Enzyme composition for removing carbohydrate antigens in donor organs, methods and uses related thereto

Abstract

The present invention provides a perfusion fluid for enzymatically cleaving A antigen from a donor organ, and related methods and uses. In particular, the perfusion fluid contains two enzymes, GalNAc deacetylase and galactosaminidase, and the perfusion fluid may further contain a buffered extracellular solution and / or a crowding agent. Furthermore, the compositions described herein have been found to be active at temperatures and pH levels suitable for cell viability. [Selected figure] Figure 2

Description

Technical Field

[0001] (Cross - Reference to Related Applications) This application claims the benefit of U.S. Provisional Patent Application No. 62 / 719,272, filed on August 17, 2018, entitled "Enzyme Compositions for Glycan Antigen Cleavage, Related Methods, Uses, Devices, and Systems".

[0002] The present invention relates to the field of enzyme compositions. In particular, the present invention relates to enzyme compositions for cleaving antigens in donor organs, as well as methods and uses for cleaving antigens using such compositions.

Background Art

[0003] Accurate blood type matching is a central requirement in transfusion medicine because plasma from people with blood type A contains antibodies against the B antigen, and vice versa, which can lead to complement activation and red blood cell (RBC) lysis by incompatible transfusions (Daniels 2010). These cell - surface antigens are glycan structures terminated with α - 1,3 - linked - N - acetylgalactosamine (GalNAc) or galactose (Gal) for blood types A and B, respectively. On the other hand, O - type red blood cells do not contain these terminal sugars and can generally be transfused (Garratty 2008). Therefore, it is necessary to adequately supply blood banks with O - type red blood cells in case of emergencies when a patient's blood type is unknown or uncertain. However, the supply is often limited.

[0004] The concept of enzymatically removing the GalNAc or Gal structure from A or B RBCs as a means of converting A or B RBCs to O was first proposed and demonstrated by Goldstein (Goldstein 1982; US4609627 and CA2272925). Using α-galactosidase from green coffee beans, B-type RBCs were converted to O-type and subsequently transfused successfully (Kruskall 2000). However, this approach was non-practical due to the amount of enzyme required. Conversion of A-type is more difficult mainly because the A blood type has many subtypes with different internal linkages (Clausen 1989). Similarly, α-galactosidase has been used for the removal of B-type antigens (see, for example, EP2243793). A major step forward towards practical conversion, including A-type conversion, was made by screening a library of bacteria for both A and B conversion activities using a tetrasaccharide substrate. Two new families of glycosidases showing high antigen cleavage activity at neutral pH values were discovered (CAZy GH109 α-N-acetylgalactosaminidase and GH110 α-galactosidase (Liu 2007)). Both enzymes completely removed their respective antigens and converted the corresponding RBCs. However, a significant amount of enzyme is required, especially for A-type conversion (60 mg enzyme / unit of blood), limiting further development. Enzymes that more efficiently remove sugar chain antigens from cells would be useful. Summary of the Invention

[0005] The present invention is based, in part, on the surprising discovery that a combination of galactosaminidase and GalNAc deacetylase is orders of magnitude more efficient than previously identified A antigen-cleaving enzymes. For example, under certain conditions, some GalNAc deacetylase and galactosaminidase enzymes can cleave A antigen at 1 μ / ml or less. Further, the cleavage efficiency by the enzyme combination is maintained at a pH suitable for maintaining the viability of red blood cells (i.e., a pH between about 6.5 and about 7.5). Further, the enzymes are found to be active at temperatures between 4°C and 37°C, which is also suitable for blood collection, washing, and storage protocols. Further, the efficiency of the enzymes is further improved by the addition of a crowding agent (e.g., dextran). It is also understood that the same two-step removal process can be applied to donor organs.

[0006] However, it will be understood by those skilled in the art that if the donor organ is perfused for a longer period, more enzyme can be used to shorten the time the donor organ can be perfused, or less enzyme can be used.

[0007] According to one embodiment, there is provided a perfusion fluid for enzymatically cleaving A antigen from a donor organ, comprising (a) a purified GalNAc deacetylase protein and (b) a purified galactosaminidase protein.

[0008] According to a further embodiment, there is provided a perfusion fluid comprising (a) a purified GalNAc deacetylase protein selected from one or more of 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 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 a further embodiment, a perfusion fluid is provided, the perfusion fluid comprising a purified enzyme having GalNAc deacetylase activity that consists essentially of an amino acid sequence that is at least 90% identical to one of the sequences of SEQ ID NOs: 2, 4, 5, 17, 23, 29, 31, and 32-35, and a purified enzyme having galactosaminidase activity that consists essentially of an amino acid sequence that is at least 90% identical to one of the sequences of SEQ ID NOs: 7, 9, 10, 19, 21, 36, and 37.

[0010] The enzyme can be selected from one or more of: (a) a purified GalNAc deacetylase protein that is the Flavonifractor plautii GalNAc deacetylase protein of SEQ ID NO: 2, SEQ ID NO: 4, and SEQ ID NO: 5; and (b) a purified galactosaminidase protein that is the Flavonifractor plautii galactosaminidase protein of SEQ ID NO: 7, SEQ ID NO: 9, and SEQ ID NO: 10. The enzyme can be selected from one or more of: (a) a purified GalNAc deacetylase protein that is the Clostridium tertium GalNAc deacetylase protein of SEQ ID NO: 17 and SEQ ID NO: 32; and (b) a purified galactosaminidase protein that is the Clostridium tertium galactosaminidase protein of SEQ ID NO: 19 and SEQ ID NO: 36. GalNAc deacetylase and galactosaminidase can cleave the A antigen at 1 μg / ml or less. GalNAc deacetylase and galactosaminidase can have A antigen cleavage activity at a pH between about 6.5 and about 7.5. GalNAc deacetylase and galactosaminidase can have A antigen cleavage activity at a temperature between 4°C and 37°C. The perfusion fluid can further contain a buffered extracellular solution. The buffered extracellular solution can be selected from Steen (trademark), Perfadex (trademark), Perfadex Plus (trademark), EuroCollins solution, histidine-tryptophan-ketoglutarate (HTK) solution, University of Wisconsin solution (UW), Celsior solution, kidney perfusion solution (KPS-1), Kyoto University solution, IGL-1 solution, and citrate solution.

[0011] According to a further embodiment, there is provided a method for enzymatically cleaving A-type antigen ex vivo from a donor organ, comprising the step of (a) perfusing a donor organ presenting A-type antigen with a fluid containing GalNAc deacetylase protein and galactosaminidase protein for a time sufficient for the enzyme to enable cleavage of A-type antigen from the donor organ, or (b) incubating a donor organ presenting A-type antigen with a fluid containing GalNAc deacetylase protein and galactosaminidase protein for a time sufficient for the enzyme to enable cleavage of A antigen from the donor organ.

[0012] GalNAc deacetylase can be a purified protein selected from one or more of 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. Galactosaminidase can be a purified protein selected from one or more 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.

[0013] A purified enzyme having GalNAc deacetylase activity can essentially comprise an amino acid sequence that is at least 90% identical to one of the sequences of SEQ ID NO: 2, 4, 5, 17, 23, 29, 31, and 32 - 35, and a purified enzyme having galactosaminidase activity can essentially comprise an amino acid sequence that is at least 90% identical to one of the sequences of SEQ ID NO: 7, 9, 10, 19, 21, 36, and 37.

[0014] GalNAc deacetylase may be the purified Flavonifractor plautii GalNAc deacetylase protein of SEQ ID NO: 4 or SEQ ID NO: 5, and galactosaminidase may be the purified Flavonifractor plautii galactosaminidase protein of SEQ ID NO: 9 or SEQ ID NO: 10.

[0015] The GalNAc deacetylase protein and the galactosaminidase protein can be present in a buffered extracellular solution. The buffered extracellular solution can be selected from Steen (trademark), Perfadex (trademark), Perfadex Plus (trademark), EuroCollins solution, histidine-tryptophan-ketoglutarate (HTK) solution, University of Wisconsin solution (UW), Celsior solution, kidney perfusion solution (KPS-1), Kyoto University solution, IGL-1 solution, and citrate solution. The donor organ can be a solid organ. The solid organ can be selected from one of the lung, kidney, liver, heart, pancreas, and intestine. The solid organ can be the lung.

[0016] The GalNAc deacetylase protein and the galactosaminidase protein are mixed with an ex vivo buffered extracellular lung solution and can be circulated through the lung, whereby the GalNAc deacetylase protein and the galactosaminidase protein contact the vascular system of the donor organ for a time sufficient to substantially remove A antigen from the vascular system of the lung. The GalNAc deacetylase protein and the galactosaminidase protein are mixed with an ex vivo buffered extracellular kidney solution and can be circulated through the kidney, whereby the GalNAc deacetylase protein and the galactosaminidase protein contact the vascular system of the donor organ for a time sufficient to substantially remove A antigen from the vascular system of the kidney. The GalNAc deacetylase protein and the galactosaminidase protein are mixed with an ex vivo buffered extracellular liver solution and can be circulated through the liver, whereby the GalNAc deacetylase protein and the galactosaminidase protein contact the vascular system of the donor organ for a time sufficient to substantially remove A antigen from the vascular system of the liver. The GalNAc deacetylase protein and the galactosaminidase protein are mixed with an ex vivo buffered extracellular heart solution and can be circulated through the heart, whereby the GalNAc deacetylase protein and the galactosaminidase protein contact the vascular system of the donor organ for a time sufficient to substantially remove A antigen from the vascular system of the heart. The GalNAc deacetylase protein and the galactosaminidase protein are mixed with an ex vivo buffered extracellular pancreas solution and can be circulated through the pancreas, whereby the GalNAc deacetylase protein and the galactosaminidase protein contact the vascular system of the donor organ for a time sufficient to substantially remove A antigen from the vascular system of the pancreas. The GalNAc deacetylase protein and the galactosaminidase protein are mixed with an ex vivo buffered extracellular intestine solution and can be circulated through the intestine, whereby the GalNAc deacetylase protein and the galactosaminidase protein contact the vascular system of the donor organ for a time sufficient to substantially remove A antigen from the vascular system of the intestine.

[0017] The time to remove A antigen from the vascular system can be about 1 hour. The time to remove A antigen from the blood vessels can be less than 1 hour. The time to remove A antigen from the vascular system is about 2 hours.

[0018] This method may further include washing the donor organ to remove GalNAc deacetylase, galactosaminidase, and cleaved A antigen. GalNAc deacetylase and galactosaminidase can cleave A antigen at 1 μg / ml or less. GalNAc deacetylase and galactosaminidase can have A antigen cleavage activity at a pH between about 6.5 and about 7.5. GalNAc deacetylase and galactosaminidase can have A antigen cleavage activity at a temperature between 4°C and 37°C.

[0019] According to a further embodiment, a composition is provided, the composition comprising a purified enzyme having GalNAc deacetylase activity consisting essentially of an amino acid sequence that is at least 85% identical to one of the sequences 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 that is at least 85% identical to one of the sequences set forth in SEQ ID NOs: 7, 9, 10, 19, 21, 36, and 37.

[0020] According to a further embodiment, a composition is provided, the composition comprising a purified enzyme having GalNAc deacetylase activity consisting essentially of an amino acid sequence that is at least 80% identical to one of the sequences 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 that is at least 80% identical to one of the sequences of SEQ ID NOs: 7, 9, 10, 19, 21, 36, and 37.

[0021] According to a further embodiment, a composition is provided, the composition comprising a purified enzyme having GalNAc deacetylase activity consisting essentially of an amino acid sequence that is at least 75% identical to one of the sequences 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 that is at least 75% identical to one of the sequences of SEQ ID NOs: 7, 9, 10, 19, 21, 36, and 37.

[0022] The composition may include (a) the purified GalNAc deacetylase and the purified galactosaminidase may be immobilized, (b) the purified GalNAc deacetylase may be immobilized, or (c) the purified galactosaminidase may be immobilized.

[0023] The immobilized enzyme may be bound to a surface, and the surface may be selected from one or more of the following: (a) beads or microspheres, (b) a container, (c) a tube, (d) a column, and (e) a matrix. The composition may further include a crowding agent. The crowding agent may be selected from one or more of dextran, dextran sulfate, dextrin, pullulan, poly(ethylene glycol), Ficoll™, and inert proteins.

[0024] According to a further embodiment, a purified enzyme comprising flavonifractor plautii GalNAc deacetylase of SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 5 is provided.

[0025] According to a further embodiment, a purified enzyme comprising flavonifractor plautii galactosaminidase of SEQ ID NO: 7, SEQ ID NO: 9, or SEQ ID NO: 10 is provided.

[0026] According to a further embodiment, a purified enzyme comprising Clostridium tertium GalNAc deacetylase of SEQ ID NO: 17 or SEQ ID NO: 32 is provided.

[0027] According to a further embodiment, a purified enzyme comprising Clostridium tertium galactosaminidase of SEQ ID NO: 19 or SEQ ID NO: 36 is provided.

[0028] Protein tags include albumin-binding protein (ABP), alkaline phosphatase (AP), AU1 epitope, AU5 epitope, AviTag, bacteriophage T7 epitope (T7-tag), bacteriophage V5 epitope (V5-tag), biotin-carboxyl carrier protein (BCCP), blue tongue virus tag (B-tag), single-domain camel 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 (trademark), 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), ketosteroid 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, SnoopTag Jr, SnoopTag, SpotTag, SpyTag (Spy-tag), streptavidin-binding peptide (SBP), staphylococcal protein A (protein A), staphylococcal protein G (protein G), Strep-tag, streptavidin (SBP-tag), Strep-tag II, Sdy-tag, small ubiquitin-like modifier (SUMO),It may be selected from one or more of 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.

[0029] According to a further embodiment, a method for enzymatically cleaving A antigen from a donor organ, comprising: (a) mixing a GalNAc deacetylase protein and a galactosaminidase protein with a donor organ presenting the A antigen; and (b) perfusing the enzyme into the donor organ blood vessels for a time sufficient for the enzyme to cleave the A antigen from the vascular lumen of the donor organ.

[0030] The method may further include adding a crowding agent. The crowding agent may be selected from one or more of dextran, dextran sulfate, dextrin, pullulan, poly(ethylene glycol), Ficoll™, hyperbranched glycerol, and inert proteins. The method may include perfusing the donor organ with an organ perfusion solution or an organ preservation solution containing the enzyme composition described herein.

[0031] The method may further include washing the donor organ and removing GalNAc deacetylase, galactosaminidase and / or the crowding agent.

[0032] GalNAc deacetylase and galactosaminidase may be able to cleave A antigen at 1 μg / ml or less. GalNAc deacetylase and galactosaminidase may have A antigen cleavage activity at a pH between about 6.5 and about 7.5. GalNAc deacetylase and galactosaminidase may have A antigen cleavage activity at a temperature between 4°C and 37°C.

[0033] GalNAc deacetylase and galactosaminidase can cleave the A antigen at 100 μg / ml or less. GalNAc deacetylase and galactosaminidase can cleave the A antigen at 90 μg / ml or less. GalNAc deacetylase and galactosaminidase can cleave the A antigen at 80 μg / ml or less. GalNAc deacetylase and galactosaminidase can cleave the A antigen at 70 μg / ml or less. GalNAc deacetylase and galactosaminidase can cleave the A antigen at 60 μg / ml or less. GalNAc deacetylase and galactosaminidase can cleave the A antigen at 50 μg / ml or less. GalNAc deacetylase and galactosaminidase can cleave the A antigen at 40 μg / ml or less. GalNAc deacetylase and galactosaminidase can cleave the A antigen at 30 μg / ml or less. GalNAc deacetylase and galactosaminidase can cleave the A antigen at 20 μg / ml or less. GalNAc deacetylase and galactosaminidase can cleave the A antigen at 15 μg / ml or less. GalNAc deacetylase and galactosaminidase can cleave the A antigen at 14 μg / ml or less. GalNAc deacetylase and galactosaminidase can cleave the A antigen at 13 μg / ml or less. GalNAc deacetylase and galactosaminidase can cleave the A antigen at 12 μg / ml or less. GalNAc deacetylase and galactosaminidase can cleave the A antigen at 11 μg / ml or less. GalNAc deacetylase and galactosaminidase can cleave the A antigen at 10 μg / ml or less. GalNAc deacetylase and galactosaminidase can cleave the A antigen at 9 μg / ml or less. GalNAc deacetylase and galactosaminidase can cleave the A antigen at 8 μg / ml or less. GalNAc deacetylase and galactosaminidase can cleave the A antigen at 7 μg / ml or less. GalNAc deacetylase and galactosaminidase can cleave the A antigen at 6 μg / ml or less. GalNAc deacetylase and galactosaminidase can cleave the A antigen at 5 μg / ml or less.GalNAc deacetylase and galactosaminidase can cleave the A antigen at 4 μg / ml or less. GalNAc deacetylase and galactosaminidase can cleave the A antigen at 3 μg / ml or less. GalNAc deacetylase and galactosaminidase can cleave the A antigen at 2 μg / ml or less. GalNAc deacetylase and galactosaminidase can cleave the A antigen at 1 μg / ml or less. GalNAc deacetylase and galactosaminidase can cleave the A antigen at 0.9 μg / ml or less. GalNAc deacetylase and galactosaminidase can cleave the A antigen at 0.8 μg / ml or less. GalNAc deacetylase and galactosaminidase can cleave the A antigen at 0.7 μg / ml or less. GalNAc deacetylase and galactosaminidase can cleave the A antigen at 0.6 μg / ml or less. GalNAc deacetylase and galactosaminidase can cleave the A antigen at 0.5 μg / ml or less. GalNAc deacetylase and galactosaminidase can cleave the A antigen at 0.4 μg / ml or less. GalNAc deacetylase and galactosaminidase can cleave the A antigen at 0.3 μg / ml or less. GalNAc deacetylase and galactosaminidase can cleave the A antigen at 0.2 μg / ml or less. GalNAc deacetylase and galactosaminidase can cleave the A antigen at 0.1 μg / ml or less. GalNAc deacetylase and galactosaminidase can cleave the A antigen at 0.09 μg / ml or less. GalNAc deacetylase and galactosaminidase can cleave the A antigen at 0.08 μg / ml or less. GalNAc deacetylase and galactosaminidase can cleave the A antigen at 0.07 μg / ml or less. GalNAc deacetylase and galactosaminidase can cleave the A antigen at 0.06 μg / ml or less. GalNAc deacetylase and galactosaminidase can cleave the A antigen at 0.05 μg / ml or less. GalNAc deacetylase and galactosaminidase can cleave the A antigen at 0.04 μg / ml or less. GalNAc deacetylase and galactosaminidase can cleave the A antigen at 0.03 μg / ml or less.GalNAc deacetylase and galactosaminidase can cleave the A antigen at 0.02 μg / ml or less. GalNAc deacetylase and galactosaminidase can cleave the A antigen at 0.01 μg / ml or less.

