US010533201B2 (12 ) United States Patent ( 10 ) Patent No.: US 10,533,201 B2 Navickas et al. (45 ) Date of Patent : Jan. 14 , 2020 (54 ) ENZYMATIC TRANSPHOSPHORYLATION ( 58 ) Field of Classification Search OF SUGAR SUBSTRATES CPC CO7H 11/04 ; CO7H 1/02 ; C12P 19/02 ; C12Y 204/01 ; C12Y 204/02007 ; C12Y ( 71 ) Applicant : BASF SE , Ludwigshafen (DE ) 301/0301 See application file for complete search history . (72 ) Inventors: Vaidotas Navickas, Mannheim (DE ) ; (56 ) References Cited Kai -Uwe Baldenius , Mannheim (DE ) ; Michael Breuer, Darmstadt (DE ) ; U.S. PATENT DOCUMENTS Patricia Wildberger, Wiener Neudorf ( AT ) ; Martin Pfeiffer , Graz ( AT ) ; 2013/0012475 Al 1/2013 Auriol et al . Bernd Nidetzky , Graz ( AT ) FOREIGN PATENT DOCUMENTS ( 73 ) Assignee: BASF SE , Ludwigshafen am Rhein EP 2150620 A2 2/2010 (DE ) WO WO - 2005073399 Al 8/2005 WO WO - 2008142155 A2 11/2008 ( * ) Notice : Subject to any disclaimer , the term of this WO WO - 2009004093 Al 1/2009 patent is extended or adjusted under 35 U.S.C. 154 ( b ) by 231 days. OTHER PUBLICATIONS Cottrill, M., et al ., “ Inositol phosphatase activity of the Escherichia (21 ) Appl. No .: 15 /509,993 coli agp - encoded acid glucose - 1 -phosphatase ” , Canadian Journal of Microbiology , vol. 48 , No. 9 , ( 2002 ) , pp . 801-809. ( 22 ) PCT Filed : Jun . 30 , 2015 Herter, T. , et al. , “ Glucose - 1 -phosphatase (AgpE ) from Enterobacter cloacae displays enhanced phytase activity ” , Applied Microbiology and Biotechnology , vol. 70 , No. 1, ( 2006 ), pp . 60-64 . ( 86 ) PCT No .: PCT /EP2015 / 064785 International Search Report for PCT/ EP2015 / 064785 dated Oct. 21, 2015 . $ 371 ( c ) ( 1 ) , Pradel, E., et al ., “ Acid Phosphatases of Escherichia coli : Molecular (2 ) Date : Mar. 9 , 2017 Cloning and Analysis of agp , the Structural Gene for a Periplasmic Acid Glucose Phosphatase ” , Journal of Bacteriology, vol. 170 , No. ( 87 ) PCT Pub . No.: WO2016 /037720 10 , ( 1988 ) , pp . 4916-4923 . Saier, M. Jr. , et al. , “ Sugar Phosphate : Sugar Transphosphorylation PCT Pub . Date : Mar. 17 , 2016 and Exchange Group Translocation Catalyzed by the Enzyme II Complexes of the Bacterial Phosphoenolpyruvate: Sugar (65 ) Prior Publication Data Phosphotransferase System ” , The Journal of Biological Chemistry , vol . 252 , No. 24 , ( 1977 ) , pp . 8899-8907 . US 2018/0148753 A1 May 31 , 2018 Written Opinion of the International Searching Authority for PCT / Foreign Application Priority Data EP2015 /064785 dated Oct. 21 , 2015 . ( 30 ) Primary Examiner Karen Cochrane Carlson Sep. 10 , 2014 ( EP ) 14184303 (74 ) Attorney , Agent, or Firm — Drinker Biddle & Reath LLP ( 51 ) Int . Ci. (57 ) ABSTRACT CO7H 11/04 ( 2006.01 ) The present invention relates to methods ofproducing sugar C12P 19/02 ( 2006.01) phosphates by enzymatic transphosphorylation , wherein the C12P 19/26 ( 2006.01 ) phosphoryl transfer from phosphate donor substrates to C12N 9/10 ( 2006.01 ) sugar substrates is catalyzed by an enzyme having alpha C12N 9/16 ( 2006.01 ) glucose - 1 - phosphatase activity . Furthermore , the present CO7H 1/02 ( 2006.01 ) invention relates to a biocatalyst having sucrose phospho (52 ) U.S. Cl. rylase as well as alpha - glucose - 1 - phosphatase activity and ??? C12P 19/02 (2013.01 ) ; C07H 1/02 its use . The production of an enzyme having alpha - glucose ( 2013.01 ) ; CO7H 11/04 ( 2013.01 ) ; C12N 1 -phosphatase activity and its use in the transphosphory 9/1051 (2013.01 ) ; C12N 9/16 ( 2013.01 ) ; C12P lation of sugar substrates is also presented herein . 