US 201700.44516A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2017/0044516 A1 Tsai et al. (43) Pub. Date: Feb. 16, 2017

(54) BIOCHEMISTRY REACTIVE MATERIAL (30) Foreign Application Priority Data AND DEVICE FOR ELMINATING ELECTRONEGATIVE LOW-DENSITY Aug. 11, 2015 (TW) ...... 104126050 LIPOPROTEIN (LDL) AND METHOD FOR Nov. 23, 2015 (CN) ...... 2O15108154214 TREATING BLOOD OR PLASMA EX VIVO O O TO ELMINATE ELECTRONEGATIVE Publication Classification LOW-DENSITY LIPOPROTEIN THEREN (51) Int. Cl. CI2N II/2 (2006.01) (71) Applicant: Industrial Technology Research A6M I/34 (2006.01) Institute, Hsinchu (TW) CI2M I/00 (2006.01) 52) U.S. C. (72) Inventors: Pei-Yi Tsai, Hsinchu City (TW). (52) CPC ...... CI2N II/I2 (2013.01): CI2M 45/09 Chih-Hung CHEN, Tainan City (TW); (2013.01); A61M I/3486 (2014.02); A61M Yi-Hung LIN, Zhubei City (TW); 2202/08 (2013.01) Chih-Chieh HUANG, Zhunan Township (TW); Hsin-Hsin SHEN, (57) ABSTRACT Zhudong Township (TW); Liang-Yin The present disclosure provides a biochemistry reactive KE, Kaohsiung City (TW); Chu-Huang - CHEN, Taichung City (TW) material, including a Substrate and an enzyme composition immobilized on the Substrate. The enzyme composition is selected from a group consisting of a first enzyme, a second (73) Assignee: Industrial Technology Research enzyme, and a combination thereof. The first enzyme is used Institute, Hsinchu (TW) for eliminating a glycan residue of an electronegative low density lipoprotein (electronegative LDL). The second enzyme is used for eliminating ceramide carried by an (21) Appl. No.: 14/984,938 electronegative low-density lipoprotein. The biochemistry reactive material is capable of eliminating electronegative (22) Filed: Dec. 30, 2015 low-density lipoprotein. Patent Application Publication Feb. 16, 2017 Sheet 1 of 26 US 2017/004.4516 A1

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BOCHEMISTRY REACTIVE MATERAL composition immobilized on the substrate. The enzyme AND DEVICE FOR ELMINATING composition is selected from a group consisting of a first ELECTRONEGATIVE LOW-DENSITY enzyme, a second enzyme, and a combination thereof. The LIPOPROTEIN (LDL) AND METHOD FOR first enzyme eliminates a glycan residue of an electronega TREATING BLOOD OR PLASMA EX VIVO tive low-density lipoprotein (LDL). The second enzyme TO ELMINATE ELECTRONEGATIVE eliminates ceramide carried by an electronegative LDL. The LOW-DENSITY LIPOPROTEIN THEREN biochemistry reactive material is capable of eliminating electronegative low-density lipoprotein. CROSS REFERENCE TO RELATED 0010. The present disclosure also provides a biochemis APPLICATION try reactive device, comprising: the biochemistry reactive 0001. The present application is based on, and claims material as mentioned above, and a container for containing priority from, Taiwan Application Serial Number the biochemistry reactive material. The container has at least 104126050, filed on Aug. 11, 2015, and China Application one inlet and at least one outlet, wherein a liquid sample Serial Number 201510815421.4, filed on Nov. 23, 2015, the enters into the biochemistry reactive device from the at least disclosure of which are hereby incorporated by reference one inlet, and flows through the biochemistry reactive mate herein in its entirety. rial to react with the biochemistry reactive material, and then flows out through the at least one outlet. INCORPORATION BY REFERENCE OF 0011. The present disclosure further provides a method SEQUENCE LISTING for ex vivo treating blood or plasma, comprising (a) ex vivo contacting a blood or plasma with an enzyme composition to 0002. A sequence listing submitted as a text file via react the enzyme composition with the blood or plasma, EFS-Web is incorporated herein by reference. The text file wherein the enzyme composition is capable of eliminating containing the sequence listing is named “O965-A24497 low-density lipoprotein. The enzyme composition is US Seq Listing..txt'; its date of creation was Dec. 29, 2015: selected from a group consisting of a first enzyme, a second and its size is 11,648 bytes. enzyme, and a combination thereof. The first enzyme is for eliminating a glycan residue of an electronegative LDL. The TECHNICAL FIELD second enzyme is for eliminating ceramide carried by an 0003. The technical field relates to a biochemistry reac electronegative low-density lipoprotein. The method also tive material and device for eliminating electronegative comprises (b) terminating the contact between the blood or low-density lipoprotein (LDL) and a method for treating plasma and the enzyme composition to terminate the reac blood or plasma ex vivo to eliminate electronegative low tion of the enzyme composition with the blood or plasma. density lipoprotein therein 0012. A detailed description is given in the following embodiments with reference to the accompanying drawings. BACKGROUND BRIEF DESCRIPTION OF DRAWINGS 0004 Low-density lipoprotein (LDL) is a kind of lipo protein that is a product of lipoprotein lipase action. Lipo 0013 The present invention can be more fully understood proteins play a role in lipid transportation. It has long been by reading the Subsequent detailed description and examples known that the level of cholesterol carried by low-density with references made to the accompanying drawings, lipoprotein is associated with the occurrence and presence of wherein: cardiovascular diseases. 0014 FIG. 1A is a schematic cross-sectional view of a 0005. At present, in the medical field, plasma LDL cho biochemistry reactive device of one embodiment of the lesterol (LDL-C) is still used as an indicator for estimating present disclosure; cardiovascular diseases. However, the low-density lipopro 0015 FIG. 1B is a schematic cross-sectional view of a tein level in the plasma of patients with acute myocardial biochemistry reactive device of another embodiment of the infarction has no tendency to increase. present disclosure; 0006. Due to external factors, such as excess oxidation 0016 FIG. 1C is a schematic cross-sectional view of a pressure, etc., low-density lipoprotein will be post-transla biochemistry reactive device of another embodiment of the tion modified, and presents higher electronegativity to present disclosure; become electronegative LDL or L5. 0017 FIG. 2 shows the transformation result for NEU2: 0007 Electronegative LDL (L5) electronegative low 0018 FIG. 3 shows the transformation result for ASAH2: density lipoprotein is a major factor for causing cardiovas 0019 FIG. 4 shows the result of gene transfection of cular disease. L5 is almost undetectable in a normal human NEU4/ASAH2 confirmed by western blot: body. In addition, it has been in vitro and in vivo verified that (0020 FIG. 5 shows the result for NEU2 purification: L5 will damages vascular endothelial cells and activate (0021 FIG. 6 shows the result for ASAH2 purification: monocytes and platelets, and result in Systemic inflamma 0022 FIG. 7 shows apoptosis of endothelial cells of tion, atherosclerosis and myocardial infraction. blood vessel co-cultured with electronegative low-density 0008. Therefore, a novel material, device and/or method lipoprotein (electronegative LDL) L5 (25 ug/mL, 50 ug/mL) for eliminating an electronegative low-density lipoprotein and L5 (1.25 g) treated by the mmobilized-NEU2 filled is/are needed. device for 2 hours (treatment temperature 37°C., pH 7.4) for 24 hours, respectively; SUMMARY 0023 FIG. 8 shows results of performing quantitative 0009. The present disclosure provides a biochemistry analysis to the LDL samples without treatment and treated reactive material, comprising a Substrate and an enzyme without enzyme at 37° C. for 2 hours or treated with NEU2 US 2017/00445 16 A1 Feb. 16, 2017 for 2 hours (treatment temperature 37° C. pH 7.4) to comprise, but are not limited to, low-density lipoproteins determine the content of L5 therein; L1, L2, L3, L4, L5, etc. In one embodiment, the electro 0024 FIGS. 9A,9B and 9C show that performing a mass negative low-density lipoprotein mentioned above may be spectrometry analysis on L5 can detect L5 specific glyco low-density lipoprotein L5. Moreover, L5 is the most elec Sylation of apoE lipoprotein; tronegative and most harmful low-density lipoprotein. 0025 FIGS. 10A-1, 10A-2, 10B-1, and 10B-2 show that 0036. The biochemistry reactive material of the present the mass spectrometry analysis result of L5 treated with disclosure may comprise, but is not limited to, a substrate NEU2, wherein apoE specific glycan residues have been and an enzyme composition, wherein the enzyme composi removed; tion is immobilized on the substrate. 0026 FIGS. 11A, 11B-1, 11B-2, 11B-3, and 11B-4 show 0037 Examples of suitable substrate may comprise silica NEU2, NEU4 immobilized on different material both are gel, cellulose, diethylaminoethyl cellulose (DEAE cellu capable of effectively eliminating glycosylation on lipopro lose), chitosan, polystyrene, polysulfone, polyetherSulfone, teins; Sequences of LDL which are most commonly glyco acrylate resin, polysaccharide, etc., but they are not limited sylated comprise: 1. (R)IGODGISTSATTNLK(C) (SEQID thereto. The substrate may have a particle structure or a NO. 3) of apoB100; 2. (K)VLVDHFGYTK(D) (SEQ ID hollow-tube structure, etc. In one embodiment, the substrate NO. 4) of apoB100; 3. (K)GVISIPR(L) (SEQ ID NO. 5) of may be a cellulose bead. In another embodiment, the sub apoB100; 4. (K)SGSSTASWIQNVDTKYQIR(I) (SEQ ID strate may be a chitosan bead. Moreover, the substrate may NO. 6) of apoB100: 5. (K)AKPALEDLRQGLLPVLESFK be a cellulose hollow fiber, a polysulfone hollow fiber, epoxy (V) (SEQ ID NO. 7) of apoB100. Furthermore, ITRI-A-01 acrylic resin or a polyethersulfone hollow fiber, etc. (NEU2), ITRI-CD-01 (NEU2), ITRI-Si-Nu-01(NEU4) all 0038. The preceding enzyme composition may comprise are capable of effectively eliminating glycan residues on a first enzyme for eliminating a glycan residue of an elec apoB; tronegative LDL, a second enzyme for eliminating ceramide 0027 FIG. 12 shows ceramide contents of L5 and L5 carried by an electronegative LDL, or a combination thereof, treated with ASAH2 for 24 hours; but it is not limited thereto. Source organisms of the first 0028 FIG. 13 shows ceramide contents of L5 and L5 enzyme and the second enzyme mentioned above have no treated with ASAH2 for 24 hours; particular limitation. In one embodiment, the first enzyme 0029 FIG. 14 shows ceramide contents of L5 and L5 and the second enzyme are bioengineered enzymes from treated with ASAH2 in the presence or absence of a buffer human genome and also possibly from animal genome. (200 mM Tris-HCl pH 8.4, 1.5 M NaCl, 25 mM CaCl) for 2 or 24 hours; 0039. The preceding first enzyme may be sialidase or 0030 FIG. 15 shows ceramide contents of L5 and L5 glycosidase. treated with ASAH2 in the presence or absence of a buffer 0040. The sialidase may comprise neuraminidase 1 (200 mM Tris-HCl pH 8.4, 1.5 M NaCl, 25 mM CaCl) for (NEU1), neuraminidase 2 (NEU2), neuraminidase 3 24 hours; (NEU3), neuraminidase 4 (NEU4) and O-sialidase bioengi 0031 FIG. 16A shows the result of quantitative analysis neered from human genome, one of the foregoing enzymes for lipid constituents of L5 and L5 treated with ASAH2 for obtained through gene transformation, expression and puri 24 hours mass spectrometry; fication, sialidase from a virus or bacterium (alias, acetyl 0032 FIG. 16B shows ceramide contents of L5 and L5 neuraminyl hydrolase), etc., but it is not limited thereto. treated with ASAH2 in the presence of a buffer (200 mM 0041 Examples of the glycosidase may comprise alpha Tris-HCl pH 8.4, 1.5 M NaCl, 25 mM CaCl2) for 2 hours: and beta-glucosidase bioengineered from human or animal 0033 FIGS. 17A, 17B-1, and 17B-2 show that immobi genome, maltase-glucoamylase and Sucrase-isomaltase, one lized ASAH2 is capable of effectively eliminating ceramide of the foregoing enzymes obtained through gene transfor and increasing a product, sphingosine; One of the most mation, expression and purification, N-glycosidase F (PN common ceramides of L5 is Cer (d18:0/25:0), and after it Gase F) and glucosidase from a virus, a bacterium or other has been catalyzed by ASAH2, a product, Sphingosine, is organism, etc., but they are not limited thereto. produced. The experimental results show that immobilized 0042. Furthermore, the second enzyme may be cerami ASAH2 (ITRI-EC-AS-01) is capable of reducing Cer (d18: dase. 0/25:0) contained by the LDL sample and increasing the 0043. The ceramidase may comprise N-acylsphingosine product sphingosine. amidohydrolase 1 (ASAH1), N-acylsphingosine amidohy drolase 2 (ASAH2), N-acylsphingosine amidohydrolase 2B DETAILED DESCRIPTION (ASAH2B), N-acylsphingosine amidohydrolase 2C 0034. In the following detailed description, for purposes (ASAH2C), N-acylethanolamine acid amidase, alkaline of explanation, numerous specific details are set forth in ceramidase 1, alkaline ceramidase 2, alkaline ceramidase 3. order to provide a thorough understanding of the disclosed but it is not limited thereto. embodiments. It will be apparent, however, that one or more 0044. In one embodiment, the enzyme composition in the embodiments may be practiced without these specific biochemistry reactive material of the present disclosure is details. In other instances, well-known structures and the first enzyme. In this embodiment, the first enzyme devices are shown schematically in order to simplify the mentioned above may be sialidase, but it is not limited drawing. thereto. 0035. In one embodiment of the present disclosure, the 0045. In another embodiment, the enzyme composition in present disclosure provides a biochemistry reactive material the biochemistry reactive material of the present disclosure which is capable of eliminating electronegative low-density is the second enzyme. In this embodiment, the second lipoprotein (electronegative LDL). Examples of the electro enzyme mentioned above may be N-acylsphingosine ami negative low-density lipoprotein mentioned above may dohydrolase 2, but it is not limited thereto. US 2017/00445 16 A1 Feb. 16, 2017