[0034] GalNAc deacetylase and galactosaminidase may have A antigen cleavage activity at a pH between about 6.5 and about 7.5. GalNAc deacetylase and galactosaminidase may have A antigen cleavage activity at a pH between about 6.0 and about 8.0. GalNAc deacetylase and galactosaminidase may have A antigen cleavage activity at a pH between about 6.8 and about 7.8. GalNAc deacetylase and galactosaminidase may have A antigen cleavage activity at a pH between about 6.9 and about 7.9. GalNAc deacetylase and galactosaminidase may have A antigen cleavage activity at a pH between about 6.4 and about 7.8.

[0035] GalNAc deacetylase and galactosaminidase may have A antigen cleavage activity at a temperature between 4°C and 37°C. GalNAc deacetylase and galactosaminidase may have A antigen cleavage activity at a temperature between 3°C and 38°C. GalNAc deacetylase and galactosaminidase may have A antigen cleavage activity at a temperature between 4°C and 40°C. GalNAc deacetylase and galactosaminidase may have A antigen cleavage activity at a temperature between 4°C and 37°C. GalNAc deacetylase and galactosaminidase may have A antigen cleavage activity at a temperature between 5°C and 37°C.

[0036] According to another embodiment, a purified enzyme comprising the flavonifractor plautii GalNAc deacetylase of SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 5 is provided.

[0037] According to another embodiment, a purified enzyme comprising the flavonifractor plautii galactosaminidase of SEQ ID NO: 7, SEQ ID NO: 9 or SEQ ID NO: 10 is provided.

[0038] According to another embodiment, a purified enzyme is provided that includes purified Clostridium tertium GalNAc deacetylase and the galactosaminidase fusion protein of SEQ ID NO: 14.

[0039] According to another embodiment, a vector is provided that includes a nucleic acid described herein and a heterologous nucleic acid sequence.

[0040] According to another embodiment, this method may be performed in vitro or ex vivo. As used herein, ex vivo means that the method is performed outside of an organism. For example, ex vivo includes ex vivo lung perfusion (EVLP) and treatment of provided blood. As used herein, ex vivo refers to an experiment or measurement or treatment performed in or on a tissue or cell from an organism in an external environment with minimal or some change from the state in which the tissue or cell (e.g., red blood cells or donor organs) was placed when it was in vivo. BRIEF DESCRIPTION OF THE DRAWINGS

[0041]

Figure 1

[0042]

Figure 2

[0043]

Figure 3

[0044]

Figure 4

[0045]

Figure 5

[0046]

Figure 6

[0047]

Figure 7

[0048]

Figure 8

[0049]

Figure 9

[0050]

Figure 10

[0051]

Figure 11

Mode for Carrying Out the Invention

[0052] The following detailed description will be better understood when read in conjunction with the accompanying drawings. To illustrate the present invention, the drawings show embodiments of the present invention. However, the present invention is not limited to the exact configurations, examples, and means shown.

[0053] Terms not directly defined herein are to be understood as having the meanings generally associated with them as understood in the technical field of the present invention.

[0054] As used herein, an "immobilized enzyme" is an enzyme attached to a surface that may be an inert insoluble material. Immobilization of the enzyme can increase its resistance to changes in conditions such as pH and temperature and assist in their removal after use and for enzyme reuse.

[0055] The immobilization of the enzyme may be achieved by various methods (e.g., affinity tag binding, surface adsorption on glass, resins, alginate beads or matrices, bead, fiber or microsphere capture, cross-linking to the surface or other enzymes, and covalent bonding to the surface).

[0056] As used herein, "affinity tag binding" refers to the immobilization of an enzyme on a surface (e.g., a porous material using a non-covalent or covalent protein tag). Affinity tag binding has been used in protein purification and more recently has been used in biocatalytic applications by EziG™ (ENGINZYME AB™ of Sweden (e.g., PCT / US 1992 / 010113, and PCT / SE 2015 / 050108)). Alternative systems for attaching active enzymes to surfaces are known in the art (see, e.g., US4088538, US4141857, US4206259, US4218363, US4229536, US4239854, US4619897, US 4748121, US4749653, US4897352, US4954444, US4978619, US5154808, US5914367, US5962279, US6030933, US6291582, US6254645, US10,016,490, and US10,041,055).

[0057] A protein tag is a peptide sequence genetically grafted onto a recombinant protein, often removable by chemical agents or enzymatic means, and is attached to the protein for various purposes. The protein tags listed in Table A are intended as examples and are not intended to be limiting in any way. Certain types of protein tags are affinity tags, added to a protein or peptide sequence so that they can be purified from crude biological sources (e.g., from an expression system organism) using affinity techniques or to facilitate immobilization of the "tagged" protein onto a surface. Examples of affinity tags include chitin-binding domain (CBD), maltose-binding protein (MBP), strep-tag, glutathione-S-transferase (GST), and polyhistidine (His-tag) that binds to a metal matrix. Another type of protein tag is an epitope tag (e.g., V5-tag, Myc-tag, HA-tag, Spot-tag, NE-tag), which is a short peptide sequence selected to facilitate the production of high-affinity antibodies and often derived from viral gene sequences to improve immunoreactivity. Epitope tags are also used for protein purification and immobilization onto a surface, but are particularly useful in Western blotting, immunofluorescence, and immunoprecipitation experiments. Yet another type of protein tag is a chromatography tag (e.g., polyanionic amino acids such as the FLAG tag), which can be used to alter the chromatographic properties of a protein to assist in separation and purification or immobilization. Still another protein tag is a solubilization tag (e.g., maltose-binding protein (MBP), glutathione S-transferase (GST), thioredoxin (TRX), and poly(NANP)) and a fluorescent tag (e.g., green fluorescent protein (GFP)). Protein tags enable specific enzymatic modifications, chemical modifications, or the binding of the protein to other components. However, depending on the type or number of tags added to the protein sequence, the native function of the protein, in this case, the enzymatic function, can be impaired by the tags.Therefore, it is necessary to select a protein tag to ensure that the activity of the enzyme is not impaired, or the protein tag may be cleaved from the protein before use.

[0058] Table A: Representative protein tags JPEG0007684216000001.jpg150165JPEG0007684216000002.jpg180144JPEG0007684216000003.jpg186138JPEG0007684216000004.jpg176142JPEG0007684216000005.jpg154148

[0059] The use of protein tags is exemplified in this application through the use of polyhistidine protein tags (His-tags) as shown in SEQ ID NOs: 5, 10, 15, 17, 19, 21, 23, 25, 27, 29, and 31. However, those skilled in the art will readily understand that depending on the purification method used and / or the surface to which the enzyme binds, any number of other protein tags can be used to purify the enzyme and / or bind the enzyme to the surface as described herein. Such protein tags can be selected from any one or more of the protein tags listed in Table A, although other such protein tags are known in the art.

[0060] Furthermore, one or more cleavage sites (e.g., the thrombin cleavage sites used in SEQ ID NOs: 15, 17, 19, 21, 23, 25, 27, 29, and 31) can be used to separate a protein tag from an enzyme or, alternatively, to cleave the enzyme. Cleavage sites can be used for removal of the N-terminal methionine, signal peptide, and / or conversion of an inactive or non-functional protein to an active protein (i.e., zymogen or proenzyme). Alternatively, cleavage sites can be used to separate two or more enzymes expressed in the same reading frame. Examples of enzymes that can cleave a protein or peptide and that would have a sequence-specific cleavage site can be selected from one or more of the following: Arg-C proteinase, Asp-N endopeptidase, Asp-N endopeptidase + N-terminal Glu BNPS-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 of [FYW] rather than before P), chymotrypsin low specificity (C-terminal of [FYWML] rather than before P), clostripain (clostridiopeptidase B), CNBr, enterokinase, factor Xa, formic acid, glutamyl endopeptidase, granzyme B, hydroxylamine, iodo-benzoic acid, LvsC, LvsN, NTCB (2-nitro-5-thiocyanobenzoic acid), neutrophil elastase, pepsin (pH 1.3), pepsin (pH > 2), proline endopeptidase, proteinase K, staphylococcal peptidase I, tobacco etch virus protease, thermolysin, thrombin, trypsin.

[0061] One skilled in the art will appreciate the importance of the combination of the active galactosaminidase enzyme and the active GalNAc deacetylase enzyme described herein that can efficiently cleave the A antigen. One skilled in the art will also understand that the addition of one or more cleavage sites and / or one or more protein tags is optional and that such modifications can be selected based on a particular expression system, purification system, and possible surface attachment strategy. Further, other modifications to the galactosaminidase sequence and the GalNAc deacetylase sequence are possible as long as the cleavage activity of the A antigen is not significantly impaired. Further, modifications of the galactosaminidase and GalNAc deacetylase are possible as long as the A antigen cleavage activity is not significantly impaired. Modifications to the galactosaminidase and GalNAc deacetylase sequences can be deletions, insertions, and / or substitutions. Substitutions can be conservative substitutions or neutral substitutions. For example, the galactosaminidase sequence and the GalNAc deacetylase sequence can share 90% or more sequence identity with the mature enzyme. For example, the galactosaminidase sequence and the GalNAc deacetylase sequence can share 85% or more sequence identity with the mature enzyme. For example, the galactosaminidase sequence and the GalNAc deacetylase sequence can share 75% or more sequence identity with the mature enzyme. Alternatively, the galactosaminidase and GalNAc deacetylase sequences can have modifications up to 5, 10, 13, 15, 20, or 25% of the amino acids.

[0062] As used herein, "adsorption to glass, alginate beads, or matrix" refers to the attachment of the enzyme to the outside of an inert substance. Generally, this type of immobilization does not occur by a chemical reaction, and the active site of the immobilized enzyme can be blocked by the surface to which it is adsorbed, which can reduce the activity of the adsorbed enzyme.

[0063] As used herein, "capture" refers to the capture of an enzyme within insoluble beads or microspheres. However, capture may impede the access of substrates and the outflow of products. As an example, mention may be made of the use as calcium alginate beads which can be produced by reacting a mixture of sodium alginate solution and enzyme solution with calcium chloride.

[0064] As used herein, "crosslinking" refers to the covalent bonding of enzymes to each other to create a matrix consisting essentially of only enzymes. When designing a crosslinked enzyme reaction, ideally the binding site does not cover the active site of the enzyme such that the activity of the enzyme is affected only by immobilization and not by occlusion of the active site of the enzyme. Nevertheless, spacer molecules such as poly(ethylene glycol) can be used to reduce steric hindrance by the substrate.

[0065] As used herein, "covalent bond" refers to the binding of an 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 possibility of the enzyme detaching from the support or surface is very low.

[0066] As used herein, "crowding agent" refers to any polymer or protein that promotes the aggregation of macromolecules by aggregating enzymes on the cell surface to improve the activity of the enzyme. Crowding agents can be, for example, dextran, dextran sulfate, dextrin, pullulan, poly(ethylene glycol), Ficoll™, hyperbranched glycerol and inert proteins (Kuznetsova, I.M et al. Int J Mol Sci. (2014) "What Macromolecular Crowding Can Do to a Protein" 15(12):23090 - 23140).

[0067] As used herein, "dextran" refers to a polysaccharide having a molecular weight of 1,000 daltons or more and having a linear backbone of α-linked d-glucopyranosyl repeating units. Dextran is divided into three structural classes (i.e., classes 1-3) based on a pyranose ring structure containing five carbon atoms and one oxygen atom. Class 1 dextran contains an α(1→6)-linked d-glucopyranosyl backbone modified with small side chains of d-glucose branches having α(1→2), α(1→3) and α(1→4) linkages. Class 1 dextran varies in three to five ways depending on its molecular weight, configuration, type and degree of branching, and length of the branched chains, as well as the microbial producing strain and culture conditions. Isomaltose and isomaltotriose are oligosaccharides having a class 1 dextran backbone structure. Class 2 dextran (alternans) contains an alternating backbone structure of α(1→3) and α(1→6)-linked d-glucopyranosyl units and α(1→3)-linked branches. Class 3 dextran (mutans) has a backbone structure of continuous α(1→3)-linked d-glucopyranosyl units having α(1→6)-linked branches.

[0068] As used herein, "pullulans" is a structural polysaccharide mainly produced from starch by the fungus Aureobasidium pullulans, consisting of repeats of α(1→6)-linked maltotriose (D-glucopyranosyl-α(1→4)-D-glucopyranosyl-α(1→4)-D-glucose) units and sometimes containing maltotetraose units.

[0069] As used herein, "dextrin" refers to D-glucopyranosyl units having a shorter chain length than dextran, starting with a single α(1→6) linkage and linearly followed by α(1→4)-linked D-glucopyranosyl units.

[0070] As used herein, "Ficoll™" is a neutral, highly branched, high-mass hydrophilic polysaccharide that dissolves readily in aqueous solution.

[0071] As used herein, "perfusion" or "perfusing" refers to the penetration of a fluid into an organ by circulating the fluid through blood vessels.

[0072] An important goal in organ preservation is to increase the number of transplantable organs available. Typically, organs have been stored refrigerated, but this has the potential for diffusion limitations, and thus hypothermic perfusion systems have been developed. Additionally, systems near normal body temperature have also been used to enhance the functional preservation of solid organs, including the liver, lung, heart, and kidney. A number of buffered extracellular solutions are used as perfusion or preservation solutions. Many buffered extracellular solutions are known. For example, Steen™, Perfadex™, Perfadex Plus™, EuroCollins solution, histidine-tryptophan-ketoglutarate (HTK) solution, University of Wisconsin solution (UW), Celsior solution, kidney perfusion solution (KPS-1), Kyoto University solution, IGL-1 solution, and citrate solution (Guibert, E.E. et al. (2011)). Many of these are commercially available, and modifications of these solutions will be apparent to those skilled in the art.

[0073] 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.

[0074] (Materials and Methods) The chemicals and commercially available enzymes used in this study were purchased from Sigma-Aldrich™, unless otherwise noted. Methylumbelliferyl monoglycoside was a generous gift from Dr. Hongming Chen, and A antigen subtype 1 penta-MU was a generous gift from Dr. David Kwan (Kwan et al. 2015).

[0075] Human fecal metagenomic library

[0076] For the generation of a human metagenomic fosmid library, a fresh human fecal sample was collected from a healthy Asian male volunteer with blood type AB + Direct DNA extraction and fosmid library construction were performed according to the procedure described in the MoE protocol (Armstrong et al. 2017).

[0077] Fosmid library screening

[0078] 51×384-well AB + Blood fosmid library plates were thawed at room temperature and replicated into 384-well plates containing 50 μl of LB medium for screening (12.5 μg / mL chloramphenicol, 25 μg / mL kanamycin, 100 μg / mL arabinose, 0.2% (v / v) maltose, 10 mM MgSO 4 ) The plates were incubated at 37 °C for 18 h in a sealed container containing a water reservoir to prevent excessive evaporation. 45 μl of reaction mixture (100 mM NaH 2 PO 4 , pH 7.4, 2% (v / v) Triton-X100, 100 μM GalNAc-α-MU, 100 μM Gal-α-MU) was added onto the grown screening plates using a QFill™ device [Genetix™]. The plates were then incubated at 37 °C for 24 h in a sealed container, and the fluorescence of each plate (e.g., 365 nm Em: 435 nm, sweep mode, gain 80) was measured at 1, 2, 4, 8, and 24 h via a SynergyH1 plate reader [BioTek™]. For all wells, the Z-score was calculated, which is given by the formula: Z-score = (median fluorescence) / standard deviation.

