Enzymatic compositions for carbohydrate antigen cleavage, methods, uses, devices and systems related thereto

Abstract

Provided herein are enzymatic compositions for carbohydrate antigen cleavage, methods, uses, devices, and systems related thereto. In particular, the composition comprises two enzymes of GalNAc deacetylase and galactosaminase, and the composition can further comprise a crowding agent. In addition, it was found that the compositions described herein have activity at temperatures and pH levels suitable for cell survival.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Patent Application Serial No. 62 / 719,272, filed on August 17, 2018, entitled "ENZYMATIC COMPOSITIONS FOR CARBOHYDRATE ANTIGEN CLEAVAGE, METHODS, USES, APPARATUSES AND SYSTEMS ASSOCIATED THEREWITH." Technical Field

[0002] The present invention relates to the field of enzyme compositions, in particular, the present invention relates to an enzyme composition for cleaving an antigen, and provides a method, use, device and system for cleaving an antigen using the composition. background

[0003] Correct matching of blood types is a major requirement in transfusion medicine, as the plasma of individuals with blood type A contains antibodies against B antigens and vice versa, and thus incompatible transfusions can lead to complement activation and red blood cell (RBC) lysis (Daniels 2010). These cell surface antigens are carbohydrate structures that terminate in α-1,3-linked-N-acetylgalactosamine (GalNAc) or galactose (Gal) for blood types A and B, respectively. On the other hand, type O RBCs do not contain these terminal sugars and can be universally transfused (Garratty 2008). Therefore, a good supply of type O RBCs is required in blood banks in emergency situations where the patient's blood type is unknown or unclear. However, the supply is usually limited.

[0004] Goldstein first proposed and demonstrated the concept of enzymatic removal of GalNAc or Gal structures from A RBC or B RBC as a means of converting ARBC or B RBC into O RBC (Goldstein 1982; US4609627 and CA2272925). Using α-galactosidase from green coffee beans, B-type RBCs were converted to O-type RBCs, followed by successful transfusions (Kruskall2000). However, the amount of enzyme required makes this method impractical. The conversion of type A is more challenging, mainly because type A blood has many subtypes with different internal connections (Clausen 1989). Similarly, α-galactosidase has been used to remove type B antigens (e.g., see EP2243793). By using a tetrasaccharide substrate to screen a bacterial library with A and B conversion activity, an important step towards actual conversion including type A has been taken. Two new glycosidase families were found to show high antigen cleavage activity at neutral pH: CAZy GH109 α-N-acetylgalactosaminidases and GH110 α-galactosidases (Liu 2007). Both enzymes transformed their corresponding RBCs, in which the respective antigens were completely removed. However, transformation still requires large amounts of enzyme, especially type A (60 mg enzyme / blood unit), which limits further development. Enzymes with higher efficiency in cleaving carbohydrate antigens from cells would be useful. Overview

[0005] The present invention is based in part on the unexpected discovery that the combination of galactosaminidase and GalNAc deacetylase as described herein is several orders of magnitude more effective than the previously identified A antigen cleavage enzyme. For example, under some conditions, some GalNAc deacetylases and galactosaminidase can cleave A antigen at or below 1 μ / ml. In addition, the cutting efficiency of the enzyme combination is maintained at a pH suitable for maintaining red blood cell viability (i.e., pH is about 6.5 to about 7.5). In addition, it is found that the enzyme is active at a temperature of 4°C to 37°C, which is also applicable to blood collection, washing and storage schemes. In addition, the efficiency of the enzyme is further improved by adding a crowding agent (e.g., dextran). It has also been recognized that the same two-step cutting process can be applied to donor organs. Enzymes as described herein are mainly tested on samples with a hematocrit of 10%, because these enzymes are better used with packaged red blood cell (rbc) bags (about 220ml) and the required amount is calculated, which contains a level of about 80% hematocrit.

[0006] In some embodiments, lacking crowding agent: 3 μg / ml, 10% hematocrit, 1 h, 37°C, >5.3 mg of each enzyme / packaged rbc bag can be used to cleave A antigen from red blood cells, and in other embodiments, with crowding agent: 0.5 μg / ml, 10% hematocrit, 1 h, 37°C, >0.9 mg of each enzyme / packaged rbc bag can be used to cleave A antigen from red blood cells. However, those skilled in the art will appreciate that more enzyme can be used to reduce the possible time to process blood, or less enzyme can be used as long as the cells are incubated for a longer time.

[0007] According to one embodiment, a composition is provided, comprising: (a) a purified GalNAc deacetylase protein; and (b) a purified galactosamidase protein.

[0008] According to one embodiment, a composition is provided, comprising: (a) a purified GalNAc deacetylase protein selected from one or more of the following: SEQ ID NO:2; SEQ ID NO:4; SEQ ID NO:5; SEQ ID NO:17; SEQ ID NO:23; SEQ ID NO:29; SEQ ID NO:31; SEQ ID NO:32; SEQ ID NO:33; SEQ ID NO:34 and SEQ ID NO:35; and (b) a purified galactosaminidase protein selected from one or more of the following: SEQ ID NO:7; SEQ ID NO:9; SEQ ID NO:10; SEQ ID NO:19; SEQ ID NO:21; SEQ ID NO:36 and SEQ ID NO:37.

[0009] According to another embodiment, a composition is provided, comprising: a purified enzyme having GalNAc deacetylase activity, which consists essentially of an amino acid sequence at least 90% identical to the sequence shown in one of SEQ ID NOs: 2, 4, 5, 17, 23, 29, 31 and 32-35; and a purified enzyme having galactosaminidase activity, which consists essentially of an amino acid sequence at least 90% identical to the sequence shown in one of SEQ ID NOs: 7, 9, 10, 19, 21, 36 and 37.

[0010] According to another embodiment, a composition is provided, comprising: a purified enzyme having GalNAc deacetylase activity, which consists essentially of an amino acid sequence at least 85% identical to the sequence shown in one of SEQ ID NOs: 2, 4, 5, 17, 23, 29, 31 and 32-35; and a purified enzyme having galactosaminidase activity, which consists essentially of an amino acid sequence at least 85% identical to the sequence shown in one of SEQ ID NOs: 7, 9, 10, 19, 21, 36 and 37.

[0011] According to another embodiment, a composition is provided, comprising: a purified enzyme having GalNAc deacetylase activity, which consists essentially of an amino acid sequence at least 80% identical to the sequence shown in one of SEQ ID NOs: 2, 4, 5, 17, 23, 29, 31 and 32-35; and a purified enzyme having galactosaminidase activity, which consists essentially of an amino acid sequence at least 80% identical to the sequence shown in one of SEQ ID NOs: 7, 9, 10, 19, 21, 36 and 37.

[0012] According to another embodiment, a composition is provided, comprising: a purified enzyme having GalNAc deacetylase activity, which consists essentially of an amino acid sequence at least 75% identical to the sequence shown in one of SEQ ID NOs: 2, 4, 5, 17, 23, 29, 31 and 32-35; and a purified enzyme having galactosaminidase activity, which consists essentially of an amino acid sequence at least 75% identical to the sequence shown in one of SEQ ID NOs: 7, 9, 10, 19, 21, 36 and 37.

[0013] According to another embodiment, a composition is provided, comprising an enzyme selected from one or more of (a), and one or more of (b): (a) the purified GalNAc deacetylase protein is the purified GalNAc deacetylase protein of Flavonifractor plautii of SEQ ID NO: 2, SEQ ID NO: 4 and SEQ ID NO: 5; and (b) the purified galactosamidase protein is the purified galactosamidase protein of Flavonifractor plautii of SEQ ID NO: 7, SEQ ID NO: 9 and SEQ ID NO: 10.

[0014] According to another embodiment, a composition is provided, comprising one or more enzymes selected from the group consisting of: (a) a purified GalNAc deacetylase protein of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 17, and SEQ ID NO: 32; and (b) the purified galactosamidase protein is a purified galactosamidase protein of Fusobacterium prausnitzii of SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 36, and SEQ ID NO: 37.

[0015] According to another embodiment, a composition is provided, comprising one or more enzymes selected from the group consisting of: (a) the purified GalNAc deacetylase protein is the purified GalNAc deacetylase protein of Clostridium tertium of SEQ ID NO: 17 and SEQ ID NO: 32; and (b) the purified galactosamidase protein is the purified galactosamidase protein of Clostridium tertium of SEQ ID NO: 19 and SEQ ID NO: 36.

[0016] The GalNAc deacetylase and galactosamidase composition may be capable of cleaving the A antigen at 1 μg / ml or less. The GalNAc deacetylase and galactosamidase composition may have A antigen cleavage activity at a pH of about 6.5 to about 7.5. The GalNAc deacetylase and galactosamidase composition may have A antigen cleavage activity at a temperature of 4°C to 37°C.

[0017] The composition may include: (a) the purified GalNAc deacetylase and the purified galactosamidase may be immobilized; (b) the purified GalNAc deacetylase may be immobilized; or (c) the purified galactosamidase may be immobilized.

[0018] The immobilized enzyme can be attached to a surface, which can be selected from one or more of the following: (a) beads or microspheres; (b) containers; (c) tubes; (d) columns; and (e) matrices. The composition can also include a crowding agent. The crowding agent can be selected from one or more of the following: dextran, dextran sulfate, dextrin, pullulan, poly(ethylene glycol), polysucrose TM and inert proteins.

[0019] According to another embodiment, a purified enzyme is provided, which includes the GalNAc deacetylase of Fusobacterium prausnitzii of SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 5.

[0020] According to another embodiment, a purified enzyme is provided, which includes the galactosamidase of Fusobacterium prausnitzii of SEQ ID NO:7, SEQ ID NO:9 or SEQ ID NO:10.

[0021] According to another embodiment, a purified enzyme is provided, which includes the GalNAc deacetylase of Clostridium tertium of SEQ ID NO: 17 or SEQ ID NO: 32.

[0022] According to another embodiment, a purified enzyme is provided, which includes the galactosamidase of Clostridium tertium of SEQ ID NO: 19 or SEQ ID NO: 36.

[0023] According to another embodiment, an isolated nucleic acid sequence encoding a GalNAc deacetylase is provided, which is selected from one or more of the following: SEQ ID NO: 1; SEQ ID NO: 3; SEQ ID NO: 16; SEQ ID NO: 24; SEQ ID NO: 26; SEQ ID NO: 28 and SEQ ID NO: 30.

[0024] According to another embodiment, an isolated nucleic acid sequence encoding a galactosamidase is provided, which is selected from one or more of the following: SEQ ID NO:6; SEQ ID NO:8; SEQ ID NO:18 and SEQ ID NO:20.

[0025] According to another embodiment, a vector is provided, comprising a nucleic acid as described herein. The vector may further comprise a heterologous nucleic acid sequence selected from one or more of the following: a protein tag and a cleavage site.

[0026] The protein tag can be selected from one or more of the following: albumin binding protein (ABP); alkaline phosphatase (AP); AU1 epitope; AU5 epitope; Avi tag; bacteriophage T7 epitope (T7 tag); bacteriophage V5 epitope (V5 tag); biotin-carboxyl carrier protein (BCCP); blue tongue virus tag (B tag); single domain camelid antibody (C tag); calmodulin binding peptide (CBP or calmodulin tag); chloramphenicol acetyltransferase (CAT); cellulose binding domain (CBP); chitin binding domain (CBD); choline binding domain (CBD); dihydrofolate reductase (DHFR); DogTag; E2 epitope; E tag; FLAG epitope (FLAG tag); galactose binding protein (GBP); green fluorescent protein (GFP); Glu-Glu (EE tag); glutathione S transferase (GST); human influenza hemagglutinin (HA); HaloTag TM ; Alternating histidine and glutamine tag (HQ tag); Alternating histidine and asparagine tag (HN tag); Histidine affinity tag (HAT); Horseradish peroxidase (HRP); HSV epitope; Isopeptag (Isopep tag); Ketosterol isomerase (KSI); KT3 epitope; LacZ; Luciferase; Maltose binding protein (MBP); Myc epitope (Myc tag); NE tag; NusA; PDZ domain; PDZ ligand; Polyarginine (Arg tag); Polyaspartic acid (Asp tag); Polycysteine ​​(Cys tag); Polyglutamic acid (Glu tag); Polyhistidine (His tag); Polyphenylalanine (Phe tag); Profinity eXact; Protein C; Rho1D4 tag; S1 tag; S tag; Softag 1; Softag 3; SnoopTagJr; SnoopTag; Spot tag; SpyTag (Spy tag); Streptavadin binding peptide (SBP); Staphylococcal protein A (Protein A); Staphylococcal protein G (Protein G); Strep tag; Streptavadin (SBP tag); Strep tag II; Sdy tag; small ubiquitin-like modifier (SUMO); tandem affinity purification (TAP); T7 epitope; tetracysteine ​​tag (TC tag); thioredoxin (Trx); TrpE; Ty tag; ubiquitin; universal; V5 tag; VSV-G or VSV tag and Xpress tag.

[0027] According to another embodiment, a method for enzymatically cleaving A antigen from blood, red blood cells or donor organs is provided, the method comprising: (a) combining a GalNAc deacetylase protein and a galactosamidase protein with: (i) blood containing type A antigen; (ii) type A or type AB red blood cells; or (iii) a donor organ displaying type A antigen; (b) incubating the enzyme with: (i) the blood; (ii) the type A or type AB red blood cells; or (iii) the donor organ for a period of time sufficient for the enzyme to cleave the A antigen from the blood or the red blood cells or the donor organ.

[0028] The GalNAc deacetylase may be a purified protein selected from one or more of the following: SEQ ID NO:2; SEQ ID NO:4; SEQ ID NO:5; SEQ ID NO:17; SEQ ID NO:23; SEQ ID NO:29; SEQ ID NO:31; SEQ ID NO:32; SEQ ID NO:33; SEQ ID NO:34 and SEQ ID NO:35; and the galactosaminidase may be a purified protein selected from one or more of the following: SEQ ID NO:7; SEQ ID NO:9; SEQ ID NO:10; SEQ ID NO:19; SEQ ID NO:21; SEQ ID NO:36 and SEQ ID NO:37.

[0029] The composition may include: a purified enzyme having GalNAc deacetylase activity, which is essentially composed of an amino acid sequence at least 90% identical to the sequence shown in one of SEQ ID NOs: 2, 4, 5, 17, 23, 29, 31 and 32-35; and a purified enzyme having galactosaminidase activity, which is essentially composed of an amino acid sequence at least 90% identical to the sequence shown in one of SEQ ID NOs: 7, 9, 10, 19, 21, 36 and 37.

[0030] The GalNAc deacetylase may be the purified GalNAc deacetylase protein of Fusobacterium prausnitzii of SEQ ID NO:4 or SEQ ID NO:5, and the galactosamidase may be the purified galactosamidase protein of Fusobacterium prausnitzii of SEQ ID NO:9 or SEQ ID NO:10.

[0031] The method may further include adding a crowding agent. The crowding agent may be selected from one or more of the following: dextran; dextran sulfate; dextrin; pullulan; poly(ethylene glycol); polysucrose TM ; Hyperbranched glycerol and inert protein.

[0032] The method may further comprise washing the blood, the red blood cells or the donor organ to remove GalNAc deacetylase, galactosamidase and the crowding agent.

[0033] The GalNAc deacetylase and the galactosamidase may be able to cleave the A antigen at 1 μg / ml or less. The GalNAc deacetylase and the galactosamidase may have A antigen cleavage activity at a pH of about 6.5 to about 7.5. The GalNAc deacetylase and the galactosamidase may have A antigen cleavage activity at a temperature of 4°C to 37°C.

[0034] According to another embodiment, a blood collection and storage system is provided, comprising: (a) a purified GalNAc deacetylase protein; and (b) a purified galactosamidase protein.

[0035] The system may further comprise a surface on which the enzyme is immobilized, the surface being selected from one or more of: (a) a bead or microsphere; (b) a container; (c) a tube; (d) a column; or (e) a matrix.

[0036] According to another embodiment, a blood collection and storage device is provided, comprising: (a) a surface; (b) a purified GalNAc deacetylase protein immobilized on the surface; and (c) a purified galactosamidase protein immobilized on the surface.