0046. In another embodiment, the enzyme composition in gene transformation, expression and purification, N-glycosi the biochemistry reactive material of the present disclosure dase F (PNGase F) and glucosidase from a virus, a bacte is a combination of the first enzyme and the second enzyme. rium or other organism, etc. In this embodiment, the first enzyme mentioned above may 0058. In addition, the second enzyme may be ceramidase. be sialidase, but it is not limited thereto, and the second The ceramidase may comprise, but is not limited to, N-acyl enzyme mentioned above may be N-acylsphingosine ami sphingosine amidohydrolase 1 (ASAH1), N-acylsphin dohydrolase 2, but it is not limited thereto. gosine amidohydrolase 2 (ASAH2), N-acylsphingosine ami 0047. In another embodiment of the present disclosure, dohydrolase 2B (ASAH2B), N-acylsphingosine the present disclosure provides a biochemistry reactive amidohydrolase 2C (ASAH2C), N-acylethanolamine acid device, and the device can be used for eliminating electro amidase, alkaline ceramidase 1, alkaline ceramidase 2, alka negative low-density lipoprotein in a liquid sample. line ceramidase 3. 0048 Examples of the foregoing liquid sample may 0059. In one embodiment, the enzyme composition in the comprise an aqueous solution, a buffer, blood, plasma, etc., biochemistry reactive material 101 mentioned above is the but they are not limited thereto. first enzyme. In this embodiment, the first enzyme men 0049. Examples of the foregoing electronegative low tioned above may be sialidase, but it is not limited thereto. density lipoprotein may comprise electronegative low-den 0060. In another embodiment, the enzyme composition in sity lipoprotein L1, L2, L3, L4 and/or L5, etc., but they are the biochemistry reactive material 101 mentioned above is not limited thereto. In one embodiment, the electronegative the second enzyme. In this embodiment, the second enzyme low-density lipoprotein mentioned above may be electro mentioned above may be N-acylsphingosine amidohydro negative low-density lipoprotein L5. lase 2, but it is not limited thereto. 0050. A cross-sectional view of a structure of the bio 0061. In another embodiment, the enzyme composition in chemistry reactive device of the present disclosure is shown the biochemistry reactive material 101 mentioned above is a in FIG. 1. combination of the first enzyme and the second enzyme. In 0051 Refer to FIG. 1A. The preceding biochemistry this embodiment, the first enzyme mentioned above may be reactive device of the present disclosure 100 may comprise sialidase, but it is not limited thereto, and the second enzyme a biochemistry reactive material 101 and a container 103 for mentioned above may be N-acylsphingosine amidohydro containing the biochemistry reactive material 101. The con lase 2, but it is not limited thereto. tainer 103 has at least one inlet 105 and at least one outlet 0062. Furthermore, a material of the container 103 of the 107. The foregoing liquid sample enters into the biochem biochemistry reactive device 100 of the present disclosure istry reactive device 100 from the inlet 105, and flows may comprise glass, acrylic, polypropylene, polyethylene, through the biochemistry reactive material 101 to react with stainless steel, titanium alloy, etc., but it is not limited the biochemistry reactive material 101, and then flows out thereto. In one embodiment, a material of the container 103 through the outlet 107. of the biochemistry reactive device 100 of the present 0052. The biochemistry reactive material 101 may com disclosure may be polypropylene. In addition, a shape of the prise, but is not limited to a substrate and an enzyme container 103 has no particular limitation, and in one composition, wherein the enzyme composition is immobi embodiment, the container 103 is a hollow column. lized on the substrate. 0063. In one embodiment, as shown in FIG. 1B, the 0053. The substrate mentioned above may comprise, but biochemistry reactive device 100 of the present disclosure is not limited to, silica gel, cellulose, diethylaminoethyl may further comprise a filtering material 109 configured in cellulose, chitosan, polystyrene, polysulfone, polyetherSul the container 103 behind the at least one inlet 105 and at fone, acrylate resin, polysaccharide, etc. The Substrate may least one outlet 107. Moreover, the pore size of the filtering have a particle structure or a hollow-tube structure, etc., but material mentioned above is smaller than the biochemistry it is not limited thereto. reactive material 101 to prevent the biochemistry reactive 0054 The enzyme composition may comprise, but is not material 101 leaking from the at least one inlet 105 and/or limited to, a first enzyme for eliminating a glycan residue of least one outlet 107, but it can allow the liquid sample to an electronegative LDL, a second enzyme for eliminating pass through. The filtering material 109 mentioned above ceramide carried by an electronegative LDL or a combina comprises filter paper, glass, acrylic, polypropylene, poly tion thereof. Source organisms of the first enzyme and the ethylene, etc., but it is not limited thereto. In this embodi second enzyme mentioned above have no particular limita ment, the substrate of the biochemistry reactive material 101 tion. In one embodiment, the first enzyme and the second may have a particle structure or a hollow-tube structure. In enzyme are human. one specific embodiment, the substrate of the biochemistry 0055. The preceding first enzyme may be sialidase or reactive material 101 has a particle structure, and in this glycosidase. specific embodiment, the substrate of the biochemistry reac 0056. The sialidase may comprise, but is not limited to, tive material 101 may be a cellulose bead or a chitosan bead, neuraminidase 1 (NEU1), neuraminidase 2 (NEU2), but it is not limited thereto. neuraminidase 3 (NEU3), neuraminidase 4 (NEU4) and 0064. When the substrate of the biochemistry reactive 0-Sialidase bioengineered from human genome, one of the material 101 is a hollow-tube structure, polyurethane (PU) foregoing enzymes obtained through gene transformation, can be used to package the device without using the filtering expression and purification, sialidase from a virus, a bacte material 109. rium or other organism, etc. 0065. In one embodiment, the container 103 may be a 0057 The glycosidase may comprise, but is not limited hollow column, and two ends of the hollow column of the to, alpha- and beta-glucosidase bioengineered from human container 103 have a first inlet 105 of the inlet mentioned or animal genome, maltase-glucoamylase and Sucrase-iso above and a first outlet 107 of the outlet mentioned above, mahase, one of the foregoing enzymes obtained through respectively. In this embodiment, the substrate of the bio US 2017/00445 16 A1 Feb. 16, 2017 chemistry reactive material 101 may have a particle struc genome, maltase-glucoamylase and Sucrase-isomaltase, one ture or a hollow-tube structure. of the foregoing enzymes obtained through gene transfor 0.066. In another embodiment, as shown in FIG. 1C, the mation, expression and purification, N-glycosidase F (PN container 103 may be a hollow column, and two ends of the Gase F) and glucosidase from a virus, a bacterium or other hollow column of the container 103 have a first inlet 105 of organism, etc., but they are not limited thereto. the inlet mentioned above and a first outlet 107 of the at 0075. Furthermore, the second enzyme mentioned above least outlet mentioned above, respectively, and a second may be ceramidase. inlet 105 of the inlet and a second outlet 107 of the outlet 0076. The ceramidase may comprise N-acylsphingosine are located at a side wall of the hollow column. In this amidohydrolase 1 (ASAH1), N-acylsphingosine amidohy embodiment, the liquid sample can enter into the biochem drolase 2 (ASAH2), N-acylsphingosine amidohydrolase 2B istry reactive device 100 from the first inlet 105 of the (ASAH2B), N-acylsphingosine amidohydrolase 2C container 103, and flows through the biochemistry reactive (ASAH2C), N-acylethanolamine acid amidase, alkaline material 101, and then flows out through the first outlet 107. ceramidase 1, alkaline ceramidase 2, alkaline ceramidase 3. Moreover, a second liquid which can be water, a dialysis but it is not limited thereto. Solution or a salt-containing aqueous Solution enters into the 0077. In one embodiment, the enzyme composition used biochemistry reactive device 100 from the first inlet 105. in the method for ex vivo treating blood or plasma of the and flows through the biochemistry reactive material 101, present disclosure is the first enzyme. In this embodiment, and then flows out through the first outlet 107. The second the first enzyme mentioned above may be sialidase, but it is liquid can bring a by-product out after the reaction or not limited thereto. dialysis. 0078. In another embodiment, the enzyme composition 0067. In this embodiment, the substrate of the biochem used in the method for ex vivo treating blood or plasma of istry reactive material 101 may have a particle structure or the present disclosure is the second enzyme. In this embodi a hollow-tube structure. In one specific embodiment, the ment, the second enzyme mentioned above may be N-acyl substrate of the biochemistry reactive material 101 has a sphingosine amidohydrolase 2, but it is not limited thereto. hollow-tube structure, and in this specific embodiment, the 0079. In another embodiment, the enzyme composition substrate of the biochemistry reactive material 101 may be used in the method for ex vivo treating blood or plasma of cellulose hollow fiber, but it is not limited thereto. the present disclosure is a combination of the first enzyme 0068. In another embodiment of the present disclosure, and the second enzyme. In this embodiment, the first the present disclosure provides a method for ex vivo treating enzyme mentioned above may be sialidase, but it is not blood or plasma. By the method for ex vivo treating blood limited thereto, and the second enzyme mentioned above or plasma, an electronegative low-density lipoprotein in may be N-acylsphingosine amidohydrolase 2, but it is not blood or plasma can be eliminated. The foregoing electro limited thereto. negative low-density lipoprotein may comprise, but is not 0080 Furthermore, in one embodiment, the enzyme com limited to, electronegative low-density lipoprotein L1, L2. position used in the method for ex vivo treating blood or L3, L4 and/or L5, etc. In one embodiment, the electronega plasma of the present disclosure can be immobilized on a tive low-density lipoprotein mentioned above is electro Substrate. Examples of the Substrate may comprise silica gel. negative low-density lipoprotein L5. cellulose, diethylaminoethyl cellulose, chitosan, polysty 0069. The method for ex vivo treating blood or plasma rene, polysulfone, polyetherSulfone, resin, polysaccharide, may comprise the following steps, but it is not limited but they are not limited thereto. Moreover, the substrate may thereto. have a particle structure or a hollow-tube structure. 0070 First, a blood or plasma ex vivo contacts with an I0081. In the method for ex vivo treating blood or plasma enzyme composition to react the enzyme composition with of the present disclosure, time for ex vivo contacting the the blood or plasma, wherein the enzyme composition is blood or plasma with the enzyme composition may be about capable of eliminating electronegative low-density lipopro 0.25-8 hours. In one embodiment, time for ex vivo contact tein. ing the blood or plasma with the enzyme composition may 0071. The preceding enzyme composition may comprise be about 2 hours. a first enzyme for eliminating a glycan residue of an elec I0082 Furthermore, in the method for ex vivo treating tronegative LDL, a second enzyme for eliminating ceramide blood or plasma of the present disclosure, temperature for ex carried by an electronegative LDL or a combination thereof, Vivo contacting the blood or plasma with the enzyme but it is not limited thereto. Source organisms of the first composition may be about 4-40° C. In one embodiment, enzyme and the second enzyme mentioned above have no temperature for ex vivo contacting the blood or plasma with particular limitation. In one embodiment, the first enzyme the enzyme composition may be about 37° C. and the second enzyme are human. I0083. In addition, in the method for ex vivo treating 0072 The preceding first enzyme may be sialidase or blood or plasma of the present disclosure, the blood or glycosidase. plasma may ex vivo contact with the enzyme composition at 0073. The sialidase may comprise neuraminidase 1 about pH 5-10. In one embodiment, the blood or plasma may (NEU1), neuraminidase 2 (NEU2), neuraminidase 3 ex vivo contact with the enzyme composition at about pH (NEU3), neuraminidase 4 (NEU4) and O-sialidase from a 74. human, or one of the foregoing enzymes obtained through I0084. Afterward, contact between the blood or plasma gene transformation, expression and purification, sialidase and the enzyme composition is terminated to terminate the from a virus, a bacterium or other organisms, etc., but it is reaction of the enzyme composition with the blood or not limited thereto. plasma. 0074 Examples of the glycosidase may comprise alpha I0085. A manner for terminating the contact between the and beta-glucosidase bioengineered from human animal blood or plasma and the enzyme composition has no par US 2017/00445 16 A1 Feb. 16, 2017 ticular limitation, for example, for terminating the contact on the injection volume. The respective fractions were between the blood or plasma and the enzyme composition, concentrated with Centriprep R filters (YM-30; EMD Mil the blood or plasma can be separated from the enzyme lipore Corp., Billerica, Mass.), dialyzed against buffer A (20 composition, or the enzyme composition can be deactivated, M, pH8.0, 0.5 M EDTA) for 24 hours (3 days) and sterilized etc. by passing through 0.22-um filters (Sartorius; Minisart(R). The isolated fractions were quantified at their protein con EXAMPLES centrations by the Lowry method and then stored at 4°C. (0096 2. Screening of NEU2 or NEU4 Example 1 (0097 (1) Transformation (Gene Cloning for pCMV6 Vector with NEU2 and NEU4 Genes) A. Methods (0098 NEU2 (neuraminidase 2) and NEU4 (neuramini I0086 1. Obtainment of Electronegative Low-Density dase 4) were purchased from Origene, RC2 19858 and Lipoprotein (Electronegative LDL) RC203948. Genes were amplified by ECOSTM 101 DH5O. 0087 (1) Purifications for Electronegative Low-Density Competent Cells (Yeastern, FYE608) according to the Lipoprotein manufacturer's directions. 0088 Blood samples to be used for LDL isolation were (0099. In short, 1 vial of competent cells with 5 uL obtained from subjects. After the initial screening, blood plasmid was Vortexed for 1 second and then incubated on ice samples were removed from the Subjects with precaution for 5 minutes. After 45 second heat-shock at 42° C., the against coagulation and ex vivo oxidation. The plasma was mixture was plate on LB agar with Kanamycin. treated with Complete Protease Inhibitor Cocktail (Roche: 0100 Colonies were checked with PCR by VP1.5 and Cat. No. 05056489001: 1 tablet/100 mL) to prevent protein XL39 primers. Procedures of the PCR comprises: 95°C. for degradation. 1 minute for pre-PCR denaturation: 2 cycles of 95°C. for 10 0089 Lipoprotein Preparation from a Human seconds, 62° C. for 20 seconds, 72° C. for 4 minutes; 2 0090. The plasma was overlaid with 2 mL Milli-Q water cycles of 95° C. for 10 seconds, 60° C. for 20 seconds, 72° and spun at 20,000 rpm for 2 hours. The upper white fraction C. for 4 minutes; 2 cycles of 95° C. for 10 seconds, 58° C. and chylomicrons were removed, and the remnant layer for 20 seconds, 72° C. for 4 minutes; 15 cycles of 95°C. for which contains VLDL, IDL, LDL and HDL was saved for a 10 seconds, 56°C. for 20 seconds, 72° C. for 4 minutes; 72 series of isolation steps. C. for 10 minutes for post-PCR incubation and holding on 0091 To progressively separate VLDL (d=0.93-1.006), 4° C. IDL (d=1.006-1.019), LDL (1.019-1,063 g/dL) and HDL 0101 (2) Plasmid Extraction (1.063-1.210 g/dL) from one another, the remnant sample 0102. After confirming the insertion of transformed colo was sequentially adjusted to d=1.006, d=1.019, d=1.063, nies, transformed cells were plate-out into 5 ml LB broth d=1.210, respectively, by adding potassium bromide, and with 25 mg/ml kanamycin, and then incubated at 37° C. then the remnant samples sequentially adjusted to d=1.006, overnight. d=1.019 and d=1063 were centrifuged at 45,000 rpm for 24 0103 Plasmid DNA was extracted according to the pro hours at 4°C., and the remnant sample sequentially adjusted tocol of Plasmid Miniprep Plus Purification Kit (GeneMark, to d=1.210 was centrifuged at 45,000 rpm for 48 hours at 4 DP01P). In short, the bacteria were centrifuged for 1 minute C. After centrifugation at each isolation step. IDL was at 14,000xg, and the media was removed. The pellet was discarded while VLDL, LDL and HDL were collected. re-suspended in 200 uL Solution I by pipetting, then 200 uL Isolated VLDL, LDL and HDL samples were treated with 5 Solution II was added therein and mixed by inverting the mM EDTA and nitrogen to avoid ex vivo oxidation. After tube. 200 uL Solution III was added to the tube and mixed that, VLDL, LDL and HDL samples were dialyzed against by inverting the tube 5 times. The lysate was centrifuged at buffer A (20 M, pH 8.0, 0.5 M EDTA) for 24 hours (x3 top speed for 5 minutes and a compact white pellet formed times) to remove excessive potassium bromide, and were along the side of the tube. The spin column was inserted into filtrated through 0.22-um filter (Sartorius; MinisartR) to a collection tube, and the clear lysate was moved to spin sterilize the samples. column and spun at top speed for 1 minute. The flow 0092 (2) LDL Subfractions through was discarded, and 500 uL Endotoxin Removal 0093. Approximately 30 mg of LDL material was Wash Solution was loaded to the spin column and kept for injected onto a UnoCR12 anion-exchange column (BioRad) 2 minutes to equilibrate the membrane, then spun at top by using the AKTA fast-protein liquid chromatography speed for 1 minute. The filtrate was discarded, and 700 LL (FPLC) pump (GE Healthcare Life Sciences, Pittsburgh, Washing Solution was added to the spin column and spun at Pa.). LDL was eluted according to electronegativity by the top speed for 1 minute, and then this step was repeated. The use of a multistep gradient of buffer B (1 mol/L NaCl in filtrate was discarded and the spin column was centrifuged buffer A) at a flow rate of 2 mL/minute. In short, samples for 5 minutes at top speed to remove residual traces of were equilibrated with buffer A for 10 minutes, followed by ethanol. The spin column was transferred into a new tube being linearly increased to 15% buffer B in 10 minutes and 35 uL HO was added to the spin column and kept for (fraction 1), linearly increased to 20% buffer B in 30 minutes 1-2 minutes and the tube was centrifuged at top speed for 2 (fraction 2, 3), kept at 20% buffer B for 10 minutes (fraction minutes to elute the DNA. The DNA quantified by 4) and linearly increased to 100% buffer B in 20 minutes microplate spectrophotometer (Epoch, BioTek). (fraction 5). Lastly, the effluents were monitored at 280 nm. 0104 (3) Transfection on HEK Cells and Protein Purifi 0094 (3) Purification of Fractionated LDL cation 0095 Based on the gradient profile, each of the LDL 0105. One day before transfection, 1.25*10 HEK293T fractions were pooled. The volume of each subfraction was cells were placed in 500 uL DMEM medium in 24-well constant. Dilution of LDL during chromatography depended plate. For each well of cells to be transfected, 1 lug of DNA US 2017/00445 16 A1 Feb. 16, 2017