[0079] All positive hits exceeding a certain threshold were rearranged into a new 384-well plate, named "simple substrate hit" plate, and stored at -70°C. Two screening plates were replicated from the "simple substrate hit" plate and rescreened for either GalNAc-α-MU or Gal-α-MU activity to confirm and deconvolute the previously detected activity.

[0080] To determine which hits could cleave the A or B antigen structure, their activities against 50 μM of A antigen subtype 1 tetra-MU or 50 μM of B antigen subtype 1tetra- MU were measured using a binding enzyme assay. This version of the binding assay was previously described by Kwan (Kwan et al. 2015). The assay was modified to be able to detect cleavage of subtype 1 A antigen by using BgaC (Jeong 2009) instead of BgaA (Singh 2014) as the binding enzyme. α-N-acetylgalactosaminidase and α-galactosidase cleave the terminal sugar, releasing H antigen subtype I tri- MU. Subsequently, α-fucosidase (AfcA (Katayarna 2004)), β-galactosidase (BgaC (Jeong 2009)), and β-hexosaminidase (SpHex (Williams 2002)) exolytically cleave the remaining sugars until 4-methylumbelliferyl alcohol is released. This can be detected as an increase in fluorescence. To achieve this, 50 μg / mL of each enzyme was added to the reaction mixture. All positive hits exceeding a certain threshold were rescreened three times, and a host cell line containing a vector lacking the insert was used as a negative control. All confirmed hits were stored separately 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 )

[0081] Fosmid Hit Sequencing

[0082] To isolate fosmid DNA for array determination, using the positive hit fosmid glycerol stock, 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 incubate overnight at 37 °C with shaking at 220 rpm. Fosmid isolation was performed using the GeneJet™ Plasmid Miniprep Kit (Thermo Fisher™). The isolated plasmid was purified from contaminating linear Escherichia coli DNA using Plasmid-Safe™ ATP-dependent DNase (Epicentre™), followed by another purification using the GeneJet™ PCR Purification Kit (Thermo Fisher™). Concentrations were calculated on a Qbit™ fluorometer (ThermoFisher™) using the Quant-iT™ dsDNA HS Assay Kit (Invitrogen™). The expected DNA size was confirmed on a 1% agarose gel. For complete fosmid sequencing, 2 ng of each fosmid was sent to the UBC Sequencing Centre (Vancouver, BC, Canada). Each fosmid was individually barcoded and sequenced using the Illumina MiSeq™ system.

[0083] Raw Illumina MiSeq (trademark) sequence data were all trimmed and assembled using Python scripts available on GitHub (trademark) at https: / / github.com / hallamlab / FabFos. Briefly, Trimmomatic was used to remove adapters and low-quality sequences from reads (Bolger 2014). These reads were screened for vectors and host sequences using BWA (Li 2013), and then filtered using Samtools (trademark) and bam2fastq scripts to remove contaminants. These high-quality and purified reads were assembled by MEGAHIT with k-mer values ranging between 71 and 241, increasing in 10 increments (Li 2015). These libraries often have a coverage of over 20,000-fold, and to prevent the accumulation of sequencing errors that interfere with proper sequence assembly, the minimum k-mer multiplicity was calculated by 1% of the estimated coverage of the fosmid. Outside of the Python script assembly that generates multiple contigs, scaffolding was performed using minimus2 (Treangen 2011). The parameterized commands can be found in both the documentation on the GitHub (trademark) page and the Python script itself.

[0084] Fosmid ORF Prediction and Hit Verification

[0085] Fosmid ORFs were identified using the metagenomic version of Prodigal (trademark) (Hyatt 2010) and compared to the CAZy (trademark) database using BLASTP (trademark) as part of the MetaPathways (trademark) v2.5 software package (Konwar 2015). MetaPathways (trademark) parameters were length > 60, BLAST score > 20, blast score ratio > 0.4, E Value <1×10 -6 is.

[0086] All predicted ORFs with annotations to members of the GH or CBM family (along with known or suspected α-galactosidase and / or α-N-acetylgalactosaminidase activity) were cloned into the pET16b plasmid using the Golden Gate™ cloning strategy (Engler 2008), and the primer sequences were set in Table B. Proteins were expressed in BL21(DE3) and cultured at 37°C and 220 rpm for 20 h in 10 mL of ZY5052 autoinduction medium (Studier 2005). Cells were harvested by centrifugation (4000×g, 4°C, 10 min) and resuspended in 1 mL of lysis buffer (100 mM NaH 2 PO 4 , pH 7.4, 2% (v / v) Triton-X™ 100, 1x Protease Inhibitor EDTA-free [Pierce™]). A ligation assay (Kwan 2015) was performed using 50 μl of crude cell lysate from the candidates, mixed with 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 A antigen subtype 1tetra- MU or 100 μM B antigen subtype 1 tetra-MU), and incubated at 37°C. All reactions were performed in triplicate in black 96-well plates. Fluorescence (365 / 435 nm) was continuously monitored for 4 h using a Synergy™ H1 plate reader [BioTek™]. Assays from crude extracts showing cleavage activity against A or B antigens were repeated this time without the conjugating enzyme, and the reaction products were isolated via an HF Bond Elut C18 column and analyzed by LC-MS and / or TLC. TLC was performed using a TLC silica gel 60 F254 TLC plate [EMD Millipore Corp.™, Billerica, MA, USA].

[0087] Table B: Primer sequences JPEG0007684216000006.jpg211139

[0088] HPAE-PAD assay

[0089] Analysis of the enzymatic release of galactosamine was performed on a HPAE-PAD (Dionex™) HPLC system. The cleavage activities of various proteins were tested for the following substrates: mucin from porcine stomach type II at 7.5 μg / μL dissolved in 100 mM NaH 2 PO 4 dissolved at pH 7.4, 5 mM A antigen subtype 1 penta- MU in 100 mM NaH 2 PO 4 present in pH 7.4, and RBCs (hematocrit 50%) from A+, B+ and O- donors present in 1xPBS pH 7.4. Samples containing 10 μg / mL of the enzyme were incubated at 37 °C for two hours and then stored at -80 °C for further analysis. A small amount of the reaction (10 μl) was diluted in H 2 O (100 μl) and analyzed on a HPAE-PAD instrument. Separation was carried out on a CarboPac PA200™ (150 mm) column with a guard column, and detection was performed using disposable gold on a polytetrafluoroethylene (PTFE) electrode and a four-potential waveform. The separation conditions were as follows: 100 mM sodium hydroxide and a sodium acetate gradient from 70 to 300 mM were applied over the first 10 minutes of the separation. The eluent was held at the final gradient conditions for 1 minute and then returned to the initial conditions over the next 1 minute. The flow rate was 1.0 ml / min and injections were made every 27 minutes. Standards of free sugars GalNAc, Gal and GalN (10 μM) were applied to the HPAE-PAD and peak elution times were determined for reference.

[0090] Kinetic assay

[0091] All kinetic assays using 4-methylumbelliferone as the leaving group were performed by fluorescence measurement. To avoid measurement errors based on the inner filter effect (Palmier 2007), the linear range of the fluorescent dye was verified using a standard curve.

[0092] Fp Galactosaminidase

[0093] Michaelis-Menten parameters for GalN antigen subtype 1 penta -MU and A antigen subtype 1 penta -MU in 100 mM NaH 2 PO 4 , pH 7.4, 37 °C. 3.4 nM Fp Galactosaminidase (5.31 nM FpGalNase_truncA) and 0.1 mg / mL SpHex, AfcA, 0.2 mg / mL BgaC and various concentrations of substrate (5 μM - 2 mM) were reacted in 100 μl. A series of four reactions with controls (without Fp Galactosaminidase) as replicates were performed. The fluorescence signal (365 / 435 nm) resulting from MU release by hydrolysis was monitored by a Synergy H1 (trademark) plate reader [BioTek (trademark)] and converted to concentration using a MU standard concentration curve measured under the same reaction conditions. The initial velocity (μM / s) was measured and plotted with Grafit7.0 (trademark) to determine kinetic parameters.

[0094] k cat / K M parameters for GalN antigen subtypes 1 / 2 / 4 tetra- MU and B antigen subtype 1 tetra -MU at pH 7.4 and 37 °C. The reaction (total volume 100 μL) was carried out in black 96-well plates and, as a binding assay, 8.63 nM Fp Galactosaminidase, 0.1 mg / mL SpHex, BgaC (BgaA for subtype 2), AfcA, various concentrations of substrate (25 μM, 20 μM, 15 μM, 10 μM, 7.5 μM, 5 μM) in 100 mM NaH 2 PO 4Performed at (pH 7.4). A series of four reactions with a control (without Fp galactosaminidase) as a replicate were carried out. The fluorescence signal (365 / 435 nm) resulting from MU release by hydrolysis was monitored by a Synergy H1™ plate reader [BioTek™] and converted to concentration using a MU standard concentration curve measured under the same reaction conditions. The initial velocity (μM / s) was determined and plotted with Grafit 7.0™ to determine the k cat / K M (s -1 *mM -1 ) parameter.

[0095] 863.2 nM of Fp galactosaminidase (100 mM NaH 2 PO 4 , at pH 7.4) or 369.9 nM of FpGH4 with various substrate concentrations (10 μM - 5 mM) in a volume of 100 μl (50 mM Tris / HCl, pH 7.4, 100 μM NAD + , 1 mM MnCl 2 , at) in a clear 96-well plate with GalN-α-pNP, the Michaelis-Menten parameters were measured at 37°C. The reaction was carried out as a series of three reactions with two controls (enzyme-free). The absorption resulting from pNP release by hydrolysis (at 405 nm) was monitored by a Synergy H1™ plate reader [BioTek™] and converted to concentration using a p-nitrophenol standard concentration curve measured under the same reaction conditions. The initial velocity (μM / s) was measured and plotted with Grafit 7.0™ to determine the kinetic parameters.

[0096] FpGalNac deacetylase

[0097] 100 mM NaH 2 PO 4 , A antigen subtype 1 at pH 7.4, 37°C penta-For MU, Michaelis-Menten parameters were measured using the aforementioned binding assay (Kwan 2015). To enable detection of cleavage of subtype 1 (later 4), BgaC (Jeong 2009) was used instead of BgaA (Singh 2014) as β-galactosidase. Also, A antigen subtype 1 penta- Since MU further contains galactose, the concentration of BgaC was increased to 0.2 mg / mL to compensate for the need to cleave both Gal-β-1,3-β-GlcNAc-β-1,3-Gal-β-MU and Gal-β-MU. Furthermore, Fp galactosaminidase was included to enable cleavage of galactosamine-containing intermediates. The reaction setup in 100 μl was 3 nM FpGalNAc deacetylase (4.52 nM FpGalNacDeAc_D1ext, 3.55 nM FpGalNacDeAc_D1+2) and 0.01 mg / mL Fp galactosaminidase, 0.1 mg / mL SpHex, AfcA, 0.2 mg / mL BgaC and various concentrations of substrate (5 μM - 2.5 mM). A series of four reactions were performed with a control (without FpGalNac deacetylase) as a replicate. The fluorescence signal (365 / 435 nm) resulting from MU release by hydrolysis was monitored with a Synergy H1™ plate reader (BioTek™) and converted to concentration using a MU standard concentration curve measured under the same reaction conditions. The initial velocity (μM / s) was determined and plotted with Grafit 7.0 to determine kinetic parameters.

[0098] k cat / K M parameters were determined for A antigen subtypes 1 / 2 / 4 tetra- For MU, they were determined at pH 7.4, 37 °C. The reaction (total volume 100 μL) was performed in black 96-well plates and as a binding assay with various concentrations of substrate (25 μM, 20 μM, 15 μM, 10 μM, 7.5 μM, 5 μM) with 12 nM FpGalNAc deacetylase 0.1 mg / mL SpHex, BgaC (BgaA for subtype II), AfcA in 100 mM NaH 2 PO 4Performed at (pH 7.4). A series of four reactions with a control (without FpGalNAc deacetylase) as a replicate were carried out. The fluorescence signal (365 / 435 nm) resulting from MU release by hydrolysis was monitored with a Synergy H1™ plate reader (BioTek™) and converted to concentration using a MU standard concentration curve measured under the same reaction conditions. The initial velocity (μM / s) was determined and plotted with Grafit™ 7.0 to determine the kcat / KM (s−1* mM−1) parameter.

[0099] GH109 subtype kinetics

[0100] A antigen subtypes 1 / 2 / 4 tetra- For MU, the kcat / KM parameter was measured at pH 7.4 and 37 °C. The reaction (total volume 100 μL) was carried out in black 96-well plates and, as a binding assay, 86.02 nM BvGH109_1 / 100.49 nM EmGH109 / 80.52 nM BvGH109_2 / 87.4 nM BsGH109 and 5 μM NAD+, each SpHex, BgaC (BgaA for subtype 2), AfcA at 0.1 mg / mL, various concentrations of substrate (25 μM, 20 μM, 15 μM, 10 μM, 7.5 μM, 5 μM) with 100 mM NaH 2 PO 4 Performed at pH 7.4. A series of four reactions with a control (without α-N-acetylgalactosaminidase) as a replicate were carried out. The fluorescence signal (365 / 435 nm) resulting from MU release by hydrolysis was monitored by a Synergy H1™ plate reader [BioTek™] and converted to concentration using a MU standard concentration curve measured under the same reaction conditions. The initial velocity (μM / s) was determined and plotted with Grafit 7.0™ to determine the kcat / KM (s−1* mM−1) parameter.

[0101] Crystal structure analysis

[0102] Before crystallization, FpGalNAcDeAc_D1ext was digested overnight with thrombin (Novagen™) at a concentration of 1 mg / mL using the manufacturer's proposed protocol. The protein was then purified by a HisTrap FF column, the flow-through fraction was collected, buffer-exchanged into 10 mM Tris pH 8.0 + 75 mM NaCl, and concentrated to 12 mg / mL.

[0103] Crystallization

[0104] FpGalNAcDeAc_D1ext (12 mg / mL) was crystallized at a protein:reservoir ratio of 1:1 using the hanging-drop vapor diffusion method with a reservoir solution consisting of 0.2 M CaCl 2 , 0.1 M MES pH 6, 18% PEG4000, and 20 mM MnCl 2 . Rapid bromide soaking was used to derivatize the crystals for phasing, and the crystals were transferred to a solution of 1 M NaBr, 25% glycerol, 18% PEG4000, 20 mM CaCl 2 , and 0.1 M Mes pH for 30 seconds and flash-frozen in liquid nitrogen. The crystal complex with blood group B antigen trisaccharide (B_tri) was prepared by pre-incubating the protein (12 mg / mL) with 10 mM B_tri for 2 hours, and then setting up the drops under the same conditions as above, but omitting MnCl 2 . The crystals were cryoprotected with the reservoir solution supplemented with 25% glycerol.

[0105] Data collection, phasing, structure determination

[0106] The dataset was collected at the Canadian Light Source (trademark). The data was integrated using XDS (Kabsch 2010) and scaled with Aimless (trademark) (Evans 2013). Phasing and automated structure solution were carried out using CRANK2 (trademark) (Skubak 2013) in the CCP4I2 (trademark) program suite (Potterton 2018). The structure was checked and refined using alternating cycles of Coot (trademark) (Emsley 2004) and Refmac (trademark) (Vagin 2004). The B_tri structural complex was solved by difference Fourier, and ligands were manually built into Coot (trademark) as well as water and metal ions. The difference density map showed the presence of Mn 2+ in the apo structure and Ca 2+ in the coordination structure. The models were validated by Coot (trademark) and Molprobity (trademark) (Chen 2010). The atomic coordinates and structure factors of the apo and B_tri complexes have been deposited in the Protein Data Bank (PDB) with the following accession numbers: Flavonifractor plautii GalNAc deacetylase protein accession number: WP_009260926.1, and Flavonifractor plautii galactosaminidase protein accession number: WP_044942952.1.

[0107] Active site mutagenesis

[0108] Based on the structural information (not shown) and sequence alignment (not shown), FpGalNAcDeAc_D1min and FpGalNase_truncA were mutated using the QuickChange™ protocol (Zhang 2004) with the primers listed in Table B. The mutants were purified via NiNTA and HIC columns as described above. The structural integrity of all mutants was confirmed by CD spectroscopy. According to that, all the tested enzymes were structurally similar to the wild type. For mutants with relatively low activity, the reactions were carried out under the same conditions as those used for the complete kinetic determination. However, k cat / K M values were measured using the substrate consumption method as described above (Vocadlo 2002). Briefly, at low substrate concentrations where [substrate]<K M (equivalent to ~1 / 5 - 1 / 10 of K m ), the k cat / K M value can be approximated by fitting the progress of the reaction to a non-linear first-order curve and dividing by the enzyme concentration.