[0037] The surface of the device on which the enzyme is immobilized may be selected from one or more of the following: (a) beads or microspheres; (b) containers; (c) tubes; (d) columns; or (e) matrices. The container may be a bag.

[0038] The GalNAc deacetylase and the galactosamidase may be able to cleave the A antigen at 100 μg / ml or less. The GalNAc deacetylase and the galactosamidase may be able to cleave the A antigen at 90 μg / ml or less. The GalNAc deacetylase and the galactosamidase may be able to cleave the A antigen at 80 μg / ml or less. The GalNAc deacetylase and the galactosamidase may be able to cleave the A antigen at 70 μg / ml or less. The GalNAc deacetylase and the galactosamidase may be able to cleave the A antigen at 60 μg / ml or less. The GalNAc deacetylase and the galactosamidase may be able to cleave the A antigen at 50 μg / ml or less. The GalNAc deacetylase and the galactosamidase may be able to cleave the A antigen at 40 μg / ml or less. The GalNAc deacetylase and the galactosamidase may be able to cleave the A antigen at 30 μg / ml or less. The GalNAc deacetylase and the galactosamidase may be able to cleave the A antigen at 20 μg / ml or less. The GalNAc deacetylase and the galactosamidase may be able to cleave the A antigen at 15 μg / ml or less. The GalNAc deacetylase and the galactosamidase may be able to cleave the A antigen at 14 μg / ml or less. The GalNAc deacetylase and the galactosamidase may be able to cleave the A antigen at 13 μg / ml or less. The GalNAc deacetylase and the galactosamidase may be able to cleave the A antigen at 12 μg / ml or less. The GalNAc deacetylase and the galactosamidase may be able to cleave the A antigen at 11 μg / ml or less. The GalNAc deacetylase and the galactosamidase may be able to cleave the A antigen at 10 μg / ml or less. The GalNAc deacetylase and the galactosamidase may be able to cleave the A antigen at 9 μg / ml or less. The GalNAc deacetylase and the galactosamidase may be able to cleave the A antigen at 8 μg / ml or less. The GalNAc deacetylase and the galactosamidase may be able to cleave the A antigen at 7 μg / ml or less. The GalNAc deacetylase and the galactosamidase may be able to cleave the A antigen at 6 μg / ml or less. The GalNAc deacetylase and the galactosamidase may be capable of cleaving the A antigen at 5 μg / ml or below.The GalNAc deacetylase and the galactosamidase may be able to cleave the A antigen at 4 μg / ml or less. The GalNAc deacetylase and the galactosamidase may be able to cleave the A antigen at 3 μg / ml or less. The GalNAc deacetylase and the galactosamidase may be able to cleave the A antigen at 2 μg / ml or less. The GalNAc deacetylase and the galactosamidase may be able to cleave the A antigen at 1 μg / ml or less. The GalNAc deacetylase and the galactosamidase may be able to cleave the A antigen at 0.9 μg / ml or less. The GalNAc deacetylase and the galactosamidase may be able to cleave the A antigen at 0.8 μg / ml or less. The GalNAc deacetylase and the galactosamidase may be able to cleave the A antigen at 0.7 μg / ml or less. The GalNAc deacetylase and the galactosamidase may be able to cleave the A antigen at 0.6 μg / ml or less. The GalNAc deacetylase and the galactosamidase may be able to cleave the A antigen at 0.5 μg / ml or less. The GalNAc deacetylase and the galactosamidase may be able to cleave the A antigen at 0.4 μg / ml or less. The GalNAc deacetylase and the galactosamidase may be able to cleave the A antigen at 0.3 μg / ml or less. The GalNAc deacetylase and the galactosamidase may be able to cleave the A antigen at 0.2 μg / ml or less. The GalNAc deacetylase and the galactosamidase may be able to cleave the A antigen at 0.1 μg / ml or less. The GalNAc deacetylase and the galactosamidase may be able to cleave the A antigen at 0.09 μg / ml or less. The GalNAc deacetylase and the galactosamidase may be able to cleave the A antigen at 0.08 μg / ml or less. The GalNAc deacetylase and the galactosamidase may be able to cleave the A antigen at 0.07 μg / ml or less. The GalNAc deacetylase and the galactosamidase may be able to cleave the A antigen at 0.06 μg / ml or less. The GalNAc deacetylase and the galactosamidase may be able to cleave the A antigen at 0.05 μg / ml or less. The GalNAc deacetylase and the galactosamidase may be capable of cleaving the A antigen at or below 0.04 μg / ml.The GalNAc deacetylase and the galactosamidase may be able to cleave the A antigen at 0.03 μg / ml or below. The GalNAc deacetylase and the galactosamidase may be able to cleave the A antigen at 0.02 μg / ml or below. The GalNAc deacetylase and the galactosamidase may be able to cleave the A antigen at 0.01 μg / ml or below.

[0039] The GalNAc deacetylase and the galactosamidase may have A antigen cleavage activity at a pH of about 6.5 to about 7.5. The GalNAc deacetylase and the galactosamidase may have A antigen cleavage activity at a pH of about 6.0 to about 8.0. The GalNAc deacetylase and the galactosamidase may have A antigen cleavage activity at a pH of about 6.8 to about 7.8. The GalNAc deacetylase and the galactosamidase may have A-antigen cleavage activity at a pH of about 6.9 to about 7.9. The GalNAc deacetylase and the galactosamidase may have A antigen cleavage activity at a pH of about 6.4 to about 7.8.

[0040] The GalNAc deacetylase and the galactosamidase may have A antigen cleavage activity at a temperature of 4°C to 37°C. The GalNAc deacetylase and the galactosamidase may have A antigen cleavage activity at a temperature of 3°C to 38°C. The GalNAc deacetylase and the galactosamidase may have A antigen cleavage activity at a temperature of 4°C to 40°C. The GalNAc deacetylase and the galactosamidase may have A antigen cleavage activity at a temperature of 4°C to 37°C. The GalNAc deacetylase and the galactosamidase may have A antigen cleavage activity at a temperature of 5°C to 37°C.

[0041] The purified GalNAc deacetylase and the purified galactosamidase may be immobilized. The purified GalNAc deacetylase may be immobilized. The purified galactosamidase may be immobilized. The immobilized enzyme may be attached to a surface. The surface may be selected from one or more of: a bead or microsphere; a container; a tube; a column; or a matrix. The surface may be selected from one or more of: a container; a tube; a column; or a matrix. The container may be a bag.

[0042] According to another embodiment, a purified enzyme of the GalNAc deacetylase of Fusobacterium prausnitzii comprising SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 5 is provided.

[0043] According to another embodiment, a purified enzyme comprising the galactosamidase of Fusobacterium prausnitzii of SEQ ID NO: 7, SEQ ID NO: 9 or SEQ ID NO: 10 is provided.

[0044] According to another embodiment, a purified enzyme is provided, which includes the purified Clostridium tertium GalNAc deacetylase and galactosaminidase fusion protein of SEQ ID NO: 14.

[0045] According to another embodiment, a vector comprising a nucleic acid described herein and a heterologous nucleic acid sequence is provided.

[0046] According to another embodiment, the method can be performed in vitro or ex vivo. As used herein, ex vivo means that the method is performed outside the organism. For example, ex vivo will include ex vivo lung perfusion (EVLP) and the processing of donated blood. As used herein, ex vivo refers to experiments or measurements or treatments performed in or on tissues or cells (e.g., red blood cells or donor organs) of an organism from an external environment, wherein the tissues or cells are under minimal or some condition changes in vivo. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 Schematic representation of the carbohydrate structures of type A, type H, and type B cell surface antigens terminating in α-1,3-linked N-acetylgalactosamine (GalNAc) or galactose (Gal), with triangles marking the cleavage sites for α-N-acetylgalactosaminidase EmGH109 and α-galactosidase BfGal110.

[0048] Figure 2 The deacetylase pathway for A antigen cleavage is shown, whereby GalNAc deacetylase from Fusobacterium prausnitzii (Fp) cleaves the terminal α-N-acetylgalactosamine (-42 m / z ) and then the galactosamine intermediate is converted to galactosamine by galactosaminidase (-161 m / z ) and analyzed by corresponding mass spectrometry (MS).

[0049] Figure 3 A treated with different concentrations of EmGH109 or GalNAc deacetylase from Fusobacterium prausnitzii (FpGalNAc deacetylase) plus galactosamidase from Fusobacterium prausnitzii (Fpgalactosamidase) + RBC or A treated at 37℃ for 1h +FACS analysis of RBCs, where for visualization, anti-H antibody (plus secondary FITC-labeled antibody) and APC-labeled anti-A antibody were used, where the region where the H antigen appears is in the box in the upper left corner. Rows AD compare EmGH109 and FpGalNAcDeAc+FpGalNase at 5μg / ml (A); 10μg / ml (B); 50μg / ml (C) and 50μg / ml+dextran 40k (D).

[0050] Figure 4 Shown is a comparison of EmGH109 and FpGalNAcDeAc+FpGalNase at various enzyme concentrations with (■) and without (◆) dextran at different temperatures, ie, 4°C, room temperature (RT), and 37°C.

[0051] Figure 5 HPAE-PAD analysis of A+B+ and O+ erythrocyte cleavage products is shown, as well as a comparison of the full-length GalNAc deacetylase (FpGalNAcDeAc) + galactosamidase (FpGalNase) enzyme from F. prausnitzii and the truncated FpGalNAcDeAc + FpGalNase enzymes on A+ erythrocytes.

[0052] Figure 6 The pH profiles of each of (A) FpGalNAc deacetylase and (B) Fpgalactosaminidase are shown.

[0053] Figure 7 Shown is the conversion of A antigen to H antigen on ARBCs analyzed by FACS for (A) A+RBC control, (B) GalNAc deacetylase (FpGalNAcDeAc) of Fusobacterium prausnitzii + galactosamidase (FpGalNase) of Fusobacterium prausnitzii (10 μg / mL), (C) FpGalNAcDeAc + Clostridium tertiotrophobia (Ct) Ct57577_GalNase (10 ug / mL), and (D) FpGalNAcDeAc + galactosamidase (Rp1021)GalNase (10 ug / mL) of Robinsoniella peoriensis (Rp). Details

[0054] The following detailed description will be better understood when read in conjunction with the accompanying drawings. For the purpose of illustrating the invention, the accompanying drawings show embodiments of the invention. However, the invention is not limited to the precise arrangements, examples and instrumentalities shown.

[0055] Any term not directly defined herein should be understood to have the meaning commonly associated therewith as understood in the field of the present invention.

[0056] As used herein, "immobilized enzymes" are enzymes attached to a surface, which may be an inert insoluble material. Immobilization of enzymes can provide enhanced resistance to changes in conditions (such as pH, temperature, etc.), and facilitate their removal after use and reuse of the enzyme.

[0057] Immobilization of enzymes can be achieved in various ways (e.g., affinity tag binding, surface adsorption on glass, resin, alginate beads or matrices, entrapment by beads, fibers or microspheres, crosslinking to surfaces or other enzymes and covalent binding to surfaces).

[0058] As used herein, "affinity tag binding" refers to the immobilization of an enzyme to a surface (e.g., a porous material using a non-covalent or covalent protein tag). Affinity tag binding has been used for protein purification and has recently been used for EziG TM (ENGINZYME AB TM , Sweden - e.g., PCT / US1992 / 010113 and PCT / SE2015 / 050108) for biocatalytic applications. Alternative systems for attaching active enzymes to surfaces are known in the art (see, e.g., U.S. Pat. No. 4,088,538; U.S. Pat. No. 4,141,857; U.S. Pat. No. 4,206,259; U.S. Pat. No. 4,218,363; U.S. Pat. No. 4,229,536; U.S. Pat. No. 4,239,854; U.S. Pat. No. 4,619,897; U.S. Pat. No. 4,748,121; U.S. Pat. No. 4,749,653; U.S. Pat. No. 4,897,352; U.S. Pat. No. 4,954,444; U.S. Pat. No. 4,978,619; U.S. Pat. No. 5,154,808; U.S. Pat. No. 5,914,367; U.S. Pat. No. 5,962,279; U.S. Pat. No. 6,030,933; U.S. Pat. No. 6,291,582; U.S. Pat. No. 6,254,645; U.S. Pat. No. 10,016,490 and U.S. Pat. No. 10,041,055).

[0059] Protein tag is a peptide sequence that gene is transplanted on recombinant protein, usually removable by chemical reagent or by enzymatic method, and is connected to protein for various purposes. The protein tag listed in Table A is intended as an example and is not intended to be limited in any way. One type of protein tag is an affinity tag, which is added to protein or peptide sequence so that they can be purified from crude biological source using affinity technology (for example, from expression system organism), or promote "labeled" protein to be fixed on the surface. Some examples of affinity tags include chitin binding domain (CBD), maltose binding protein (MBP), Strep tag, glutathione-S-transferase (GST) and polyhistidine (His tag) combined with metal matrix. Another type of protein tag is an epitope tag (for example, including V5 tag, Myc tag, HA tag, Spot tag and NE tag), which are short peptide sequences selected for easy production of high-affinity antibodies, and are usually derived from viral gene sequences to improve immunoreactivity. Epitope tags are particularly useful in Western blotting, immunofluorescence and immunoprecipitation experiments, although they are also used for purification and immobilization of proteins to surfaces. Another type of protein tag is a chromatographic tag (e.g., a polyanionic amino acid, such as a FLAG tag), which can be used to change the chromatographic properties of a protein to help separation and purification or immobilization. Other protein tags are solubilizing tags (e.g., maltose binding protein (MBP), glutathione S-transferase (GST), thioredoxin (TRX) and poly-(NANP)) and fluorescent tags (e.g., green fluorescent protein (GFP)). Protein tags can allow specific enzymatic modification, chemical modification or protein to be connected to other components. However, depending on the type or quantity of the tag added to the protein sequence, the natural function of the protein, in this case, the enzymatic function may be impaired by the tag. Therefore, it is necessary to select a protein tag to ensure that the activity of the enzyme is not impaired, or alternatively, the protein tag can be cut from the protein before use.

[0060] Table A: Exemplary protein tags

[0061] In the present application, the use of protein tags is exemplified by using polyhistidine protein tags (His tags) as shown in SEQ ID NO: 5, 10, 15, 17, 19, 21, 23, 25, 27, 29 and 31, but those skilled in the art will readily appreciate that any number of other protein tags can be used to purify the enzyme and / or to attach the enzyme to a surface as described herein, depending on the purification method used and / or the surface to which the enzyme is attached. Such protein tags can be selected from any one or more of the protein tags listed in Table A, but other such protein tags are known in the art.

[0062] In addition, one or more cleavage sites (e.g., thrombin cleavage sites as used in SEQ ID NOs: 15, 17, 19, 21, 23, 25, 27, 29, and 31) can be used to release protein tags from enzymes or otherwise cleave enzymes. Cleavage sites can be used to remove N-terminal methionines, signal peptides, and / or convert inactive or non-functional proteins into active proteins (i.e., zymogens / proenzymes). Alternatively, cleavage sites can be used to separate two or more enzymes expressed in the same reading frame. Examples of enzymes that are capable of cleaving proteins or peptides and will have a sequence-specific cleavage site may be selected from one or more of the following: Arg-C protease; Asp-N endopeptidase; Asp-N endopeptidase + N-terminal GluBNPS-skatole; Caspase 1; Caspase 2; Caspase 3; Caspase 4; Caspase 5; Caspase 6; Caspase 7; Caspase 8; Caspase 9; Caspase 10; Chymotrypsin - high specificity (C-terminal to [FYW], not before P); Chymotrypsin - low specificity Isotropic (C-terminal to [FYWML], not before P); clostripain (clostridial peptidase B); CNBr; enterokinase; factor Xa; formate; glutamyl endopeptidase; granzyme B; hydroxylamine; iodoacetic acid; LysC; LysN; NTCB (2-nitro-5-thiocyanatobenzoic acid); neutrophil elastase; pepsin (pH 1.3); pepsin (pH>2); proline-endopeptidase; proteinase K; staphylococcal peptidase I; tobacco etch virus protease; thermolysin; thrombin and trypsin.

