15 Pareth-7 Empilan KCL 7 3 (Sodium) Dodecylben- NANSA SSA F 7.5 zenesulfonate K-Cocoate NANSA PC 38F 3 Sodium Laureth Sulfate Empicol ESB3/M6 9 Builder Sodium Citrate Sodium Citrate 3 Tribasic Dihydrate Liquid Sorbitol D-Sorbitol 0.8 properties/ Propylene glycol 1,2-Propanediol 2.5 stability Glycerin Glycerol 0.8 Triethanolamine Triethanolamine 0.5 Methylisothiazolinone 2-Methyl-4- 0.01 isothiazolin-3-one Ethanol 1 Neutralizer Sodium hydroxide (4M) 5.27

TABLE-US-00003 TABLE 1.2 Composition of Test HDL2 Detergent Formula Component Ingredient Trade name Wt. % Solvent Water (total) QS Propylene glycol 5 Surfactant Sodium Alkyl Aryl Polystep B27 12 Ether Sulfate (30% active) Sodium alkylbenzene Nacconal 90G 1.2 sulfonate Builder Trisodium citrate 0.5 Disodium ethylenedi- 0.25 aminetatraacetate Liquid Sodium formate 0.25 properties/ Acrylic acid Sokalan PA 30 0.5 stability homopolymer CL (50% active) Oleic acid 1 Calcium chloride 0.1 Brightener Tinopal CBS-X 0.25 Preservative Bioban 425 0.04 (25% active) Neutralizer Sodium carbonate 0.25 Sodium Hydroxide 0.25 (50%) to pH 8.5

Cleaning Performance in Detergent

[0159] Variants were tested for cleaning performance relative to BspE04637-T1 parent protease on various microswatches: BMI (blood/milk/ink on cotton, EMPA-116), egg yolk with pigment (aged by heat, CFT CS-38), and POM (pigment/oil/milk, CFT-C10). Pre-punched (to fit on MTP), rinsed, and filled swatch-containing plates (Corning 3641) were prepared by Center for Testmaterials BV, Vlaardingen, Netherlands. Detergents were dosed as described in Table 1.3, and were added to the MTPs prior to enzyme addition.

TABLE-US-00004 TABLE 1.3 List of Wash Conditions Used for Performance Assays Final Wash Hardness Set Detergent Conc, (g/L) Conc. (ppm) Buffer pH Test HDL1 7.5 250 5 mM sodium HEPES 8 Test HDL2 0.84 150 5 mM sodium HEPES 8

[0160] Aliquots of enzyme were added to the detergent-filled MTPs to reach a final volume of 200 uL with a final enzyme concentration of 1.25 ppm in the wash. Laundry cleaning assays were carried out at 25 C. When testing CS-10 and CS-38 stains using Test HDL1, the assay was carried out for 25 min. When testing EMPA-116 in Test HDL1 or CS-10 and CS-38 in Test HDL2, tests were carried out over 50 min. When EMPA-116 was tested in Test HDL2, the assay was carried out over 65 min. Following these incubations, 130 uL aliquots of each supernatant was transferred to a fresh MTP and a SpectraMax plate reader was used to measure the absorbance at 600 nm for EMPA-116 swatches and at 405 nm for CFT CS-38 and CFT CS-10 swatches. Absorbance results were obtained by subtracting the value for a blank (no enzyme added) from each sample value. The cleaning PI for each assay condition was obtained by dividing the absorbance values for a given variant by that of the BspE04637-T1 parent protease tested at the same concentration.

Thermostability Assay in Detergent

[0161] The variants were tested for stability under stress conditions using 10% Test HDL1 detergent, diluted in deionized water (hereinafter stressor) by measuring the residual activity following incubation at elevated temperature. The elevated temperature was set to obtain approximately 30% residual activity of the stressed sample compared to the unstressed (baseline) sample.

[0162] For the unstressed condition, the diluted enzyme sample was mixed in 10% Test HDL1 detergent (diluted in deionized water) and baseline protease activity (no elevated temperature stress) was measured immediately upon dilution by the DMC assay described above. For the stressed condition, these enzyme samples diluted in stressor were incubated in a 96-well plate at 72 C. for 5 min using a thermal cycler. The plate was then cooled and the resulting stressed protease activity was measured by the DMC method. Percent (%) residual activities for each sample were calculated by determining the ratio of the stressed over the baseline protease activity and multiplying the value by 100.