[0109] GH36 phylogenetic mapping

[0110] The reference sequences of GH36 were downloaded from the CAZy™ database using the SACCHARIS™ cazy_extract.pl script (Jones 2018). A reference tree was constructed using the phylogeny-based protein profiling software, TreeSAPP™ (available at https: / / github.com / hallamlab / TreeSAPP), and the sequences were mapped to these trees. Briefly, protein family domains were extracted from all full-length sequences downloaded from CAZy™ (Yin 2012) using HMMs from dbCAN. These sequences were then clustered at 70% sequence similarity using UCLUST™ to remove redundant sequence space and reduce the size of the trees (Edgar 2010). The reference tree was constructed using RAxML™ version 8.2.0, determining the time to end the bootstrap before 1000 replicates were executed by "--autoMRE", and selecting the optimal protein model by PROTGAMMAAUTO™ (Stamatakis 2006 and Stamatakis 2008).

[0111] Next, the query sequences were mapped to these reference trees using TreeSAPP™. Briefly, the protein sequences were aligned to the HMMs using hmmsearch™ and the aligned regions were extracted (Eddy 1998). hmmalign™ was used to include the new query sequences in the reference multiple alignment, and TrimAl™ removed the positions that were not conserved from the alignment file (Capella-Gutierrez 2009). RAxML™ was used to classify the query sequences in the reference tree by insertion. The placement of each query sequence was filtered and concatenated singly. The Jplace™ file before display in iTOL™ (Matsen 2012 and Letunic 2016).

[0112] RBC assay

[0113] Using a protocol approved by the University of British Columbia's Clinical Ethics Committee, whole blood was collected from healthy consenting donors into citrate Vacutainers. Tubes were rotated at 1000×g for 4 minutes at room temperature to separate RBCs, which were then washed three times with 1xPBS pH 7.4. For assays in the presence of dextran 40k, washed RBCs (200 μL, 10% hematocrit) were placed in tubes, the supernatant was partially removed, and replaced with 1xPBS pH 7.4 in the presence and absence of dextran 40k (final concentration 300 mg / mL). Additionally, some assays were performed in 1xPBS pH 7.4 + 25% plasma or 100% plasma. RBCs were gently mixed and placed on an orbital shaker for 30 seconds. Next, the diluted enzyme solution was added to make a final volume of 200 μL. Tubes were very gently vortexed and placed on an orbital shaker at the set temperature for a predetermined time.

[0114] MTS card

[0115] After the reaction, RBCs were washed three times with excess 1xPBS pH 7.4 and analyzed using a Micro Typing System™ (MTS) card [MTS™, Florida, USA]. RBCs (12 μl, 5% hematocrit) suspended in diluent [MTS, Florida, USA] were carefully added to the mini gel column, leaving a space between the blood and the contents of the mini gel. The MTS card was centrifuged at 156×g for 6 minutes at room temperature using a Beckman Coulter Allegra X-22R™ centrifuge with a sample holder modified as recommended. The degree of antigen removal from the RBC surface was evaluated from the position of the RBCs in the mini gel after rotation. RBCs with high surface antigen concentrations aggregated with the monoclonal antibodies present in the gel column and could not penetrate (MTS™ score 4). RBCs without surface antigen did not aggregate and migrated to the bottom of the mini gel (MTS score 0). RBCs that underwent partial removal of surface antigen migrated to a position between these and were assigned a score between 0 (absent) and 4 (present) according to the manufacturer's instructions.

[0116] Agglutination assay of H antigen

[0117] To analyze the conversion of A antigen to H antigen after enzymatic treatment, washed A-ECO-RBCs were mixed in equal amounts with 2 μg / mL of anti-H antibody (anti-blood group Hab antigen antibody [97-I]: cat no. ab24213 (Abcam™)), and the appearance of agglutination within a 30-minute time frame was monitored. RBCs agglutinated with anti-H antibody were assigned a score between 0 (no agglutination within 1800 seconds) and 5 (agglutination within 120 seconds).

[0118] FACS

[0119] Enzymatically treated RBCs were washed twice with 1xPBS pH 7.4, and 1% hematocrit ECO-RBCs were treated with 1 / 100 APC-anti-A antibody (Alexa Fluor™ 647 mouse anti-human blood group A: cat no. 565384 (BD Pharmingen™)) and / or anti-H antibody (anti-blood group Hab antigen antibody [97-I]: cat no. ab24213 (Abcam™)) at room temperature for 30 minutes, then washed twice with 1xPBS pH 7.4. For the detection of anti-H antibody, a secondary FITC-labeled antibody (goat F(ab′)2 anti-mouse IgM mu chain (FITC): cat no. ab5926 (Abcam™)) was used at a 1 / 500 concentration. The data were evaluated after reconstitution in 1xPBS pH 7.4 (hematocrit 1%) using a flow cytometer (CytoFLEX™ (Beckman Coulter™)).

[0120] Enzyme adsorption and antigenicity

[0121] To test whether the enzyme could be easily removed from the treated RBCs, the adsorbability was evaluated. FpGalNAc deacetylase and FpGalNase (F / P = 1) labeled with Pacific Blue were incubated with RBCs at 37 °C for 1 hour, and after several washes, the residual fluorescence was measured with a flow cytometer (CytoFLEX™ (Beckman Coulter™)).

[0122] RBCs were incubated with each enzyme at 50 μg / mL, and the enzyme-treated RBCs were mixed with homologous or autologous serum, and the antigenicity was tested by observing the possibility of aggregation. In addition, to evaluate potential anti-IgG, -C3d exposure, the treated RBCs were tested with anti-IgG, -C3d MTS™ cards [MTS™, Florida, USA]. The incubation time was 30 minutes at 37°C.

[0123] Synthesis of antigen subtypes

[0124] Synthesis of A and B antigen subtypes 1 / 2 / 4 tetra-MU was performed using the modified protocol described by Kwan (Kwan et al. 2015).

[0125] Two-step H antigen subtype 1 / 2 / 4 tri-MU synthesis

[0126] All three syntheses were carried out on a 20 mg scale of GalNAc-α-MU / GlcNAc-α-MU in 10 mL of 50 mM Tris / HCl, 200 mM NaCl, pH 7.4, 10 mM MnCl 2 , 50 U alkaline phosphatase, 1.5 equivalents of UDP-Gal, 1.2 equivalents of GDP-Fuc (scaled with LacNAc-MU product). Depending on the desired product, various glycosyltransferases were added at a concentration of 100 μg / mL. That is, for subtype I, CgtBS42 and Te2FT, for subtype II, HP0826 and WbgL, and for subtype IV, LgtD and Te2FT. The reaction was carried out at 37°C, and the progress was controlled by TLC (mobile phase EtAc:MeOH:H 2 O ratio of 6:2:1), and 4-methylumbelliferone was 10% H 2 SO 4It was hydrolyzed from the compound via [specific means] and detected by UV (360 nm). After no further increase in the product was observed, the reaction was applied to an HF Bond Elut C18 column, washed with several column volumes of 5% methanol, and the product was eluted with 25% methanol. Then, the solvent was removed under reduced pressure.

[0127] A antigen subtype 1 / 2 / 4 tetra- MU synthesis

[0128] 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 tri- on the scale of MU, 25 U alkaline phospholylase, 1.5 equivalents of UDP-GalNAc and 100 μg / mL BgtA. After no further increase in the product was observed, the reaction was applied to an HF Bond Elut C18 column via TLC, washed with several column volumes of 5% methanol, and the product was eluted with 25% methanol. Then, the solvent was removed under reduced pressure. The final product was further purified on a 1.5×46 cm HW-40F size exclusion column and then lyophilized. 2 、25U alkaline phospholylase, 1.5 equivalents of UDP-GalNAc and 100 μg / mL BgtA at 37°C. After no further increase in the product was observed, the reaction was applied to an HF Bond Elut C18 column via TLC, washed with several column volumes of 5% methanol, and the product was eluted with 25% methanol. Then, the solvent was removed under reduced pressure. The final product was further purified on a 1.5×46 cm HW-40F size exclusion column and then lyophilized.

[0129] B antigen subtype 1 / 2 / 4 tetra- MU synthesis

[0130] The final synthesis step was 10 mg of H antigen subtype 1 / 2 / 4 tri- on the scale of MU, carried out at 37°C in 5 mL of 50 mM Tris / HCl, 200 mM NaCl, pH 7.4, 25 U alkaline phospholylase, 1.5 equivalents of UDP-Gal and 100 μg / mL BoGT6a. After no further increase in the product was observed, the progress was tracked via TLC, the reaction was applied to an HF Bond Elut C18 column, washed with several column volumes of 5% methanol, and the product was eluted with 25% methanol. Then, the solvent was removed under reduced pressure. The final product was further purified on a 1.5×46 cm HW-40F size exclusion column and then lyophilized.

[0131] GalN antigen subtype 1 penta- MU synthesis

[0132] 10 mg of A antigen subtype 1 penta- MU was incubated with 1 μg / mL of FpGalNAc deacetylase and 5 mL of 100 mM NaH 2 PO 4 at 37 °C for 30 minutes, then 1 mM of EDTA was added to stop the reaction, complete conversion of the substrate was confirmed by TLC, the reaction mixture was applied to an 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 under reduced pressure.

[0133] Protein purification

[0134] All proteins and their cleavages were cloned into pET16b or pET28a via Golden Gate™ cloning (Engler 2008) or PIPE cloning (Klock 2008). Primer sequences are listed in Table B.

[0135] Protein production for extended characterization was performed in BL21(DE3) cells, cultured in 200 mL of ZY5052 autoinduction medium (Studier 2005) at 37 °C, 220 rpm for 20 hours, and inoculated with 100 μl of an overnight LB culture. Cells were harvested by centrifugation (4000 xg, 40 °C, 10 minutes) 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, 1x protease inhibitor EDTA-free (Pierce™), 2 U benzonase (Novagen™), 0.3 mg / mL lysozyme, 10 mM MgCl 2) was resuspended and then sonicated on ice (3-minute pulse time; 5-second pulses, 10-second intervals, 35% amplitude). After removal of cell debris by centrifugation (14000×g, 4°C, 30 minutes), the supernatant was collected and loaded onto a nickel affinity chromatography column (5 mL HisTrapHP™ column (GE™)) using a peristaltic pump. Elution was performed on an AKTApurifier™ system (GE™) and monitored via SDS-PAGE with a 10–75% gradient of 50 mM Tris / HCl, 400 mM imidazole, pH 7.4, 2 mM DTT. Fractions containing protein were identified and then pooled. Buffer exchange and concentration into 50 mM Tris / HCl, 150 mM NaCl, pH 7.4, 2 mM DTT were performed in Amicon Ultra-15 Centrifugal Filter Units™ MWCO 10 kDa (Millipore™).

[0136] FpGalNAc deacetylase, Fp galactosaminidase and its cleavage require a second round of purification and the buffer was exchanged using Amicon Ultra-15 Centrifugal Filter Units MWCO 10 kDa (Millipore™) prior to loading the protein onto a hydrophobic interaction chromatography column (10 mL phenyl sepharose high performance column (Pharmacia Biotech™)). Column loading, washing and elution (0–100% gradient) were processed through an AKTApurifier (GE™) using the following buffer conditions: FpGalNAc deacetylase, binding 1xPBS, 800 mM NH 2 PO 4, pH 7.4 and elution in 1x PBS, pH 7.4 and Fp galactosaminidase, binding in 25 mM Tris / HCl, 1 M NaCl, pH 7.4, elution in 25 mM Tris / HCl pH 7.4. Fractions containing the protein were identified by SDS-PAGE and then pooled. Buffer exchange and concentration into 50 mM Tris / HCl, 150 mM NaCl, pH 7.4 were performed in Amicon Ultra-15 Centrifugal Filter Units™ MWCO 10 kDa (Millipore™).

[0137] Characterization of the protein

[0138] Optimal pH value

[0139] A antigen subtype 1 penta- MU and GalN antigen subtype 1 penta- The general pH ranges of the activities of FpGalNAc deacetylase and Fp galactosaminidase towards MU were determined by the appearance of products on TLC plates for different pH values. Reactions were carried out on a 100 μl scale, at 37 °C, with 50 μM substrate and 1 μg / mL enzyme in the appropriate buffer systems. Buffers for pH 4 - 6 were based on 50 mM citric acid / sodium citrate buffer, for pH 6 - 8 on 50 mM sodium phosphate buffer and for pH 8 - 10 on 50 mM glycine / sodium hydroxide buffer.

[0140] To determine the optimal pH value, 5 μg / mL of Fp galactosaminidase was incubated with 100 μl of 50 mM sodium phosphate buffer in the pH range (5.8 - 8.0) and 200 μM of GalN-α-pNP, and the absorption resulting from pNP release (at 405 nm) was monitored at 37 °C for 1 hour by a Synergy H1™ plate reader (BioTek™).

[0141] 5 μg / mL of FpGalNAc deacetylase and 50 μM of A antigen subtype Ipenta-MU were pre-incubated for 10 minutes at 37 °C in 25 mM sodium phosphate buffer over various pH ranges (5.8 - 10.0). The reaction was quenched in 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) resulting from MU release by hydrolysis was monitored for 30 minutes at 37 °C by a Synergy H1™ plate reader (BioTek™).

[0142] Protein stability

[0143] FpGalNAc deacetylase and FpGalNase were stored at 4 °C in 1xPBS buffer pH 7.4. After 2 weeks and 12 weeks, A antigen subtype 1 penta- As described for the optimal pH for MU, the enzyme activity was tested in the binding enzyme reaction of GalN-α-pNP to FpGalNAc deacetylase and FpGalNase.

[0144] FpGalNAc deacetylase inhibition

[0145] FpGalNAc deacetylase was tested against different potential inhibitors in a 96-well plate format as a binding assay. At 37 °C in a 100 μL scale, using 50 μM of A antigen subtype 1penta-MU and 5 μg / mL of FpGalNAc deacetylase, 100 mM of NaH 2 PO 4Reactions were carried out at pH 7.4 with 10 μg / mL of Fp galactosaminidase, 50 μg / mL of SpHex, 50 μg / mL of AfcA, and 50 μg / mL of BgaC. As inhibitors, EDTA (1, 10, 100 μM), Marimastat (1, 10, 100, 1000 μM), DMSO (2%, 4%), and Protease Inhibitor Cocktail EDTA-free (Pierce™) (1x, 2x, and 4x) were tested. Fluorescence (365 / 435 nm) was continuously monitored for 1 hour using a Synergy H1™ plate reader (BioTek™). Additives showing strong effects were retested without the binding enzyme, and product formation was analyzed by TLC.

[0146] Limited proteolysis

[0147] Limited proteolysis was performed to investigate whether there is a smaller and more stable subdomain of Fp galactosaminidase. Fp galactosaminidase was treated with thermolysin (protein:protease mass ratio 10:1) at various temperatures (20°C, 37°C, 42°C, 50°C, and 65°C) for 1.5 hours. The samples were then electrophoresed on an SDS-PAGE gel, and a stable fragment migrating at approximately 70 kDa (from the initial 118 kDa) was identified, and nearly complete degradation was achieved at an incubation temperature of 50°C. This fragment was sent to the UBC Proteomics Core Facility for peptide identification and was determined to be the C-terminal cleavage form of the full-length protein with a cleavage site between amino acids 690 and 700.

[0148] Glycan array screening

[0149] For glycan array screening, 500 μg of FpGalNAcDeAc_D2ext was labeled with fluorescein isothiocyanate (FITC) at an F / P ratio of 1 using the Fluorotag™ FITC conjugation kit (Sigma™). Screening was performed on the printed array of version 5.3 of the Protein-Glycan Interaction Core Facility™ of the CFG, which consists of 600 glycans in 6 replicates for protein concentrations of 5 and 50 μg / mL. Analysis of the binding motif was performed using the web tools of Emory University (https: / / glycopattern.emory.edu / ).

[0150] Enzyme tests in buffered extracellular solution

[0151] Using compositions containing purified GalNAc deacetylase enzyme (SEQ ID NO: 5) and purified galactosaminidase enzyme (SEQ ID NO: 10), the compatibility with buffered extracellular solutions PBS, Steen™, and Perfadex™ was tested at 37°C, 37°C, and 4°C, respectively. Human type A red blood cells (RBCs) were incubated in PBS, Steen™, and Perfadex™ at various doses of the enzyme compositions to measure the ability of the enzyme to cleave the A antigen from the red blood cells. A 1% RBC solution was treated with various doses of the enzyme in PBS, Steen™, and Perfadex™ solutions, and the level of antigen removal at the end of the treatment was analyzed by flow cytometry.