[0063] It will be appreciated by those skilled in the art that the combination of active galactosamidase and active GalNAc deacetylase that can effectively cut A antigen as described herein is important, and it will also be appreciated by those skilled in the art that adding one or more cleavage sites and / or one or more protein tags is optional, and such modifications can be selected based on specific expression systems, purification systems, and possible surface connection strategies. In addition, other modifications to galactosamidase and GalNAc deacetylase sequences are also possible, as long as the cleavage activity of A antigen is not significantly impaired. In addition, modifications to galactosamidase and GalNAc deacetylase are possible, as long as the cleavage activity of A antigen is not significantly impaired. Modifications to galactosamidase and GalNAc deacetylase sequences can be deletions, insertions, and / or substitutions. Substitutions can be conservative substitutions or neutral substitutions. For example, galactosamidase and GalNAc deacetylase sequences may share 90% or higher sequence identity with mature enzymes. For example, galactosamidase and GalNAc deacetylase sequences may share 85% or higher sequence identity with mature enzymes. For example, the galactosamidase and GalNAc deacetylase sequences may share 75% or more sequence identity with the mature enzyme. Alternatively, the galactosamidase and GalNAc deacetylase sequences may be modified in 5%, 10%, 13%, 15%, 20% or up to 25% of the amino acids.

[0064] As used herein, "adsorbed on glass, alginate beads or matrix" refers to connecting the enzyme to the outside of an inert material. Typically, this type of immobilization is not caused by a chemical reaction, and the active sites of the immobilized enzyme may be blocked by the surface to which it is absorbed, which may reduce the activity of the absorbed enzyme.

[0065] As used herein, "entrapment" refers to the capture of an enzyme within an insoluble bead or microsphere. However, entrapment may hinder the arrival of the substrate and the departure of the product. An example is the use of calcium alginate beads, which can be produced by reacting a mixture of a sodium alginate solution and an enzyme solution with calcium chloride.

[0066] As used herein, "cross-linking" refers to the covalent bonding of enzymes to each other to produce a matrix consisting almost exclusively of enzymes. When designing a cross-linked enzyme reaction, the binding site ideally does not cover the active site of the enzyme, so that the activity of the enzyme is only affected by the immobilization, rather than the closure of the enzyme active site. However, spacer molecules (such as poly (ethylene glycol)) can be used to reduce the steric hindrance of the substrate.

[0067] As used herein, "covalent bonding" refers to the bonding of the enzyme to an insoluble support or surface (e.g., silica gel) via a covalent bond. Due to the strength of the covalent bond between the enzyme and the support or surface, the enzyme is much less likely to detach from the support or surface.

[0068] As used herein, "crowding agent" refers to any polymer or protein that promotes crowding of macromolecules by concentrating enzymes on the cell surface to improve the activity of the enzymes. Crowding agents can be, for example, dextran, dextran sulfate, dextrin, pullulan, poly(ethylene glycol), polysucrose, TM , hyperbranched glycerol and inert proteins. (Kuznetsova, IM et al., Int J Mol Sci. (2014) “What Macromolecular Crowding Can Do to a Protein” 15(12): 23090–23140).

[0069] As used herein, "dextran" refers to a polysaccharide having a molecular weight of ≥1,000 Daltons and a linear backbone of d-glucopyranose repeating units connected by α-. Dextran can be divided into 3 structural categories (i.e., Class 1-3) based on the pyranose ring structure, which contains five carbon atoms and one oxygen atom. Class 1 dextran contains a d-glucopyranose backbone modified with a small side chain of d-glucose branches with α (1→2), α (1→3) and α (1→4) linkages. Class 1 dextran varies in their molecular weight, spatial arrangement, branching type and degree, and branch length, 3-5 depending on the microbial production strain and culture conditions. Isomaltose and isomaltotriose are oligosaccharides with a Class 1 dextran backbone structure. Class 2 dextran (alternating) contains a backbone structure of d-glucopyranose units connected by alternating α (1→3) and α (1→6) branches with α (1→3) connections. Class 3 dextran (mutans) have a backbone structure of consecutive α(1→3) linked d-glucopyranosyl units with α(1→6) linked branches.

[0070] "Pullulan" as used herein is a structural polysaccharide produced primarily from starch by the fungus Aureobasidium pullulans and consists of repeating α(1→6)-linked maltotriose (D-glucopyranosyl-α(1→4)-D-glucopyranosyl-α(1→4)-D-glucose) units with occasional maltotetraose units.

[0071] As used herein, "dextrin" refers to a D-glucopyranosyl unit having a shorter chain length than dextran, which begins with a single α(1→6) bond but has a linear continuation of α(1→4) linked D-glucopyranosyl units.

[0072] "Dextran sulfate" as used herein is derived from dextran via sulfation.

[0073] As used herein, "polysucrose TM" is a neutral, highly branched, high-quality hydrophilic polysaccharide that is easily soluble in aqueous solutions.

[0074] Various alternative embodiments and examples are described herein. These embodiments and examples are illustrative and should not be construed as limiting the scope of the invention. Materials and methods

[0075] Unless otherwise stated, chemicals and commercial enzymes used in this study were purchased from Sigma-Aldrich TM The monosaccharide methylumbelliferyl glycoside was a generous gift from Dr. Hongming Chen, and the subtype 1A antigen 五 -MU was a generous gift from Dr. David Kwan (Kwan et al., 2015).

[0076] Human fecal metagenomic library

[0077] To generate a human metagenomics fosmid library, + Fresh human fecal samples were collected from healthy Asian male volunteers with blood types. Direct DNA extraction and fosmid library creation were performed according to the procedures described in the MoE protocol (Armstrong et al., 2017).

[0078] Fosmid library screening

[0079] 51×384 holes AB + The blood Fosmid library plate was thawed at room temperature and replicated in 50 μL of screening LB medium (12.5 μg / mL chloramphenicol, 25 μg / mL kanamycin, 100 μg / mL arabinose, 0.2% (v / v) maltose, 10 mM MgSO 4 ) in a 384-well plate. The plate was incubated at 37°C for 18 hours in a sealed container with a water reservoir to prevent excessive evaporation. TM Instruments [Genetix TM ] 45 μl of reaction mixture (100 mM NaH 2 PO 4 , pH 7.4, 2% (v / v) Triton-X 100, 100 μM GalNAc-α-MU, 100 μM Galα-MU) were added to the growing screening plate. The plate was then incubated at 37°C in a sealed container for 24 h and read at 1, 2, 4, 8 and 24 hours via a Synergy H1 plate reader [BioTek TM] The fluorescence of each plate was measured (Ex: 365 nm, Em: 435 nm, scanning mode, gain 80). For all wells, a Z score was calculated, which was given by the following formula: Z score = (fluorescence value - median) / standard deviation.

[0080] All positive hits above a certain threshold were re-arrayed in a new 384-well plate, referred to as the “simple substrate hit” plate, and stored at -70°C. Two screening plates were replicated from the “simple substrate hit” plate and re-screened for GalNAc-α-MU or Gal-α-MU activity to validate and deconvolute previously detected activities.

[0081] To determine which hits could cleave the A or B antigen structures, a coupled enzyme assay was used to determine their activity against 50 μM of subtype 1A antigen. 四 -MU or 50 μM subtype 1B antigen 四 -MU activity. A version of this coupled assay was previously described by Kwan (Kwan et al., 2015). Our assay was modified to also detect cleavage of subtype 1A antigens by using BgaC (Jeong 2009) instead of BgaA (Singh 2014) as the coupling enzyme. Potential α-N-acetylgalactosaminidase or α-galactosidase will cleave the terminal sugar, releasing the subtype IH antigen 三 -MU. Subsequently, α-fucosidase (AfcA (Katayarna 2004)), β-galactosidase (BgaC (Jeong 2009)), and β-hexosaminidase (SpHex (Williams 2002)) will cleave the remaining sugars in an outward manner until 4-methylumbelliferyl alcohol is released; this is detectable as an increase in fluorescence. To achieve this, 50 μg / mL of each enzyme was added to the reaction mixture. All positive hits above a certain threshold were screened again in triplicate, and a host cell line containing a vector lacking any insert was used as a negative control. All validated hits were stored at -70°C in LB medium (12.5 μg / mL chloramphenicol, 25 μg / mL kanamycin, 15% (v / v) glycerol, 0.2% (v / v) maltose, 10 mM MgSO 4 )middle.

[0082] Fosmid hit sequencing

[0083] To isolate fosmid DNA for sequencing, glycerol stocks of positive hit fosmids were used to inoculate 5 mL of TB medium (12.5 μg / mL chloramphenicol, 25 μg / mL kanamycin, 100 μg / mL arabinose, 0.2% (v / v) maltose, 10 mM MgSO 4) and incubated overnight at 37°C and 220 rpm. TM Plasmid miniprep kit (Thermo Fisher TM ) for Fosmid separation. TM ATP-dependent DNA enzyme (Epicentre TM ) Fosmid was purified from contaminating linear E. coli DNA and then isolated using GeneJet TM PCR purification kit (Thermo Fisher TM ) for another round of purification. TM Fluorometer (ThermoFisher TM ) using Quant-iT TM dsDNAHS assay kit (Invitrogen TM ) to calculate the concentration. Expected DNA size was verified on a 1% agarose gel. For complete fosmid sequencing, 2 ng of each fosmid was sent to the UBC Sequencing Center (Vancouver, BC, Canada). TM Each fosmid is individually barcoded and sequenced by the system.

[0084] Use on GitHub TM The python script available at (https: / / github.com / hallamlab / FabFos) is used for all Illumina MiSeq TM The raw sequence data were trimmed and assembled. Briefly, Trimmomatic was used to remove adapters and low-quality sequences from the reads (Bolger 2014). These reads were screened for vector and host sequences using BWA (Li 2013) and then analyzed using Samtools. TM and bam2fastq scripts to remove contaminants. These high-quality and purified reads were assembled by MEGAHIT with k-mer values ​​ranging from 71 to 241, increasing in increments of 10 (Li 2015). Since these libraries typically have more than 20,000-fold coverage, and to prevent the accumulation of sequencing errors that interfere with the assembly of correct sequences, the minimum k-mer multiplicity was calculated by 1% of the estimated coverage of the fosmid. More than one contig was then generated using the minimus2 (Treangen2011) python script assembly in addition to the python script assembly. Parameterized commands can be found on GitHub TM page and the documentation for the python script itself.

[0085] Fosmid ORF prediction and hit validation

[0086] Using Prodigal TM Fosmid ORFs were identified using the metagenomic version of (Hyatt 2010) and used as a MetaPathway TM BLASTP as part of the v2.5 package (Konwar 2015) TM CAZy TM MetaPathway TM Parameters: length>60, BLAST score>20, blast score ratio>0.4, E 值 <1×10-6.

[0087] Using Golden Gate TM Cloning strategy (Engler 2008) All predicted ORFs with annotations for GH or CBM family members (with known or suspected α-galactosidase and / or α-N-acetylgalactosaminidase activity) were cloned into pET16b plasmid, and the primer sequences are listed in Table B. Proteins were expressed in 10 mL of ZY5052 auto-induction medium (Studier 2005) in BL21 (DE3) cultured at 37°C, 220 rpm for 20 h. Cells were harvested by centrifugation (4000×g, 4°C, 10 min) and resuspended in 1 mL of lysis buffer (100 mM NaHCO3). 2 PO 4 , pH 7.4, 2% (v / v) Triton-X TM 100, 1× protease inhibitor without EDTA [Pierce TM ]). Use 50 μL of assay buffer (100 mM NaH 2 PO 4 , pH 7.4, 50 μg / mL Sphex, 50 μg / mL AfcA, 50 μg / mL BgaC, 100 μM subtype 1A antigen 四 -MU or 100 μM of subtype 1B antigen 四 The coupling assay was performed with 50 μL of crude cell lysate of the candidate mixed with 10 μL of 1 μM-MU and incubated at 37°C (Kwan 2015). All reactions were performed in triplicate on a black 96-well plate. Synergy TM H1 plate reader [BioTek TM] The fluorescence (365 / 435 nm) was continuously monitored for 4 hours. The assay was repeated from crude extracts showing A or B antigen cleavage activity, this time without the coupled enzyme, and the reaction products were separated via a HF Bond Elut C18 column and analyzed by LC-MS and / or TLC. TLC silica gel 60 F254 TLC plates [EMD Millipore Corp. TM , Billerica, Ma, USA] for TLC.

[0088] Table B: Primer sequences

[0089] HPAE-PAD assay

[0090] Analysis of enzymatic release of galactosamine was performed on HPAE-PAD (Dionex TM The cleavage activity of different proteins was tested on the following substrates: 100 mM NaH 2 PO 4 7.5 μg / μL of type II mucin from porcine stomach in 100 mM NaH 2 PO 4 (pH 7.4) at 5 mM subtype 1A antigen 五 -MU and RBCs from A+ donors, B+ donors, and O donors in 1× PBS (pH 7.4) (50% hematocrit). Samples containing 10 μg / mL enzyme were incubated at 37°C for 2 hours and then stored at -80°C for further analysis. A small aliquot (10 μl) of the reaction was diluted in H 2 O (100 μl) and analyzed on a HPAE-PAD instrument. TM (150mm) column, and detection was performed using disposable gold and four potential waveforms on polytetrafluoroethylene (PTFE) electrodes. The separation conditions were as follows: 100mM sodium hydroxide and sodium acetate gradient from 70 to 300mM in the first 10 minutes of separation. The eluent was maintained at the final gradient conditions for 1min and then returned to the starting conditions within the next minute. The flow rate was 1.0ml / min, and injections were performed every 27 minutes. Standards of free sugars GalNAc, Gal1 and GalN (10μM) were also applied to HPAE-PAD to determine the peak elution time for reference.

[0091] Kinetic assay

[0092] All kinetic assays using 4-methylumbelliferone as leaving group were performed by fluorescence measurements. In order to avoid measurement errors based on the inner filter effect (Palmier 2007), a standard curve was used to verify the linear range of the fluorophore.

[0093] Fp-galactosaminidase

[0094] In 100 mM NaH 2 PO 4 (pH 7.4) at 37°C to determine subtype 1 GalN antigen 五 -MU and subtype 1A antigens 五 -MU Michaelis-Menten parameters. Reactions were performed in 100 μL with 3.4 nM Fp galactosaminidase (5.31 nM FpGalNase_truncated) and 0.1 mg / mL Sphex, AfcA, 0.2 mg / mL BgaC and different concentrations of substrate (5 μM-2 mM). Reactions were run in a series of four replicates, with the control (no Fp galactosaminidase) in duplicate. TM Plate reader [BioTek TM The fluorescence signal (365 / 435 nm) generated by the release of MU by hydrolysis was monitored and converted to concentration using a standard concentration curve of MU determined under the same reaction conditions. The initial rate (μM / s) was determined and analyzed in Grafit7.0. TM The kinetic parameters were determined by plotting the graphs.

[0095] Determination of subtype 1 / 2 / 4 GalN antigen at pH 7.4 and 37°C 四 -MU and subtype 1B antigens 四 -MU's K cat / K M Parameters. Reactions were performed in black 96-well plates (total volume 100 μL) and were performed as 2 PO 4 The coupled assay was performed in 4% NaHCO (pH 7.4) with 100 mM NaH 2 PO 4 The reaction was run in a series of four replicates, with the control (no Fp-galactosaminidase) in duplicate. The reaction was performed by Synergy H1 TM Plate reader [BioTek TMThe fluorescence signal (365 / 435 nm) generated by the release of MU by hydrolysis was monitored and converted to concentration using a standard concentration curve of MU determined under the same reaction conditions. The initial rate (μM / s) was determined and analyzed in Grafit7.0. TM Draw a graph to determine k cat / K M (s -1* mM -1 )parameter.

[0096] In a clear 96-well plate at 37°C, 863.2 nM Fp-galactosaminidase (in 100 mM NaH 2 PO 4 , pH 7.4) or 369.9 nM FpGH4 (in 50 mM Tris / HCl (pH 7.4), 100 μM NAD+, 1 mM MnCl 2 The Michaelis-Menten parameters of GalN-α-pNP were determined in a volume of 100 μl with different concentrations of substrate (10 μM-5 mM). The reactions were run in a series of three replicates, two of which were controls (no enzyme). TM Plate reader [BioTek TM The absorption (at 405 nm) generated by the hydrolysis release of pNP was monitored and converted to concentration using a standard concentration curve of p-nitrophenol determined under the same reaction conditions. The initial rate (μM / s) was determined and analyzed in Grafit7.0 TM The kinetic parameters were determined by plotting the graphs.

