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18 Methods for Bioencapsulation of Enzymes and Cells Thomas M. S. Chang Summary Methods to microencapsulate enzymes and cells including recombinant enzymes, stem cells, and genetically engineered cells have been described in this chapter. More specific examples of enzyme encapsulation include the microencapsulation of xanthine oxidase for Lesch Nyhan disease, phenylalanine ammonia lyase for phenylketonuria, and microencapsulation of multienzyme systems with cofactor recycling for multistep enzyme conversions. Methods for cell encapsulation include the details for encapsulating hepatocytes for liver failure and for gene therapy. This also includes the details of a novel two-step method for encapsulation of high concentrations of smaller cells. The recent co-encapsulation of bone marrow stem cells with hepatocytes is also included. Another new approach is the detailed method for encapsulation of genetically engineered Escherichia coli DH5 cells and other microorganisms for the removal or conversion metabolites. Another new approach is the nanoencapsulation of hemoglobin and enzymes to form 150-nm diameter nanocapsules; the details are available elsewhere. Key Words: Artificial cells; bioencapsulation; microencapsulation; nanoencapsulation; enzyme; cells; genetically engineered cells; blood substitutes; liver cells; stem cells; microorganisms. 1. Introduction Microencapsulation of biologically active material in the form of “artificial cells” was reported as early as 1964 (1–4). However, it is only in the last 10 yr that many centers are developing this process extensively (5,6). More recently we have concentrated on artificial cells for blood substitutes, enzyme therapy, and cell therapy. Space allows only a few examples to be given here. HIV has stimulated extensive development in the last 10 yr. The early idea of cross-linked hemoglobin (1,4) has now been developed as a first-generation blood substitute now in phase III clinical trials by a number of groups (6–9). New generations of hemoglobin-based blood substitutes are also being developed (9). In this research center we are looking at a polyhemoglobin blood substitute with antioxidant enzyme activities (10–14). This is prepared by From: Methods in Biotechnology, Vol. 17: Microbial Enzymes and Biotransformations Edited by: J. L. Barredo © Humana Press Inc., Totowa, NJ 289
290 Chang crosslinking hemoglobin (PolyHb) with superoxide dismutase (SOD) and catalase (CAT) into PolyHb-SOD-CAT. This is important in cases with potentials for oxidant damage, as in severe sustained hemorrhagic shock or in stroke and other conditions. We are also developing a further generation of blood substitutes based on the use of nanotechnology to prepare 150-nm diameter biodegradable polymeric membrane nanocapsules containing hemoglobin and enzymes. The details for this are available elsewhere (12–15). Injection of enzyme artificial cells is effective in enzyme therapy for inborn errors of metabolism (16) and for cancer therapy (17). However, accumulation of the implanted artificial cells is a problem. This has been recently by giving the enzyme artificial cells orally as in the following example. Lesch Nyhan is a very rare disease consisting of an inborn error of metabolism with accumulation of hypoxanthine. Being lipid-soluble hypoxanthine can diffuse rapidly into the intestine. Therefore the enzyme microcapsules can be given orally to avoid the need for injection. Daily oral administration of microencapsulated xanthine oxidase resulted in the lowering of hypoxanthine in the plasma and cerebralspinal fluid in a patient with Lesch Nyhan disease (18,19). Phenylketonuria (PKU) is the most common inborn error of metabolism with enzyme defects resulting in the elevation of the amino acid phenylalanine. However, amino acids from the body do not diffuse rapidly into the intestine. This can now be solved based on recent new findings of extensive enterorecirculation of amino acids between the body and the intestine (20,21). Large volumes of digestive juice containing enzymes and other proteins are secreted into the intestine. These are digested by tryptic enzymes in the intestine into amino acids. The amino acids formed this way in the intestine are reabsorbed into the body. It has been shown for the first time that amino acids formed from this source are hundreds of time higher than those from protein in ingested food (20,21). This means that when given orally, microcapsules containing a specific enzyme can remove the corresponding specific amino acids in the intestine and therefore prevent them from returning to the body. The net result is a depletion of this specific amino acid from the body. For example, microencapsulated phenylalanine ammonia lyase given orally once a day can selectively remove phenylalanine from the enterorecirculating amino acids. This explains earlier observation of the effectiveness of this approach for lowering the elevated systemic phenylalanine in the PKU rats (22) and more recently in the ENU2 phenylketonuric mice (23). This approach can also be used to remove any of the other 26 amino acids in the body with potential for treating other inborn errors of metabolism and for amino-acid-dependent tumors. This is now being developed for clinical trials. This has resulted in renewed interest in the microencapsulation of enzymes. For example, our recent studies show that microencapsulated tyrosinase can be giving orally in rats to lower
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TM METHODS IN BIOTECHNOLOGY 17 Mic
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M E T H O D S I N B I O T E C H N O
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© 2005 Humana Press Inc. 999 River
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Contents Preface ..................