19/26 ( 2013.01) ; C12Y 204/01 ( 2013.01 ) ; C12Y 204/01007 ( 2013.01 ) ; C12Y 301/0301 9 Claims, 10 Drawing Sheets ( 2013.01 ) Specification includes a Sequence Listing . U.S. Patent Jan. 14 , 2020 Sheet 1 of 10 US 10,533,201 B2 HO ? bilo??? ?? . HPO 2 + HOR im OR HOS Lokaa B HO . HON + ATP deleo ADP 'OH HO . "OH ATP regeneration C ozi ?? . + P2074 6 HPO 2 OH HOV hOH mass action effect Figure 1 U.S. Patent Jan. 14 , 2020 Sheet 2 of 10 US 10,533,201 B2 ?? + His18 N H NH His18 N HO HO Asp290 OH . PO HO O. H OH Asp290 Fax ?? His18 NH His18 *O HO OH Asp290 H Asp290 HO HO Glucosecose Glucose1 NH NH His18 His18 0. OH THO O HO Asp290 HO HO -OH Asp290 HO OH A B Figure2 U.S. Patent Jan. 14 , 2020 Sheet 3 of 10 US 10,533,201 B2 40x103 30x103 20x103 )dmolldegcm2ellipticity.(Molar 10x103 0 www -10x103 -20x103 180 200 220 240 260 Wavelength [nm ] Figure 3 1 MASWSHPQFE KIEGRQTVPE GYQLQQVLMM SRHNLRAPLA NNGSVLEQST 51 PNKWPEWDVP GGQLTTKGGV LEVYMGHYMR EWLAEQGMVK SGECPPPDTV 101 YAYANSLQRT VATAQFFITG AFPGCDIPVH HQEKMGTMDP TENPVITDDS 151 AAFSEKAVAA MEKELSKLQL TDSYQLLEKI VNYKDSPACK EKQQCSLVDG 201 KNTFSAKYQQ EPGVSGPLKV GNSLVDAFTL QYYEGEPMDQ VAWGEIKSDQ 251 QWKVLSKLKN GYQDSLFTSP EVARNVAKPL VSYIDKALVT DRTSAPKITV 301 LVGHDSNIAS LLTALDFKPY QLHDQNERTP IGGKIVFQRW HDSKANRDLM 351 KIEYVYQSAE QLRNADALTL QAPAQRVTLE LSGCPIDANG FCPMDKFDSV 401 LNEAVK Figure 4 U.S. Patent Jan. 14 , 2020 Sheet 4 of 10 US 10,533,201 B2 ? 18 16 141 Productconcentrations[mm] 20 40 09 80 Time [mm ] B 1 1 2 Productconcentrations[mm] 1 8 0 20 40 09 08 Time [mm ] Figure 5 1 ww Substrate,productconcentrations[MM] 20 60 80 100 120 140 1160 Time [min ] Figure 6 U.S. Patent Jan. 14 , 2020 Sheet 5 of 10 US 10,533,201 B2 Figure7 60 60 50 40 40 Time[min] 30 30 Time[min] 20 Www 20 10 10 F1PG6P/ F6PF1P/ F1P/P F1PIG6P F1P/F6P F1P/P 40 30 20 50 50 40 20 10 Ratio Ratio 60 60 50 50 40 40 Time[min] 30 Time[min] 20 20 10 10 0 25 20 15 10 25 20 15 10 ] MM[ concentrations product , Substrate ] mm[ concentrations product , Substrate B U.S. Patent Jan. 14 , 2020 Sheet 6 of 10 US 10,533,201 B2 Figure7 60 60 50 40 40 Time[min] Time[min] 20 20 *** 10 F1P/PF6PF1P/ F1P/G6Pa F1PIP, F6PF1P/ F1PIGOP 0 50 40 20 10 50 40 30 20 10 Ratio Ratio 60 60 50 50 40 40 30 Time[min] 30 Timemin[] 20 20 10 10 0. 