[0097] FpGalNAc deacetylase

[0098] The PCR products were analyzed using a previously described coupled assay (Kwan 2015) at 37°C in 100 mM NaH 2 PO 4 (pH 7.4) to identify subtype 1A antigen 五 -Michaelis-Menten parameters for MU. The assay was modified to allow detection of cleavage of subtype 1 (and later 4) by using BgaC (Jeong 2009) instead of BgaA (Singh 2014) as β-galactosidase. In addition, since subtype 1A antigen 五-MU contains additional galactose, and the concentration of BgaC is increased to 0.2 mg / mL to compensate for its need to cut both Gal-β-1, 3-β-GlcNAc-β-1, 3-Gal-β-MU and Gal-β-MU. In addition, Fp galactosaminidase is included to allow the cleavage of galactosamine-containing intermediates. The reaction settings in 100μ are 3nM FpGalNAc deacetylase (4.52nM FpGalNacDeAc_D1ext, 3.55nM FpGalNacDeAc_D1+2) and 0.01mg / mL Fp galactosaminidase, 0.1mg / mL Sphex, AfcA, 0.2mg / mL BgaC and different concentrations of substrate (5μM-2.5mM). The reaction was performed in a series of four replicates, with the control (no FpGalNac deacetylase) in duplicate. By Synergy H1 TM Plate reader [BioTek TM The fluorescence signal (365 / 435 nm) generated by the release of MU by hydrolysis was monitored and converted to concentration using a standard concentration curve of MU determined under the same reaction conditions. The initial rate (μM / s) was determined and plotted in Grafit7.0 to determine the kinetic parameters.

[0099] Determine subtype 1 / 2 / 4A antigens at pH 7.4 and 37°C 四 -MU's K cat / K M Parameters. Reactions were performed in black 96-well plates (total volume 100 μL) and were performed as 2 PO 4 The coupled assay was performed in 4% NaHCO (pH 7.4) with 100 mM NaH 2 PO 4 The reaction was performed in a series of four replicates with 12 nM FpGalNac deacetylase, 0.1 mg / mL Sphex, BgaC (BgaA of subtype II), AfcA, and different concentrations of substrate (25 μM, 20 μM, 15 μM, 10 μM, 7.5 μM, 5 μM). The reaction was run in duplicate with the control (no FpGalNac deacetylase). The reaction was performed in duplicate with Synergy H1 TM Plate reader [BioTek TM The fluorescence signal (365 / 435 nm) generated by the release of MU by hydrolysis was monitored and converted to concentration using a standard concentration curve of MU determined under the same reaction conditions. The initial rate (μM / s) was determined and analyzed in Grafit7.0. TM Draw a graph to determine k cat / K M (s -1*mM -1 )parameter.

[0100] Dynamics of GH109 isoforms

[0101] Determine subtype 1 / 2 / 4A antigens at pH 7.4 and 37°C 四 -MU's K cat / K M Parameters. Reactions were performed in black 96-well plates (total volume 100 μL) and were performed as 2 PO 4 The coupled assay was performed in 4% NaHCO (pH 7.4) with 100 mM NaH 2 PO 4 (pH 7.4) with 86.02nM BvGH109_1 / 100.49nM EmGH109 / 80.52nM BvGH109_2 / 87.4nM BsGH109 and 5μM NAD+, 0.1mg / mL each of Sphex, BgaC (BgaA of subtype 2), AfcA, and different concentrations of substrate (25μM, 20μM, 15μM, 10μM, 7.5μM, 5μM). The reaction was run in a series of four replicates, with the control (no α-N-galactosaminidase) in duplicate. The reaction was performed by Synergy H1 TM Plate reader [BioTek TM The fluorescence signal (365 / 435 nm) generated by the release of MU by hydrolysis was monitored and converted to concentration using a standard concentration curve of MU determined under the same reaction conditions. The initial rate (μM / s) was determined and analyzed in Grafit7.0. TM Draw a graph to determine k cat / K M (s -1* mM -1 )parameter.

[0102] Crystallography

[0103] Prior to crystallization, thrombin (Novagen TM ) was digested with FpGalNAcDeAc_D1ext overnight. The protein was then purified by HisTrap FF column and the flow-through was collected, buffer exchanged into 10 mM Tris (pH 8.0) + 75 mM NaCl, and concentrated to 12 mg / mL.

[0104] crystallization

[0105] Using the hanging drop diffusion method, 0.2 M CaCl 2, 0.1 M MES (pH 6), 18% PEG 4000 and 20 mM MnCl 2 FpGalNAcDeAc_D1ext (12 mg / mL) was crystallized at a 1:1 protein:stock ratio. Crystals were derivatized using a rapid bromide soak for phasing and the crystals were transferred to 1 M NaBr, 25% glycerol, 18% PEG4000, 20 mM CaCl 2 A 0.1 M Mes solution was prepared by heating for 30 seconds and flash freezing in liquid nitrogen. 2 Crystal complexes with B-type blood antigen trisaccharide (B_tris) were prepared by pre-incubating the protein (12 mg / mL) with 10 mM B_tris for 2 h before setting up the droplets. Crystals were cryoprotected with a stock solution supplemented with 25% glycerol.

[0106] Data acquisition, phasing, and structure determination

[0107] Canadian Light Source TM (Canadian Light Source TM ) were used to collect the data set. The data were integrated using XDS (Kabsch 2010) and Aimless TM (Evans 2013) Determine the proportion. Use CRANK2 TM (Skubak 2013) in CCP4I2 TM Phase determination and automated structure solution were performed using the Coot TM (Emsley 2004) and Refmac TM (Vagin 2004) by alternating cycles of checking and refining the structure. The B_triplex was solved by the difference Fourier method, and the ligands were TM Water and metal ions are also constructed manually in Coot TM The difference density map confirmed the presence of Mn in the apo structure. 2+ and the presence of Ca in the ligand structure 2+ By Coot TM and Molprobity TM (Chen 2010) validated the model. The atomic coordinates and structure factors of the apo and B_tri complex have been deposited in the Protein Data Bank (PDB) with accession numbers: SEQ ID NO: WP_009260926.1 of the GalNAc deacetylase protein of Fusobacterium prausnitzii; and SEQ ID NO of the galactosamidase protein of Fusobacterium prausnitzii: WP_044942952.1

[0108] Active site mutagenesis

[0109] Based on structural information (not shown) and sequence alignment (not shown), Quickchange TM Protocol (Zhang 2004), using the primers shown in Table B, mutations of FpGalNAcDeAc_D1min and FpGalNase_Truncate were made. Mutants were purified via NiNTA and HIC columns as described above. The structural integrity of all mutants was checked via CD spectroscopy; all tested enzymes were structurally similar to their wild-type. For mutants with relatively low activity, reactions were performed under the same conditions used for the full kinetic assay; however, kcat / KM values ​​were determined using the substrate consumption method as previously described (Vocadlo 2002). In brief: in which [substrate] <K M (equivalent to K m At low substrate concentrations (~1 / 5-1 / 10 of the cat / K M Values ​​can be approximated by nonlinearly fitting the reaction time course to a first-order curve and dividing by the enzyme concentration.

[0110] GH36 phylogeny

[0111] Using Saccharis TM cazy_extract.pl script (Jones 2018) from CAZy TM The reference sequence of GH36 was downloaded from the database. The phylogeny-based protein analysis software TreeSAPP was used TM (available at https: / / github.com / hallamlab / TreeSAPP) to build reference trees and map sequences to these trees. Briefly, HMMs from dbCAN were used to map sequences from CAZy TM (Yin 2012) All full-length sequences were downloaded to extract protein family domains. Then, UCLUST was used to TM The sequences were clustered at 70% sequence similarity to remove redundant sequence space and reduce the size of the tree (Edgar 2010). TM 8.2.0 version to build reference trees with "--autoMRE" to decide when to exit bootstrapping before 1000 repetitions, and PROTGAMMAAUTO TM The best protein model was selected (Stamatakis 2006 and Stamatakis 2008).

[0112] Then use TreeSAPP TM Map query sequences to these reference trees. Briefly, use hmmsearch TM Align protein sequences with the HMM and extract aligned regions (Eddy 1998). hmmalign TM was used to include new query sequences in the reference multiple alignment, and then TrimA1 TM Non-conserved positions were removed from the alignment file (Capella-Gutierrez 2009). Using RAxML TM Sort the query sequence in the reference tree by insertion. Filter the position of each query sequence and concatenate them into a single query sequence. TM Jplace before visualization TM documents (Matsen 2012 and Letunic 2016).

[0113] RBC determination

[0114] Whole blood from healthy consenting donors was collected into citrate vacuum blood collection tubes using a protocol approved by the Clinical Ethics Committee of the University of British Columbia. The tube was spun at 1000 × g for 4 min at RT to separate RBCs and washed 3 times with 1 × PBS (pH 7.4). For the determination in the presence of dextran 40k, washed RBCs (200 μL, 10% hematocrit) were placed in a tube, the supernatant was partially removed, and replaced with 1 × PBS (pH 7.4) containing or not containing dextran 40k (final concentration of 300 mg / mL). In addition, some determinations were performed in 1 × PBS (pH 7.4) + 25% plasma or 100% plasma. RBCs were carefully mixed and placed on an orbital shaker for 30 s. The diluted enzyme solution was then added to a final volume of 200 μL. The tube was vortexed very lightly and placed on an orbital shaker for a defined time at a set temperature.

[0115] MTS Card

[0116] After the reaction, RBCs were washed three times with excess 1× PBS (pH 7.4) and lysed using a microprinting system. TM (MicroTyping System TM ,MTS) card [MTS TM, Florida, USA] for analysis. RBCs (12 μl, 5% hematocrit) suspended in diluent [MTS, Florida, USA] were carefully added to the minigel column, leaving space between the blood and the contents of the minigel. The Beckman Coulter AllegraX-22R with a modified sample holder was used as recommended. TM Centrifuge the MTS card at 156 × g for 6 min at RT. Assess the extent of antigen removal from the RBC surface from the position of the RBCs in the mini-gel after the spin according to the manufacturer's instructions. RBCs with high surface antigen concentrations agglutinate and are unable to permeate when interacting with the monoclonal antibodies present in the gel column (MTS TM RBCs without surface antigens did not agglutinate and migrated to the bottom of the minigel (MTS score of 0). RBCs with partial removal of surface antigens that migrated to a location between these antigens were assigned a score of 0 (absent) to 4 (present) according to the manufacturer's instructions.

[0117] H antigen agglutination assay

[0118] To analyze the conversion of A antigen to H antigen after enzymatic treatment, washed A-ECO-RBC were incubated in equal parts with 2 μg / mL of anti-H antibody (anti-H ab blood group antigen antibody [97-1]: Catalog number: ab24213 (Abcam TM )) were mixed and the agglutination phenomenon was monitored over a 30-minute period. RBCs agglutinated with anti-H antibodies were given a score of 0 (no agglutination within 1800 sec) to 5 (agglutination within 120 sec).

[0119] FACS

[0120] The enzyme-treated RBCs were washed twice with 1×PBS (pH 7.4), and ECO-RBCs with a 1% hematocrit were incubated with 1 / 100 APC-anti-A antibody (AlexaFluor TM 647 Mouse Anti-Human Blood Type A: Catalog Number: 565384 (BD Pharmingen TM )) and / or anti-H antibody (anti-H ab blood group antigen antibody [97-1]: Catalog number: ab24213 (Abcam TM )) was treated at RT for 30 minutes and then washed twice with 1× PBS (pH 7.4). For detection of anti-H antibodies, a secondary FITC-labeled antibody (goat F(ab')2 anti-mouse IgM mu chain (FITC)) was used at a concentration of 1 / 500: Catalog number: ab5926 (Abcam TM )). Using a flow cytometer (CytoFLEX TM(Beckman Coulter TM )) After reconstitution into 1×PBS (pH 7.4) (1% hematocrit), the data were evaluated.

[0121] Enzyme adsorption and antigenicity

[0122] To test whether the enzymes could be easily removed from RBCs after treatment, potential adsorption was assessed. Pacific Blue labeled FpGalNAc deacetylase and FpGalNase (F / P = 1) were incubated with RBCs individually at 37°C for 1 h and after several washing steps, the RBCs were analyzed by flow cytometry (CytoFLEX TM (Beckman Coulter TM )) to measure the residual fluorescence.

[0123] Antigenicity was tested by incubating RBCs with 50 μg / mL of each enzyme and mixing the enzyme-treated RBCs with allogeneic or autologous serum and observing potential agglutination. In addition, to assess potential anti-IgG, -C3d exposure, anti-IgG, -C3d MTS TM Card [MTS TM , Florida, USA]. The incubation time was 30 minutes at 37°C.

[0124] Antigen subtype synthesis

[0125] Subtype 1 / 2 / 4A and B antigens 四 The synthesis of -MU was performed using a modified protocol as described by Kwan (Kwan et al., 2015).

[0126] Two-step subtype 1 / 2 / 4H antigen 三 - Synthesis of MU

[0127] All three syntheses were prepared in 10 mL of 50 mM Tris / HCl, 200 mM NaCl, pH 7.4, 10 mM MnCl 2 , 50U of alkaline phosphatase, 1.5 equivalents of UDP-Gal, 1.2 equivalents of GDP-Fuc (determined by LacNAc-MU product) 20mg of GalNAc-α-MU / GlcNAc-α-MU scale. Depending on the desired product, for subtype I CgtBS42 and Te2FT, for subtype II HP0826 and WbgL, for subtype IV LgtD and Te2FT, different glycosyltransferases were added at a concentration of 100μg / mL. The reaction was carried out at 37°C and analyzed by TLC (mobile phase, EtAc: MeOH: H 2 O, the ratio was 6:2:1) to control the process, and 4-methylumbelliferone was reacted with 10% H2 SO 4 The compound was hydrolyzed and detected via UV (360 nm). After no further product increase was observed, the reaction was applied to a HF Bond Elut C18 column, washed with several column volumes of 5% methanol, and the product was eluted with 25% methanol. The solvent was then removed in vacuo.

[0128] Subtype 1 / 2 / 4A antigen 四 - Synthesis of MU

[0129] The final synthesis step was carried out at 37°C in 5 mL of 50 mM Tris / HCl, 200 mM NaCl, pH 7.4, 10 mM MnCl 2 , 25 U of alkaline phosphatase, 1.5 equivalents of UDP-Gal and 100 μg / mL of BgtA in 10 mg of subtype 1 / 2 / 4H antigen 三 -MU scale. Progress was followed via TLC and after no further product increase was observed, the reaction was applied to a HF Bond Elut C18 column, washed with several column volumes of 5% methanol, and the product eluted with 25% methanol. The solvent was then removed in vacuo. The final product was further purified on a 1.5×46 cm HW-40F size exclusion column and then freeze-dried.

[0130] Subtype 1 / 2 / 4B antigen 四 - Synthesis of MU

[0131] The final synthesis step was carried out at 37°C with 10 mg of subtype 1 / 2 / 4H antigen in 5 mL of 50 mM Tris / HCl, 200 mM NaCl, pH 7.4, 25 U of alkaline phosphatase, 1.5 equivalents of UDP-Gal and 100 μg / mL of BoGT6a. 三 -MU scale. Progress was followed via TLC and after no further product increase was observed, the reaction was applied to a HF BondElut C18 column, washed with several column volumes of 5% methanol, and the product eluted with 25% methanol. The solvent was then removed in vacuo. The final product was further purified on a 1.5×46 cm HW-40F size exclusion column and then freeze-dried.

[0132] Subtype 1GalN antigen 五 -MU Synthesis

[0133] 10 mg of subtype 1A antigen 五 -MU in 5 mL of 100 mM NaH 2 PO 41 μg / mL FpGalNAc deacetylase in 5% ethanol was incubated at 37°C for 30 min and then terminated by adding 1 mM EDTA. The complete conversion of the substrate was checked via TLC and the reaction was applied to a HF Bond Elut C18 column, washed with several column volumes of 2% methanol, and the product was eluted with 10% methanol. The solvent was then removed in vacuo.