[0152] Immunohistochemical analysis of arterial biopsies

[0153] To test the dose-increasing effect of an enzyme composition containing purified GalNAc deacetylase enzyme (SEQ ID NO: 5) and purified galactosaminidase enzyme (SEQ ID NO: 10), A-type human arteries in STEEN (trademark) solution were used for testing, and the proportion of A-type antigen was quantified by immunohistochemical analysis of biopsies taken from untreated (control), treated (processed) A-type arteries, and O-type arteries as negative controls. Area quantification software was used and normalized against the control group using the following formula. JPEG0007684216000007.jpg17152The residual positive level of A-type antigen quantified in the O-type group explains the artifacts generated during the process.

[0154] Enzyme treatment on human arteries was tested in human pulmonary arteries (static treatment). The dose in this case was prepared as the unit of the weight of the enzyme relative to the volume of the STEEN (trademark) solution. The arteries were biopsied, treated, and analyzed by immunohistochemistry with double staining by CD31 (staining endothelial cells) and BTA (staining blood type A antigen). Enzyme treatment of human arteries was performed for 4 hours at both 1 μg / mL and 10 μg / mL. Immunohistochemical staining of artery biopsies at 20 times magnification of arteries treated without enzyme (control) and arteries treated with enzyme (processed) was imaged. CD31 indicated the location of endothelial cells (blood vessels), and BTA indicated the location of blood type A antigen. BTA in untreated arteries was present together with endothelial cells (CD31 positive), and BTA was not present in treated arteries.

[0155] Human donor lung test

[0156] Regarding the effect of 1-hour enzyme treatment on ex vivo perfused human donor lungs, the expression level of A-type antigen was quantified using immunohistochemical analysis of lung tissue biopsies and area quantification software normalized against pre-treatment biopsies using the following formula. JPEG0007684216000008.jpg26153

[0157] The effects of 1-hour and 3-hour enzyme treatments (i.e., an enzyme composition containing purified GalNAc deacetylase enzyme (SEQ ID NO: 5) and purified galactosaminidase enzyme (SEQ ID NO: 10)) on ex vivo perfused human donor lungs were tested. Immunohistochemical staining of biopsied human donor lungs was imaged at 20x magnification to measure the effects on lungs treated with the enzyme composition. CD31 indicates the location of endothelial cells (blood vessels). BTA indicates the location of blood group A antigen. Pre-treatment images showed that blood group antigens were localized in both blood vessels and airways. In post-treatment images of the right upper dependent (RUD), right upper non-dependent (RUND), right middle non-dependent (RMND), right middle dependent (RMD), right lower non-dependent (RLND), and right lower dependent (RLD) regions of the lungs, blood group A antigen was not present in blood vessels.

[0158] In this study, two separate ex vivo perfused human donor lungs were tested, and the results are shown in Figures 10 and 11 corresponding to 1 hour and 3 hours, respectively.

[0159] (Example) Example 1: Construction and Screening of Metagenomic Library

[0160] AB +A metagenomic library containing large (35 - 65 kb) fragments of DNA extracted from fecal samples provided by male donors of blood type was constructed. Such libraries contain multiple genes per bacterium, increasing the probability of expression of at least some of these genes and enabling the expression of small “pathways” of multiple genes. The inventors' library contains approximately 19,500 clones in 51×384 well plates and may contain approximately 800,000 genes. Thus, initial screening of such libraries using expensive A antigen substrates was not practical. Instead, screening was performed using the simple and highly sensitive fluorogenic substrates methylumbelliferyl α - glycosides of galactose and N - acetyl - galactosamine (Gal - α - MU and GalNAc - α - MU). In the first screening with a mixture of these two substrates, a subset of 226 hits was obtained. These were rescreened against each individual substrate, and 44 were identified as having GalNAcase activity and 166 as having galactosidase activity. The second round of screening was performed on these hits using the A antigen and B antigen tetrasaccharide glycoside substrates shown in Figure 1, along with a control group without substrate, using a binding enzyme assay (Kwan 2015): The binding enzyme can act and release MU only when the first Gal or GalNAc is cleaved. Eleven of these hits contained A antigen cleavage activity, one of which also cleaved the B antigen, while six produced fluorescence in the absence of substrate and thus encoded a pathway that produces an irrelevant fluorescent product.

[0161] Example 2: Sequencing and initial analysis of hits

[0162] Eleven plasmids were sequenced on an Illumina MiSeq™, and the ORFs present in the CAZy™ database (http: / / www.cazy.org / ) (Lombard 2014) were identified using Metapathways™ software (Konwar 2015). Due to the fairly deep sequencing of the currently available human microbiome, the organisms from which all fosmids were derived could be identified. Of the 11 that were derived from overlapping fragments of two genomes of the genus Bacteroides, their sequences could be classified into five clusters. The only gene common to all fosmids in cluster B was the GH109 enzyme (B. vulgatus). Cluster A also contained GH109 (B. stercoris), but GH109 was the only CAZy gene found in fosmids from other Bacteroides (B. vulgatus). The fosmid N08 from the obligate anaerobic bacterium Flavonifractor plautii (Li 2015) contained three ORFs found within CAZy, namely the putative carbohydrate-binding module CBM32 and two potential glycoside hydrolases GH36 and GH4. Finally, the fosmid K05 from Collinsella sp., presumably Collinsella tanakaei, did not contain CAZy-related ORFs. Here, generation of a sub-library of fosmid K05 enabled the identification of an ORF with A-cleavage activity, which was later identified as GH36 (not shown).

[0163] Example 3: Analysis of the GH109 Enzyme

[0164] The GH109 family was found based on the A antigen cleavage activity of some of its members. These enzymes have an unusual NAD +It uses a dependency mechanism, which was first discovered in the enzyme in GH4 Add Yip Ref(2004)J.Amer.Chem.Soc.126,8354-8355 showing the mechanism (Varrot 2005 and Liu 2007). The three GH109 genes identified here were cloned with a His tag after removal of the signal peptide and expressed in Escherichia coli BL21(DE3). These three proteins, BsGH109, BvGH109_1 and BvGH109_2 (not shown), were purified together with the standard GH109 (EmGH109) from Elizabethkingia meningosepticum as a standard (Liu 2007), and kinetic parameters were determined for each of them. The three novel enzymes showed similar catalytic efficiencies to each of the three A-subtype substrates tested and mainly reflected the kinetic parameters of the EmGH109 standard. In contrast, when their A antigen cleavage activities were tested on RBCs using an approved MTS card, unfortunately only EmGH109 showed significant activity. The test was performed in the presence of dextran 40K as a crowding agent, which has been shown to increase activity by aggregating the enzyme on the cell surface (Chapanian 2014). In its absence, even 150 μg / mL of EmGH109 showed no effect, but in the presence of 300 mg / mL of dextran 40K, 15 μg / mL of the enzyme was sufficient (see Figures 3 and 4). Previous studies have also shown that low ionic strength also enhances the activity of EmGH109 on cells (Liu 2007). Therefore, EmGH109 is not effective throughout the blood. + When their A antigen cleavage activities were tested on RBCs using an approved MTS card, unfortunately only EmGH109 showed significant activity. The test was performed in the presence of dextran 40K as a crowding agent, which has been shown to increase activity by aggregating the enzyme on the cell surface (Chapanian 2014). In its absence, even 150 μg / mL of EmGH109 showed no effect, but in the presence of 300 mg / mL of dextran 40K, 15 μg / mL of the enzyme was sufficient (see Figures 3 and 4). Previous studies have also shown that low ionic strength also enhances the activity of EmGH109 on cells (Liu 2007). Therefore, EmGH109 is not effective throughout the blood.

[0165] Example 4: Analysis of GH36 from fosmid K05 derived from Collinsella sp.

[0166] The GH36 protein identified in fosmid K05 (named K05GH36) showed activity against GalNAc-α-MU and the A antigen tetrasaccharide. This is consistent with it being a member of the GH36 family, which mainly contains α-galactosidase and α-N-acetylgalactosaminidase and performs hydrolysis by a double-displacement mechanism involving a covalent β-glycosyl enzyme intermediate (comfort 2007). Phylogenetic analysis placed its sequence within cluster 4 of the GH36 subfamily (Fredslund 2011). Interestingly, this cluster also contains a characterized GH36 from Clostridium perfringens that is also known to cleave the A antigen structure (Calcutt 2002). However, when testing the ability of K05GH36 to remove the A antigen from red blood cells, its activity was unexpected and only scored 3 even when used in combination with a crowding agent.

[0167] Example 5: Analysis of fosmid N08 from Flavonifractor plautii

[0168] Since these novel enzymes did not offer advantages, attention was turned to the N08 fosmid from F. prausnitzii, especially because its gene product cleaves both A and B antigens. Three CAZ-related genes were cloned, their signal peptide sequences removed, expressed in E. coli BL21(DE3), and the resulting enzymes purified at yields up to 140 mg / L. Surprisingly, when the individual purified proteins were tested against A and B tetrasaccharide substrates, the only cleavage observed was that of the B antigen by N08GH36, and there was no cleavage of the A antigen by any of them. Therefore, combinations of pairs of these enzymes were tested, and it was surprising to find that a mixture of N08CBM32 and N08GH36 rapidly cleaved the A antigen tetrasaccharide. TLC analysis of the reaction mixtures with the individual enzymes revealed that N08CBM32 catalyzed the conversion of the A antigen to a more polar but still UV-active product, while subsequent addition of N08GH36 released a sugar product that co-migrated with galactosamine along with the H antigen trisaccharide. MS analysis of the reaction mixtures showed that N08CBM32 was an A antigen deacetylase, thus a decrease at 42 m / z and of the more polar product, while N08GH36 was a galactosaminidase and was shown to have a new activity in this family (Figure 2). This was further confirmed by high-performance anion-exchange chromatography with pulsed amperometric detection (HPAE-PAD) analysis of the reaction (Figure 5), which showed that treatment of the A antigen by both enzymes released galactosamine, which was not released by the individual enzymes. Similar results were obtained with the gastric mucin substrate, although this enzyme was presumably evolved against this substrate. Therefore, these two enzymes are hereafter referred to as FpGalNAc deacetylase (FpGalNAcDeAc) and Fp galactosaminidase (FpGalNase).

[0169] This pathway of A antigen degradation has not been characterized so far, but interestingly, it was suggested more than 50 years ago as an explanation for the so-called "acquired" B phenomenon. In this phenomenon, A-type patients infected with Clostridium tertium showed an apparent change in blood type to B, similar to forensic samples of human tissues submerged in the Thames River (Ref Judd and Annesley https: / / doi.org / 10.1016 / S0887-7963(96)80087-3, Transfusion medicine reviews(1996)10,111-117) (Gerbal 1975). This was probably because the anti-B antibody used for typing could not distinguish between terminal Gal and GalN.

[0170] When GH4, the third enzyme of the fosmid, was examined, it was found to hydrolyze Gal-α-pNP, GalN-α-pNP, and GlcN-α-pNP, but not to cleave substrates based on A antigen. Therefore, it does not seem to be directly involved in the conversion of A antigen. However, these glycosaminidases show new activities within the GH4 family.

[0171] Example 6: Characterization of FpGalNAc deacetylase

[0172] More detailed bioinformatics analysis of this gene by Phyre2 (trademark) (Kelley 2015) showed that it has an approximately 308 amino acid domain with unknown function at the N-terminus, an approximately 145 amino acid CBM32 near the C-terminus, and a linker region in between. Since all constructs containing the unprocessed deacetylase domain actually showed catalytic activity (Table 2), this basic structure was confirmed by cleavage analysis. Therefore, this protein is classified as the founding member of a new family of carbohydrate esterases, CExx.

[0173] Acetamidoglycan deacetylase has been proven to be a metalloenzyme that requires all divalent metal ions (Blair 2005). Consistent with this, treatment with 100 μM EDTA almost abolished the enzyme activity, but the activity increased with the addition of Mn 2+ , Co 2+ , Ni 2+ or Zn 2+ . Other inhibitors of (non-metallic) amidase had no effect. The optimal pH of this enzyme was around 8 (Figure 6), and its substrate specificity was narrow, being limited to different A subtypes and shorter versions thereof. However, among these subtypes, it was not very distinguishable, and there was only about a two-fold difference in specific activity among all of these subtypes (Table 2). Such a pH-dependence and specificity profile is ideal for RBC conversion because all subtypes of A are deacetylated while others are not.

[0174] The specificity of the CBM portion of the protein was examined using the glycan array of the Consortium for Functional Glycomics (CFG). The preferred target was a glycan having a repeating N-acetyl lactosamine (LacNAc) structure, as also seen for the founding member of the CBM32 family, i.e., N-acetylglucosaminidase from Clostridium perfringens (Ficko-Blean 2006). However, unlike CBM, our composition does not show high-affinity binding to blood antigen structures. The repeating LacNAc structure is a common component of the cell surface, like some O-glycans and glycolipids, as well as a general component of complex and hybrid N-glycans (Cohen 2009). In our case, these probably act as anchor points for the deacetylase domain to bind. Thereby, its catalytic domain comes close to the A antigen without competing with its own substrate. Supporting this model, removal of the domain decreased the RBC activity without affecting the cleavage rate of the soluble substrate (Table 2).

[0175] Example 7: Crystal Structure Analysis of FpGalNAc Deacetylase

[0176] To provide structural insights into this novel enzymatic activity, the truncated protein was subjected to crystallization trials and it was found that FpGalNAcDeAc_D1ext produced crystals that diffracted to the best resolution. The solution of this structure revealed a catalytic domain adopting a five-bladed β-propeller structure with an active site containing a divalent metal ion coordinated by D100 and H252. The binding mode was revealed by co-crystallization of the B-antigen trisaccharide as an analogue of the reaction product with the enzyme. At the base of the active site pocket, the non-reducing terminal galactosyl moiety, which is the differentiating group between the A- and B-antigens, makes hydrogen bond interactions with H97, E64 and two metal-coordinated water molecules. The remaining ligands are surface-exposed and polar interactions between the fucosyl group and the side chains of S61 and D121 were identified. Since the C1-OH group of the reducing terminal galactosyl moiety is exposed to the solvent, elongation to the substrate (i.e., at GlcNAc) is readily regulated by the enzyme. By modeling the N-acetyl group of the A-trisaccharide onto this structure, rational mutations of neighboring amino acids that might be involved in substrate deacetylation could be made. Residue E64 was found to be important for activity as both mutants were inactive, and it is suggested that it probably plays a direct role in the activation of the nucleophilic water molecule (Table 1). Residues coordinating the divalent metals D100, Y315 and H252 were also found to be important, and any mutations that resulted in a rate decrease of about 5000-fold were consistent with their apparent role in the binding of the divalent metal ion. By analogy with other acetamido sugar deacetylases, we propose that FpGalNAc deacetylase catalyzes hydrolysis by a mechanism that polarizes the carbonyl and activates a water molecule for nucleophilic attack on the carbonyl to form a tetrahedral intermediate. The breakdown of this intermediate is facilitated by proton donation to the sugar nitrogen atom by His100.

[0177] Table 1 | A antigen Type2 tetra Specific activity of the mutants of FpGalNAcDeAc_D1min against the cleavage of -MU JPEG0007684216000009.jpg77153

[0178] Example 8: Characterization of FpGalNAcDeAc and FpGalNase

[0179] In the phylogenetic analysis of the sequences, FpGalNase was placed in a new subgroup (5) of the GH36 family (Fredslund 2011). The 390 amino acid catalytic domain is located in the center of this large (1079 amino acid) protein and has a potential carbohydrate-binding domain at the C-terminus. Removal of this C-terminal domain did not affect the kinetic parameters of the enzyme with soluble substrates (Table 2), but + reduced the cleavage efficiency of RBC. This enzyme is specific for galactosamine-containing sugars and does not cleave GalNAc residues in any of the situations tested. However, it has a rather broad specificity for the cleavage of de-N-acetylated galactosaminides ranging from simple aryl glycoside GalN-α-pNP to higher order ones. In fact, the k cat / K M values of the three A subtypes tested (Table 2) were all similar to each other and also similar to those of the deacetylase. The k cat / K M value for the cleavage of B antigen was more than 2000-fold lower than that for the corresponding GalN antigen, yet it was sufficient to produce a positive hit on the original screen. This specificity for deacetylated α-galactose-containing substrates, combined with its optimal pH of about 6.5 - 7.0, makes it suitable for use in blood type conversion together with deacetylases (Figure 6).

[0180] Table 2 | Kinetic parameters of FpGalNAcDeAc and FpGalNase constructs against different antigenic substrates JPEG0007684216000010.jpg149160

[0181] Example 9: Cleavage of A antigen from RBC

[0182] A + , B + and O + type RBCs were incubated with FpGalNAcDeAc and FpGalNase respectively and as a mixture, and the released sugars were analyzed by HPAE-PAD ion chromatogram. None of the enzymes used individually released sugar products. However, when using the mixture of both, galactosamine was clearly released from type A + RBCs, but not from B + or O + and showed high specificity only for the A antigen. This is very important as it shows that GalNAc is not released from the RBC surface in other situations. The cleaved form of FpGalNase was also effective, but the activity was slightly lower.