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Contributors OLGA ABIAN • Departa
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Contributors xi CHANDRAN SANDHYA
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2 Adrio and Demain The revolutionar
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4 Adrio and Demain is conducted und
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6 Adrio and Demain pharmacological
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8 Adrio and Demain a component of s
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10 Adrio and Demain of enantiopure
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12 Adrio and Demain encoding surfac
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14 Adrio and Demain 1994. DNA vacci
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16 Adrio and Demain was further eng
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18 Adrio and Demain removes food st
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20 Adrio and Demain Recently, a new
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22 Adrio and Demain 5. Demain, A. L
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24 Adrio and Demain 47. Ostergaard,
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26 Adrio and Demain 84. Shimaoka, M
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2 Enzyme Biosensors Steven J. Setfo
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Enzyme Biosensors 31 mal enzyme pro
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Enzyme Biosensors 33 Table 1 Defini
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Enzyme Biosensors 35 vidual sensors
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Enzyme Biosensors 37 2.5.3. Screen-
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Enzyme Biosensors 39 2.6.2. Stabili
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Enzyme Biosensors 41 Table 3 Commer
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Enzyme Biosensors 43 Fig. 4. Bayer
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Enzyme Biosensors 45 David Klonoff,
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Enzyme Biosensors 47 Fig. 7. Format
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Enzyme Biosensors 49 stability and
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Enzyme Biosensors 51 materials and,
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Enzyme Biosensors 53 Fig. 10. Biodo
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Enzyme Biosensors 55 Fig. 11. DiagN
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Enzyme Biosensors 57 Fig. 13. Holog
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Enzyme Biosensors 59 sis system sui
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3 Directed Evolution of Enzymes How
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Fig. 1. Flow process chart of direc
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Improving Nucleic Acid Polymerases
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4 L-Glutaminase as a Therapeutic En
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Therapeutic Enzymes 77 bial enzymes
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Therapeutic Enzymes 79 properties.
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Therapeutic Enzymes 81 ume with dis
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Therapeutic Enzymes 83 Table 2 L-gl
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Therapeutic Enzymes 85 3.2.5.8. INO
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Therapeutic Enzymes 87 5. Determine
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Therapeutic Enzymes 89 Table 5 Char
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5 Enzymatic Production of D-Amino A
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93 Fig. 1. Enzymatic route for the
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Enzymatic Production of D-Amino Aci
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106 Bañuelos et al. high sucrose c
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108 Bañuelos et al. 1. Culture the
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110 Bañuelos et al. 1. Grow Asperg
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112 Bañuelos et al. 3. Inject 20
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7 Food-Grade Corynebacteria for Enz
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Corynebacteria and Enzyme Productio
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142 Asano Fig. 1. Synthesis of (S)-
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144 Asano 2. Hydrolyze the ethyl es
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146 Asano 3.2.5. Purification of Ph
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148 Asano formate dehydrogenase, Ph
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150 Asano 3. Asano, Y., Nakazawa, A
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152 Ito et al. Purafect ® (Genenco
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154 Ito et al. 22. Bacillus subtili
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156 Ito et al. 3. The reaction was
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158 Ito et al. 3.2.3. Purification
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160 Ito et al. 3.3.3. Purification
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162 Ito et al. 3. Saeki, K., Okuda,
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10 Microbial Proteases Chandran San
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Microbial Proteases 167 Table 1. In
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Microbial Proteases 169 1.3.1. Ammo
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Microbial Proteases 171 3. Crude en
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Microbial Proteases 173 Effect of t
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Microbial Proteases 175 Table 2 Eff
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Microbial Proteases 177 4. Notes 1.
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Microbial Proteases 179 References
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182 Mellado et al. Microorganisms i
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184 Mellado et al. feed industries.
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186 Mellado et al. 1. Prepare sampl
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188 Mellado et al. 3. Incubate in d
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190 Mellado et al. 10. Mellado, E.,
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192 Tewari et al. of α-1,4-glycosi
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194 Tewari et al. 6. Incubate the f
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196 Tewari et al. 3.8.2. Enzyme Pro
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198 Tewari et al. 3.8.13. Vitamins
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200 Tewari et al. 3.10.3. Optimal T
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202 Tewari et al. 3. Suspend the ce
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204 Tewari et al. 2. Adjust differe
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206 Tewari et al. 9. As a standard
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208 Tewari et al. 19. Sharma, H. S.
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210 Lei and Kim from phosphorus pol
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212 Lei and Kim 3. 0.2 M glycine-HC
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214 Lei and Kim
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216 Lei and Kim 3. Pipet up and dow
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218 Lei and Kim 5. Collect samples
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220 Lei and Kim Fig. 2. Western blo
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222 Lei and Kim 3.5.6. In Vitro Hyd
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224 Lei and Kim 7. Rodriguez, E., H
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226 Weckbecker and Hummel Glucose d
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228 Weckbecker and Hummel Fig. 1. C
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234 Fig. 6. Comparison of the long-
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236 Weckbecker and Hummel 3. Heilma
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Page 287 and 288: 274 Mateo et al. with other molecul
Page 289 and 290: 276 Mateo et al. or than randomly i
Page 291 and 292: 278 Mateo et al. of penicillin G ac
Page 293 and 294: 280 Mateo et al. 18. Water-MIBK bip
Page 295 and 296: 282 Fig. 4. Thermal inactivation of
Page 297 and 298: 284 Mateo et al. Fig. 6. Stability
Page 299 and 300: 286 Mateo et al. 3.10. Inactivation
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Page 305 and 306: 292 Chang Therefore a new method ha
Page 307 and 308: 294 Chang 2. Collagenase perfusion
Page 309 and 310: 296 Chang 7. Allow the beaker to st
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Page 315 and 316: 302 Chang the range of 0.00724 to 0
Page 317 and 318: 304 Chang 12. Chang, T. M. S. and Y
Page 319 and 320: 306 Chang 45. Friedman, E. A. (1999
Page 321 and 322: 308 Index Acetyl-phosphate, 241 Acr
Page 323 and 324: 310 Index catalase, 290 hemoglobin-
Page 325 and 326: 312 Index food amylases, 8 protease
Page 327 and 328: 314 Index cells agar, 78 sodium alg
Page 329 and 330: 316 Index applications, 203 charact
Page 331 and 332: 318 Index RNA, 61, 62 Roche Diagnos
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