EXAMPLE 2

Generation of BspE04637-Clade Subtilisins

[0163] DNA manipulations to generate BspE04637-clade subtilisins were carried out via molecular biology techniques known in the art. The BspE04637-clade subtilisins are more fully described in PCT International Application No. PCT/US2015/057512, filed Oct. 27, 2015. The predicted and observed sequences of the naturally occurring BspE04637 wild-type protease are set forth in PCT International Application No. PCT/US2015/057512. A series of artificial DNA sequences were synthesized (by Integrated DNA Technologies, Inc.) to generate the genes encoding the truncated version of the BspE04637 protease sequence of BspE04637-T1 (SEQ ID NO:1). Molecular biology techniques known in the art were used to introduce multiple amino acid modifications into this sequence, which consists of a C-terminal truncation of the naturally occurring sequence, yielding a mature protein of 278 amino acids (SEQ ID NO:1).

[0164] A DNA fragment containing the following elements: the BPN terminator, chloramphenicol resistance marker from Staphylococcus aureus (CAT), B. subtilis aprE promoter, B. subtilis aprE signal peptide sequence, and native BspE04637 protease pro-peptide was constructed. The pool of genes of interest (encoding the various BspE04637-T1 variants) and the DNA fragment described above were assembled using Gibson technology to form an integration cassette that targets the B. Subtilis aprE locus. These expression cassettes consisted of: B. subtilis aprE promoter (SEQ ID NO:2), the B. subtilis aprE signal peptide sequence (SEQ ID NO:3 for nucleotide sequence and SEQ ID NO:4 for the amino acid sequence), the native BspE04637 protease pro-peptide (SEQ ID NO: 5 for nucleotide sequence and SEQ ID NO: 6 for amino acid sequence), the region encoding the mature protease sequences for BspE04637-T1 parent (SEQ ID NO:1) or variants thereof, and a B amyloliquefaciens terminator (SEQ ID NO:7). The nucleotide sequences of the BspE04637-T1 variants are set forth in SEQ ID NOs: 8-17 and the amino acid sequences of the BspE04637-T1 variants are set forth in SEQ ID NOs: 18-27.

[0165] A suitable B. subtilis strain was transformed with the reaction products of the above mentioned Gibson assembly reaction and the transformed cells were plated on Luria's Agar+5 ppm chloramphenicol containing skim milk. Single colonies from the agar plates were picked into individual wells of a 96 well MTP that contained 150 microliters of Luria's broth+5 ppm chloramphenicol in each well. The cultures were grown overnight at 37 C. and 250 RPM. 75 microliters of 50% glycerol (diluted in water) was added to each well and the MTPs for long-term storage at 80 C.

[0166] To produce the recombinant proteases, the B. subtilis carrying each gene of interest was cultivated in an enriched semi-defined media for 2 days at 37 C. and 250 RPM. Cultures were subjected to centrifugation and filtration, and the clarified supernatants were used for the screening assays described below.

EXAMPLE 3

Evaluation of BspE04637-Clade Protease Variants for Cleaning and Stability

[0167] BspE04637-clade protease variants listed in Table 4 were tested for cleaning performance relative to BspE04637-T1 parent protease on the following microswatches: BMI (EMPA-116), egg yolk with pigment (aged by heat, CFT CS-38), and POM (pigment/oil/milk, CFT-C10).

TABLE-US-00005 TABLE 4 BspE04637-clade Protease Variants Sequence Substitutions Relative to No. Mutations Amino Acid Sample ID BspE04637-T1 Parent vs Parent Sequence BSPE-01299 R37A-D53G-S101R-N120R-I143R-D166N-D184S-P197K- 12 SEQ ID NO: 18 L238K-E248Q-D255N-R272N BSPE-01314 D15K-D53R-N56K-S105K-D122H-D166N-D184S-P197K- 11 SEQ ID NO: 19 E248Q-D255N-V274E BSPE-01329 D15K-A55R-S101R-N120R-I143R-D166N-D184S-P197K- 12 SEQ ID NO: 20 L238K-E248Q-N268K-V274E BSPE-01346 H48R-A55R-S101R-N120R-S133R-R164N-L238K-E248Q- 10 SEQ ID NO: 21 D255N-N268K BSPE-01420 Q12K-D15K-R37A-D122H-R164N-G169D-E248Q- 9 SEQ ID NO: 22 N268K-D275S BSPE-01443 D15K-H48R-A55R-S101R-N120R-I143R-G165R-D175N- 12 SEQ ID NO: 23 A198E-N243R-N254K-D275K BSPE-01483 Q12K-V26R-R37A-A55R-N78D-S105K-I143R-A155V- 13 SEQ ID NO: 24 D184S-A198E-N243R-N259R-D275K BSPE-01560 T14D-D53R-S101R-I143R-R164N-D175S-A198E-N243R- 9 SEQ ID NO: 25 D275K BSPE-01579 D15K-D53R-S101R-N120R-I143R-G165R-D175S-N243R- 11 SEQ ID NO: 26 D255N-N264K-D275K BSPE-01581 Q12K-V26R-R37A-H48R-A55R-N78D-S101R-D122H- 16 SEQ ID NO: 27 I143R-D166N-D175P-D184S-Y220R-N243R-D255N- R272N