was diluted in 100 uL serum-free medium, and 1.5 LL of 0.115. In brief, total proteins isolated from each LDL Lipofectamine 2000 Transfection Reagent (Invitrogen) was Subfraction were first digested with trypsin, and the resulting add thereto and mixed gently and incubated for 30 minutes tryptic peptides were chromatographically separated on a at room temperature. After incubation, the complex was Nano-Acquity separations module (Waters Corporation, added to each well containing cells and mixed gently. The MA, USA) incorporating a 50 fmol-on-column tryptic digest cells were incubated at 37° C. in a CO, incubator for 20 of yeast alcohol dehydrogenase as the internally spiked hours. The transfected cells were lysed by RIPA which protein quantification standard. Peptide elution will be containing protease inhibitor to prepare to purify the pro executed through a 75 imx25 cm BEH C-18 column under teins. gradient conditions at a flow rate of 300 nL/minute over 30 0106. In short, 80 uL ANTI-FLAG M2 Magnetic Beads minutes at 35° C. The mobile phase was composed of (Sigma-Aldrich) were equilibrated for one-well cell lysate acetonitrile as the organic modifier and formic acid (0.1% purification. After protein-resin binding at 4°C. overnight, V/v) for molecule protonation. Mass spectrometry was per the bound FLAG fusion protein was eluted by competitive formed on a XevoR G2-XS QTof instrument equipped with elution with 150 lug/ml 3x FLAG peptide for 2 times, the a nano-electrospray ionization interface and operated in the eluate was collected, and the protein checked by western data-independent collection mode (MSE). Parallel ion frag blot. mentation was programmed to Switch between low (4 eV) 0107 3. Efficacy Test for NEU2 or NEU4 and high (15-45 eV) energies in the collision cell, and data 0108 (1) Protein Quantification was collected from 50 to 2000 m/z utilizing glu-fibrinopep 0109 Pierce BCA Protein Assay Kit (Thermo) was used tide B as the separate data channel lock mass calibrant. Data for protein quantification according to the manufacturers was processed with ProteinLynx GlobalServer v2.4 (Wa directions. ters). Deisotoped results were searched for protein associa 0110. In short, 25 uL serial diluted BSA standard and 5 tion from the Uniprot (www.uniprot.org) human protein LL sample in 20 uIl sample diluent were pipetted into a database. 96-well microplate. To prepare BCA working reagent, 50 0116 4. Screening of ASAH2 parts of BCA Reagent A was mixed with 1 part of BCA 0117 (1) Transformation (Gene Cloning for pCMV6 Reagent B and placed on ice until use. 200 uL of the BCA Vector with ASAH2 Genes): working reagent was added to each well and mixed thor 0118 ASAH2 (N-acylsphingosine amidohydrolase 2) oughly, and the plate was covered and incubated at 37° C. was purchased from Origene, RC203706. Genes were for 30 minutes. The absorbance at 562 nm was measured by amplified by ECOSTM 101 DH5C. Competent Cells (Yeast spectrophotometer (Epoch, BioTek). ern, FYE608) according to the manufacturer's directions. 0111 (2) Apoptosis Measurements 0119. In short, 1 vial of competent cells with 5 L 0112 Endothelial cells were used after 3 or 4 passages plasmid was Vortexed for 1 second and then incubated on ice and maintained in DMEM (InvitrogenTM, Thermo Fisher for 5 minutes. After 45 second heat-shock at 42° C., the Scientific) containing 10% FBS. During treatment, FBS was mixture was plate on LB agar with Kanamycin. reduced to 5% in DMEM. 1x10" cells were seeded in I0120 Colonies were checked with PCR by VP1.5 and 96-well plate for 24 hours for subconfluent cultures, and the XL39 primers. Procedures of the PCR comprise: 95°C. for cultured cells were exposed to PBS (lipoprotein-free, nega 1 minute for pre-PCR denaturation: 2 cycles of 95°C. for 10 tive control) or graded (25, 50, and 100 lug/mL) LDL seconds, 62° C. for 20 seconds, 72° C. for 4 minutes; 2 Subfractions, unfractionated normolipidemic LDL, and cycles of 95° C. for 10 seconds, 60° C. for 20 seconds, 72° LDL/L1/L5 incubated with sialidase for 24 hours. Apoptosis C. for 4 minutes; 2 cycles of 95° C. for 10 seconds, 58° C. was assessed with visualization by a Zeiss Axiovert 200 for 20 seconds, 72° C. for 4 minutes; 15 cycles of 95°C. for fluorescence microscope and filters to capture digital images 10 seconds, 56°C. for 20 seconds, 72° C. for 4 minutes; 72 based on Hoechst 33342, propidium iodide (red), and cal C. for 10 minutes for post-PCR incubation and holding on cein AM (green) staining of nuclear, apoptotic DNA mem 40 C. brane integrity and cytoplasm respectively according to the I0121 (2) Plasmid Extraction protocol of the manufacturer (InvitrogenTM, Thermo Fisher I0122. After confirming the insertion of transformed colo Scientific). nies, transformed cells were plate-out into 5 ml LB broth 0113 (3) LC/MS Analysis for Protein Composition with 25 mg/ml kanamycin, and then incubated at 37° C. 0114 LDL subfractions were quantified the protein con overnight. tents by use of quantitative proteomics techniques utilizing I0123 Plasmid DNA was extracted according to the pro serially coupled liquid chromatography data-independent tocol of Plasmid Miniprep Plus Purification Kit (GeneMark, parallel-fragmentation mass spectrometry (LC/MS). Such DP01P). In short, the bacteria were centrifuged for 1 minute analysis has been shown to be highly quantitative with at 14,000x g, and the media was removed. The pellet was respect to both relative and/or absolute (when incorporating re-suspended in 200 uL Solution I by pipetting, then 200 uL spiked internal peptide standards in the data collection/ Solution II was added therein and mixed by inverting the analysis procedures) protein abundance in complex protein tube. 200 uL Solution III was added to the tube and mixed mixtures. Quantitative analysis was performed essentially as by inverting the tube 5 times. The lysate was centrifuged at previously described (PMCID: PMC3816395; Pure Appl top speed for 5 minutes and a compact white pellet formed Chem. 2011; 83(9): 10.1351/PAC-CON-10-12-07. Chemi along the side of the tube. The spin column was inserted into cal composition—oriented receptor selectivity of L5, a natu a collection tube, and the clear lysate was removed to spin rally occurring atherogenic low-density lipoprotein), except column and spun at top speed for 1 minute. The flow on a Waters nanoACQUITY UPLC System and Xevo(R) through was discarded, and 500 uL Endotoxin Removal G2-XS QTof mass spectrometer (Waters Corporation, MA, Wash Solution was loaded to the spin column and kept for USA). 2 minutes to equilibrate the membrane, then spun at top US 2017/00445 16 A1 Feb. 16, 2017