[0183] Next, tests were conducted to remove antigens from RBCs using industry-standard MTS (trademark) cards. RBCs were loaded onto these antibody-binding columns and centrifuged in a centrifuge. RBCs without antigens move to the bottom of the column and are scored as 0, while untreated RBCs containing the corresponding antigens adhere to the top and are scored as 4, and the degree of antigen removal is ranked with intermediate scores. Treatment with FpGalNase alone did not remove A or B antigenicity at the concentrations in (Table 3), which is consistent with its inactivity towards the GalNAc substrate and low activity towards Gal. Incubation with FpGalNAcDeAc removes antigenicity by conversion of the acetamide to an amine and weakens the binding of the anti-A antibody used. The minimum amount of enzyme required for complete antigen deacetylation was evaluated with FpGalNAcDeAc alone and in combination with FpGalNase, both in the presence and absence of 300 mg / ml dextran as a crowding agent. An amount of FpGalNase up to 3 μg / ml was sufficient without dextran, but the required loading was reduced to 0.5 μg / ml by containing 300 mg / ml dextran (Table 3). The conventional best enzyme, EmGH109, was ineffective in the absence of dextran unless a low-salt buffer was used, but the minimum effective concentration was 15 μg / ml in the presence of dextran, which was a 30-fold higher loading. The version of FpGalNAcDeAc lacking the CBM was much less effective.

[0184] Table 3|A + , B + and AB + Results of MTS cards when RBCs were treated with EmGH109, FpGalNAcDeAc and FpGalNase JPEG0007684216000011.jpg140156

[0185] Since the MTS (trademark) card test does not evaluate the complete conversion of the A antigen and no antibody was available to detect the GalN antigen, the detection of the newly formed H antigen on the treated RBCs was focused on. FpGalNase was functional at a concentration as low as 5 μg / ml and, as confirmed by FACS analysis shown in Figure 3, brought about an increase in H antigen levels concomitant with the loss of the A antigen. By measuring the agglutination time in the presence of anti-H antibody, the functionality of both enzymes was demonstrated for several A + RBC donors and, under whole blood reaction conditions, an ability that could not be achieved with other blood-converting enzymes. Thus, this pair of enzymes converts A + RBCs to O-type "universal donor" RBCs at an enzyme loading much lower than that required for the best conventional enzymes. However, before these RBCs are transfused to patients, it is most likely by washing the cells after centrifugation, but it is recommended to remove all of the trace amounts of enzymes used in the conversion to avoid harmful immune reactions. To confirm that this can be achieved, A + RBCs were treated with fluorescently labeled samples of FpGalNAcDeAc and FpGalNase and then, using FACS analysis, it was confirmed that simple washing was effective (Figure 3).

[0186] Further characterization of the generated A-ECO RBCs may be useful to evaluate their full feasibility for use in transfusion medicine, but the possibility of including the enzyme directly in the plasma, potentially during blood collection, may enable an easy and cost-effective implementation into existing automation routines for blood collection and storage, away from the process. In particular, as shown in Table 4, the stability of the enzymes was tested. Table 4: Storage stability of galactosaminidase and GalNAc deacetylase JPEG0007684216000012.jpg92157

[0187] Example 10: GalNAc deacetylase and galactosaminidase fusion from Clostridium tertium

[0188] In the search for similar enzymes, a novel Clostridium tertium natural fusion of galactosaminidase and GalNAc deacetylase linked by CBM (GH36 domain - CBM - deacetylation domain) was identified. In the first test, this enzyme was shown to cleave the A antigen of red blood cells (the same mechanism of first deacetylation and then galactosamine cleavage), but it was not very efficient (i.e., similar to EmGH109). The Clostridium tertium deacetylation domain is not as efficient as the F. plautii GalNAc deacetylase, but when assisted by the F. plautii GalNAc deacetylase, the Clostridium tertium galactosaminidase domain exhibits activity similar to that of the F. plautii galactosaminidase on red blood cells.

[0189] Example 11: Alternative GalNAc deacetylase and galactosaminidase enzymes

[0190] Data show that Clostridium tertium, galactosaminidase (Ct5757_GalNAse) and Rp1021 have equivalent enzyme activity against the conversion of the GalN antigen to the H antigen (the second reaction step).

[0191] Data on alternative GalNAc deacetylase and galactosaminidase enzymes were also collected and the alternative enzymes were compared to the GalNAc deacetylase of Flavonifractor plautii and the galactosaminidase of Flavonifractor plautii. As shown in Table 5, the MTS scores for anti-A antibodies on treated A RBCs are shown for the Clostridium tertium natural fusion of galactosaminidase and GalNAc deacetylase, which requires the presence of dextran to effectively cleave the A antigen and also shows good activity (Ct5757_DeAcase) when combined with Flavonifractor plautii galactosaminidase (FpGalNase). Also, in Table 6, the data show that Robinsoniella peoriensis (Rp) Rp3672 and Rp3671 can deacetylate the A antigen on RBCs but are less efficient than FpGalNAcDeAcase and the activity was achieved only in the presence of a crowding agent (i.e., dextran 40k).

[0192] Table 5: MTS scores for anti-A antibodies on treated A RBCs JPEG0007684216000013.jpg53153

[0193] Table 6: MTS scores for Robinsoniella peoriensis (Rp) 3671 and 3672 JPEG0007684216000014.jpg36148

[0194] Figure 7 shows the conversion of the A antigen on A RBCs to the H antigen, analyzed by FACS sorting, for (A) A+RBC control, (B) flavonifractor plautii GalNAc deacetylase (FpGalNAcDeAc) + flavonifractor plautii galactosaminidase (FpGalNase) (10 μg / mL), (C) FpGalNAcDeAc + Clostridium tertium (Ct) Ct5757_GalNase (10 μg / mL), and (D) FpGalNAcDeAc + Robinsoniella peoriensis (Rp) galactosaminidase (Rp1021) GalNase (10 μg / mL). The data show that Clostridium tertium (Ct) Ct5757_GalNase and Robinsoniella peoriensis (Rp) galactosaminidase (Rp1021) GalNase exhibit enzyme activity comparable to that of flavonifractor plautii galactosaminidase for the conversion of the GalN antigen to the H antigen (second reaction step).

[0195] Example 12: Compatibility of Enzyme Composition with Perfusion / Preservation Fluid

[0196] To confirm that the enzyme composition is compatible with the EVLP system, first, the functions of the enzymes (purified flavonifractor plautiae GalNAc deacetylase protein of SEQ ID NO: 5 and purified flavonifractor plautiae galactosaminidase protein of SEQ ID NO: 10) in organ perfusion / preservation fluids (STEEN™ and Perfadex™, XVIVO perfusion) were tested. Compatibility was evaluated based on the ability of the enzyme composition to remove blood group A antigens on erythrocytes in STEEN™ at 37°C or Perfadex™ at 4°C. Since PBS is one of the standard solutions used for blood treatment, phosphate-buffered saline (PBS) at 37°C was used as a control group. The temperatures examined for STEEN™ and Perfadex™ were based on the working temperatures in clinical practice. The antigen removal level was analyzed by flow cytometry. To help predict the appropriate dose in organs, dose escalation tests in STEEN™ and Perfadex™ were performed (see Figure 8). The unit of the dose used throughout the test was defined as the weight of the enzyme (μg) relative to the volume of the solution (mL).

[0197] The enzyme composition was found to be fully compatible with STEEN™ and Perfadex™ perfusion / preservation fluids, and the perfusion / preservation fluids were shown to enhance the efficiency of the enzyme composition compared to PBS. The enzyme composition was able to remove more than 90% of the antigens in STEEN™ and Perfadex™ at a total enzyme concentration of 1 μg / mL, while the same effect was achieved at a dose of 4 μg / mL in PBS (Figure 8).

[0198] Example 13: Static Treatment of Human Arteries

[0199] To test the effectiveness of the enzymes (purified flavonifractor plautii GalNAc deacetylase protein of SEQ ID NO: 5 and purified flavonifractor plautii galactosaminidase protein of SEQ ID NO: 10) at the tissue level, an in vitro model of human arteries was used. The pulmonary arteries of the same human donor were divided into a control group (STEEN™ solution) and a treatment group (enzyme composition + STEEN™ solution), and statically incubated at 37 °C for 4 hours. Biopsies were performed at the end of the incubation for both groups. The enzyme composition was administered at 1 μg / mL and 10 μg / mL. Changes in blood group antigens were analyzed immunohistochemically. Serial sections of the biopsies were double-stained with CD31 (a marker for endothelial cells) to show the location of the vascular inner surface, and with BTA to show the expression of blood group antigens.

[0200] The expression level of blood group A antigen in the treatment group was significantly decreased compared to the control group. The dose effects of 1 μg / mL and 10 μg / mL were the same as those in the treated arteries. If the total concentration (dose) of the enzyme was lower than 1 μg / mL, the enzyme might also act. When the staining images of BTA were compared with CD31, the disappearance of blood group antigens was confirmed (Figure 9).

[0201] Example 14: Ex vivo perfusion of human lungs

[0202] The effectiveness of the enzyme-containing STEEN™ solution in removing tissue blood group antigens in human organs (e.g., lungs) was tested in the Toronto EVLP setting. Lungs from human donors were evaluated by clinical ex vivo lung perfusion (EVLP) and were determined to be unsuitable for transplantation and thus suitable for testing the enzyme composition. After the lungs declined, the enzyme composition (purified flavonifractor plautii GalNAc deacetylase protein of SEQ ID NO: 5 and purified flavonifractor plautii galactosaminidase protein of SEQ ID NO: 10) was added to the STEEN™ perfusion fluid to initiate the treatment. The dose was 1 μg / mL. Biopsies were performed before and after the treatment. Changes in the expression of blood group antigens were analyzed immunohistochemically. The function and physiology of the lungs were also monitored every hour during the experimental period to confirm that there were no acute side effects due to the treatment.

[0203] In the human lung, 1.5 L of perfusion fluid is required for single-lung EVLP and 2 L for bilateral-lung EVLP. In the first test of right single-lung EVLP (Figure 10), 1.5 mg of the enzyme composition was added to the perfusion fluid to a concentration of 1 μg / mL. The lung was treated for 1 hour. Immunohistochemical analysis showed a significant decrease in the amount of blood group A antigen after treatment (Figure 10). Comparing pre-treatment biopsy sections double-stained for blood group antigen and blood vessels, the lung antigen was localized not only on the surface of the blood vessel wall but also in the airways. Comparison of double-stained post-treatment biopsies showed that the intravascular antigen was effectively removed.

[0204] In the second test (Figure 11), right single-lung EVLP was treated with 1.5 mg of the enzyme composition in STEEN (trademark) perfusion fluid to a concentration of 1 μg / mL. The lung was treated for 3 hours. Immunohistochemical analysis showed a significant decrease in the expression level of blood group A antigen. Comparing pre-treatment biopsies double-stained for blood group antigen and blood vessels, the blood group antigen in the lung was localized not only on the blood vessel surface but also in the airways (Figure 11). Comparison of double-stained post-treatment biopsies showed that the intravascular antigen was effectively removed (Figure 11). No acute side effects on the physiology and function of the lung were observed after the start of enzyme treatment.

[0205] The results show that at a dose of 1 μg / mL, the enzyme shows an effect within 1 hour in the perfused human lung.

[0206] In this specification, various embodiments of the present invention are disclosed. Within the scope of the present invention, many adaptations and modifications can be made in accordance with the general knowledge of those skilled in the art, and such modifications include the substitution of known equivalents for any aspect of the present invention in order to achieve substantially the same results in substantially the same manner. Numerical ranges include the numerical values defining the range. In this specification, the term "comprising" is used as a non-limiting term that is substantially equivalent to the phrase "including but not limited to", and the word "comprises" has the corresponding meaning. As used herein, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, a reference to "a thing" includes a plurality of such things. The citation of references herein does not admit that such references are prior art to the embodiments of the present invention. The present invention includes all embodiments and variations substantially as described above with reference to the examples and drawings.

[0207] (Array) The flavonifractor proutii DNA sequence was modified from the naturally occurring DNA sequence (GalNAc deacetylase 2311 / 2319 nt / galactosaminidase 3228 / 3237 nt). In particular, there is a difference in the length of the sequence used for protein purification, whereby the signal peptide was removed and an N-terminal His tag was added via the vector backbone.

[0208] Informal Sequence List

[0209] SEQ ID NO: 2

[0210] Description: Flavonifractor proutii GalNAc deacetylase (protein sequence) MRNRRKAVSLLTGLLVTAQLFPTAALAADSSESALNKAPGYQDFPAYYSDSAHADDQVTHPDVVVLEEPWNGYRYWAVYTPNVMRISIYENPSIVASSDGVHWVEPEGLSNPIEPQPPSTRYHNCDADMVYNAEYDAMMAYWNWADDQGGGVGAEVRLRISYDGVHWGVPVTYDEMTRVWSKPTSDAERQVADGEDDFITAIASPDRYDMLSPTIVYDDFRDVFILWANNTGDVGYQNGQANFVEMRYSDDGITWGEPVRVNGFLGLDENGQQLAPWHQDVQYVPDLKEFVCISQCFAGRNPDGSVLHLTTSKDGVNWEQVGTKPLLSPGPDGSWDDFQIYRSSFYYEPGSSAGDGTMRVWYSALQKDTNNKMVADSSGNLTIQAKSEDDRIWRIGYAENSFVEMMRVLLDDPGYTTPALVSGNSLMLSAETTSLPTGDVMKLETSFAPVDTSDQVVKYTSSDPDVATVDEFGTITGVSVGSARIMAETREGLSDDLEIAVVENPYTLIPQSNMTATATSVYGGTTEGPASNVLDGNVRTIWHTNYAPKDELPQSITVSFDQPYTVGRFVYTPRQNGTNGIISEYELYAIHQDGSKDLVASGSDWALDAKDKTVSFAPVEAVGLELKAIAGAGGFGTAAELNVYAYGPIEPAPVYVPVDDRDASLVFTGAWNSDSNGSFYEGTARYTNEIGASVEFTFVGTAIRWYGQNDVNFGAAEVYVDGVLAGEVNVYGPAAAQQLLFEADGLAYGKHTIRIVCVSPVVDFDYFSYVGE

[0211] Sequence number 4

[0212] Description: Flavonifractor plautii GalNAc deacetylase (removing signal peptide protein sequence) ADSSESALNKAPGYQDFPAYYSDSAHADDQVTHPDVVVLEEPWNGYRYWAVYTPNVMRISIYENPSIVASSDGVHWVEPEGLSNPIEPQPPSTRYHNCDADMVYNAEYDAMMAYWNWADDQGGGVGAEVRLRISYDGVHWGVPVTYDEMTRVWSKPTSDAERQVADGEDDFITAIASPDRYDMLSPTIVYDDFRDVFILWANNTGDVGYQNGQANFVEMRYSDDGITWGEPVRVNGFLGLDENGQQLAPWHQDVQYVPDLKEFVCISQCFAGRNPDGSVLHLTTSKDGVNWEQVGTKPLLSPGPDGSWDDFQIYRSSFYYEPGSSAGDGTMRVWYSALQKDTNNKMVADSSGNLTIQAKSEDDRIWRIGYAENSFVEMMRVLLDDPGYTTPALVSGNSLMLSAETTSLPTGDVMKLETSFAPVDTSDQVVKYTSSDPDVATVDEFGTITGVSVGSARIMAETREGLSDDLEIAVVENPYTLIPQSNMTATATSVYGGTTEGPASNVLDGNVRTIWHTNYAPKDELPQSITVSFDQPYTVGRFVYTPRQNGTNGIISEYELYAIHQDGSKDLVASGSDWALDAKDKTVSFAPVEAVGLELKAIAGAGGFGTAAELNVYAYGPIEPAPVYVPVDDRDASLVFTGAWNSDSNGSFYEGTARYTNEIGASVEFTFVGTAIRWYGQNDVNFGAAEVYVDGVLAGEVNVYGPAAAQQLLFEADGLAYGKHTIRIVCVSPVVDFDYFSYVGE