[0168] Table 5 shows the results obtained when the cleaning of various BspE04637-clade protease variants were compared to BspE04637-T1 parent protease in Test HDL1 and Test HDL2 detergents.

TABLE-US-00006 TABLE 5 Cleaning of BspE04637-clade Protease Variants Compared to BspE04637-T1 Parent Protease, Reported as PI EMPA-116 CFT CS-38 CFT C-10 Test Test Test Test Test Test Sample ID HDL1 HDL2 HDL1 HDL2 HDL1 HDL2 BSPE-01443 1.83 3.56 1.18 3.24 1.87 2.05 BSPE-01581 2.06 3.44 1.18 2.47 1.84 2.08 BSPE-01483 2.15 3.37 1.26 3.10 1.85 1.84 BSPE-01560 2.24 3.68 1.19 2.97 1.79 1.84 BSPE-01314 2.26 3.89 1.29 3.47 1.92 2.23 BSPE-01346 1.75 3.54 1.24 3.35 1.74 1.99 BSPE-01329 1.84 3.76 1.24 3.11 1.75 2.04 BSPE-01299 2.02 3.67 1.26 2.62 1.91 2.01 BspE04637-T1 1.00 1.00 1.00 1.00 1.00 1.00 parent
Table 6 shows the stability of various BspE04637 protease variants in 10% Test HDL 1 compared to BspE04637-T1 parent protease as measured in the Stability Assay described in Example 1 hereinabove.

TABLE-US-00007 TABLE 6 Stability of BspE04637-T1 and Variants, Reported as % Residual Activity % Residual Activity in 10% Test Sample ID HDL 1 (72 C. for 5 min) BSPE-01579 60 BSPE-01420 52 BSPE-01443 31 BSPE-01581 31 BSPE-01483 19 BSPE-01560 20 BSPE-01314 22 BSPE-01346 14 BSPE-01329 19 BSPE-01299 16 BspE04637-T1 51

EXAMPLE 4

Sequence Alignment of BspE04637-T1 and Variants Thereof

[0169] An alignment of the amino acid sequences of BspE04637-T1 parent protease (SEQ ID NO:1) and protease variants thereof: BSPE-01299 (SEQ ID NO:18), BSPE-01314 (SEQ ID NO:19), BSPE-01329 (SEQ ID NO: 20), BSPE-01346 (SEQ ID NO:21), BSPE-01420 (SEQ ID NO:22), BSPE-01443 (SEQ ID NO:23), BSPE-01483 (SEQ ID NO:24), BSPE-01560 (SEQ ID NO:25), BSPE-01579 (SEQ ID NO:26), and BSPE-01581 (SEQ ID NO:27) with the naturally truncated form of BspE04637 (SEQ ID NO:34) (which is further described in PCT International Application No. PCT/US2015/057512); previously disclosed BspE04637-clade subtilisins: SWT183_1430046 (SEQ ID NO: 28) and Bacillus sp. KSM-LD1 BAD02409.1 (SEQ ID NO: 29); and the sequences of the mature forms of the following subtilisins: Bacillus amyloliquefaciens_CAA24990 (SEQ ID NO: 30), Bacillus lentus_P29000 (SEQ ID NO: 31), Bacillus sp. SprC_AAC43580 (SEQ ID NO: 32), and Bacillus licheniformis_CAJ70731 (SEQ ID NO: 33), is shown in FIG. 1. A box has been drawn around the motif of the BspE04637-clade proteases. The sequences were aligned with default parameters using the MUSCLE program from Geneious software (Biomatters Ltd.) (Robert C. Edgar, MUSCLE: multiple sequence alignment with high accuracy and high throughput, Nucl. Acids Res. (2004) 32 (5): 1792-1797).

[0170] A phylogenetic tree for amino acid sequences of the mature forms of the subtilisins from FIG. 1 was built using the Geneious Tree builder program, and is set forth in FIG. 2.