[0134] Protein purification

[0135] Via Golden Gate TM All proteins and truncations thereof were cloned into pET16b or pET28a by cloning (Engler 2008) or PIPE cloning (Klock 2008). Primer sequences are listed in Table B.

[0136] The production of proteins for extended characterization was performed in BL21 (DE3) cells in 200 mL of ZY5052 auto-induction medium (Studier 2005) at 37°C, 220 rpm for 20 h, inoculated with 100 μl of an overnight LB culture. Cells were harvested by centrifugation (4000 × g, 40°C, 10 min) and resuspended in 10 mL of lysis buffer (50 mM Tris / HCl, 150 mM NaCl, 1% (v / v) glycerol, 40 mM imidazole, pH 7.4, 2 mM DTT, 1× EDTA-free protease inhibitor (Pierce TM ), 2U Benzonase (Novagen TM ), 0.3 mg / mL lysozyme, 10 mM MgCl 2 ) and then sonicated on ice (3 min pulse time; 5 sec pulse, 10 sec pause, 35% amplitude). After removing cell debris by centrifugation (14000×g, 4°C, 30 min), the supernatant was collected and loaded onto a nickel affinity chromatography column (5 mL of HisTrap HP TM Column (GE TM )) on AEKTApurifier TM System (GE TM Elution was performed and monitored on an Amicon Ultra-15 centrifugal filter unit using a 10-75% gradient of 50 mM Tris / HCl, 400 mM imidazole, pH 7.4, 2 mM DTT, and protein-containing fractions were identified by SDS-PAGE and then pooled. TM MWCO 10kDa (Millipore TM), the buffer was exchanged into 50 mM Tris / HCl, 150 mM NaCl, pH 7.4, 2 mM DTT, and concentrated.

[0137] FpGalNAc deacetylase, Fpgalactosaminidase and truncations thereof had to undergo a second round of purification before loading the proteins onto a hydrophobic interaction chromatography column (10 mL of Phenyl Sepharose High Performance Column (Pharmacia Biotech TM )) Before use, use Amicon Ultra-15 centrifugal filter device TM MWCO 10kDa (Millipore TM ) Exchange buffer. Column loading, washing and elution (gradient 0-100%) were performed by AEKTApurifier TM System (GE TM ) were treated with the following buffer conditions: FpGalNAc deacetylase; combined with 1× PBS, 800 mM NH 2 PO 4 , pH 7.4 and elution with 1× PBS (pH 7.4) and Fp-galactosaminidase; combined with 25 mM Tris / HCl, 1 M NaCl, pH 7.4 and elution with 25 mM Tris / HCl (pH 7.4). Fractions containing protein were identified by SDS-PAGE and then combined. TM MWCO10kDa(Millipore TM ), the buffer was exchanged into 50 mM Tris / HCl, 150 mM NaCl, pH 7.4, and concentrated.

[0138] Protein characterization

[0139] Optimum pH

[0140] For subtype 1A antigen 五 -MU and subtype 1GalN antigens 五 The general pH range for the activity of -MU, FpGalNAc deacetylase and Fpgalactosaminidase was determined by the appearance of products on TLC plates for changes in pH, respectively. Reactions were performed at 37°C with 50 μM substrate and 1 μg / mL enzyme in an appropriate buffer system at a scale of 100 μl. Buffers for pH 4-6 were based on 50 mM citric acid / sodium citrate buffer, buffers for pH 6-8 were based on 50 mM sodium phosphate buffer and buffers for pH 8-10 were based on 50 mM glycine / sodium hydroxide buffer.

[0141] To determine the optimal pH, 5 μg / mL of Fp-galactosaminidase was incubated in 100 μL of 50 mM sodium phosphate buffer with different pH ranges (5.8-8.0) and 200 μM GalN-α-pNP. The absorption (at 405 nm) generated by pNP release was measured by Synergy H1 TM Plate reader (BioTek TM ) were monitored at 37°C for 1 h.

[0142] 5 μg / mL FpGalNAc deacetylase and 50 μM subtype IA antigen 五 -MU was pre-incubated in 25 mM sodium phosphate buffer with different pH ranges (5.8-10.0) at 37°C for 10 min. The reaction was quenched with 100 mM sodium phosphate buffer (pH 7.5), 100 μM EDTA, 5 μg / mL Fp galactosaminidase, 50 μg / mL Sphex, 50 μg / mL AfcA and 50 μg / mL BgaC (final volume 100 μl). The fluorescence signal (365 / 435 nm) generated by the hydrolysis release of MU was detected by Synergy H1 TM Plate reader (BioTek TM ) were monitored at 37°C for 30 min.

[0143] Protein stability

[0144] FpGalNAc deacetylase and FpGalNase were stored in 1× PBS buffer (pH 7.4) at 4° C. After 2 and 12 weeks, subtype IA antigens were added as in the coupled enzyme reaction for FpGalNAc deacetylase. 五 The enzyme activity was tested according to the optimal pH conditions for the coupled enzyme reaction of GalN-α-pNP with -MU and FpGalNase.

[0145] Inhibition of FpGalNAc deacetylase

[0146] FpGalNAc deacetylase was tested against different potential inhibitors as a coupled assay in a 96-well plate format. Reactions were performed at 37°C on a 100 μL scale in 100 mM NaHCO 400 μL with 10 μg / mL FpGalNAc deacetylase, 50 μg / mL Sphex, 50 μg / mL AfcA, 50 μg / mL BgaC. 2 PO 4 (pH 7.4) at 50 μM subtype 1A antigen 五-MU and 5 μg / mL FpGalNAc deacetylase. As inhibitors, EDTA (1, 10, 100 μM), Marimastat (1, 10, 100, 1000 μM), DMSO (2%, 4%), EDTA-free protease inhibitor cocktail (Pierce TM )(1×, 2× and 4×). Using Synergy H1 TM Plate reader (BioTek TM ) Fluorescence (365 / 435 nm) was continuously monitored for 1 hour. Additives showing strong effects were run again without coupled enzyme and analyzed for product formation via TLC.

[0147] Limited proteolysis

[0148] To investigate whether there is a smaller, stable subdomain of Fp galactosaminidase, limited proteolysis was performed. Fp galactosaminidase was treated with thermolysin (10:1 protein: protease mass ratio) for 1.5 hr at various temperatures (20°C, 37°C, 42°C, 50°C and 65°C). The samples were then run on SDS-PAGE gels and a stable fragment running at approximately 70 kDa (down from an initial 118 kDa) was identified, with almost complete digestion achieved at an incubation temperature of 50°C. The fragment was sent to the UBC Proteomics Core Facility for peptide identification and determined to be a C-terminal truncated form of the full-length protein with a cleavage site between amino acids 690-700.

[0149] Glycan array screening

[0150] For glycan array screening, use Fluorotag TM FITC conjugation kit (Sigma TM ) 500 μg of FpGalNAcDeAc_D2ext was labeled with fluorescein isothiocyanate (FITC) at an F / P ratio of 1. Screening was performed at the CFG Protein-Glycan Interaction Core Facility TM (the CFG's Protein-Glycan Interaction Core Facility TM ), where the printed arrays were version 5.3, consisting of 600 glycans in 6 replicate samples at protein concentrations of 5 μg / mL and 50 μg / mL. Analysis of binding motifs was performed using a web tool from Emory University (https: / / glycopattern.emory.edu / ). Example

[0151] Example 1: Metagenomic library construction and screening

[0152] We constructed a metagenomic library containing + Large (35-65 kb) DNA fragments extracted from fecal samples provided by male donors with blood types. Such libraries contain multiple genes per bacterium, which increases the probability that at least some of these genes are expressed and allows expression of small "pathways" of multiple genes. Our library contains ~19,500 clones in 51×384 well plates, and potentially ~800,000 genes, so initial screening of such libraries with expensive A antigen substrates is impractical. Instead, we first screened with simple, sensitive, fluorescent substrates - galactose and methylumbelliferyl α-glycosides of N-acetyl-galactosamine (Gal-α-MU and GalNAc-α-MU). This initial screen with a mixture of the two substrates produced a subset of 226 hits. These substrates were rescreened against each individual substrate, identifying 44 with GalNACase and 166 with galactosidase activity. Using Figure 1 A second round of screening of these hits was performed using the coupled enzyme assay (Kwan2015) with the A and B antigen tetrasaccharide glycoside substrates shown in , as well as a no-substrate control: the coupled enzyme can function and release MU only when the initial Gal or GalNAc is cleaved. Eleven of these hits contained A antigen cleavage activity, one of which also cleaved B antigen, while six produced fluorescence in the absence of substrate, thus encoding pathways that produce unrelated fluorescent products.

[0153] Example 2: Sequencing and initial analysis of hits

[0154] In Illumina MiSeq TM Eleven fosmids were sequenced and analyzed using Metapathway TM Software (Konwar 2015) identified the presence of CAZy TMORFs in the CAZy database (http: / / www.cazy.org / ) (Lombard 2014). Due to the considerable depth of human microbiome sequencing now available, the organisms from which all fosmids originated can be identified. Their sequences can be divided into five clusters, because eight of the eleven are derived from overlapping fragments of the genomes of only two Bacteroides sp. The only gene common to all fosmids in cluster B is the GH109 enzyme (B. vulgatus); cluster A also contains GH109 (B. stercoris), which is the only CAZy gene found in a fosmid derived from another Bacteroides genus (B. vulgatus). Fosmid No8 from the obligate anaerobe Fusobacterium prausnitzii (Li 2015) contains three ORFs found in CAZy: the apparent carbohydrate binding module CBM32 and two potential glycoside hydrolases - GH36 and GH4. Finally, fosmid K05 from Collinsella sp. (probably Collinsella tanakaei) does not contain the CAZy-associated ORF. Here, the generation of a sublibrary of fosmid K05 allowed the identification of an ORF with A-cleavage activity, which was subsequently identified as GH36 (not shown).

[0155] Example 3: Analysis of GH109 enzyme

[0156] The GH109 family was established based on the A antigen cleavage activity of several of its members. These enzymes use an unusual NAD + The mechanism of dependence was first found in the enzyme from GH4 Add Yip Ref (2004) J. Amer. Chem. Soc., 126, 8354-8355, because this is the one that shows the mechanism (Varrot 2005; and Liu 2007). After removing the signal peptide, the three GH109 genes identified here were cloned with a His tag and expressed in Escherichia coli BL21 (DE3). The three proteins (BsGH109, ​​BvGH109_1 and BvGH109_2) were purified (not shown), as well as the classic GH109 (EmGH109) from Elizabethkingia menosepticum (Liu 2007) as a standard, and the kinetic parameters of each protein were determined. These three new enzymes showed similar catalytic efficiency in each of the three subtype A substrates tested, which largely reflected the kinetic parameters of the EmGH109 standard. In contrast, when using an approved MTS card in A +When their A antigen removal activity was tested on RBCs, disappointingly only EmGH109 was significantly active. Testing was done in the presence of dextran 40K as a crowding agent, which we have shown to increase activity by concentrating the enzyme on the cell surface (Chapanian 2014). In its absence, even 150 μg / mL of EmGH109 was ineffective, whereas in the presence of 300 mg / mL dextran 40K, 15 μg / mL of the enzyme was sufficient (see Figure 3 and Figure 4 ). Previous studies have shown that low ionic strength also enhances the activity of EmGH109 on cells (Liu 2007). Therefore, EmGH109 is ineffective in whole blood.

[0157] Example 4: Analysis of GH36 from Fosmid K05 of Collinsella

[0158] The identified GH36 protein in fosmid K05 (designated K05GH36) is active against GalNAc-α-MU and A antigen tetrasaccharides. This is consistent with members of the GH36 family, which primarily contain α-galactosidases and α-N-acetylgalactosaminidase and hydrolyze via a double displacement mechanism involving a covalent β-glycosylase intermediate (Comfort 2007). Phylogenetic analysis aligned its sequence within cluster 4 of the GH36 subfamily (Fredslund 2011). Interestingly, this cluster also contains the very close characterized GH36 from Clostridium perfringens, which is also known to cleave A antigen structures (Calcutt 2002). However, when we tested the ability of K05GH36 to remove A antigen from red blood cells, its activity was disappointing, scoring only 3 even when used in combination with a crowding agent.

[0159] Example 5: Analysis of Fosmid No 8 from Fusobacterium prausnitzii

[0160] Since these new enzymes offered no advantages, our attention turned to the No8 fosmid from F. plautii, especially because its gene product cleaves both A and B antigens. Three CAZy-related genes were cloned, their signal peptide sequences removed, expressed in E. coli BL21(DE3), and the resulting enzymes purified with yields as high as 140 mg / L. Surprisingly, when we tested the purified proteins individually against A and B tetrasaccharide substrates, the only cleavage observed was that of B antigen by No8GH36, while A antigen was not cleaved by either of them. We therefore tested combinations of these enzymes in pairs and surprisingly found that a mixture of No8CBM32 and No8GH36 rapidly cleaved the A antigen tetrasaccharide. TLC analysis of the reaction mixtures with the individual enzymes showed that No8CBM32 catalyzed the conversion of the A antigen to a more polar but still UV-active product, while the subsequent addition of No8GH36 released a sugar product that co-migrated with galactosamine as well as the H antigen trisaccharide. MS analysis of the reaction mixture showed that No8CBM32 is an A antigen deacetylase, thus having a lower m / z of 42 and being more polar, while No8GH36 is a galactosaminidase, a new activity for this family ( Figure 2 This was further confirmed by high performance anion exchange chromatography (HPAE-PAD) analysis of the reaction ( Figure 5 ), which showed that treatment of the A antigen with both enzymes released galactosamine, whereas the enzyme alone did not. Similar results were obtained with a gastric mucin substrate, for which the enzymes were presumed to release galactosamine. Therefore, the two enzymes are hereafter referred to as FpGalNAc deacetylase (FpGalNAcDeAc) and Fp galactosaminidase (FpGalNase).

[0161] Although this pathway for degradation of the A antigen has not been previously characterized, it is fascinating that an explanation was proposed more than 50 years ago to explain the so-called “acquired” B phenomenon, in which the blood type of type A patients infected with Clostridium difficile changed significantly to type B (Gerbal 1975), as did forensic samples of human tissue submerged in the Thames (Ref Judd and Annesley https: / / doi.org / 10.1016 / S0887-7963(96)80087-3, Transfusion medicine reviews (1996) 10, 111-117). This was presumably because the anti-B antibodies used for typing could not distinguish between terminal Gal and GalN.

[0162] Studies of the third enzyme in the fosmid GH4 showed that although it hydrolyzes Gal-α-pNP, GalN-α-pNP, and GlcN-α-pNP, it does not cleave any A-antigen-based substrates and, therefore, does not appear to play a direct role in the conversion of A-antigen. However, these glycosaminidase enzymes do represent novel activities within the GH4 family.

[0163] Example 6: Characterization of FpGalNAc deacetylase

[0164] Using Phyre2 TM (Kelley 2015) Closer bioinformatic analysis of the gene revealed a domain of ∼308 amino acids of previously unknown function at the N-terminus and a CBM32 of ∼145 amino acids near the C-terminus, with a linker region between them. Truncation analysis confirmed this basic structure, as all constructs containing the complete deacetylase domain were indeed catalytically active (Table 2). Therefore, this protein was classified as a founding member of a new carbohydrate esterase family, CExx.

[0165] Acetamidosugar deacetylases have been shown to be metalloenzymes that require divalent metal ions (Blair 2005). Accordingly, treatment with 100 μM EDTA largely abolished the enzyme activity, while addition of Mn 2+ 、Co 2+ 、Ni 2+ or Zn 2+ Increased enzyme activity. Other (non-metallic) amidase inhibitors had no effect. The enzyme has a broad pH distribution with an optimum around pH 8 ( Figure 6 ), and has a narrow substrate specificity, limited to the different A subtypes and their shorter forms. However, among those subtypes, it is not very discriminating, with the specific activity between all of them differing only ~2-fold (Table 2). This pH dependence and specificity profile is ideal for RBC conversion, since all subtypes of A are deacetylated, but no others.