speed for 1 minute. The filtrate was discarded, and 700 LL 0.136. In brief, lipids were chromatographically separated Washing Solution was added to the spin column and spun at on a ACQUITY UPLC System (Waters Corporation, MA, top speed for 1 minute, and then this step was repeated. The USA) incorporating a CSHTM 1.7 um, 2.1 mmx10 cm C-18 filtrate was discarded and the spin column was centrifuged column under gradient conditions at a flow rate of 400 for 5 minutes at top speed to remove residual traces of uL/minute over 18 minutes at 55° C. The mobile phase A ethanol. The spin column was transferred into a new tube will be composed of 10 mM NH4HCO, in ACN/HO and 35 uL H2O was added to the spin column and kept for (60/40) and 0.1% formic acid (0.1% v/v), mobile phase B 1-2 minutes and the tube was centrifuged at top speed for 2 will be composed of 10 mM NHHCO, in IPA/ACN (90/10) minutes to elute the DNA. The DNA quantified by and 0.1% formic acid (0.1% V/v) for molecule protonation. microplate spectrophotometer (Epoch, BioTek). Mass spectrometry was performed on a XevoR G2-XS QT (0.124 (3) Transfection on HEK Cells and Protein Purifi of instrument equipped with an electrospray ionization inter cation face and operated in the data-independent collection mode 0125 One day before transfection, 1.25*10 HEK293T (MSE). Parallel ion fragmentation was programmed to cells were placed in 500 uL DMEM medium in 24-well switch between low (4 eV) and high (35-55 eV) energies in plate. For each well of cells to be transfected, 1 lug of DNA the collision cell, and data was collected from 50 to 1600 was diluted in 100 uL serum-free medium, and 1.5 ul, of m/Z utilizing leucin as the separate data channel lock mass Lipofectamine 2000 Transfection Reagent (Invitrogen) was calibrant. Data was processed with MarkerLynx (Waters). add thereto and mixed gently and incubated for 30 minutes at room temperature. After incubation, the complex was B. Results added to each well containing cells and mixed gently. The cells were incubated at 37° C. in a CO, incubator for 20 0.137 1. Transformation hours. The transfected cells were lysed by RIPA which 0.138 (1) NEU2 containing protease inhibitor to prepare to purify the pro 0.139 Transformation result for NEU2 is shown in FIG. teins. 2 0126. In short, 80 uL ANTI-FLAG M2 Magnetic Beads 0140. According to FIG. 2, it is known that NEU2 (Sigma-Aldrich) were equilibrated for one-well cell lysate transformations for colonies 3, 5 and 6 (see lane 3, 5 and 6. purification. respectively) were successful. Therefore, colonies 3, 5 and 6 0127. After protein-resin binding at 4°C. overnight, the were selected to be amplified, and plasmid of NEU2 was bound FLAG fusion protein was eluted by competitive stocked. elution with 150 ug/ml 3x FLAG peptide for 2 times, the eluate was collected, and the protein checked by western 0141 (2) ASAH2 blot. 0.142 Transformation result for ASAH2 is shown in FIG. 0128 5. Efficacy Test for ASAH2 3 0129 (1) Protein Quantification 0.143 Colony 7 was selected to be amplified, and plasmid 0130 Pierce BCA Protein Assay Kit (Thermo) was used of ASAH2 was stocked. for protein quantification according to the manufacturers 0144. 2. Transfection directions. 0145 Transfection of NEU4/ASAH2 genes was con 0131. In short, 25 uL serial diluted BSA standard and 5 firmed by western blot, and the result is shown in FIG. 4. LL sample in 20 uIl sample diluent were pipetted into a 0146 Conditions for the gene transfection are shown in 96-well microplate. To prepare BCA working reagent, 50 the following: parts of BCA Reagent A was mixed with 1 part of BCA 0147 HEK293T 1.25x10 cells in 24 well Reagent B and placed on ice until use. 200 uL of the BCA 0148 Plasmid: NEU4 and ASAH2 working reagent was added to each well and mixed thor 0149 DNA amount: 1 ug oughly, and the plate was covered and incubated at 37° C. (O150 Transfected by Lipofectamine for 30 minutes. The absorbance at 562 nm was measured by spectrophotometer (Epoch, BioTek). 0151 SDS-PAGE: using 5 ul sample (0132 (2) Lipid Extraction 0152 Primary antibody: anti-DDK (1:2000) 0.133 30 ug LDL/L1/L5 were incubated with 5 ug 0153. 3. Protein Purification ASAH2 in ASAH2 buffer (200 mM Tris-HCl at pH 8.4, 1.5 0154 (1) NEU2 Purification M. NaCl, 25 mM CaCl) at 37° C. After 2 or 24 hours (O155 The result for NEU2 purification is shown in FIG. incubation, Samples were transferred to a glass tube. 1 mL 5. FIG. 5 shows that NEU2 was indeed purified. The amino H2O, 2.5 mL methanol and 1.25 mL CHCl were added to acid sequence of NEU2 is shown as SEQ ID NO. 1. samples, and vortexed for 15 seconds. Then, additional 0.9 0156 (2) ASAH2 Purification mL HO and 1.25 mL CHCl were applied to samples, (O157. The result for ASAH2 purification is shown in FIG. vortexed for 15 seconds, and centrifuged at 3000 rpm for 10 6. FIG. 6 shows that ASAH2 was indeed purified (extract 1 minutes. Bottom layer organic solvents were transferred to a 2.0 mL. glass tube using a glass syringe. Each sample was and extract 2 are proteins obtained from different batches). flushed with nitrogen until dry pallets, and dissolved with The amino acid sequence of ASAH2 is shown as SEQ ID 0.25 mL sample solution (isopropanol/acetonitrile/HO-2: NO. 2. 1:1). 0134 (3) LC/MS Analysis for Lipid Composition Example 2 0135 Total lipids, phospholipids, neutral lipids and free fatty acid from each subfractions of LDL were quantified the Enzyme Immobilization lipid contents by use of liquid chromatography data-inde 0158 Method 1 pendent parallel-fragmentation mass spectrometry (LC/ 0159 0.4454g heat-activated silica gel was placed in 7 MS). Quantitative analysis was performed essentially as mL CHCl, and APTS was added therein by a weight of 1/5 previously described. weight of heat-activated silica gel to form a mixture. After US 2017/00445 16 A1 Feb. 16, 2017

