US010533165B2 (12 ) United States Patent ( 10 ) Patent No.: US 10,533,165 B2 Kolkman et al. (45 ) Date of Patent : Jan. 14 , 2020 (54 ) SERINE PROTEASES OF THE BACILLUS (52 ) U.S. CI. GIBSONII -CLADE CPC C12N 9/54 (2013.01 ) ; A47L 15/0002 (2013.01 ) ; CIID 3/386 (2013.01 ) ; C12Y ( 71 ) Applicant : Danisco US Inc., Palo Alto , CA (US ) 304/21 ( 2013.01) ; A47L 2601/20 ( 2013.01) (58 ) Field of Classification Search (72 ) Inventors : Marc Kolkman , Oegstgeest (NL ) ; Rie CPC combination set ( s ) only . Mejldal, Ostbirk (DK ) ; Frits See application file for complete search history. Goedegebuur, Vlaardingen (NL ) ; Lilia Maria Babe , Emerald Hills , CA (US ) ; ( 56 ) References Cited Anja Hemmingsen Kellett -Smith , Arhuc (DK ) ; Harm Mulder , Oegstgeest U.S. PATENT DOCUMENTS (NL ) ; Richard R. Bott , Burlingame, 2005/0113273 A1 5/2005 Weber et al . CA (US ) ; Miles Christopher Scotcher , 2014/0017763 Al 1/2014 Wieland et al. Hayward , CA (US ) FOREIGN PATENT DOCUMENTS ( 73 ) Assignee: DANISCO US INC , Palo Alto , CA (US ) DE 102100005345 A1 9/2012 ( * ) Notice: Subject to any disclaimer, the term of this OTHER PUBLICATIONS patent is extended or adjusted under 35 Deng et al ., " Secretory Expression , Functional Characterization , U.S.C. 154 ( b ) by 20 days . and Molecular Genetic Analysis of Novel Halo - Solvent - Tolerant (21 ) Appl. No.: 15 / 104,219 Protease from Bacillus gibsonii, ” J. Microbiol. Biotechnol ., 2014 , vol . 24 , No. 2 , pp . 197-208 . (22 ) PCT Filed : Dec. 12 , 2014 Database Genese [Online ] Nov. 8 , 2012 ( Nov. 8 , 2012 ) , “ Bacillus gibsonii protease variant G12L / 121V /MI22L / A222S , SEQ ID : 6.” , PCT/ US2014 /070107 retrieved from EBI accession No. GSP :BAA09836 Database acces (86 ) PCT No.: sion No. BAA09836 . $ 371 ( c ) ( 1 ) , Database Geneseq [Online ] Oct. 15 , 2003 (Oct. 15 , 2003 ), “ B (2 ) Date : Jun . 13 , 2016 gibsonii protease. ” , retrieved from EBI accession No.GSP : ABR63713 Database accession No. ABR63713 . (87 ) PCT Pub . No .: WO2015 /089447 Database UniProt [Online ] Oct. 16 , 2013 ( Oct. 16 , 2013 ) , “ SubName: Full = AprBG {ECO :0000313 : EMBL. : AGS78407.1 } ; EC = 3.4.21.19 PCT Pub . Date : Jun . 