[0166] The glycan arrays of the Consortium for Functional Glycomics (CFG) were used to explore the specificity of the CBM portion of the protein. The preferred targets are glycans with a repetitive N-acetyllactosamine (LacNAc) structure; it is in the founding member of the CBM32 family; it can also be seen in the N-acetylglucosaminidase of Clostridium perfringens (Ficko-Blean 2006). However, unlike this CBM, ours did not show high affinity binding to blood antigen structures. Repeating LacNAc structures are common components of cell surfaces (Cohen 2009), as a universal component of complex and hybrid N-glycans as well as some O-glycans and glycolipids. In our case, they may serve as an anchor point for the attachment of the deacetylase domain. This would bring its catalytic domain very close to the A antigen without competing for its own substrate. In support of this model, removal of the domain resulted in reduced RBC activity without affecting the cleavage rate of soluble substrates (Table 2).

[0167] Example 7: Crystallization analysis of FpGalNAc deacetylase

[0168] To provide structural insight into this novel enzymatic activity, crystallization trials were performed on truncated proteins and it was found that FpGalNAcDeAc_D1ext produced crystals that diffracted to the best resolution. Solution of this structure revealed a catalytic domain adopting a 5-fold β-propeller structure with an active site containing a divalent metal ion coordinated by D100 and H252. Co-crystallization of the enzyme with a close analog of the B-antigen trisaccharide as the reaction product revealed its binding mode. At the basis of the active site pocket, the non-reducing terminal galactosyl moiety, as a distinguishing group between the A-antigen and the B-antigen, forms hydrogen bonding interactions with H97, E64 and the two metal-coordinated waters. The rest of the ligand is surface exposed and polar interactions are identified between the fucosyl and S61 and D121 side chains. The C1-OH group of the reducing terminal galactosyl moiety is solvent exposed, so the enzyme is easily adapted to extension of the substrate (i.e., with GlcNAc). Mimicking the N-acetyl group of the A-trisaccharide on this structure allowed us to make rational mutations of nearby amino acids that may be involved in substrate deacetylation. The two mutants are inactive, so residue E64 is proved to be crucial for activity, which shows a direct effect (table 1) in the activation of nucleophilic water molecules. The residues D100, Y315 and H252 of the coordination divalent metal are also proved to be important, wherein any mutation causes a rate reduction of ~ 5000 times, consistent with their apparent effect in conjunction with divalent metal ions. Similar to other acetylamino sugar deacetylases, we propose that FpGalNAc deacetylase is hydrolyzed by the following mechanism, wherein metal is used for polarized carbonyl and activated water molecules form tetrahedral intermediates with nucleophilic attack carbonyl. The proton supply of sugar nitrogen atoms by His 100 promotes the decomposition of this intermediate.

[0169] Table 1 | FpGalNAcDeAc_D1min and its mutants cleave type 2 A antigen 四 -Specific activity of MU ND = no detectable activity

[0170] Example 8: Characterization of FpGalNAcDeAc and FpGalNase

[0171] Phylogenetic analysis of the sequence placed FpGalNase in a new subgroup of the GH36 family (5) (Fredslund 2011). The 390 amino acid catalytic domain is located in the center of this large (1079 amino acid) protein, with a potential carbohydrate binding domain at the C-terminus. Removal of this C-terminal domain had no effect on the kinetic parameters of the enzyme with soluble substrates (Table 2), but resulted in a decrease in the deacetylated A +The cleavage efficiency of RBC was reduced. The enzyme is specific for galactosamine-containing sugars and will not cleave GalNAc residues in any context tested. However, it has a fairly broad specificity for cleavage of de-N-acetylated galactosamine from simple aryl glycosides GalN-α-pNP upwards. In fact (Table 2) the k cat / K M values ​​with each other and with those of the deacetylase k cat / K M The values ​​are similar. cat / K M The value is more than 2000-fold lower than that of the corresponding GalN antigen, but still sufficient to generate a positive hit in the original screen. This specificity for deacetylated α-galactose conformation substrates combined with its pH optimum of ~6.5-7.0 is well suited for blood group conversion in conjunction with deacetylase ( Figure 6 ).

[0172] Table 2 | Kinetic parameters of FpGalNAcDeAc and FpGalNase constructs for different antigenic substrates

[0173] Example 9: Cleavage of A antigen from RBC

[0174] A + , B + and O + Type A RBCs were incubated with FpGalNAcDeAc and FpGalNase alone and as a mixture, and the released sugars were analyzed on HPAE-PAD ion chromatograms. None of the enzymes used released any sugar products alone. However, when a mixture of the two was used, galactosamine was clearly released from A + type RBC release, rather than from B + or + Release, thus having high specificity only for the A antigen. This is very important because it shows that GalNAc is not released from the RBC surface under any other circumstances. A truncated form of FpGalNase is also effective, but slightly less active.

[0175] We then proceeded to use the industry standard MTS TMCard test removes antigen from RBC.The column of these antibody conjugates is loaded with RBC and rotated in a centrifuge.Antigen-free RBC migrates to the bottom of the column and is scored as 0, while untreated RBC carries the corresponding antigen rod at the top and is scored as 4, and the intermediate score ranks the degree of antigen removal.Separately, FpGalNase treatment can not remove A or B antigenicity (Table 3) at the concentration adopted, which is consistent with its inactivity to GalNAc substrate and low activity to Gal.Hatching with FpGalNAcDeAc removes the antigenicity caused by acetamide being converted into amine, thereby impairing the combination of the anti-A antibody adopted.In the absence and presence of 300mg / ml of dextran as crowding agent, the minimum amount of enzyme required for the complete antigen deacetylation of independent FpGalNAcDeAc and combined with FpGalNase was assessed. Without assistance from dextran, amounts of FpGalNase down to 3 μg / ml were sufficient, while inclusion of 300 mg / ml of dextran reduced the required loading to 0.5 μg / ml (Table 3). By comparison with the best previous enzyme, EmGH109 was ineffective in the absence of dextran unless a low salt buffer was used, while the minimum effective concentration in the presence of dextran was 15 μg / ml (30-fold higher loading). The FpGalNAcDeAc form lacking a CBM was much less efficient.

[0176] Table 3 | Treatment of A with EmGH109, ​​FpGalNAcDeAc and FpGalNase + , B + and AB + MTS card results from RBC.

[0177] Due to the MTS TM The card test does not assess the complete conversion of the A antigen, and since no antibodies are available to detect the GalN antigen, we focused on detecting the newly formed H antigen on the treated RBCs. FpGalNase is functional at a concentration of only 5 μg / ml, which results in an increase in H antigen levels consistent with the loss of the A antigen, as shown in Figure 2. Figure 3 By measuring the aggregation time in the presence of anti-H-antibody, we demonstrated that both enzymes are active against several A + The functionality of the RBC donor, also under whole blood reaction conditions, has not been achieved before with other blood conversion enzymes. As a result, this pair of enzymes uses a much lower enzyme loading than the best previous enzymes required to convert A +RBCs are converted to O-type "universal donor" RBCs. However, before these RBCs are delivered to patients, it is recommended to remove all traces of the enzyme used in the conversion to avoid adverse immune responses, most preferably by washing the cells after centrifugation. To confirm that this is achievable, we treated A samples with fluorescently labeled FpGalNAcDeAc and FpGalNase + RBCs were then analyzed using FACS to confirm that simple washing was indeed effective ( Figure 3 ).

[0178] Further characterization of the A-ECO RBCs produced may be used to assess their full viability for use in transfusion medicine, but the possibility of including the enzyme directly in the plasma when collecting donated blood may allow for easy, cost-effective separation of the process from existing automated procedures for blood collection and storage. Specifically, the stability of the enzymes was tested as shown in Table 4. Table 4: Storage stability of galactosaminidase and GalNAc deacetylase Substrate: 100 μM GalN-α-pNP Substrate: 100uM T1 A antigen 四 -MU

[0179] Example 10: Fusion of GalNac deacetylase and galactosaminidase from Clostridium tertium

[0180] In the search for similar enzymes, a new native fusion of a galactosaminidase and a GalNAc deacetylase linked by a CBM (GH36_domain-CBM-deacetylating_domain) was identified. Initial tests showed that the enzyme cleaves the A antigen of red blood cells (same mechanism, first deacetylation, then galactosamine cleavage), but not very efficiently (i.e. similar to EmGH109). The deacetylation domain of C. tertius is not as effective as the GalNAc deacetylase of F. prausnitzii, but if complemented with the GalNAc deacetylase of F. prausnitzii, the galactosaminidase domain of C. tertius shows similar activity on red blood cells as the galactosaminidase of F. prausnitzii.

[0181] Example 11: Alternative GalNAc deacetylase and galactosaminidase

[0182] The data showed that the galactosaminidase of C. difficile (Ct5757_GalNAse) and Rp1021 did have comparable enzymatic activities for the conversion of GalN antigen to H antigen (the second reaction step).

[0183] The data of alternative GalNAc deacetylase and galactosaminidase were also collected, and alternative enzymes were compared with the GalNAc deacetylase of Fusobacterium prausnitzii and the galactosaminidase of Fusobacterium prausnitzii. As shown in Table 5, the MTS scores of the anti-A antibodies of the natural fusion of the third clostridium bacillus of galactosidase and GalNAc deacetylase on treated A RBC are shown, which requires the presence of dextran to effectively cut the A antigen, and when combined with the galactosaminidase (FpGalNase) of Fusobacterium prausnitzii, the good activity of the GalNAc deacetylase (Ct5757_DeAcase) of the third clostridium bacillus is also shown. Also in Table 6, data show that Rp3672 and Rp3671 of Robinsoniella peoriensis (Rp) can deacetylate the A antigen on RBC, but efficiency is lower than FpGalNAcDeAase, and activity is only achieved when there is a crowding agent (i.e. dextran 40k).

[0184] Table 5: MTS scores for anti-A antibodies on treated A RBCs

[0185] Table 6: MTS scores of 3671 and 3672 of Robinsoniella peoriensis (Rp) sample Anti-A MTS score A RBC control 4 Rp3671 (50 μg / mL) + dextran 40k 3 Rp3672 (50 μg / mL) + dextran 40k 1

[0186] Figure 7Shown is the conversion of A antigen to H antigen on ARBCs analyzed by FACS sorting for (A) A+RBC control, (B) GalNAc deacetylase (FpGalNAcDeAc) of Fusobacterium prausnitzii + galactosamidase (FpGalNase) of Fusobacterium prausnitzii (10 μg / mL), (C) FpGalNAcDeAc + Ct57577_GalNase of Clostridium tertiotrophobia (Ct) (10 ug / mL), and (D) FpGalNAcDeAc + galactosamidase (Rp1021)GalNase (10 ug / mL) of Robinsoniella peoriensis (Rp). The data showed that galactosamidase (Rp1021) GalNase from Clostridium tertiotrophoside (Ct) Ct5757_GalNase and Robinsoniella peoriensis (Rp) had enzyme activities comparable to galactosamidase (FpGalNase) from Fusobacterium peoriensis for converting GalN antigen to H antigen (second reaction step).

[0187] Although various embodiments of the present invention are disclosed herein, many adaptations and modifications can be made within the scope of the present invention according to the common knowledge of those skilled in the art. Such modifications include replacing any aspect of the present invention with known equivalents so as to obtain the same results in substantially the same manner. Numerical ranges include the numbers defining the range. The word "comprising" is used as an open term in this article, which is substantially equivalent to the phrase "including but not limited to", and the word "comprise" has a corresponding meaning. As used herein, the singular forms "a / an" and "the" include plural indicators unless the context clearly specifies otherwise. Therefore, for example, referring to "things" includes more than one such thing. The references cited herein are not prior art to admit that these references are embodiments of the present invention. The present invention includes all embodiments and variations substantially as described above and with reference to the examples and drawings. sequence

[0188] The DNA sequence of F. prausnitzii was modified from the naturally occurring DNA sequence (GalNAc deacetylase 2311 / 2319nt / galactosamidase 3228 / 3237nt). In particular, the length of the sequence used for protein purification varied, thereby removing the signal peptide and adding an N-terminal His tag via the vector backbone.

[0189] Informal Sequence Listing

[0190] SEQ ID NO: 2

[0191] Description: GalNAc deacetylase from Fusobacterium prausnitzii (protein sequence) MRNRRKAVSLLTGLLVTAQLFPTAALAADSSESALNKAPGYQDFPAYYSDSAHADDQVTHPDVVVLEEPWNGYRYWAVYTPNVMRISIYENPSIVASSDGVHWVEPEGLSNPIEPQPPSTRYHNCDADMVYNAEYDAMMAYWNWADDQGGGVGAEVRLRISYDGVHWGVPVTYDEMTRVWSKPTSDAERQVAD GEDDFITAIASPDRYDMLSPTIVYDDFRDVFILWANNTGDVGYQNGQANFVEMRYSDDGITWGEPVRVNGFLGLDENGQQLAPWHQDVQYVPDLKE FVCISQCFAGRNPDGSVLHLTTSKDGVNWEQVGTKPLLSPGPDGSWDDFQIYRSSFYYEPGSSAGDGTMRVWYSALQKDTNNKMVADSSGLTIQAK SEDDRIWRIGYAENSFVEMMRVLLDDPGYTTPALVSGNSLMLSAETTSLPTGDVMKLETSFAPVDTSDQVVKYTSSDPDVATVDEFGTITGVSVGSARIMAETREGLSDDLEIAVVENPYTLIPQSNMTATATSVYGGTTEGPASNVLDGNVRTIWHTNYAPKDELPQSITVSFDQPYTVGRFVYTPRQNGTN GIISEYELYAIHQDGSKDLVASGSDWALDAKDKTVSFAPVEAVGLELKAIAGAGGFGTAAELNVYAYGPIEPAPVYVPVDDRDASLVFTGAWNSDSNGSFYEGTARYTNEIGASVEFTFVGTAIRWYGQNDVNFGAAEVYVDGVLAGEVNVYGPAAAQQLLFEADGLAYGKHTIRIVCVSPVVDFDYFSYVGE

[0192] SEQ ID NO: 4

[0193] Description: GalNAc deacetylase from Fusobacterium prausnitzii (protein sequence that removes the signal peptide) ADSSESALNKAPGYQDFPAYYSDSAHADDQVTHPDVVVLEEPWNGYRYWAVYTPNVMRISIYENPSIVASSDGVHWVEPEGLSNPIEPQPPSTRYHNCDADMVYNAEYDAMMAYWNWADDQGGGVGAEVRLRISYDGVHWGVPVTYDEMTRVWSKPTSDAERQVADGEDDFITAIASPDRYDMLSPTIVYDDFRDVFILWANNTGDVGYQNGQANFVEMRYSDDGITWGEPVRVNGFLGLDENGQQLAPWHQDVQYVPDLKEFVCISQCFAGRNPDGSVLHLTTSKDGVNWEQVGTKPLLSPGPDGSWDDFQIYRSSFYYEPGSSAGDGTMRVWYSALQKDTNNKMVADSSGNLTIQAKSEDDRIWRIGYAENSFVEMMRVLLDDPGYTTPALVSGNSLMLSAETTSLPTGDVMKLETSFAPVDTSDQVVKYTSSDPDVATVDEFGTITGVSVGSARIMAETREGLSDDLEIAVVENPYTLIPQSNMTATATSVYGGTTEGPASNVLDGNVRTIWHTNYAPKDELPQSITVSFDQPYTVGRFVYTPRQNGTNGIISEYELYAIHQDGSKDLVASGSDWALDAKDKTVSFAPVEAVGLELKAIAGAGGFGTAAELNVYAYGPIEPAPVYVPVDDRDASLVFTGAWNSDSNGSFYEGTARYTNEIGASVEFTFVGTAIRWYGQNDVNFGAAEVYVDGVLAGEVNVYGPAAAQQLLFEADGLAYGKHTIRIVCVSPVVDFDYFSYVGE