stirring at room temperature for 24 hours, the mixture was suspension and stirred for 30 minutes and then the solid filtered. The obtained solid was drained in vacuum at 50° C. therein was filtered out. The solid was washed with dioxane, After the solid was drained, 5% glutaraldehyde (phosphate water and acetone in order and dried under reduced pressure Buffer, pH=8, IXTBS) was added to the solid, and stirred for to forman activated solid support. After that, NEU2 1/10000 21 hours to form a solution. The solid in the solution was (wt %) was added to the activated solid support and stirred filtered out and washed with water and a solid substance was for 18 hours. Afterward, the activated solid support was obtained. NEU2 (1/100-10000 wt %) was added to the solid filtered out and washed in water to obtain an enzyme substance and diluted with phosphate buffer, 1xTBS pH=8 immobilized product. to a volume of 2 mL, and reacted with the solid substance (0170 Method 7 at room temperature for 24 hours. Finally, the solid sub (0171 0.5 g chitosan beads were added to 10 mL 0.5% stance was filtered out and washed with phosphate buffer glutaraldehyde, and stirred at room temperature for 1 hour, (pH-8) and an enzyme immobilized product (ITRI-Siw-Nu and then washed with water, continuously and thoroughly to 01) was obtained. form activated beads. After that, the activated beads were (0160 Method 2 reacted with NEU2 1/3500 (wt %) at room temperature for 0161 3-glycidoxypropyltrimethoxysilane was added to 2 hours, filtered out and then washed with deionized water heat-activated silica gel in toluene by a weight of 1/5 weight to obtain an enzyme immobilized product. of heat-activated silica gel, refluxed for 20 hours, and then (0172 Method 8 filtered. The obtained solid was washed with acetone and 0173 1 g cellulose hollow fiber, as per the procedures in then drained in vacuum. NEU2 (1/100-10000 wt %) was Method 4, was activated by APTS and glutaraldehyde, and added to the solid and stirred in phosphate buffer for 2 hours then reacted with NEU23/10000 (wt %) in phosphate buffer and 15 minutes, and then the solid was filtered out. The solid (pH=8), stirred overnight, and washed with a phosphate was washed with deionized water and a buffer (pH 8) to buffer (pH-8) to obtain an enzyme immobilized product. obtain an enzyme immobilized product. 0.174 Method 9 (0162 Method 3 0.175 1 g cellulose hollow fiber, as per the procedures in 0163. 1 g cellulose beads in 15 mL water were adjusted Method 3, was activated by cyanogen bromide, and then to about pH 11 by a NaOH solution, and then 1 g cyanogen reacted with NEU2 in phosphate buffer (pH=8), stirred bromide was added therein at room temperature. After about overnight, washed with a phosphate buffer (pH-8) to obtain 30 minutes, the cellulose beads were washed in deionized an enzyme immobilized product. water and a phosphate buffer (pH 8) in order. NEU2 in a (0176 Method 10 phosphate buffer was added to the cellulose beads by a 0177 ECR-8204F epoxy-acrylate resin was washed in weight ratio of 1/600, and stirred overnight. After that, the deionized water, reacted with ASAH2 1/10000 (wt %), cellulose beads were washed in a phosphate buffer (pH 8) to adjusted to a volume of 2 mL with a 0.2 M sodium phosphate obtain an enzyme immobilized product. buffer, and then stirred for 24 hours. After that, epoxy (0164 Method 4 acrylate resin was filtered out and washed in deionized water 0.165 0.5 g cellulose beads in 1.5 mL water were refluxed and 2M phosphate buffer (pH-8) to obtain about 52 mg of in 10 mL toluene, and then cellulose beads were filtered out enzyme immobilized product (ITRI-EC-AS-01). and washed with acetone and a phosphate buffer (pH 8). 0.178 Method 11 After that, the cellulose beads were added to 5% (w/v) 0179 Iontosorb MT200 cellulose beads were washed in glutaraldehyde (phosphate buffer, pH 8) and stirred at room deionized water. Next, the cellulose beads were washed with temperature for 21 hours. Afterward, the cellulose beads 3:7 water/dioxane, 7:3 water/dioxane, 100% dioxane in were filtered out, and washed in a phosphate buffer (pH 8) order. After that, dioxane was added to the cellulose beads, to obtain glutaraldehyde-activated-cellulose beads. NEU2 in and CDI was added therein by a weight of 1/3 weight of a phosphate buffer was added to the cellulose beads by a cellulose beads, and stirred for about 0.5-1 hour to form a weight ratio of 2/1000, and stirred overnight. After that, the solution. The dioxane in the solution was removed under cellulose beads were washed in a phosphate buffer (pH 8) to reduced pressure, and then NEU 2 was immediately added obtain an enzyme immobilized product. therein and stirred for about 2 hours and 15 minutes. After (0166 Method 5 the reaction, the cellulose beads in the solution were filtered (0167 Hypogel(R) 200NH, were added to 5% (w/v) glut out and were washed in a buffer (pH=6.5) to obtain a wet araldehyde (phosphate buffer, pH 8) and stirred at room product about 0.2 g (ITRI-CD-01). temperature for 21 hours. Afterward, the solid substance was 0180 Method 12 filtered out, and washed with a phosphate buffer (pH 8) to 0181 0.5 lug NEU2 was added to 2% w/v alginate aque obtain glutaraldehyde-activated gel. NEU2 (1/10000 wt %) ous solution to form a mixture solution. Next, the mixture was diluted with a phosphate buffer (pH-8) to a volume of Solution was dropped into a stirring 2% CaCl (w/v) aqueous 15 mL, and mixed with 1.13 g of the glutaraldehyde solution by a syringe needle. After that, the CaCl aqueous activated gel at room temperature for 20 hours. Finally, the solution was continuously stirred for 30 minutes, and then solid was filtered out and washed in a phosphate buffer particles formed in the CaCl aqueous solution were filtered (pH-8) to obtain an enzyme immobilized product. out and washed in deionized water to obtain a wet product (0168 Method 6 (ITRI-A-01). 0169. 1 g diethylaminoethyl cellulose (DEAE cellulose) was washed in water, suspended in an NaOH solution (1 M Example 3 aqueous solution), stirred for 10 minutes, and then filtered out and washed in water. The obtained solid substance was Efficacy of Immobilized-NEU2 Filled Device Suspended in 10 mL dioxane to form a Suspension. 2 g 0182 NEU2 was immobilized by Method 2 in Example cyanuric chloride and 10 mL toluene were added to the 2, and then the immobilized NEU2 was filled into a tube to US 2017/00445 16 A1 Feb. 16, 2017

form a biochemistry reactive device (immobilized-NEU2 content in the L5 samples mentioned above (for the detailed filled device) shown in FIG. 1B. experimental methods, please see “5. Efficacy test for 0183 (1) Determination of Apoptosis ASAH2 in “A. Method” of Example 1). 0184 Endothelial cells of blood vessel were co-cultured (0197) The results for LC/MS analysis are shown in with electronegative low-density lipoprotein (electronega Table 1 (the four values shown in each group were obtained tive LDL) L5 (25 ug/mL: 50 lug/mL) and L5 (1.25 ug) which from determining the same sample four times). Conversion was treated by the mmobilized-NEU2 filled device for 2 was performed to signal of each sample in Table 1 to obtain hours (treatment temperature 37° C. pH 7.4) for 24 hours, ceramide content percentage of each sample (the highest respectively. After that, apoptosis of the endothelial cells signal of the L5 without treatment was set as 100%), and the was determined, and the results are shown in FIG. 7. results are shown in FIG. 12. ASAH2H1 and ASAH2H2 0185. According to FIG. 7, it is known that 25 g/mL L5 shown in Table 1 and FIG. 12 are ASAH2 obtained from results in apoptosis to about 15% endothelial cells and 50 different batches. ug/mL L5 results in apoptosis to about 30% endothelial cells while after the treatment of 1.25 ug NEU2, apoptosis effect TABLE 1. of L5 to endothelial cells is reduced. 0186 (2) Quantitative Analysis for Electronegative Low LC/MS analysis results for L5 without treatment and L5 treated with Density Lipoprotein (Electronegative LDL) ASAH2 for 24 hours 0187 LDL samples were obtained from a heart disease 24 hour baseline ASAH2#1 treatment ASAH2#2 treatment patient. Quantitative analysis for L5 was performed on the Signal 459.6464 295.3353 2O2.1154 LDL samples without treatment and those treated without 443.4776 236.9632 177.1598 enzyme at 37° C. for 2 hours or treated with NEU2 for 2 449.82O1 230.7273 173.031 hours (treatment temperature 37° C. pH 7.4) to determine 451.5772 249.0337 175.7823 the content of L5 in the samples mentioned above. The Mean 451.1303 253.014.9 182.0221 results are shown in FIG. 8. Standard 6.658431 29.21906 13. SOSO3 Deviation 0188 According to FIG. 8, it is known that after being Decrease 43.9 59.7 treated with NEU2 enzyme for 2 hours, L5 content of the LDL sample was decreased from 12.4% to 8.48%. (0189 (3) Mass Spectrometry 24-hour baseline represents ceramide content of L5 without 0190. Mass spectrometry analysis was performed on L5 treatment. and L5 treated with NEU2 for 2 hours (treatment tempera 0198 According to Table 1 and FIG. 12, it is known that ture 37° C., pH 7.4). The results are shown in FIGS. 9A, 9B after L5 was treated with ASAH2 for 24 hours, the ceramide and 9C. content of L5 decreased significantly. 0191 It has been known that the feature of L5 is that (0199 (2) LC/MS Analysis for L5 Treated with ASAH2 serine and threonine of apolipoprotein E (apoE) are usually for 24 Hours (for the Detailed Experimental Methods, glycosylated. Please See “5. Efficacy Test for ASAH2 in “A. Method” of (0192 Refer to FIGS. 9A and 9B. Molecular weight 1497 Example 1) indicates non-toxic LDL. Molecular weight of LDL with one glycosyl molecule is 1700, molecular weight of LDL with (0200 L5 was treated with ASAH2 for 24 hours. LC/MS two glycosyl molecules is 1884, and molecular weight of analysis was performed on L5 without treatment and L5 LDL with three glycosyl molecules is 2154. FIG.9C shows with the preceding treatment to determine the ceramide that the amino acid sequence of apolipoprotein E is glyco content in the L5 samples mentioned above. Sylated, and that results in the charge-to-mass ratio of the 0201 The results for LC/MS analysis are shown in original peptide chain being increased from 1497.8009 to Table 2 (the four values shown in each group were obtained 1700.8868, 1884.9021 and 2154.0300. from determining the same sample four times). Conversion 0193 FIGS. 10A1-2 show that there is no molecule with was performed to signal of each sample in Table 2 to obtain a charge-to-mass ratio of 1700, 1884 or 2154 that is detected ceramide content percentage of each sample (the highest for L5 treated by the immobilized-NEU2 filled device for 2 signal of the L5 without treatment was set as 100%), and the hours, and that indicates that there is no glycosylation on results are shown in FIG. 13. ASAH2H1 and ASAH2H2 serine and threonine of apolipoprotein E, i.e., the glycan shown in Table 2 and FIG. 13 are ASAH2 obtained from residues of LDL have been removed. different batches. (0194 Similarly, FIGS. 10B1-2 show that for L5 treated by the immobilized-NEU2 filled device for 2 hours, there is TABLE 2 no glycosylation on other sites of apolipoprotein E, and that LC/MS analysis results for L5 without treatment and L5 treated with indicates that the glycan residues of LDL have been ASAH2 for 24 hours removed. 24 hour baseline ASAH2#1 treatment ASAH2#2 treatment Example 4 Signal 2008.465 1827.823 1638.186 2007.321 1747.422 1627.067 Efficacy of ASAH2 1946.98S 1725.032 1622.848 1989.728 1688.382 1616.651 (0195 (1) LC/MS Analysis for L5 Treated with ASAH2 Mean 1988.125 1747.16S 1626.188 for 24 Hours Standard 28.73594 59.02271 9.07O668 Deviation (0196) L5 was treated with ASAH2 for 24 hours. LC/MS Decrease 12.1.1997 18.20495 analysis was performed on L5 without treatment and L5 with the preceding treatment to determine the ceramide US 2017/00445 16 A1 Feb. 16, 2017