[0213] Sequence number 5

[0214] Description: His tag Flavonifractor plautii GalNAc deacetylase having (pET16a - protein sequence) MG HHHHHHHHHHSSGADSSESALNKAPGYQDFPAYYSDSAHADDQVTHPDVVVLEEPWNGYRYWAVYTPNVMRISIYENPSIVASSDGVHWVEPEGLSNPIEPQPPSTRYHNCDADMVYNAEYDAMMAYWNWADDQGGGVGAEVRLRISYDGVHWGVPVTYDEMTRVWSKPTSDAERQVADGEDDFITAIASPDRYDMLSPTIVYDDFRDVFILWANNTGDVGYQNGQANFVEMRYSDDGITWGEPVRVNGFLGLDENGQQLAPWHQDVQYVPDLKEFVCISQCFAGRNPDGSVLHLTTSKDGVNWEQVGTKPLLSPGPDGSWDDFQIYRSSFYYEPGSSAGDGTMRVWYSALQKDTNNKMVADSSGNLTIQAKSEDDRIWRIGYAENSFVEMMRVLLDDPGYTTPALVSGNSLMLSAETTSLPTGDVMKLETSFAPVDTSDQVVKYTSSDPDVATVDEFGTITGVSVGSARIMAETREGLSDDLEIAVVENPYTLIPQSNMTATATSVYGGTTEGPASNVLDGNVRTIWHTNYAPKDELPQSITVSFDQPYTVGRFVYTPRQNGTNGIISEYELYAIHQDGSKDLVASGSDWALDAKDKTVSFAPVEAVGLELKAIAGAGGFGTAAELNVYAYGPIEPAPVYVPVDDRDASLVFTGAWNSDSNGSFYEGTARYTNEIGASVEFTFVGTAIRWYGQNDVNFGAAEVYVDGVLAGEVNVYGPAAAQQLLFEADGLAYGKHTIRIVCVSPVVDFDYFSYVGE

[0215] Sequence number 7

[0216] Description: flavonifractor plautigallactosaminidase

[0217] Sequence number 9

[0218] Description: flavonifractor plautii galactosaminidase (signal peptide removed protein sequence)

[0219] Array number 10

[0220] Description: His tag Flavonifractor plautigallactosaminidase having (pET16a - protein sequence) MG HHHHHHHHHH

[0221] Accession number 12

[0222] Description: Clostridium tertium isolated protein sequence 099345757.1 - Ct5757 (a fusion of galactosaminidase and GalNAc deacetylase linked with CBM (original protein sequence))

[0223] Accession number 14

[0224] Description: Clostridium tertium 5757 (Ct5757) isolated protein sequence without signal peptide (Accession number 099345757.1 - Ct5757)

[0225] Array number 15

[0226] Description: His tag and a Clostridium tertium 5757 (Ct5757) fusion protein sequence expression construct having a thrombin cleavage site (in the pET28a vector) MGSS HHHHHH

[0227] Sequence number 17

[0228] Description: His tag And Clostridium tertium 5757 (Ct5757) GalNAc deacetylase protein sequence expression construct having a thrombin cleavage site (in pET28a vector) MGSS HHHHHH SSGLVPRGSHSGQYWLVFQPDNDVLQTKTNPSSMKQSANNNPYNYNILPNSFPIGTGYNAYKGDVSFYATFKEASSQAIPQNSWALKYVDSEETTGENGRATNAFDGNNNTIWHTKYSGGNAAPMPHEIQIDLRGVYNINQINYLPRQDGGTNGTIKDYEVYLSLDGVNWGQPISKGTFESNSTEKIVKFNETKSRYVKLKALSEINNKQFTTVADLKVFGWEISKIEKPLQNAETYLNIPTYDGLNQSTHPDVKYFKNGWNGYKYWMIMTPNRTGSSVAENPSILASDDGINWEVPAGVTNPIAPMPQVGHNCDVDMIYNEATDELWVYWVESDDITKGWVKLIKSKDGVNWSSQQVVVDDNRAKYSTLSPSIIFKDNKYYMWSVNTGNSGWNNQSNKVELRESSDGVNWSNPTVVNTLAQDGSQIWHVNVEYIPSKNEYWAIYPAYKNGTGSDKTELYYAKSSDGVNWTTYKNPILSKGTSGKWDDMEIYRSCFVYDEDTNMIKVWYGAVSQNPQIWKIGFTENDYDKFIEGLTQ

[0229] Sequence number 19

[0230] Description: His tag And Clostridium tertium 5757 (Ct5757) protein sequence galactosaminidase expression construct having a thrombin cleavage site (in pET28a vector) MGSS HHHHHHSSGLVPRGSHYNLIDNISVEKLDTDISQANENVFLNGNGIALEVDNRGATCIYLVDENGVKTKATTSLDTADFSGYPIIGGQKIRDFVIISKNLEENINSILGVGNRLTIISKSSSTNLIRKIVFETSNSNPGAIYSTVSYKAESNDLLVDSFHENEYTMSLGQGPFLAYQGCADQQGANTIVNVTNGYNHNSGQNNYSVGVPFSYVYNSVGGIGIGDASTSRREFKLPIIGKDNTVSLGMEWNGQTLKKGAETAIGTSVITTTNGDYYSGLKSYAEVMKDKGISAPASIPDIAYDSRWESWGFEFDFTIEKIVNKLDELKAMGIKQITLDDGWYTYAGDWKLSPQKFPNGNADMKYLTDEIHKRGMTAILWWRPVDGGINSKLVSEHPEWFIKNSQGNMVRLPGPGGGNGGTAGYALCPNSEGSIQHHKDFVTVALEEWGFDGFKEDYVWGIPKCYDSSHKHSSLSDTLENQYKFYEAIYEQSIAINPDTFIELCNCGTPQDFYSTPYVNHAPTADPISRVQTRTRVKAFKAIFGDDFPVTTDHNSVWLPSALGTGSVMITKHTTLSSSDREQYNKYFGLARDLELAKGEFIGNLYKYGIDPLESYVIRKGEDIYYSFYKDNSSYSGNIEIKGLDSNATYRIEDYVNNRVIARGVKGPTATINTSFTDNLLVRAIPDDTPAEVTTFDVGNNTILSSTDSGNSKYLNAVSTTLEKTATIDSLSIYIGNNSENGKLQIAIYDDNNGKPGTKKAYVEEFVPTKNSWNTKKVVNSVTLPSGQYWLVFQPDNDVLQTKTNPSSMKQSANNNPYNYNILPNSFPIGTGYNAYKGDVSFYATFKEASSQAIPQNSWALKYVDSEETTGENGRATNAFDGNNNTIWHTKYSGGNAAPMPHEIQIDLRGVYNINQINYLPRQDGGTNGTIKDYEVYLSLDGVNWGQPISKGTFESNSTEKIVKFNETKSRYVKLKALSEINNKQFTTVADLKVFGWEISKIEK

[0231] Array number 21

[0232] Description: His tag And a Robin soniera peoriensis Rp1021 galactosaminidase protein expression construct having a thrombin cleavage site (in the pET28a vector) MGSS HHHHHH

[0233] Sequence number 23

[0234] Description: His tag And Ruthenibacterium lactatiformans R18755 GalNAc deacetylase protein sequence expression construct having a thrombin cleavage site (in pET28a vector) MGSS HHHHHH MGSS

[0235] Sequence number 25

[0236] Description: His tag And Robinsoniella peoriensis Rp 3671 GalNAc deacetylase protein expression construct having a thrombin cleavage site (in pET28a vector) MGSS HHHHHH

[0237] Array number 27

[0238] Description: His tag And a Robin soniera peoriensis Rp 3672 GalNAc deacetylase protein expression construct having a thrombin cleavage site (in the pET28a vector) MGSS HHHHHH

[0239] Array number 29

[0240] Description: His tag and a Robin soniela peoriensis Rp 3671 GalNAc deacetylase protein having a thrombin cleavage site, Rp3671 expression construct (in the pET28 a vector) MGSS HHHHHHSSGLVPRGSHSPLSAAAESGTGTRLVKGQTGYLTEEQAIRNQEQTTEEREQKLTGEETAEVLMEGTKDSGIVQTEEVQTKEMQTEDAQTEEVQTEEMQTEDAQTKEVQTEEMQTEDAQTEEVQTKEEPAEETHMKEIQTQGTKKASDRNGKARVTEILEDAQDPANRIVYLSDLQWKSENHTVDSELPTRKDKSFGGGKITLKVDGTVTEFDKGIGTQTDSTIVYDLEGKGYTKFETYVGVDYSQKENIPGEVCDVKFRVKIDDKIVSETGVLDPLSNAVKISVNIPDTAKTLTLYADKVTETWSDHANWADAKFYQALPEPENVAFKKTVVTRKTSDNSEAPVNPDSAVNSSKAVDGVIDSSSYFDFGDQANSGAVRESLYMEVDLKGSYLLSDIQLWRYWKDGRTYAATAIVVAEDENFENAAVIYNSDTTGEIHHLGAGSDMLYAETESGKTFPVPENTKARYIRVYTYGVNGTSGVTNHIVELKVNAYVFGDEILPEKPDDSKIFPNAVNPLKLQGPGTNDQVTHPDVTVFDEPWNGYKYWMAYTPNKPGSSYFENPCIAASNDGVNWEFPAQNPVQPRYDSEIENQNEHNCDTDIVYDPVNDRLIMYWEWAQDEAVNGKTHRSEIRYRVSYDGINWGVEDKTGVLMTGPTDHGCAIATEGERYSDLSPTVVYDKTEKIYKMWANDAGDVGYENKQNNKVWYRTSQDGISNWSDKTYVENFLGVNEDGLQMYPWHQDIQWVEEFQEYWALQQAFPAGSGPDNSSLRFSKSKDGLHWEPVSEKALITVGAPGTWDAGQIYRSTFWYEPGGAKGNGTFHIWYAALAEGQSHWDIGYTSANYADAMYKLTGSR

[0241] Sequence number 31

[0242] Description: His tagand a Robinso niela peoriensis Rp 3672_GalNAc deacetylase_protein expression construct having a thrombin cleavage site (in the pET28a vector) MGSS HHHHHH

[0243] Sequence number 32

[0244] Description: Clostridium tertium 5757 (Ct5757) GalNAc deacetylase protein sequence HSGQYWLVFQPDNDVLQTKTNPSSMKQSANNNPYNYNILPNSFPIGTGYNAYKGDVSFYATFKEASSQAIPQNSWALKYVDSEETTGENGRATNAFDGNNNTIWHTKYSGGNAAPMPHEIQIDLRGVYNINQINYLPRQDGGTNGTIKDYEVYLSLDGVNWGQPISKGTFESNSTEKIVKFNETKSRYVKLKALSEINNKQFTTVADLKVFGWEISKIEKPLQNAETYLNIPTYDGLNQSTHPDVKYFKNGWNGYKYWMIMTPNRTGSSVAENPSILASDDGINWEVPAGVTNPIAPMPQVGHNCDVDMIYNEATDELWVYWVESDDITKGWVKLIKSKDGVNWSSQQVVVDDNRAKYSTLSPSIIFKDNKYYMWSVNTGNSGWNNQSNKVELRESSDGVNWSNPTVVNTLAQDGSQIWHVNVEYIPSKNEYWAIYPAYKNGTGSDKTELYYAKSSDGVNWTTYKNPILSKGTSGKWDDMEIYRSCFVYDEDTNMIKVWYGAVSQNPQIWKIGFTENDYDKFIEGLTQ

[0245] Sequence number 33

[0246] Description: Ruthenibacterium lactatiformans R18755 GalNAc deacetylase protein sequence HEETDLLVNGGFETGDSTGWNWFNNAVVDSAAPHSGNYCAKVAKNSSYEQVVTVSPDTKYVLTGWAKSEGSSVMTLGVKNYGGQETFSATLSADYQQLAVTFTTGPNAQTATIYGYRQNSGSGAGYFDDVELTAVQDFAPYQPLANAIAPQAIPTYDGANQPTHPSVVKFEQPWNGYLYWMAMTPYPFNDGSYENPSIVASNDGENWIVPEGVSNPLAGTPSPGHNCDVDLVYVPASDELRMYYVEADDIISSRVKMISSRDGVHWSEPQVVMQDLVRKYSILSPSIEILPDGTYMMWYVDTGNAGWNSQNNQVKYRTSADGIKWSGAVTCTDFVQPGYQIWHIDVHYDTSSGAYYAVYPAYPNGTDCDHCNLFFAVNRTGKQWETFSRPILKPSTEGGWDDFCIYRSSMLIDDGMLKVWYGAKKQEDSSWHTGLTMRDFSEFMKILER

[0247] Sequence number 34

[0248] Description: Robinsoniella peoriensis Rp3671 GalNAc deacetylase protein HSPLSAAAESGTGTRLVKGQTGYLTEEQAIRNQEQTTEEREQKLTGEETAEVLMEGTKDSGIVQTEEVQTKEMQTEDAQTEEVQTEEMQTEDAQTKEVQTEEMQTEDAQTEEVQTKEEPAEETHMKEIQTQGTKKASDRNGKARVTEILEDAQDPANRIVYLSDLQWKSENHTVDSELPTRKDKSFGGGKITLKVDGTVTEFDKGIGTQTDSTIVYDLEGKGYTKFETYVGVDYSQKENIPGEVCDVKFRVKIDDKIVSETGVLDPLSNAVKISVNIPDTAKTLTLYADKVTETWSDHANWADAKFYQALPEPENVAFKKTVVTRKTSDNSEAPVNPDSAVNSSKAVDGVIDSSSYFDFGDQANSGAVRESLYMEVDLKGSYLLSDIQLWRYWKDGRTYAATAIVVAEDENFENAAVIYNSDTTGEIHHLGAGSDMLYAETESGKTFPVPENTKARYIRVYTYGVNGTSGVTNHIVELKVNAYVFGDEILPEKPDDSKIFPNAVNPLKLQGPGTNDQVTHPDVTVFDEPWNGYKYWMAYTPNKPGSSYFENPCIAASNDGVNWEFPAQNPVQPRYDSEIENQNEHNCDTDIVYDPVNDRLIMYWEWAQDEAVNGKTHRSEIRYRVSYDGINWGVEDKTGVLMTGPTDHGCAIATEGERYSDLSPTVVYDKTEKIYKMWANDAGDVGYENKQNNKVWYRTSQDGISNWSDKTYVENFLGVNEDGLQMYPWHQDIQWVEEFQEYWALQQAFPAGSGPDNSSLRFSKSKDGLHWEPVSEKALITVGAPGTWDAGQIYRSTFWYEPGGAKGNGTFHIWYAALAEGQSHWDIGYTSANYADAMYKLTGSR

[0249] Sequence number 35

[0250] Description: Robinsoniella peoriensis Rp3672_GalNAc deacetylase protein

[0251] Array number 36

[0252] Description: Clostridium tertium 5757 (Ct5757) galactosaminidase protein sequence HYNLIDNISVEKLDTDISQANENVFLNGNGIALEVDNRGATCIYLVDENGVKTKATTSLDTADFSGYPIIGGQKIRDFVIISKNLEENINSILGVGNRLTIISKSSSTNLIRKIVFETSNSNPGAIYSTVSYKAESNDLLVDSFHENEYTMSLGQGPFLAYQGCADQQGANTIVNVTNGYNHNSGQNNYSVGVPFSYVYNSVGGIGIGDASTSRREFKLPIIGKDNTVSLGMEWNGQTLKKGAETAIGTSVITTTNGDYYSGLKSYAEVMKDKGISAPASIPDIAYDSRWESWGFEFDFTIEKIVNKLDELKAMGIKQITLDDGWYTYAGDWKLSPQKFPNGNADMKYLTDEIHKRGMTAILWWRPVDGGINSKLVSEHPEWFIKNSQGNMVRLPGPGGGNGGTAGYALCPNSEGSIQHHKDFVTVALEEWGFDGFKEDYVWGIPKCYDSSHKHSSLSDTLENQYKFYEAIYEQSIAINPDTFIELCNCGTPQDFYSTPYVNHAPTADPISRVQTRTRVKAFKAIFGDDFPVTTDHNSVWLPSALGTGSVMITKHTTLSSSDREQYNKYFGLARDLELAKGEFIGNLYKYGIDPLESYVIRKGEDIYYSFYKDNSSYSGNIEIKGLDSNATYRIEDYVNNRVIARGVKGPTATINTSFTDNLLVRAIPDDTPAEVTTFDVGNNTILSSTDSGNSKYLNAVSTTLEKTATIDSLSIYIGNNSENGKLQIAIYDDNNGKPGTKKAYVEEFVPTKNSWNTKKVVNSVTLPSGQYWLVFQPDNDVLQTKTNPSSMKQSANNNPYNYNILPNSFPIGTGYNAYKGDVSFYATFKEASSQAIPQNSWALKYVDSEETTGENGRATNAFDGNNNTIWHTKYSGGNAAPMPHEIQIDLRGVYNINQINYLPRQDGGTNGTIKDYEVYLSLDGVNWGQPISKGTFESNSTEKIVKFNETKSRYVKLKALSEINNKQFTTVADLKVFGWEISKIEK

[0253] Accession number 37

[0254] Description: Robiniera peoriensis Rp1021 galactosaminidase protein sequence