[0194] SEQ ID NO: 5

[0195] Description: GalNAc deacetylase of His tag Clostridium propionicum (pET16a - protein sequence) with MG HHHHHHHHHHSSGADSSESALNKAPGYQDFPAYYSDSAHADDQVTHPDVVVLEEPWNGYRYWAVYTPNVMRISIYENPSIVASSDGVHWVEPEGLSNPIEPQPPSTRYHNCDADMVYNAEYDAMMAYWNWADDQGGGVGAEVRLRISYDGVHWGVPVTYDEMTRVWSKPTSDAERQVADGEDDFITAIASPDRYDMLSPTIVYDDFRDVFILWANNTGDVGYQNGQANFVEMRYSDDGITWGEPVRVNGFLGLDENGQQLAPWHQDVQYVPDLKEFVCISQCFAGRNPDGSVLHLTTSKDGVNWEQVGTKPLLSPGPDGSWDDFQIYRSSFYYEPGSSAGDGTMRVWYSALQKDTNNKMVADSSGNLTIQAKSEDDRIWRIGYAENSFVEMMRVLLDDPGYTTPALVSGNSLMLSAETTSLPTGDVMKLETSFAPVDTSDQVVKYTSSDPDVATVDEFGTITGVSVGSARIMAETREGLSDDLEIAVVENPYTLIPQSNMTATATSVYGGTTEGPASNVLDGNVRTIWHTNYAPKDELPQSITVSFDQPYTVGRFVYTPRQNGTNGIISEYELYAIHQDGSKDLVASGSDWALDAKDKTVSFAPVEAVGLELKAIAGAGGFGTAAELNVYAYGPIEPAPVYVPVDDRDASLVFTGAWNSDSNGSFYEGTARYTNEIGASVEFTFVGTAIRWYGQNDVNFGAAEVYVDGVLAGEVNVYGPAAAQQLLFEADGLAYGKHTIRIVCVSPVVDFDYFSYVGE

[0196] SEQ ID NO: 7

[0197] Description: Galactosaminidase of Clostridium pectinovorum

[0198] SEQ ID NO: 9

[0199] Description: Galactoamidase from Fusobacterium prausnitzii (protein sequence with signal peptide removed)

[0200] SEQ ID NO: 10

[0201] Description: Has His tag Galactosamidase from Fusobacterium prausnitzii (pET16a-protein sequence) MG HHHHHHHHHH

[0202] SEQ ID NO: 12

[0203] Description: Protein sequence isolated from Clostridium difficile identified as 99345757.1-Ct5757 (fusion of galactosidase and GalNAcDe acetylase linked via a CBM) (original protein sequence)

[0204] SEQ ID NO: 14

[0205] Description: Isolated protein sequence of Clostridium difficile 5757 (Ct5757) with signal peptide removed (identifier 099345757.1-Ct5757)

[0206] SEQ ID NO: 15

[0207] Description: With His tag and thrombin cleavage site of the third Clostridium 5757 (Ct5757) fusion protein sequence expression construct (in pET28a vector) MGSS HHHHHH

[0208] SEQ ID NO: 17

[0209] Description: With His tag and thrombin cleavage site of the third Clostridium 5757 (Ct5757) GalNAc deacetylase protein sequence_expression construct (in pET28a vector) MGSS HHHHHH SSGLVPRGSHSGQYWLVFQPDNDVLQTKTNPSSMKQSANNNPYNYNILPNSFPIGTGYNAYKGDVSFYATFKEASSQAIPQNSWALKYVDSEETTGENGRATNAFDGNNNTIWHTKYSGGNAAPMPHEIQIDLR GVYNINQINYLPRQDGGTNGTIKDYEVYLSLDGVNWGQPISKGTFESNSTEKIVKFNETKSRYVKLKALSEINNKQFTTVADLKVFGWEISKIEKPLQNAETYLNIPTYDGLNQSTHPDVKYFKNGWNGYKYWM IMTPNRTGSSVAENPSILASDDGINWEVPAGVTNPIAPMPQVGHNCDVDMIYNEATDELWVYWVESDDITKGWVKLIKSKDGVNWSSQQVVVDDNRAKYSTLSSPIIFKDNKYYMWSVNTGNSGWNNQSNKVEL RESSDGVNWSNPTVVNTLAQDGSQIWHVNVEYIPSKNEYWAIYPAYKNGTGSDKTELYYAKSSDGVNWTTYKNPILSKGTSGKWDDMEIYRSCFVYDEDTNMIKVWYGAVSQNPQIWKIGFTENDYDKFIEGLTQ

[0210] SEQ ID NO: 19

[0211] Description: With His tag and a third Clostridium difficile 5757 (Ct5757) protein sequence galactosamidase_expression construct (in pET28a vector) MGSS HHHHHHSSGLVPRGSHYNLIDNISVEKLDTDISQANENVFLNGNGIALEVDNRGATCIYLVDENGVKTKATTSLDTADFSGYPIIGGQKIRDFVIISKNLEENINSILGVGNRLTIISKSSSTNLIRKIVFETSNSNPGAIYSTVSYKAESNDLLVDSFHENEYTMSLGQGPFLAYQGCADQQGANTIVNVTNGYNHNSGQNNYSVGVPFSYVYNSVGGIGIDDASTSRREFKLPIIGKDNTVSLGMEWNGQTLKKGAETAIGTSVITTTNGDYYSGLKSYAEVMKDKGISAPASIPDIAYDSRWESWGFEFDFTIEKIVNKLDELKAMGIKQITLDDGWYTYAGDWKLSPQKFPNGNADMKYLTDEIHKRGMTAILWWRPPVDGGINSKLVSEHPEWFIKNSQGNMVRLPGPGGNGGTAGYALCPNSEGSIQHHKDFVTVALEEWGFDGFKEDYVWGIPKCYDSSHKHSSLSDTLENQYKFYEAIYEQSIAINPDTFIELCN CGTPQDFYSTPYVNHAPTADPISRVQTRTRVKAFKAIFGDDFPVTTDHNSVWLPSALGTGSVMITKHTTLSSDRQYNKYFGLARDLELAKGEFIGNLYKYGIDPLESYVIRKGEDIYYSFYKDNSSYSGNIEIKGLDSNATYRIEDYVNNRVIARGVKGPTATINTSFTDNLLVRAIPDDTPAEVTTFDVGNNTILSSTDSGNSKYLNAVSTTLEKTATIDSLSIYIGNNSENGKLQIAIYDDNNGKPGTKKAYVEEFVPTKNSWNTKKVNSVNTLPSGQYWLVFQPDNDVLQTKTNPSSMKQSANNNPYNYNILPNSFPIGTGYNAYKGDVSFYATFKEASSQAIPQNSWALKYVDSEETTGENGRATNAFDGNNTIWHTKYSGGNAAPMPHEIQIDLRGVYNINQINYLPRQDGGTNGTIKDYEVYLSLDGVNWGQPISKGTFESNSTEKIVKFNETKSRYVKLKALSEINNKQFTTVADLKVFGWEISKIEK

[0212] SEQ ID NO: 21

[0213] Description: With His tag Galactoamidase protein expression construct of Robinsoniella peoriensis Rp1021 with thrombin cleavage site (in pET28a vector) MGSS HHHHHH

[0214] SEQ ID NO: 23

[0215] Description: With His tag GalNAc deacetylase protein sequence expression construct of Ruthenibacterium lactatiformans R18755 with a thrombin cleavage site (in pET28a vector) MGSS HHHHHH SSGLVPRGSHEETDLLVNGGFETGDSTGWNWFNNAVVDSAAPHSGNYCAKVAKNSSYEQVVTVSPDTKYVLTGWAKSEGSSVMTLGVKNYGGQETFSATLSADYQQLAVTFTTG PNAQTATIYGYRQNSGSGAGYFDDVELTAVQDFAPYQPLANAIAPQAIPTYDGANQPTHPSVVKFEQPWNGYLYWMAMTPYPFNDGSYENPSIVASNDGENWIVPEGVSNPLAGT PSPGHNCDVDLVYVPASDELRMYYVEADDIISSRVKMISSRDGVHWSEPQVVMQDLVRKYSILSPSIEILPDGTYMMWYVDTGNAGWNSQNNQVKYRTSADGIKWSGAVTCTDF VQPGYQIWHIDVHYDTSSGAYYAVYPAYPNGTDCDHCNLFFAVNRTGKQWETFSRPILKPSTEGGWDDFCIYRSSMLIDDGMLKVWYGAKKQEDSSWHTGLTMRDFSEFMKILER

[0216] SEQ ID NO: 25

[0217] Description: With His tag GalNAc deacetylase protein of Robinsoniella peoriensis Rp3671 with a thrombin cleavage site_expression construct (in pET28a vector) MGSS HHHHHH

[0218] SEQ ID NO: 27

[0219] Description: With His tag GalNAc deacetylase protein of Robinsoniella peoriensis Rp3672 with a thrombin cleavage site_expression construct (in pET28a vector) MGSS HHHHHH

[0220] SEQ ID NO: 29

[0221] Description: With His tag GalNAc deacetylase protein Rp3671 of Robinsoniella peoriensis Rp3671 with a thrombin cleavage site (in pET28a vector) MGSS HHHHHHSSGLVPRGSHSPLSAAAESGTGTRLVKGQTGYLTEEQAIRNQEQTTEEREQKLTGEETAEVLMEGTKDSGIVQTEEVQTKEMQTEDAQTEEVQTEEMQTEDAQTKEVQTEEMQTEDAQTEEVQTKEEPAEETHMKEIQTQGTKKASDRNGKARVTEILEDAQDPANRIVYLSDLQWKSENHTVDSELPTRKDKSFGGGKITLKVDGTVTEFDKGIGTQTDSTIVYDLEGKGYTKFETYVGVDYSQKENIPGEVCDVKFRVKIDDKIVSETGVLDPLSNAVKISVNIPDTAKTLTLYADKVTETWSDHANWADAKFYQALPEPENVAFKKTVVTRKTSDNSEAPVNPDSAVNSSKAVDGVIDSSSYFDFGDQANSGAVRESLYMEVDLKGSYLLSDIQLWRYWKDGRTYAATAIVVAEDENFENAAVIYNSDTTGEIHHLGAGSDMLYAETESGKTFPVPENTKARYIRVYTYGVNGTSGVTNHIVELKVNAYVFGDEILPEKPDDSKIFPNAVNPLKLQGPGTNDQVTHPDVTVFDEPWNGYKYWMAYTPNKPGSSYFENPCIAASNDGVNWEFPAQNPVQPRYDSEIENQNEHNCDTDIVYDPVNDRLIMYWEWAQDEAVNGKTHRSEIRYRVSYDGINWGVEDKTGVLMTGPTDHGCAIATEGERYSDLSPTVVYDKTEKIYKMWANDAGDVGYENKQNNKVWYRTSQDGISNWSDKTYVENFLGVNEDGLQMYPWHQDIQWVEEFQEYWALQQAFPAGSGPDNSSLRFSKSKDGLHWEPVSEKALITVGAPGTWDAGQIYRSTFWYEPGGAKGNGTFHIWYAALAEGQSHWDIGYTSANYADAMYKLTGSR

[0222] SEQ ID NO: 31

[0223] Description: Having His tagRobinsoniella peoriensis Rp3672_GalNAc deacetylase_protein expression construct with thrombin cleavage site (in pET28a vector) MGSS HHHHHH

[0224] SEQ ID NO: 32

[0225] Description: GalNAc deacetylase protein sequence of Clostridium tertiotrophicum 5757 (Ct5757) HSGQYWLVFQPDNDVLQTKTNPSSMKQSANNNPYNYNILPNSFPIGTGYNAYKGDVSFYATFKEASSQAIPQNSWALKYVDSEETTGENGRATNAFDGNNNTIWHTKYSGGNAAPMPHEIQIDLRGVYNINQ INYLPRQDGGTNGTIKDYEVYLSLDGVNWGQPISKGTFESNSTEKIVKFNETKSRYVKLKALSEINNKQFTTVADLKVFGWEISKIEKPLQNAETYLNIPTYDGLNQSTHPDVKYFKNGWNGYKYWMIMTPN RTGSSVAENPSILASDDGINWEVPAGVTNPIAPMPQVGHNCDVDMIYNEATDELWVYWVESDDITKGWVKLIKSKDGVNWSSQQVVVDDNRAKYSTLSSPIIFKDNKYYMWSVNTGNSGWNNQSNKVELRES SDGVNWSNPTVVNTLAQDGSQIWHVNVEYIPSKNEYWAIYPAYKNGTGSDKTELYYAKSSDGVNWTTYKNPILSKGTSGKWDDMEIYRSCFVYDEDTNMIKVWYGAVSQNPQIWKIGFTENDYDKFIEGLTQ

[0226] SEQ ID NO: 33

[0227] Description: GalNAc deacetylase protein sequence of Ruthenibacterium lactatiformans R18755 HEETDLLVNGGFETGDSTGWNWFNNAVVDSAAPHSGNYCAKVAKNSSYEQVVTVSPDTKYVLTGWAKSEGSSVMTLGVKNYGGQETFSATLSADYQQLAVTFTTGPNAQTATIYGYRQNSGSGAGYFDDVELTAVQDFAPYQPLANAIAPQAIPTYDGANQPTHPSVVKFEQPWNGYLYWMAMTPYPFNDGSYENPSIVASNDGENWIVPEGVSNPLAGTPSPGHNCDVDLVYVPASDELRMYYVEADDIISSRVKMISSRDGVHWSEPQVVMQDLVRKYSILSPSIEILPDGTYMMWYVDTGNAGWNSQNNQVKYRTSADGIKWSGAVTCTDFVQPGYQIWHIDVHYDTSSGAYYAVYPAYPNGTDCDHCNLFFAVNRTGKQWETFSRPILKPSTEGGWDDFCIYRSSMLIDDGMLKVWYGAKKQEDSSWHTGLTMRDFSEFMKILER

[0228] SEQ ID NO: 34

[0229] Description: GalNAc deacetylase protein of Robinsoniella peoriensis Rp3671 HSPLSAAAESGTGTRLVKGQTGYLTEEQAIRNQEQTTEEREQKLTGEETAEVLMEGTKDSGIVQTEEVQTKEMQTEDAQTEEVQTEEMQTEDAQTKEVQTEEMQTEDAQTEEVQTKEEPAEETHMKEIQTQGTKKASDRNGKARVTEILEDAQDPANRIVYLSDLQWKSENHTVDSELPTRKDKSFGGGKITLKVDGTVTEFDKGIGTQTDSTIVYDLEGKGYTKFETYVGVDYSQKENIPGEVCDVKFRVKIDDKIVSETGVLDPLSNAVKISVNIPDTAKTLTLYADKVTETWSDHANWADAKFYQALPEPENVAFKKTVVTRKTSDNSEAPVNPDSAVNSSKAVDGVIDSSSYFDFGDQANSGAVRESLYMEVDLKGSYLLSDIQLWRYWKDGRTYAATAIVVAEDENFENAAVIYNSDTTGEIHHLGAGSDMLYAETESGKTFPVPENTKARYIRVYTYGVNGTSGVTNHIVELKVNAYVFGDEILPEKPDDSKIFPNAVNPLKLQGPGTNDQVTHPDVTVFDEPWNGYKYWMAYTPNKPGSSYFENPCIAASNDGVNWEFPAQNPVQPRYDSEIENQNEHNCDTDIVYDPVNDRLIMYWEWAQDEAVNGKTHRSEIRYRVSYDGINWGVEDKTGVLMTGPTDHGCAIATEGERYSDLSPTVVYDKTEKIYKMWANDAGDVGYENKQNNKVWYRTSQDGISNWSDKTYVENFLGVNEDGLQMYPWHQDIQWVEEFQEYWALQQAFPAGSGPDNSSLRFSKSKDGLHWEPVSEKALITVGAPGTWDAGQIYRSTFWYEPGGAKGNGTFHIWYAALAEGQSHWDIGYTSANYADAMYKLTGSR