24 hour baseline represents ceramide content of L5 without TABLE 3-continued treatment. 0202 According to Table 2 and FIG. 13, it is known that LC/MS analysis results for L5 without treatment and L5 treated with after L5 was treated with ASAH2 for 24 hours, the ceramide ASAH2 in the presence or absence of a buffer for 24 hours content of L5 decreased significantly. 24 hour baseline ASAH2#1 treatment ASAH2#2 treatment 0203 (3) LC/MS Analysis for L5 Treated with ASAH2 Mean 217.07 120.85 125.93 in the Presence or Absence of a Buffer for 2 or 24 Hours Standard 2.31 2.51 7.11 0204. In the presence or absence of a buffer (200 mM Deviation Tris-HCl pH 8.4, 1.5 M NaCl, 25 mM CaCl), L5 was Decrease 44.33 41.99 treated with ASAH2 for 2 or 24 hours. LC/MS analysis was performed on L5 without treatment and L5 with the preced 24 hour baseline represents ceramide content of L5 without ing treatment to determine the ceramide content in the L5 treatment. samples mentioned above (for the detailed experimental 0210. According to Table 3 and FIG. 15, it is known that methods, please see “5. Efficacy test for ASAH2 in “A. in the presence or absence of a buffer, after L5 was treated Method of Example 1 except the part of mixing with the with ASAH2 for 24 hours, the ceramide content of L5 both buffer or not). decreased significantly. 0205 Conversion was performed to signal of each 0211 (5) LC/MS Analysis for L5 Treated with ASAH2 sample to obtain the ceramide content percentage of each for 24 Hours sample (the highest signal of the L5 without treatment and 0212 Quantitative analysis for lipid constituents was kept for 2 hours was set as 100%), and the results are shown performed on L5 and L5 treated with ASAH2 for 24 hours in FIG. 14. ASAH2H1 and ASAH2H2 shown in FIG. 14 are by mass spectrometry, and the ceramide contents of the L5 ASAH2 obtained from different batches. In FIG. 14, LDL samples mentioned above were compared. The results are baseline represents ceramide content of L5 without treat shown in FIG. 16A. ment and kept for 0 hour; LDL 2 hours represents ceramide 0213 (6) LC/MS Analysis for L5 Treated with ASAH2 content of L5 without treatment and kept for 2 hour; LDL 24 in the Presence of a Buffer for 2 Hours hours represents ceramide content of L5 without treatment 0214. In the presence of a buffer (200 mM Tris-HCl pH and kept for 24 hour. 8.4, 1.5 M NaCl, 25 mM CaCl), L5 was treated with 0206. According to FIG. 14, it is known that, in the ASAH2 for 2 hours. LC/MS analysis was performed on L5 presence of a buffer, after L5 was treated with ASAH2 for without treatment and L5 with the preceding treatment to 2 hours, the ceramide content of L5 decreased significantly. determine the ceramide content in the L5 samples mentioned Moreover, in the presence or absence of a buffer, after L5 above (for the detailed experimental methods, please see “5. was treated with ASAH2 for 24 hours, the ceramide content Efficacy test for ASAH2 in “A. Method” of Example 1 of L5 both decreased significantly. except the part of mixing with the buffer or not). The results 0207 (4) LC/MS Analysis for L5 Treated with ASAH2 for LC/MSE analysis are shown in Table 4 (the four values in the Presence or Absence of a Buffer for 24 Hours shown in each group were obtained from determining the 0208. In the presence or absence of a buffer (200 mM same sample four times). Conversion was performed to Tris-HCl pH 8.4, 1.5 M NaCl, 25 mM CaCl), L5 was signal of each sample in Table 4 to obtain ceramide content treated with ASAH2 for 24 hours. LC/MS analysis was percentage of each sample (the highest signal of the L5 performed on L5 without treatment and L5 with the preced without treatment was set as 100%), and the results are ing treatment to determine the ceramide content in the L5 shown in FIG. 16B. samples mentioned above (for the detailed experimental methods, please see “5. Efficacy test for ASAH2 in “A. TABLE 4 Method of Example 1 except the part of mixing with the LC/MS analysis results for L5 without treatment and L5 treated with buffer or not). ASAH2 in the presence or absence of a buffer for 2 hours 0209. The results for LC/MS analysis are shown in Table 3 (the four values shown in each group were obtained Name of sample Signal from determining the same sample four times). Conversion L5 0 hour 529.0532 was performed to signal of each sample in Table 3 to obtain L5 0 hour 498.5066 L5 0 hour 478.2745 ceramide content percentage of each sample (the highest L5 0 hour 432.8346 signal of the L5 without treatment was set as 100%), and the L5 + ASAH 2 hours 266.8874 results are shown in FIG. 15. ASAH2H1 and ASAH2H2 L5 + ASAH 2 hours 276.2790 shown in Table 3 and FIG. 15 are ASAH2 obtained from L5 + ASAH 2 hour 282.97.67 different batches. L5 + ASAH 2 hour 283.6284 TABLE 3 0215. According to Table 4 and FIG. 16B, it is known that in the presence of a buffer, after L5 was treated with LC/MS analysis results for L5 without treatment and L5 treated with ASAH2 for 2 hours, the ceramide content of L5 decreased ASAH2 in the presence or absence of a buffer for 24 hours significantly. 24 hour baseline ASAH2#1 treatment ASAH2#2 treatment 0216. It will be apparent to those skilled in the art that Signal 217.36 12240 121.87 various modifications and variations can be made to the 22O16 122.08 121.65 disclosed embodiments. It is intended that the specification 214.80 117.11 123.68 and examples be considered as exemplary only, with the true 215.96 121.81 136.51 scope of the disclosure being indicated by the following claims and their equivalents. US 2017/00445 16 A1 Feb. 16, 2017 11

SEQUENCE LISTING

<16O is NUMBER OF SEO ID NOS: 7

<210s, SEQ ID NO 1 &211s LENGTH: 38O 212. TYPE: PRT <213> ORGANISM: Homo sapiens

<4 OOs, SEQUENCE: 1 Met Ala Ser Leu Pro Val Lieu Gln Lys Glu Ser Val Phe Glin Ser Gly 1. 5 1O 15 Ala His Ala Tyr Arg Ile Pro Ala Lieu. Lieu. Tyr Lieu Pro Gly Glin Glin 2O 25 3O Ser Lieu. Lieu Ala Phe Ala Glu Glin Arg Ala Ser Llys Lys Asp Glu. His 35 4 O 45 Ala Glu Lieu. Ile Val Lieu. Arg Arg Gly Asp Tyr Asp Ala Pro Thr His SO 55 6 O Glin Val Glin Trp Glin Ala Glin Glu Val Val Ala Glin Ala Arg Lieu. Asp 65 70 7s 8O Gly His Arg Ser Met Asn Pro Cys Pro Leu Tyr Asp Ala Glin Thr Gly 85 90 95 Thr Lieu Phe Leu Phe Phe Ile Ala Ile Pro Gly Glin Val Thr Glu Gln 1OO 105 11 O Glin Glin Lieu. Glin Thr Arg Ala Asn Val Thir Arg Lieu. Cys Glin Val Thr 115 12 O 125 Ser Thr Asp His Gly Arg Thir Trp Ser Ser Pro Arg Asp Lieu. Thir Asp 13 O 135 14 O Ala Ala Ile Gly Pro Ala Tyr Arg Glu Trp Ser Thr Phe Ala Val Gly 145 150 155 160 Pro Gly His Cys Lieu. Glin Lieu. His Asp Arg Ala Arg Ser Lieu Val Val 1.65 17O 17s Pro Ala Tyr Ala Tyr Arg Llys Lieu. His Pro Ile Glin Arg Pro Ile Pro 18O 185 19 O Ser Ala Phe Cys Phe Lieu. Ser His Asp His Gly Arg Thir Trp Ala Arg 195 2OO 2O5 Gly His Phe Val Ala Glin Asp Thir Lieu. Glu. Cys Glin Val Ala Glu Val 21 O 215 22O Glu Thr Gly Glu Glin Arg Val Val Thir Lieu. Asn Ala Arg Ser His Lieu. 225 23 O 235 24 O Arg Ala Arg Val Glin Ala Glin Ser Thr Asn Asp Gly Lieu. Asp Phe Glin 245 250 255 Glu Ser Gln Leu Val Lys Llys Lieu Val Glu Pro Pro Pro Glin Gly Cys 26 O 265 27 O Gln Gly Ser Val Ile Ser Phe Pro Ser Pro Arg Ser Gly Pro Gly Ser 27s 28O 285 Pro Ala Gln Trp Lieu. Leu Tyr Thr His Pro Thr His Ser Trp Glin Arg 29 O 295 3 OO Ala Asp Lieu. Gly Ala Tyr Lieu. Asn Pro Arg Pro Pro Ala Pro Glu Ala 3. OS 310 315 32O Trp Ser Glu Pro Val Lieu. Lieu Ala Lys Gly Ser Cys Ala Tyr Ser Asp 3.25 330 335 Lieu. Glin Ser Met Gly Thr Gly Pro Asp Gly Ser Pro Leu Phe Gly Cys 34 O 345 35. O US 2017/00445 16 A1 Feb. 16, 2017 12