25 20 15 10 25 20 15 10 0 ] mm[ concentrations product , Substrate ] MM[ concentrations product , Substrate U.S. Patent Jan. 14 , 2020 Sheet 7 of 10 US 10,533,201 B2 600 DO 7 Fru[mm] pH 200400 5 4 100 3 2.5 2.0 1.5 1.0 0.5 16 14 12 10 6 2 logikcat_app ] mM( concentration Product B 1000 80 800 60 600 Fru[mm] 40 Time[min] 400 200 20 20 15 10 consumption/ Transphosphorylation ) MM( concentration product , Substrate Figure8 U.S. Patent Jan. 14 , 2020 Sheet 8 of 10 US 10,533,201 B2 OH OH OH HO OH SPase OH -OH HO OH HO OH re OH OH HO OH AG1Pase HO + OH OH OH Figure 9 0.8 36 0.6 32 islip 0.4 Productyields 6 4 0.2 2 0.0 D-GlcNAca D -Man L -Sorb D-Fru D -GION D-Gal a-Olx L-Ara D -Xolº L-Aol L- Fuc ' Figure 10 U.S. Patent Jan. 14 , 2020 Sheet 9 of 10 US 10,533,201 B2 1.0 18 16 0.8 14 0.6 12 10 D-Man/aGic1P D-Man1PYields 8 0.4 T 6 0.2 4 2 0.0 0 0 200 400 600 800 1000 Initial aGic 1 - P concentrations [mm ] Figure 11 U.S. Patent Jan. 14 , 2020 Sheet 10 of 10 US 10,533,201 B2 100 mM sucrose A 100 mM phosphate 800 mM D -mannose 1.3 MM SPase /0.1 uM aG1Pase 70 60 50 Substrate,productconcentrations[MM] 40 30 20 10 0B 0 20 40 60 80 100 120 Time [min ] B 200 mM sucrose 200 mM phosphate 800 mM D -mannose 1.3 MM SPase 0.1 uM aG1Pase 200 60 50 150 Phosphateglucose,1-phosphate[MM] 40 Reactionproducts[mm] 100 30 20 50 10 0 0 0 50 100 150 200 250 Time [min ] Figure 12 US 10,533,201 B2 1 2 ENZYMATIC TRANSPHOSPHORYLATION 1B ) ( 34 , 35 ). This makes the biocatalytic phosphoryl trans OF SUGAR SUBSTRATES fer a technically complex overall transformation of currently limited use in sugar phosphate synthesis . CROSS - REFERENCE TO RELATED Transphosphorylation catalyzed by phosphatases in an APPLICATIONS 5 alteration of their natural phosphomonoester hydrolysis reaction has been considered as alternative route towards This application is a national stage application ( under sugar phosphates ( FIG . 1C ) ( 36-39 ) . Under conditions 35 U.S.C. § 371 ) of PCT/ EP2015 /064785 , filed Jun . 30 , 2015 , which claims benefit of European Application No. where sugar was present in a concentration high enough to 14184303.7 , filed Sep. 10 , 2014 , both of which are incor- 10 effectively outcompete the reaction with water , some phos porated herein by reference in their entirety . phatases ( e.g. acid phosphatase ) promoted sugar phosphate The present invention relates to methods of producing formation in moderate ( e.g. D -mannose 6 -phosphate ; 15 % ) sugar phosphates by enzymatic transphosphorylation , to excellent yields ( e.g. Glc -6 - P ; up to 95 % ) ( 37 ) . Usage of wherein the phosphoryl transfer from phosphate donor sub expedient phosphoryl donor substrates such as inorganic strates to sugar substrates is catalyzed by an enzyme having 15 pyro- or oligo - phosphate was advantageous . However , high alpha -glucose - 1 -phosphatase activity . In another aspect , the preponderance of donor substrate hydrolysis , fast secondary present invention relates to a biocatalyst having sucrose hydrolysis of sugar phosphate product, and combined inhi phosphorylase as well as alpha - glucose - 1 -phosphatase bition and mass action effects of the released phosphates activity and its use . According to a further aspect, the present (FIG . 1C ) are critical issues of phosphatase - catalyzed syn invention also relates to the production of an enzymehaving 20 thesis (40 , 41) . Transphosphorylation systems with alpha- glucose - 1 -phosphatase activity and its use in the improved synthetic efficacy therefore still need to be iden transphosphorylation of sugar substrates.