[0230] SEQ ID NO: 35

[0231] Description: Robinsoniella peoriensis Rp3672 GalNAc deacetylase protein

[0232] SEQ ID NO: 36

[0233] Description: Galactosamidase protein sequence of Clostridium tertiotrophicum 5757 (Ct5757) HYNLIDNISVEKLDTDISQANENVFLNGNIALEVDNRGATCIYLVDENGVKTKATTSLDTADFSGYPIIGGQKIRDFVIISKNLEENINSILGVGNRLTIISKSSSTNLIRKIVFETSNSNPGAIYSTVSYKAESNDLLVDSFHENEYTMSLGQGPFLAYQGCADQQGANTIVNVTNGYNHNSGQNNYSVGVPFSYVYNSVGGIGIDDASTSRREFKLPIIGKDNTVSLGMEWNGQTLKKGAETAIGTSVITTTNGDYYSGLKSYAEVMKDKGISAPASIPDIAYDSRWESWGFEFDFTIEKIVNKLDELKAMGIKQITLDDGWYTYAGDWKLSPQKFPNGNADMKYLTDEIHKRGMTAILWWRPPVDGGINSKLVSEHPEWFIKNSQGNMVRLPGPGGNGGTAGYALCPNSEGSIQHHKDFVTVALEEWGFDGFKEDYVWGIPKCYDSSHKHSSLSDTLENQYKFYEAIYEQSIAINPDTFIELCNCGTPQ DFYSTPYVNHAPTADPISRVQTRTRVKAFKAIFGDDFPVTTDHNSVWLPSALGTGSVMITKHTTLSSDRQYNKYFGLARDLELAKGEFIGNLYKYGIDPLESYVIRKGEDIYYSFYKDNSSYSGNIEIKGLDSNATYRIEDYVNNRVIARGVKGPTATINTSFTNLLVRAIPDDTPAEVTTFDVGNNTILSSTDSGNSKYLNAVSTTLEKTATIDSLSIYIGNNSENGKLQIAIYDDNNGKPGTKKAYVEEFVPTKNSWNTKKVNSVNTLPSGQYWLVFQPDNDVLQTKTNPSSMKQSANNNPYNYNILPNSFPIGTGYNAYKGDVSFYATFKEASSQAIPQNSWALKYVDSEETTGENGRATNAFDGNNTIWHTKYSGGNAAPMPHEIQIDLRGVYNINQINYLPRQDGGTNGTIKDYEVYLSLDGVNWGQPISKGTFESNSTEKIVKFNETKSRYVKLKALSEINNKQFTTVADLKVFGWEISKIEK

[0234] SEQ ID NO: 37

[0233] Description: Galactosamidase protein sequence of Robinsoniella peoriensis Rp1021 References Kuznetsova, I.M et al. Int J Mol Sci. (2014) “What Macromolecular Crowding Can Do to a Protein” 15(12):23090–23140. Marcus, D, M.et al. Biochem (1964) “Immunochemical Studies on Blood Groups. XXXI. Destruction of Blood Group A Activity by an Enzyme from Clostridium tertium Which Deacetylates N-Acetylgalactosamine in Intact Blood Group Substances” (4)437-443. Daniels, G.and Reid M.E. Transfusion (2010) “Blood groups: the past 50 years.” 50(2):281-9.doi:10.1111 / j.1537-2995.2009.02456.x.Epub 2009 Nov 9 Vox Sang. 2011 Nov;101(4):327-32.doi:10.1111 / j.1423-0410.2011.01540.x.Epub 2011 Sep 6. Garratty, G. Vox Sang. (2008) “Modulating the red cell membrane to produce universal / stealth donor red cells suitable for transfusion.” 94(2):87-95. Epub 2007 Nov 22. Goldstein et al. 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Claims

1. A composition comprising: (a) purified GalNAc deacetylase protein; and (b) Purified galactosamidase protein.

2. The composition according to claim 1, wherein the composition is selected from one or more of the following: (a) the purified GalNAc deacetylase protein is selected from one or more of the following: SEQ ID NO: 2; SEQ ID NO: 4; SEQ ID NO: 5; SEQ ID NO: 17; SEQ ID NO: 23; SEQ ID NO: 29; SEQ ID NO: 31; SEQ ID NO: 32; SEQ ID NO: 33; SEQ ID NO: 34 and SEQ ID NO: 35; and (b) The purified galactosamidase protein is selected from one or more of the following: SEQ ID NO:7; SEQ ID NO:9; SEQ ID NO:10; SEQ ID NO:19; SEQ ID NO:21; SEQ ID NO:36 and SEQ ID NO:

37.

3. The composition of claim 1, wherein the composition comprises: a purified enzyme having GalNAc deacetylase activity, which consists essentially of an amino acid sequence at least 90% identical to a sequence shown in one of SEQ ID NOs: 2, 4, 5, 17, 23, 29, 31, and 32-35; and a purified enzyme having galactosaminidase activity, which consists essentially of an amino acid sequence at least 90% identical to a sequence shown in one of SEQ ID NOs: 7, 9, 10, 19, 21, 36, and 37.

4. The composition according to claim 1 or 2, wherein the composition comprises one or more enzymes selected from the group consisting of: (a) the purified GalNAc deacetylase protein is the purified GalNAc deacetylase protein of Flavonifractor plautii of SEQ ID NO: 2, SEQ ID NO: 4 and SEQ ID NO: 5; and (b) The purified galactosamidase protein is the purified galactosamidase protein of Fusobacterium prausnitzii of SEQ ID NO:7, SEQ ID NO:9 and SEQ ID NO:

10.

5. The composition according to claim 1 or 2, wherein the composition is selected from one or more of the following: (a) the purified GalNAc deacetylase protein is the purified GalNAc deacetylase protein of Clostridium tertium of SEQ ID NO: 17 or SEQ ID NO: 32; and (b) The purified galactosamidase protein is the purified galactosamidase protein of Clostridium tertium of SEQ ID NO: 19 or SEQ ID NO:

36.

6. The composition according to any one of claims 1 to 5, wherein the GalNAc deacetylase and galactosaminidase are capable of cleaving the A antigen at or below 1 μg / ml.

7. The composition of any one of claims 1-6, wherein the GalNAc deacetylase and galactosaminidase have A antigen cleavage activity at a pH of about 6.5 to about 7.

5.

8. The composition according to any one of claims 1 to 7, wherein the GalNAc deacetylase and galactosaminidase have A antigen cleavage activity at a temperature of 4°C to 37°C.

9. The composition according to any one of claims 1 to 8, wherein (a) the purified GalNAc deacetylase and the purified galactosaminidase are immobilized; (b) the purified GalNAc deacetylase is immobilized; or (c) The purified galactosaminidase is immobilized.

10. The composition of claim 9, wherein the immobilized enzyme is attached to a surface selected from one or more of the following: (a) beads or microspheres; (b) containers; (c) pipe; (d) a column; or (e) Matrix.

11. The composition of any one of claims 1-10, wherein the composition further comprises a crowding agent.

12. The composition of claim 11, wherein the crowding agent is selected from one or more of the following: dextran, dextran sulfate, dextrin, pullulan, poly(ethylene glycol), polysucrose TM and inert proteins.

13. A purified enzyme comprising the GalNAc deacetylase of Fusobacterium prausnitzii of SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO:

5.

14. A purified enzyme comprising the galactosamidase of Fusobacterium prausnitzii of SEQ ID NO:7, SEQ ID NO:9 or SEQ ID NO:

10.

15. A purified enzyme comprising the GalNAc deacetylase protein of Clostridium tertium of SEQ ID NO: 17 or SEQ ID NO:

32.

16. A purified enzyme comprising the galactosamidase protein of Clostridium tertium of SEQ ID NO: 19 or SEQ ID NO:

36.

17. An isolated nucleic acid sequence encoding a GalNAc deacetylase selected from one or more of the following: SEQ ID NO: 1; SEQ ID NO: 3; SEQ ID NO: 16; SEQ ID NO: 24; SEQ ID NO: 26; SEQ ID NO: 28 and SEQ ID NO:

30.

18. An isolated nucleic acid sequence encoding a galactosamidase selected from one or more of the following: SEQ ID NO: 6; SEQ ID NO: 8; SEQ ID NO: 18 and SEQ ID NO:

20.

19. A vector comprising the nucleic acid of claim 17 or 18 and a heterologous nucleic acid sequence.

20. The vector according to claim 19, wherein the heterologous nucleic acid sequence is selected from one or more of the following: a protein tag and a cleavage site.

21. The vector according to claim 20, wherein the protein tag is selected from one or more of the following: albumin binding protein (ABP); alkaline phosphatase (AP); AU1 epitope; AU5 epitope; Avi tag; bacteriophage T7 epitope (T7 tag); bacteriophage V5 epitope (V5 tag); biotin-carboxy carrier protein (BCCP); blue tongue virus tag (B tag); single domain camelid antibody (C tag); calmodulin binding peptide (CBP or calmodulin tag) ; Chloramphenicol acetyltransferase (CAT); Cellulose binding domain (CBP); Chitin binding domain (CBD); Choline binding domain (CBD); Dihydrofolate reductase (DHFR); DogTag; E2 epitope; E tag; FLAG epitope (FLAG tag); Galactose binding protein (GBP); Green fluorescent protein (GFP); Glu-Glu (EE tag); Glutathione S-transferase (GST); Human influenza hemagglutinin (HA); HaloTag TM ; Alternating histidine and glutamine tag (HQ tag); Alternating histidine and asparagine tag (HN tag); Histidine affinity tag (HAT); Horseradish peroxidase (HRP); HSV epitope; Isopeptag (Isopep tag); Ketosterol isomerase (KSI); KT3 epitope; LacZ; Luciferase; Maltose binding protein (MBP); Myc epitope (Myc tag); NE tag; NusA; PDZ domain; PDZ ligand; Polyarginine (Arg tag); Polyaspartic acid (Asp tag); Polycysteine ​​(Cys tag); Polyglutamic acid (Glu tag); Polyhistidine (His tag); Polyphenylalanine (Phe tag); Profinity eXact; Protein C; Rho1D4 tag; S1 tag; S tag; Softag1; Softag 3; SnoopTagJr; SnoopTag; Spot tag; SpyTag (Spy tag); Streptavadin binding peptide (SBP); Staphylococcal protein A (Protein A); Staphylococcal protein G (Protein G); Strep tag; Streptavadin (SBP tag); Strep tag II; Sdy tag; small ubiquitin-like modifier (SUMO); tandem affinity purification (TAP); T7 epitope; tetracysteine ​​tag (TC tag); thioredoxin (Trx); TrpE; Ty tag; ubiquitin; universal; V5 tag; VSV-G or VSV tag; and Xpress tag.

22. A vector comprising the nucleic acid of claim 17 or 18.

23. A method for enzymatically cleaving A antigen from red blood cells, the method comprising: (a) combining a GalNAc deacetylase protein and a galactosamidase protein with: (i) blood containing type A antigen; or (ii) type A or AB red blood cells; (b) incubating the enzyme with (i) the blood; or (ii) the type A or type AB red blood cells for a period of time sufficient for the enzyme to cleave the A antigen from the blood or the red blood cells.

24. The method of claim 23, wherein the GalNAc deacetylase is a purified protein selected from one or more of the following: SEQ ID NO:2; SEQ ID NO:4; SEQ ID NO:5; SEQ ID NO:17; SEQ ID NO:23; SEQ ID NO:29; SEQ ID NO:31; SEQ ID NO:32; SEQ ID NO:33; SEQ ID NO:34 and SEQ ID NO:35; and the galactosaminidase is a purified protein selected from one or more of the following: SEQ ID NO:7; SEQ ID NO:9; SEQ ID NO:10; SEQ ID NO:19; SEQ ID NO:21; SEQ ID NO:36 and SEQ ID NO:

37.

25. The method of claim 23, wherein the composition comprises: a purified enzyme having GalNAc deacetylase activity consisting essentially of an amino acid sequence at least 90% identical to a sequence as shown in one of SEQ ID NOs: 2, 4, 5, 17, 23, 29, 31, and 32-35; and a purified enzyme having galactosaminidase activity consisting essentially of an amino acid sequence at least 90% identical to a sequence as shown in one of SEQ ID NOs: 7, 9, 10, 19, 21, 36, and 37.

26. The method of claim 23, wherein the GalNAc deacetylase is a purified GalNAc deacetylase protein of Fusobacterium prausnitzii of SEQ ID NO: 4 or SEQ ID NO: 5, and the galactosamidase is a purified galactosamidase protein of Fusobacterium prausnitzii of SEQ ID NO: 9 or SEQ ID NO:

10.

27. The method of any one of claims 23-26, further comprising adding a crowding agent.

28. The method of claim 27, wherein the crowding agent is selected from one or more of the following: dextran; dextran sulfate; dextrin; pullulan; poly(ethylene glycol); polysucrose TM ; Hyperbranched glycerol and inert protein.

29. The method of any one of claims 23-28, further comprising washing the blood or the red blood cells to remove GalNAc deacetylase, galactosamidase, and the crowding agent.

30. The method of any one of claims 23-29, wherein the GalNAc deacetylase and galactosaminidase are capable of cleaving the A antigen at or below 1 μg / ml.

31. The method of any one of claims 23-30, wherein the GalNAc deacetylase and galactosaminidase have A antigen cleavage activity at a pH of about 6.5 to about 7.

5.

32. The method according to any one of claims 23-31, wherein the GalNAc deacetylase and galactosaminidase have A antigen cleavage activity at a temperature of 4°C to 37°C.

33. A blood collection and storage system comprising: (a) purified GalNAc deacetylase protein; and (b) Purified galactosamidase protein.

34. The system of claim 33, wherein: (a) the GalNAc deacetylase is a purified protein selected from one or more of the following: SEQ ID NO: 2; SEQ ID NO: 4; SEQ ID NO: 5; SEQ ID NO: 17; SEQ ID NO: 23; SEQ ID NO: 29; SEQ ID NO: 31; SEQ ID NO: 32; SEQ ID NO: 33; SEQ ID NO: 34 and SEQ ID NO: 35; and (b) the galactosamidase is a purified protein selected from one or more of the following: SEQ ID NO:7; SEQ ID NO:9; SEQ ID NO:10; SEQ ID NO:19; SEQ ID NO:21; SEQ ID NO:36 and SEQ ID NO:

37.

35. The system of claim 33, wherein the composition comprises: a purified enzyme having GalNAc deacetylase activity consisting essentially of an amino acid sequence at least 90% identical to a sequence as set forth in one of SEQ ID NOs: 2, 4, 5, 17, 23, 29, 31, and 32-35; and a purified enzyme having galactosaminidase activity consisting essentially of an amino acid sequence at least 90% identical to a sequence as set forth in one of SEQ ID NOs: 7, 9, 10, 19, 21, 36, and 37.

36. The system of claim 33, 34 or 35, wherein the system further comprises a surface on which the enzyme is immobilized, the surface being selected from one or more of the following: (a) beads or microspheres; (b) containers; (c) pipe; (d) a column; or (e) Matrix.

37. A blood collection and storage device, the device comprising: (a) Surface; (b) purified GalNAc deacetylase protein immobilized on the surface; and (c) Purified galactosamidase protein immobilized on the surface.

38. The apparatus of claim 37, wherein: The GalNAc deacetylase is a purified protein selected from one or more of the following: SEQ ID NO: 2; SEQ ID NO: 4; SEQ ID NO: 5; SEQ ID NO: 17; SEQ ID NO: 23; SEQ ID NO: 29; SEQ ID NO: 31; SEQ ID NO: 32; SEQ ID NO: 33; SEQ ID NO: 34 and SEQ ID NO: 35; and (b) the galactosamidase is a purified protein selected from one or more of the following: SEQ ID NO:7; SEQ ID NO:9; SEQ ID NO:10; SEQ ID NO:19; SEQ ID NO:21; SEQ ID NO:36 and SEQ ID NO:

37.

39. The apparatus of claim 37, wherein the composition comprises: a purified enzyme having GalNAc deacetylase activity consisting essentially of an amino acid sequence at least 90% identical to a sequence as shown in one of SEQ ID NOs: 2, 4, 5, 17, 23, 29, 31, and 32-35; and a purified enzyme having galactosaminidase activity consisting essentially of an amino acid sequence at least 90% identical to a sequence as shown in one of SEQ ID NOs: 7, 9, 10, 19, 21, 36, and 37.

40. The device of claim 37, wherein the enzyme is immobilized on a surface selected from one or more of: (a) beads or microspheres; (b) containers; (c) pipe; (d) a column; or (e) Matrix.

41. Apparatus according to claim 40, wherein said container is a bag.

42. The composition of claim 10, wherein the container is a bag.

43. The system of claim 36, wherein the container is a bag.

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