- Continued

Lieu. Tyr Glu Ala Asn Asp Tyr Glu Glu Ile Val Phe Leu Met Phe Thr 355 360 365 Lieu Lys Glin Ala Phe Pro Ala Glu Tyr Lieu Pro Glin 37 O 375 38O

<210s, SEQ ID NO 2 &211s LENGTH: 78O 212. TYPE: PRT <213> ORGANISM: Homo sapiens

<4 OOs, SEQUENCE: 2 Met Ala Lys Arg Thr Phe Ser Asn Lieu. Glu Thir Phe Lieu. Ile Phe Leu 1. 5 1O 15

Lieu Wal Met Met Ser Ala Ile Thr Wall Ala Lieu Lleu Ser Lieu. Lieu. Phe 2O 25 3O Ile Thr Ser Gly Thr Ile Glu Asn His Lys Asp Leu Gly Gly His Phe 35 4 O 45 Phe Ser Thr Thr Glin Ser Pro Pro Ala Thr Glin Gly Ser Thr Ala Ala SO 55 6 O Glin Arg Ser Thr Ala Thr Gln His Ser Thr Ala Thr Glin Ser Ser Thr 65 70 7s 8O

Ala Thr Glin. Thir Ser Pro Wall Pro Lieu. Thir Pro Glu Ser Pro Leu Phe 85 90 95 Gln Asin Phe Ser Gly Tyr His Ile Gly Val Gly Arg Ala Asp Cys Thr 1OO 105 11 O Gly Glin Val Ala Asp Ile Asn Lieu Met Gly Tyr Gly Llys Ser Gly Glin 115 12 O 125 Asn Ala Glin Gly Ile Lieu. Thir Arg Lieu. Tyr Ser Arg Ala Phe Ile Met 13 O 135 14 O Ala Glu Pro Asp Gly Ser Asn Arg Thr Val Phe Val Ser Ile Asp Ile 145 150 155 160 Gly Met Val Ser Glin Arg Lieu. Arg Lieu. Glu Val Lieu. Asn Arg Lieu. Glin 1.65 17O 17s Ser Lys Tyr Gly Ser Lieu. Tyr Arg Arg Asp Asn Val Ile Lieu. Ser Gly 18O 185 19 O Thr His Thr His Ser Gly Pro Ala Gly Tyr Phe Glin Tyr Thr Val Phe 195 2OO 2O5 Val Ile Ala Ser Glu Gly Phe Ser Asn Gln Thr Phe Gln His Met Val 21 O 215 22O Thr Gly Ile Leu Lys Ser Ile Asp Ile Ala His Thr Asn Met Llys Pro 225 23 O 235 24 O Gly Lys Ile Phe Ile Asn Lys Gly Asn. Wall Asp Gly Val Glin Ile Asn 245 250 255

Arg Ser Pro Tyr Ser Tyr Lieu. Glin Asn Pro Glin Ser Glu Arg Ala Arg 26 O 265 27 O

Tyr Ser Ser Asn. Thir Asp Llys Glu Met Ile Val Lieu Lys Met Val Asp 27s 28O 285 Lieu. Asn Gly Asp Asp Lieu. Gly Lieu. Ile Ser Trp Phe Ala Ile His Pro 29 O 295 3 OO

Val Ser Met Asn. Asn. Ser Asn His Lieu Val Asn. Ser Asp Asn Val Gly 3. OS 310 315 32O

Tyr Ala Ser Tyr Lieu. Lieu. Glu Glin Glu Lys Asn Lys Gly Tyr Lieu Pro 3.25 330 335 US 2017/00445 16 A1 Feb. 16, 2017 13

- Continued

Gly Glin Gly Pro Phe Val Ala Ala Phe Ala Ser Ser Asn Lieu. Gly Asp 34 O 345 35. O Val Ser Pro Asn Ile Lieu. Gly Pro Arg Cys Ile Asn Thr Gly Glu Ser 355 360 365 Cys Asp Asn Ala Asn Ser Thr Cys Pro Ile Gly Gly Pro Ser Met Cys 37 O 375 38O Ile Ala Lys Gly Pro Gly Glin Asp Met Phe Asp Ser Thr Glin Ile Ile 385 390 395 4 OO Gly Arg Ala Met Tyr Glin Arg Ala Lys Glu Lieu. Tyr Ala Ser Ala Ser 4 OS 41O 415 Gln Glu Val Thr Gly Pro Leu Ala Ser Ala His Gln Trp Val Asp Met 42O 425 43 O Thr Asp Val Thr Val Trp Lieu. Asn Ser Thr His Ala Ser Lys Thr Cys 435 44 O 445 Llys Pro Ala Lieu. Gly Tyr Ser Phe Ala Ala Gly. Thir Ile Asp Gly Val 450 45.5 460 Gly Gly Lieu. Asn Phe Thr Glin Gly Lys Thr Glu Gly Asp Pro Phe Trp 465 470 47s 48O Asp Thir Ile Arg Asp Glin Ile Lieu. Gly Llys Pro Ser Glu Glu Ile Llys 485 490 495 Glu Cys His Llys Pro Llys Pro Ile Lieu. Lieu. His Thr Gly Glu Lieu. Ser SOO 505 51O Llys Pro His Pro Trp His Pro Asp Ile Val Asp Val Glin Ile Ile Thr 515 52O 525 Lieu. Gly Ser Leu Ala Ile Thr Ala Ile Pro Gly Glu Phe Thr Thr Met 53 O 535 54 O Ser Gly Arg Arg Lieu. Arg Glu Ala Val Glin Ala Glu Phe Ala Ser His 5.45 550 555 560 Gly Met Glin Asn Met Thr Val Val Ile Ser Gly Lieu. Cys Asn Val Tyr 565 st O sts Thr His Tyr Ile Thr Thr Tyr Glu Glu Tyr Glin Ala Glin Arg Tyr Glu 58O 585 59 O Ala Ala Ser Thr Ile Tyr Gly Pro His Thr Lieu Ser Ala Tyr Ile Glin 595 6OO 605 Lieu. Phe Arg Asn Lieu Ala Lys Ala Ile Ala Thr Asp Thr Val Ala Asn 610 615 62O Lieu. Ser Arg Gly Pro Glu Pro Pro Phe Phe Lys Gln Lieu. Ile Val Pro 625 630 635 64 O Lieu. Ile Pro Ser Ile Val Asp Arg Ala Pro Lys Gly Arg Thr Phe Gly 645 650 655 Asp Val Lieu. Glin Pro Ala Lys Pro Glu Tyr Arg Val Gly Glu Val Ala 660 665 67 O

Glu Val Ile Phe Val Gly Ala Asn Pro Lys Asn. Ser Val Glin Asn Glin 675 68O 685 Thr His Glin Thr Phe Lieu. Thr Val Glu Lys Tyr Glu Ala Thr Ser Thr 69 O. 695 7 OO

Ser Trp Glin Ile Val Cys Asn Asp Ala Ser Trp Glu Thr Arg Phe Tyr 7 Os 71O 71s 72O Trp His Lys Gly Lieu. Lieu. Gly Lieu. Ser Asn Ala Thr Val Glu Trp His 72 73 O 73 US 2017/00445 16 A1 Feb. 16, 2017 14

- Continued

Ile Pro Asp Thr Ala Gln Pro Gly Ile Tyr Arg Ile Arg Tyr Phe Gly 740 74. 7 O

His Asn Arg Lys Glin Asp Ile Lieu Lys Pro Ala Val Ile Lieu. Ser Phe 7ss 760 765

Glu Gly. Thir Ser Pro Ala Phe Glu Val Val Thir Ile 770 775 78O

<210s, SEQ ID NO 3 &211s LENGTH: 17 212. TYPE: PRT <213> ORGANISM: Homo sapiens

<4 OOs, SEQUENCE: 3 Arg Ile Gly Glin Asp Gly Ile Ser Thir Ser Ala Thir Thr Asn Lieu Lys 1. 5 1O 15

Cys

<210s, SEQ ID NO 4 &211s LENGTH: 12 212. TYPE: PRT <213> ORGANISM: Homo sapiens

<4 OOs, SEQUENCE: 4 Llys Val Lieu Val Asp His Phe Gly Tyr Thir Lys Asp 1. 5 1O

<210s, SEQ ID NO 5 &211s LENGTH: 9 212. TYPE: PRT <213> ORGANISM: Homo sapiens

<4 OOs, SEQUENCE: 5 Lys Gly Val Ile Ser Ile Pro Arg Lieu. 1. 5

<210s, SEQ ID NO 6 &211s LENGTH: 21 212. TYPE: PRT <213> ORGANISM: Homo sapiens

<4 OOs, SEQUENCE: 6 Lys Ser Gly Ser Ser Thr Ala Ser Trp Ile Glin Asn Val Asp Thir Lys 1. 5 1O 15

Tyr Glin Ile Arg Ile 2O

<210s, SEQ ID NO 7 &211s LENGTH: 22 212. TYPE: PRT <213> ORGANISM: Homo sapiens

<4 OO > SEQUENCE: 7 Lys Ala Lys Pro Ala Lieu. Glu Asp Lieu. Arg Glin Gly Lieu. Lieu Pro Val 1. 5 1O 15