18 , 2015 { EC0 :0000313 : EMBL .: AGS78407.I } ; Flags: Fragment ; ” , XP002737457 , retrieved from EBI accession No. UNIPROT: S5VEFO (65 ) Prior Publication Data Database accession No. S5VEFO . International Search Report and the Written Opinion — PCT/ US2014 / US 2016/0319266 A1 Nov. 3 , 2016 070107 — dated Apr. 9 , 2015 . Primary Examiner Kagnew H Gebreyesus Related U.S. Application Data (57 ) ABSTRACT (60 ) Provisional application No.61 / 915,737 , filed on Dec. The present disclosure relates to serine proteases cloned 13, 2013 , provisional application No. 62 / 069,200 , from Bacillus gibsonii , and variants thereof. Compositions filed on Oct. 27 , 2014 . containing the serine proteases are suitable for use in clean ing fabrics and hard surfaces, as well as in a variety of (51 ) Int. Ci. industrial applications. C12N 9/54 ( 2006.01) A47L 15/00 (2006.01 ) 17 Claims, 18 Drawing Sheets CI1D 3/386 ( 2006.01 ) Specification includes a Sequence Listing . U.S. Patent Jan. 14 , 2020 Sheet 1 of 18 US 10,533,165 B2 Term - lambda apre promoter region signal peptide apre Tet R PRO Bgi02446 RBS 1 precursor Apre SP - Bgi0244 6 -10 Signal 1 mature chain Sgi02446 35 Signal 1 Rep ori (PAMalpha - 1 ) PHYT - B9102446 STOP codon 6870 1 RBS 2 terminator 110 Signal 2 35 Signal 2 -10 Signal 3 Gram + Origin 2 -35 Signal 3 -35 Signal 4 Amp R ( Tn3 from PACYC177 ) -10 Signal 4 RBS 3 FIG . 1 ppm protease FIG.2 U.S. Patent Jan. 14 , 2020 Sheet 2 of 18 US 10,533,165 B2 & Krachtig Kirkland Ultra 00900 0.10 0.05 10 protease (ppm ) FIG.3A 0.254 OMO color 0.20 Kirkland Uitra 0.15 00900 0.10 0.05 protease (ppm ) FIG.3B U.S. Patent Jan. 14 , 2020 Sheet 3 of 18 US 10,533,165 B2 0.8 0.6 PH 10.5 0.2 0 Protease (ppm ] FIG . 3C U.S. Patent Jan. 14 , 2020 Sheet 4 of 18 US 10,533,165 B2 hai 50 WO20071.31657 - CAS91385 ( 1 ) QOTVPWGITRVOAPAVHNRGVTGSGVRVAILDSGISQ - HSDLTIRGGASF WO03054184 - CAE48421 ( 1 ) QQTVPWGITRVQAPAVHNRGVTGSGVRVAILDSGIST - HSDLTIRGGASF WO2008086916 - CAV33594 ( 1 ) QQTVPWGITRVOAPAVHNRGVTGSGVRVAILDSGISQ - HSDLTIRGGASF Bgi02446 ( 1 ) QQTVPWGITRVQAPAVHNRGITGSGVRVAILDSGISA - HSDLNIRGGASF WOO3054185 - CAE 48424 ( 1 ) QQTVPWGITRVQAPTVANRGITGSGVKVAILDTGIAC - HSDLTIRGGASF US7642080-0004 ( 1 ) OQSVPWGITRVQAPAAINRGTNGSGVRAAVLDTGIST - HSDLTIRGGASF US 7642080-0006 ( 1 ) QQSVPWGITRVOAPAAINRGTNGSGVRVAVLDTGIST - HSDLTIRGGASF US 7642080-0002 ( 1 ) QQTVPWGIQRVQAPAVINRGINGSGVRVAVLDSGISS - HSDLSISGGVSF Blentus P29600 ( 1 ) AQSVPWGISRVQAPAAHNRGLTGSGVKVAVLDTGIST - HPDLNIRGGASF Bsp.Sendai BAA06157 ( 1 ) NOVTPWGITRVQAPTAWTRGYTGIGVRVAVLDTGIST - HPDLNIRGGVSF Bacillus sp BAA05540 ( 1 ) SQTVPWGISFINTQQAHNRGIFGNGARVAVLDTGIAS - HPDLRIAGGASF B_amyloliquefaciens_CAA24990 ( 1 ) AQSVPYGVSQIKAPALHSQGYTGSNVKVAVIDSGIDSSHPDLKVAGGASM G_stearothermophilus ABY25856 ( 1 ) AQSVPYGVSQIKAPALHSOGFTGSNVKVAVIDSGIDSSHPDLKVAGGASM Buatrophaeus YP003972439 ( 1 ) AQSVPYGISQIKAPAVHSQGYTGSNVKVAVIDSGIDSSHPDLKVSGGASF Busubtilis BAN09118 ( 1 ) AQSVPYGISQIKAPALHSQGYTGSNVKVAVIDSGIDSSHPDLNVRGGASE B.circulans ADN04910 ( 1 ) AQTVPYGIPQIKAPAVHAQGYKGANVKVAVLDTGIHAAHPDLNVAGGASF Bstratosphericus WP_007497196 ( 1 ) AQTVPYGIPQIKAPAVHAQGYKGANVKVAVLDTGIHAAHPDLNVAGGASF Blicheniformis CAJ 70731.1 ( 1 ) AQTVPYGIPLIKADKVQAQGFKGANVKVAVLDTGIQASHPDLNVVGGASF Bupumilus ADK11996 ( 1 ) AQTVPYGIPOIKAPAVHAQGYKGANVKVAVLDTGIHAAHPDLNVAGGASF B_Sp.sprD AAC43581 ( 1 ) AQTVPYGVPRIKADVARAQNVTGSGVKVAVLDTGIDASHEDLRVVGGASF Bacillus SP - LG12 sprc_AAC43580 ( 1 ) AQTVPWGIPHIKADKAHAAGVTGSGVKVAILDTGIDANHADLNVKGGASF Bacillus sp BAD11988 ( 1 ) AQTTPWGVTHINAHRAHSSGVTGSGVKVAILDTGIHASHPDLNVRGGASF * * : . * :: * : ** : * ** . * : Consensus ( 1 ) AQTVPWGI I APAVH RG TGSGVKVAVLDTGI HPDL RGGASE 51 100 WO2007131657 - CAS91385 (50 ) IPGEP - TTADLNGHGTHVAGTVAALNNSIGVIGVAPSADLYAVKVLGANG WO03054184 - CAE 48421 ( 50 ) VPGEP - TTADLNGHGTHVAGTVAALNNSIGVIGVAPSADLYAVKVLGANG WO2008086916 - CAV33594 ( 50 ) VPGEP - TTEDLNGHGTHVAGTVAALNNSFGVIGVAPSADLYAVKVLGAGG Bgi02446 (50 ) VPGEP - ITADLNGHGTHVAGTVAALNNSIGVIGVAPNAELYAVKVLGANG W003054185 - CAE48424 (50 ) VPGES - ITADLNGHGTHVAGTVAALNNSIGVIGVAPSADLYAVKVLGANG US7642080-0004 ( 50 ) VPGEP - NTSDLNGHGTHVAGTIAALNNSIGVVGVAPNADLYAVKVLGANG US7642080-0006 ( 50 ) VPGEP - NTSDLNGHGTHVAGTIAALNNSIGVVGVAPNADLYAVKVLGANG US 7642030-0002 ( 50 ) VPGEP - TIADGNGHGTHVAGTIAALNNSIGVVGVAPNAQIYGVKVLGANG Blentus P29600 ( 50 ) VPGEP - STQDGNGHGTHVAGTIAALNNSIGVLGVAPSAELYAVKVLGASG Bsp Sendai BAA06157 ( 50 ) VPGEP - SYQDGNGHGTHVAGTIAALNNSIGVVGVAPNAELYAVKVLGANG Bacillus_sp_BAA05540 ( 50 ) ISSEP - SYHDNNGHGTHVAGTIAALNNSIGVLGVAPSADLYAVKVLDRNG Byamyloliquefaciens.CAA24990 ( 51 ) VPSETNPFOONNSHGTHVAGTVAALNNSIGVLGVAPSASLYAVKVLGADG Gustearothermophilus ABY25856 ( 51 ) VPSETNPFODNNSHGTHVAGTVAALNNSVGVLGVAPSASLYAVKVLGADG Buatrophaeus.YP003972439 (51 ) VPSEPNPFODGNSHGTHVAGTVAALNNSVGVLGVAPSASLYAVKVLSSSG Busubtilis BANO9118 ( 51 ) VPSETNPYODGSSHGTHVAGTVAALNNTIGVLGVAPSASLYAVKVLDSTG B.circulans ADN04910 ( 51 ) VPSEPNATODFOSHGTHVAGTIAALDNTIGVLGVAPSASLYAVKVLDRNG B_stratosphericus WP_007497196 ( 51 ) VPSEPNATQDFQSHGTHVAGTIAALDNTIGVLGVAPSASLYAVKVLORNG