[0255] <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. Science (1982) “Group B erythrocytes enzymatically converted to group O survive normally in A,B,and O individuals.” 215(4529):168-70. US4609627;and CA2272925 Kruskall M.S. et al. Transfusion (2000) “Transfusion to blood group A and O patients of group B RBCs that have been enzymatically converted to group O.” 40(11):1290-8. Clausen, H and Hakomori, S. Vox Sang. (1989) “ABH and related histo-blood group antigens; immunochemical differences in carrier isotypes and their distribution.” 56(1):1-20. EP2243793 Liu, Q.P. et al. J Biol Chem.(2008)“Identification of a GH110 subfamily of alpha 1,3-galactosidases: novel enzymes for removal of the alpha 3Gal xenotransplantation antigen.” 283(13):8545-54. doi:10.1074 / jbc.M709020200. Epub 2008 Jan 28. PCT / US1992 / 010113; and PCT / SE2015 / 050108 US4088538; US4141857; US4206259; US4218363; US4229536; US4239854; US4619897; US4748121; US4749653; US4897352; US4954444; US4978619; US5154808; US5914367; US5962279; US6030933; US6291582; US6254645; US10,016,490; and US10,041,055 Jeong, J.K. et al. J Bacteriol.(2009) “Characterization of the Streptococcus pneumoniae BgaC protein as a novel surface beta-galactosidase with specific hydrolysis activity for the Galbeta1-3GlcNAc moiety of oligosaccharides.” 191(9):3011-23. doi:10.1128 / JB.01601-08. Epub 2009 Mar 6. Singh, A.K. et al. PLoS Pathog.(2014) “Unravelling the multiple functions of the architecturally intricate Streptococcus pneumoniae β-galactosidase, BgaA.” 10(9):e1004364. doi:10.1371 / journal.ppat.1004364. eCollection 2014 Sep. Katayarna, T. et al. J Bacteriol.(2004) “Molecular cloning and characterization of Bifidobacterium bifidum 1,2-alpha-L-fucosidase (AfcA), a novel inverting glycosidase (glycoside hydrolase family 95).” 186(15):4885-93. Williams, S.J. et al. J Biol Chem.(2002) “Aspartate 313 in the Streptomyces plicatus hexosaminidase plays a critical role in substrate-assisted catalysis by orienting the 2-acetamido group and stabilizing the transition state.” 277(42):40055-65. Epub 2002 Aug 8. Bolger, A.M. et al. Bioinformatics.(2014) “Trimmomatic: a flexible trimmer for Illumina sequence data.” 30(15):2114-20. doi: 10.1093 / bioinformatics / btu170. Epub 2014 Apr 1. Li 2013 Treangen, T.J. et al. Curr Protoc Bioinformatics (2011) “Next generation sequence assembly with AMOS.” Chapter 11:Unit 11.8. doi: 10.1002 / 0471250953.bi1108s33. Hyatt, D. et al. BMC Bioinformatics.(2010) “Prodigal: prokaryotic gene recognition and translation initiation site identification.” 11:119. doi:10.1186 / 1471-2105-11-119. Konwar, K.M. et al. Bioinformatics.(2015) “MetaPathways v2.5: quantitative functional, taxonomic and usability improvements.” 31(20):3345-7. doi:10.1093 / bioinformatics / btv361. Epub 2015 Jun 15. Studier, F.W. Protein Expr Purif.(2005) “Protein production by auto-induction in high density shaking cultures.” 41(1):207-34. Palmier M.O. and Van Doren S.R. Anal Biochem. (2007) “Rapid determination of enzyme kinetics from fluorescence: overcoming the inner filter effect.” 371(1):43-51. Epub 2007 Jul 18. Kabsch, W. Acta Crystallogr D Biol Crystallogr.(2010) “XDS” 66(Pt 2):125-32. doi: 10.1107 / S0907444909047337. Epub 2010 Jan 22. Evans, P.R. and Murshudov, G.N. Acta Crystallogr D Biol Crystallogr. (2013) “How good are my data and what is the resolution?” 69(Pt 7):1204-14. doi: 10.1107 / S0907444913000061. Epub 2013 Jun 13. Skubak, P. and Pannu, N.S. Nat Commun. (2013) “Automatic protein structure solution from weak X-ray data.” 4:2777. doi: 10.1038 / ncomms3777. Potterton, L. et al. Acta Crystallogr D Struct Biol. (2018) “CCP4i2: the new graphical user interface to the CCP4 program suite.” 74(Pt 2):68-84. doi: 10.1107 / S2059798317016035. Epub 2018 Feb 1. Emsley, P. and Cowtan, K. Acta Crystallogr D Biol Crystallogr. (2004) “Coot: model-building tools for molecular graphics.” 60(Pt 12 Pt 1):2126-32. Epub 2004 Nov 26. Vagin, A.A. et al. Acta Crystallogr D Biol Crystallogr. (2004) “REFMAC5 dictionary: organization of prior chemical knowledge and guidelines for its use.” 60(Pt 12 Pt 1):2184-95. Epub 2004 Nov 26. Chen, V.B. et al. Acta Crystallogr D Biol Crystallogr.(2010) “MolProbity: all-atom structure validation for macromolecular crystallography.” 66(Pt 1):12-21. doi: 10.1107 / S0907444909042073. Epub 2009 Dec 21. Zhang 2004 Vocadlo, D.J. et al. Biochemistry.(2002) “A case for reverse protonation: identification of Glu160 as an acid / base catalyst in Thermoanaerobacterium saccharolyticum beta-xylosidase and detailed kinetic analysis of a site-directed mutant.” 41(31):9736-46. Jones, D.R. et al. Biotechnol Biofuels.(2018) “SACCHARIS: an automated pipeline to streamline discovery of carbohydrate active enzyme activities within polyspecific families and de novo sequence datasets.” 11:27. doi: 10.1186 / s13068-018-1027-x. eCollection 2018. Yin, Y. et al. Nucleic Acids Res.(2012) “dbCAN: a web resource for automated carbohydrate-active enzyme annotation.” 40(Web Server issue):W445-51. doi: 10.1093 / nar / gks479. Epub 2012 May 29. Edgar, R.C. Bioinformatics.(2010) “Search and clustering orders of magnitude faster than BLAST.” 26(19):2460-1. doi: 10.1093 / bioinformatics / btq461. Epub 2010 Aug 12. Stamatakis, A. Bioinformatics. (2006) “RAxML-VI-HPC: maximum likelihood-based phylogenetic analyses with thousands of taxa and mixed models.” 22:2688-2690. doi:10.1093 / bioinformatics / btl446. Stamatakis, A. and Ott, M. Philos Trans R Soc Lond B Biol Sci. (2008) “Efficient computation of the phylogenetic likelihood function on multi-gene alignments and multi-core architectures.” 363(1512):3977-84. doi: 10.1098 / rstb.2008.0163. Eddy, S.R. Bioinformatics. (1998) “Profile hidden Markov models.” 14(9):755-63. Review. Capella-Gutierrez, S. et al. Bioinformatics. (2009) “trimAl: a tool for automated alignment trimming in large-scale phylogenetic analyses.” 25(15):1972-3. doi: 10.1093 / bioinformatics / btp348. Epub 2009 Jun 8. Matsen, F.A. et al. PLoS One. (2012) “A format for phylogenetic placements.” 7(2):e31009. doi: 10.1371 / journal.pone.0031009. Epub 2012 Feb 22. Letunic, I. and Bork, P. Nucleic Acids Res. (2016) “Interactive tree of life (iTOL) v3: an online tool for the display and annotation of phylogenetic and other trees.” 44(W1):W242-5. doi: 10.1093 / nar / gkw290. Epub 2016 Apr 19. Engler, C. et al. PLoS One. (2008) “A one pot, one step, precision cloning method with high throughput capability.” 3(11):e3647. doi: 10.1371 / journal.pone.0003647. Epub 2008 Nov 5. Kwan, D.H. et al. J Am Chem Soc. (2015) “Toward Efficient Enzymes for the Generation of Universal Blood through Structure-Guided Directed Evolution.” 137(17):5695-705. doi: 10.1021 / ja5116088. Epub 2015 Apr 24. The eleven fosmids were sequenced on an Illumina MiSeq TM and ORFs therein that are present in the CAZy TM database (http: / / www.cazy.org / )(Lombard 2014 Konwar, K.M. et al. Bioinformatics. (2015) “MetaPathways v2.5: quantitative functional, taxonomic and usability improvements.” 31(20):3345-7. doi: 10.1093 / bioinformatics / btv361. Epub 2015 Jun 15. Li, D. et al. Bioinformatics. (2015) “MEGAHIT: an ultra-fast single-node solution for large and complex metagenomics assembly via succinct de Bruijn graph.” 31(10):1674-6. doi: 10.1093 / bioinformatics / btv033. Epub 2015 Jan 20. enzymes from GH4 Add Yip, V.L. and Withers, S.G. J. Amer. Chem. Soc. (2006) “Mechanistic analysis of the unusual redox-elimination sequence employed by Thermotoga maritima BglT: a 6-phospho-beta-glucosidase from glycoside hydrolase family 4.” 126, 8354-8355 Chapanian, R. et al. Nat Commun. (2014) “Enhancement of biological reactions on cell surfaces via macromolecular crowding.” 5:4683. doi: 10.1038 / ncomms5683. Varrot, A. et al. J Mol Biol. (2005) “NAD+ and metal-ion dependent hydrolysis by family 4 glycosidases: structural insight into specificity for phospho-beta-D-glucosides.” 346(2):423-35. Epub 2005 Jan 7. Liu, Q.P. et al. Nat Biotechnol. (2007) “Bacterial glycosidases for the production of universal red blood cells.” 25(4):454-64. Epub 2007 Apr 1. Comfort, D.A. et al. Biochemistry (2007) “Biochemical analysis of Thermotoga maritima GH36 alpha-galactosidase (TmGalA) confirms the mechanistic commonality of clan GH-D glycoside hydrolases.” 46(11):3319-30. Epub 2007 Feb 27. Calcutt, M.J. et al. FEMS Microbiol Lett. (2002) “Identification, molecular cloning and expression of an alpha-N-acetylgalactosaminidase gene from Clostridium perfringens.” 214(1):77-80. Gerbal, A., Maslet, C. and Salmon, C. Vox Sang. (1975) “Immunological aspects of the acquired B antigen.” 28(5):398-403. Judd, W.J. and Annesley, T.M. Transfusion medicine reviews (1996) “The acquired-B phenomenon.” 10, 111-117. Kelley, L.A. et al. Nat Protoc. (2015) “The Phyre2 web portal for protein modeling, prediction and analysis.” 10(6):845-58. doi: 10.1038 / nprot.2015.053. Epub 2015 May 7. Ficko-Blean, E. and Boraston, AB. J Biol Chem. (2006) “The interaction of a carbohydrate-binding module from a Clostridium perfringens N-acetyl-beta-hexosaminidase with its carbohydrate receptor.” 281(49):37748-57. Epub 2006 Sep 21. Cohen, M. et al. Blood. (2009) “ABO blood group glycans modulate sialic acid recognition on erythrocytes.” 114(17):3668-76. doi: 10.1182 / blood-2009-06-227041. Epub 2009 Aug 24. Fredslund, F. et al. J Mol Biol. (2011) “Crystal structure of α-galactosidase from Lactobacillus acidophilus NCFM: insight into tetramer formation and substrate binding.” 412(3):466-80. doi: 10.1016 / j.jmb.2011.07.057. Epub 2011 Jul 30. Guibert, E. E. et al. Transfus Med Hemother. (2011) “Organ Preservation: Current Concepts and New Strategies for the Next Decade” 38(2): 125-142.

Claims

1. A perfusion fluid for enzymatically cleaving A antigen from a donor organ, wherein the perfusion fluid comprises: (a) one or more purified flavonifractor plautii GalNAc deacetylases selected from SEQ ID NO: 2, SEQ ID NO: 4, and SEQ ID NO: 5; (b) one or more purified flavonifractor plautii galactosaminidases selected from SEQ ID NO: 7, SEQ ID NO: 9, and SEQ ID NO:

10. A perfusion fluid comprising the same.

2. The perfusion fluid according to claim 1, wherein the perfusion fluid comprises a purified enzyme having GalNAc deacetylase activity that is essentially composed of an amino acid sequence that is at least 90% identical to one of the sequences of SEQ ID NO: 2, 4, and 5, and a purified enzyme having galactosaminidase activity that is essentially composed of an amino acid sequence that is at least 90% identical to one of the sequences of SEQ ID NO: 7, 9, and 10.

3. The perfusion fluid according to claim 1 or 2, wherein the perfusion fluid comprises: (a) one or more of the purified GalNAc deacetylase proteins selected from the purified flavonifractor plautii GalNAc deacetylase proteins of SEQ ID NO: 2, SEQ ID NO: 4, and SEQ ID NO: 5; (b) one or more of the purified galactosaminidase proteins selected from the purified flavonifractor plautii galactosaminidase proteins of SEQ ID NO: 7, SEQ ID NO: 9, and SEQ ID NO:

10. A perfusion fluid comprising the same.

4. The perfusion fluid according to any one of claims 1 to 3, wherein the perfusion fluid satisfies at least one of the following (i) to (iii): (i) capable of cleaving A antigen at a concentration of 1 μg / ml or less; (ii) having A antigen cleavage activity at a pH between 6.5 and 7.5; (iii) having A antigen cleavage activity at a temperature between 4°C and 37°C.

5. The perfusion fluid according to any one of claims 1 to 4, further comprising a buffered extracellular solution selected from Steen (trademark), Perfadex (trademark), Perfadex Plus (trademark), EuroCollins solution, histidine-tryptophan-ketoglutaric acid (HTK) solution, University of Wisconsin solution (UW), Celsior solution, kidney perfusion solution (KPS-1), Kyoto University solution, IGL-1 solution, and citrate solution.

6. A method for enzymatically cleaving A antigen ex vivo from a donor organ, comprising: (a) perfusing an A antigen-presenting donor organ with a fluid containing a flavonifractor plautii GalNAc deacetylase protein and a flavonifractor plautii galactosaminidase protein for a time sufficient for the enzyme to enable cleavage of the A antigen from the donor organ, or, (b) incubating an A antigen-presenting donor organ with a fluid containing a flavonifractor plautii GalNAc deacetylase protein and a flavonifractor plautii galactosaminidase protein for a time sufficient for the enzyme to enable cleavage of the A antigen from the donor organ, the method comprising: the flavonifractor plautii GalNAc deacetylase protein being one or more selected from SEQ ID NO: 2, SEQ ID NO: 4, and SEQ ID NO: 5; and the flavonifractor plautii galactosaminidase protein being one or more selected from SEQ ID NO: 7, SEQ ID NO: 9, and SEQ ID NO:

10. **Claim 7** The method according to claim 6, wherein the fluid comprises a purified enzyme having GalNAc deacetylase activity consisting essentially of an amino acid sequence that is at least 90% identical to one of the sequences of SEQ ID NO: 2, 4, and 5, and a purified enzyme having galactosaminidase activity consisting essentially of an amino acid sequence that is at least 90% identical to one of the sequences of SEQ ID NO: 7, 9, and 10. **Claim 8** The method according to claim 6, wherein the flavonifractor plautii GalNAc deacetylase is the purified flavonifractor plautii GalNAc deacetylase protein of SEQ ID NO: 4 or SEQ ID NO: 5, and the flavonifractor plautii galactosaminidase is the purified flavonifractor plautii galactosaminidase protein of SEQ ID NO: 9 or SEQ ID NO:

10. **Claim 9** The method according to any one of claims 6 to 8, wherein the flavonifractor plautii GalNAc deacetylase protein and the flavonifractor plautii galactosaminidase protein are present in a buffered extracellular solution selected from Steen (trademark), Perfadex (trademark), Perfadex Plus (trademark), EuroCollins solution, histidine-tryptophan-ketoglutaric acid (HTK) solution, University of Wisconsin solution (UW), Celsior solution, kidney perfusion solution (KPS-1), Kyoto University solution, IGL-1 solution, and citrate solution.

10. The method according to any one of claims 6 to 9, wherein the donor organ is a solid organ.

11. The method according to claim 10, wherein the solid organ is selected from lung, kidney, liver, heart, pancreas, and intestine.

12. The solid organ is a lung, and the flavonifractor plautii GalNAc deacetylase protein and the flavonifractor plautii galactosaminidase protein are mixed ex vivo with a buffered extracellular solution containing the lung and circulated through the lung, whereby the GalNAc deacetylase protein and the galactosaminidase protein are in contact with the vascular system of the lung for a time sufficient to substantially remove A antigen from the vascular system of the lung. The method according to claim 11.

13. The method according to claim 12, wherein the time for removing A antigen from the vascular system of the lung is within 1 hour.

14. The method according to any one of claims 6 to 13, further comprising a step of washing the donor organ to remove flavonifractor plautii GalNAc deacetylase, flavonifractor plautii galactosaminidase, and cleaved A antigen.

15. The fluid is the following (i) to (iii) (i) capable of cleaving A antigen at 1 μg / ml or less (ii) having A antigen cleavage activity at a pH between 6.5 and 7.5, (iii) having A antigen cleavage activity at a temperature between 4°C and 37°C, The method according to any one of claims 6 to 14, which satisfies at least one of the above.

Images