Lieu. Glu Ser Phe Llys Val 2O US 2017/00445 16 A1 Feb. 16, 2017 15

What is claimed is: 12. The biochemistry reactive material as claimed in 1. A biochemistry reactive material, comprising: claim 1, wherein the enzyme composition is the first a Substrate; and enzyme. an enzyme composition immobilized on the Substrate, 13. The biochemistry reactive material as claimed in wherein the enzyme composition is selected from a claim 12, wherein the first enzyme is neuraminidase 2. group consisting of 14. The biochemistry reactive material as claimed in a first enzyme for eliminating a glycan residue of an claim 1, wherein the enzyme composition is the second electronegative low-density lipoprotein (electro enzyme. negative LDL): 15. The biochemistry reactive material as claimed in a second enzyme for eliminating ceramide carried by claim 14, wherein the second enzyme is N-acylsphingosine an electronegative low-density lipoprotein; and amidohydrolase 2. 16. The biochemistry reactive material as claimed in a combination thereof, claim 1, wherein the enzyme composition is the combination wherein the biochemistry reactive material is capable of of the first enzyme and the second enzyme. eliminating electronegative low-density lipoprotein. 17. The biochemistry reactive material as claimed in 2. The biochemistry reactive material as claimed in claim claim 16, wherein the first enzyme is neuraminidase 2, and 1, wherein the Substrate comprises silica gel, cellulose, the second enzyme is N-acylsphingosine amidohydrolase 2. diethylaminoethyl cellulose (DEAE cellulose), chitosan, 18. The biochemistry reactive material as claimed in polystyrene, polysulfone, polyetherSulfone, acrylate resin or claim 1, wherein the electronegative low-density lipoprotein polysaccharide. comprises electronegative low-density lipoprotein L1, L2. 3. The biochemistry reactive material as claimed in claim L3, L4 or L5. 1, wherein the substrate has a particle structure or a hollow 19. The biochemistry reactive material as claimed in tube structure. claim 18, wherein the electronegative low-density lipopro 4. The biochemistry reactive material as claimed in claim tein is electronegative low-density lipoprotein L5. 1, wherein the substrate is a cellulose bead. 20. A biochemistry reactive device, comprising: 5. The biochemistry reactive material as claimed in claim the biochemistry reactive material as claimed in claim 1: 1, wherein the substrate is a chitosan bead. and 6. The biochemistry reactive material as claimed in claim a container for containing the biochemistry reactive mate 1, wherein the Substrate is a cellulose hollow fiber, a rial, wherein the container has at least one inlet and at polysulfone hollow fiber, epoxy acrylic resin or a polyether least one outlet, sulfone hollow fiber. wherein a liquid sample enters into the biochemistry 7. The biochemistry reactive material as claimed in claim reactive device from the at least one inlet, and flows 1, wherein the first enzyme is sialidase or glycosidase. through the biochemistry reactive material to react with the biochemistry reactive material, and then flows out 8. The biochemistry reactive material as claimed in claim through the at least one outlet. 7, wherein the sialidase is selected from a group consisting 21. The biochemistry reactive device as claimed in claim of: 20, wherein a material of the container comprises glass, neuraminidase 1 (NEU1), neuraminidase 2 (NEU2), acrylic, polypropylene, polyethylene, stainless steel or tita neuraminidase 3 (NEU3), neuraminidase 4 (NEU4) and nium alloy. O-Sialidase bioengineered from human genome, one of 22. The biochemistry reactive device as claimed in claim the foregoing enzymes obtained through gene transfor 20, further comprising: mation, expression and purification, and sialidase from a filtering material configured in the container behind the a virus or bacterium (alias, acetylneuraminyl hydro at least one inlet and at least one outlet, lase). wherein a pore size of the filtering material is smaller than 9. The biochemistry reactive material as claimed in claim the biochemistry reactive material to prevent the bio 7, wherein the glycosidase is selected from a group consist chemistry reactive material leaking from the at least ing of one inlet and/or least one outlet. alpha- and beta-glucosidase bioengineered from human or 23. The biochemistry reactive device as claimed in claim animal genome, maltase-glucoamylase and Sucrase 20, wherein a material of the filtering material comprises isomaltase, one of the foregoing enzymes obtained filter paper, glass, acrylic, polypropylene or polyethylene. through gene transformation, expression and purifica 24. The biochemistry reactive device as claimed in claim tion, and N-glycosidase F (PNGase F) and glucosidase 20, wherein the container is a hollow column, and two ends from a virus or bacterium. of the container have a first inlet of the at least one inlet and 10. The biochemistry reactive material as claimed in a first outlet of the at least outlet, respectively. claim 1, wherein the second enzyme is ceramidase. 25. The biochemistry reactive device as claimed in claim 11. The biochemistry reactive material as claimed in claim 24, a second inlet of the at least one inlet and a second outlet 10, wherein the ceramidase is selected from a group con of the at least outlet are located at a side wall of the hollow sisting of: column. N-acylsphingosine amidohydrolase 1 (ASAH1), N-acyl 26. The biochemistry reactive device as claimed in claim sphingosine amidohydrolase 2 (ASAH2), N-acylsphin 20, wherein the substrate has a particle structure or a gosine amidohydrolase 2B (ASAH2B), N-acylsphin hollow-tube structure. gosine amidohydrolase 2C (ASAH2C), 27. The biochemistry reactive device as claimed in claim N-acylethanolamine acid amidase, alkaline ceramidase 22, wherein the substrate has a particle structure or a 1, alkaline ceramidase 2 and alkaline ceramidase 3. hollow-tube structure. US 2017/00445 16 A1 Feb. 16, 2017

28. The biochemistry reactive device as claimed in claim 43. The biochemistry reactive device as claimed in claim 22, wherein the substrate has a particle structure. 20, wherein the electronegative low-density lipoprotein 29. The biochemistry reactive device as claimed in claim comprises electronegative low-density lipoprotein L1, L2. 24, wherein the substrate has a particle structure or a L3, L4 or L5. hollow-tube structure. 44. The biochemistry reactive device as claimed in claim 30. The biochemistry reactive device as claimed in claim 20, wherein the electronegative low-density lipoprotein is 25, wherein the substrate has a hollow-tube structure. electronegative low-density lipoprotein L5. 31. The biochemistry reactive device as claimed in claim 45. The biochemistry reactive device as claimed in claim 20, wherein the Substrate comprises silica gel, cellulose, 20, wherein the liquid sample comprises aqueous solution, diethylaminoethyl cellulose, chitosan, polystyrene, polysul blood or plasma. fone, polyetherSulfone, acrylate resin or polysaccharide. 46. A method for ex vivo treating blood or plasma, comprising: 32. The biochemistry reactive device as claimed in claim (a) ex vivo contacting a blood or plasma with an enzyme 28, wherein the substrate is a cellulose bead. composition to react the enzyme composition with the 33. The biochemistry reactive device as claimed in claim blood or plasma, wherein the enzyme composition is 28, wherein the substrate is a chitosan bead. capable of eliminating electronegative low-density 34. The biochemistry reactive device as claimed in claim lipoprotein, and the enzyme composition is selected 30, wherein the substrate is a cellulose hollow fiber, a from a group consisting of: polysulfone hollow fiber, epoxy acrylic resin or a polyether a first enzyme for eliminating a glycan residue of an sulfone hollow fiber. electronegative low-density lipoprotein (LDL): 35. The biochemistry reactive device as claimed in claim a second enzyme for eliminating ceramide carried by a 20, wherein the first enzyme is sialidase or glycosidase. electronegative low-density lipoprotein (LDL); and 36. The biochemistry reactive device as claimed in claim a combination thereof, and 35, wherein the sialidase is selected from a group consisting (b) terminating contact between the blood or plasma and of: the enzyme composition to terminate the reaction of the neuraminidase 1 (NEU1), neuraminidase 2 (NEU2), enzyme composition with the blood or plasma. neuraminidase 3 (NEU3), neuraminidase 4 (NEU4) and 47. The method for ex vivo treating blood or plasma as O-Sialidase bioengineered from human genome, one of claimed in claim 46, wherein the step (a) is performed for the foregoing enzymes obtained through gene transfor about 0.25-8 hours. mation, expression and purification, and sialidase from 48. The method for ex vivo treating blood or plasma as a virus or bacterium (alias, acetylneuraminyl hydro claimed in claim 46, wherein the step (a) is performed at lase). about 4-40° C. 37. The biochemistry reactive device as claimed in claim 49. The method for ex vivo treating blood or plasma as 35, wherein the glycosidase is selected from a group con claimed in claim 46, wherein the step (a) is performed at sisting of: about pH 5-10. alpha- and beta-glucosidase bioengineered from human or 50. The method for ex vivo treating blood or plasma as animal genome, maltase-glucoamylase and Sucrase claimed in claim 46, wherein the first enzyme is sialidase or isomaltase, one of the foregoing enzymes obtained glycosidase. through gene transformation, expression and purifica 51. The method for ex vivo treating blood or plasma as tion, and N-glycosidase F (PNGase F) and glucosidase claimed in claim 50, wherein the sialidase is selected from from a virus or bacterium. a group consisting of 38. The biochemistry reactive device as claimed in claim neuraminidase 1 (NEU1), neuraminidase 2 (NEU2), 20, wherein the second enzyme is ceramidase. neuraminidase 3 (NEU3), neuraminidase 4 (NEU4) and 39. The biochemistry reactive device as claimed in claim O-Sialidase bioengineered from human genome, one of 38, wherein the ceramidase is selected from a group con the foregoing enzymes obtained through gene transfor sisting of: mation, expression and purification, and sialidase from N-acylsphingosine amidohydrolase 1, N-acylsphingosine a virus or bacterium (alias, acetylneuraminyl hydro amidohydrolase 2, N-acylsphingosine amidohydrolase lase). 2B, N-acylsphingosine amidohydrolase 2C, N-acyle 52. The method for ex vivo treating blood or plasma as thanolamine acid amidase, alkaline ceramidase 1, alka claimed in claim 50, wherein the glycosidase is selected line ceramidase 2 and alkaline ceramidase 3. from a group consisting of: 40. The biochemistry reactive device as claimed in claim alpha- and beta-glucosidase bioengineered from human or 20, wherein the enzyme composition is the first enzyme, and animal genome, maltase-glucoamylase and Sucrase the first enzyme is neuraminidase 2. isomaltase, one of the foregoing enzymes obtained 41. The biochemistry reactive device as claimed in claim through gene transformation, expression and purifica 20, wherein the enzyme composition is the second enzyme, tion, and N-glycosidase F (PNGase F) and glucosidase and the second enzyme is N-acylsphingosine amidohydro from a virus or bacterium. lase 2. 53. The method for ex vivo treating blood or plasma as 42. The biochemistry reactive device as claimed in claim claimed in claim 46, wherein the second enzyme is cerami 20, wherein the enzyme composition is the combination of dase. the first enzyme and the second enzyme, and the first 54. The method for ex vivo treating blood or plasma as enzyme is neuraminidase 2 and the second enzyme is claimed in claim 53, wherein the ceramidase is selected from N-acylsphingosine amidohydrolase 2. a group consisting of US 2017/00445 16 A1 Feb. 16, 2017

N-acylsphingosine amidohydrolase 1, N-acylsphingosine 59. The method for ex vivo treating blood or plasma as amidohydrolase 2, N-acylsphingosine amidohydrolase claimed in claim 46, wherein the enzyme composition is the 2B, N-acylsphingosine amidohydrolase 2C, N-acyle combination of the first enzyme and the second enzyme. thanolamine acid amidase, alkaline ceramidase 1, alka 60. The method for ex vivo treating blood or plasma as line ceramidase 2 and alkaline ceramidase 3. claimed in claim 59, wherein the first enzyme is neuramini dase 2, and the second enzyme is N-acylsphingosine ami 55. The method for ex vivo treating blood or plasma as dohydrolase 2. claimed in claim 46, wherein the enzyme composition is the 61. The method for ex vivo treating blood or plasma as first enzyme. claimed in claim 46, wherein the enzyme composition is 56. The method for ex vivo treating blood or plasma as immobilized on the substrate. claimed in claim 55, wherein the first enzyme is neuramini 62. The method for ex vivo treating blood or plasma as dase 2. claimed in claim 61, the Substrate comprises silica gel. 57. The method for ex vivo treating blood or plasma as cellulose, diethylaminoethyl cellulose, chitosan, polysty claimed in claim 46, wherein the enzyme composition is the rene, polysulfone, polyetherSulfone, resin or polysaccharide. second enzyme. 63. The method for ex vivo treating blood or plasma as 58. The method for ex vivo treating blood or plasma as claimed in claim 61, the substrate has a particle structure or claimed in claim 57, wherein the second enzyme is N-acyl a hollow-tube structure. sphingosine amidohydrolase 2. k k k k k