Blicheniformis CAJ70731.1 ( 51 ) VAGEA - YNTDGNGHGTHVAGTVAALDNTTGVLGVAPSVSLYAVKVLNSSG Bupumilos_ADK11996 (51 ) VPSEPNATODFQSHGTHVAGTIAALDNTIGVLGVAPSASLYAVKVLDRNG B.sp.sprD_AAC43581 (51 ) VSEEPDALTDGNGHGTHVAGTIAALNNNVGVLGVSYDVDLYAVKVLSAGG Bacillus sp LG12 sprc_AAC43580 ( 51 ) VSGEPNALQDGNGHGTHVAGTVAALNNTTGVLGVAYNADLYAVKVLSASG Bacillus sp BAD11988 ( 51 ) ISGESNPYIDSNGHGTHVAGTVAALNNTVGVLGVAYNAELYAVKVLSASG ******** **** ** : ** : Consensus (51 ) VPGEP T DNGHGTHVAGTVAALNNSIGVLGVAPSA LYAVKVLGANG FIG . 4A U.S. Patent Jan. 14 , 2020 Sheet 5 of 18 US 10,533,165 B2 101 150 WO2007131657 - CAS91385 ( 99 ) RGSVSGIAQGLEWAAANNMHIANMSLGADAPSSTLERAVNYATSOGVLVI WO03054184 - CAE 48421 ( 99 ) RGSVSGIAQGLEWAAANNMHIANMSLGSDAPSITLERAVNYATSQGVLVI WO2008086916 - CAV33594 ( 99 ) RGSVSGIAQGLEWAAANNMHIANMSLGADAPSSTLERAVNYATSRGVLVI Bgi02446 ( 99 ) SGSVSGIAQGLEWAATNNMHIANMSLGSDFPSSTLERAVNYATSRDVLVI W003054185 - CAE 48424 ( 99 ) RGSVSGIAOGLEWAATNNMHIANMSLGSDAPSTTLERAVNYATSRGVLVI US7642080-0004 ( 99 ) RGSIGGIAQGLEWAAANNMHIANLSLGSDAPSSTLEQAVNYATSRGVLVI US 7642080-0006 ( 99 ) RGSIGGIAQGLGWAAANNMHIANLSLGSDAPSSTLEQAVNYATSRGVLVI US7642080-0002 ( 99 ) RGSVSGIAQGLEWAATNNMDIANLSLGSDAPSSTLEQAVNFATSRGVLVV Bulentus P29600 ( 99 ) SGSVSSIAQGLEWAGNNGMHVANLSLGSPSPSATLEQAVNSATSRGVLVV B_sp_Sendai BAA06157 ( 99 ) SGSVSSIAQGLQWTAQNNIHVANLSLGSPVGSQTLELAVNQATNAGVLVV Bacillus sp BAA05540 ( 99 ) SGSLASVAQGIEWAINNNMHIINMSLGSTSGSSTLELAVNRANNAGILLV Bmamyloliquefaciens_CAA24990 ( 101 ) SGOYSWIINGIEWAIANNMDVINMSLGGPSGSAALKAAVDKAVASGVVVV G - stearothermophilus_ABY25856 ( 101 ) SGQYSWIINGIEWAIAYNMDVINMSLGGPSGSAALKAAVDKAVASGIVVV Buatrophaeus_YP003972439 ( 101 ) SGDYSWIINGIEWAISNNMDVINMSLGGPOGSTALKAVVDKAVSQGIVVV B_subtilis BAN09118 ( 101 ) SGOYSWIINGIEWAISNNMDVINMSLGGPIGSTALKTVVDKAVASGIVVV B.circulans ADN04910 ( 101 ) DGQYSWIISGIEWAVANNMDVINMSLGGPNGSTALKNAVDTANNRGVVVV Bustratosphericus WP_007497196 ( 101 ) DGQYSWIISGIEWAVANNMDVINMSLGGPSGSTALKNAVDTANNRGVVVV Blicheniformis CAJ70731.1 ( 100 ) SGSYSGIVSGIEWATTNGMDVINMSLGGASGSTAMKQAVDNAYARGVVVV Bupumilus ADK11995 ( 101 ) DGQYSWIISGIEWAVANNMDVINMSLGGASGSTALKNAVDTANNRGVVVV B._SP_sprD AAC43581 ( 101 ) SGTLAGIAQGIEWAIDNNMDVINMSLGGSTGSTTLKQASDNAYNSGIVVI Bacillus_sp_LG12 sprc_AAC43580 ( 101 ) SGTLSGIAQGIEWSISNGMNVINMSLGGSSGSTALQQACNNAYNRGIVVI Bacillus_sp_BAD11988 ( 101 ) SGTLSGIAQGVEWSIANKMDVINMSLGGSSGSTALQRAVDNAYRNNIVVV . * : * : * * Consensus ( 101 ) SGS SGIAQGIEWA ANNMDVINMSLGG