Enzyme Capsules Research Articles

Oral Administration of Therapeutic Enzyme Capsule for the Management of Inflammatory Bowel Disease.

BackgroundOral administration of proteins/peptides is challenging in clinical application due to their instability and susceptibility in the gastrointestinal tract.Materials and MethodsThe in situ polymerization on the surface of enzymes was used to encapsulate antioxidant enzymes (superoxide dismutase (SOD) and catalase (CAT)) in polymeric shells, and the reactive oxygen species (ROS) scavenging ability was monitored based on DCFH-DA probe using flow cytometry and confocal laser scanning microscopy. The mRNA expression level of pro-inflammatory factors was assessed by real-time qPCR, using lipopolysaccharide-induced RAW264.7 cells as a model. Finally, the enzyme capsules were orally administered for the treatment of inflammatory bowel disease using dextran sodium sulfate (DSS)-induced colitis mice as a model, based on the evaluation of the disease-associated index, ROS level and pro-inflammatory cytokines’ expression.ResultsThe enzyme capsules could effectively scavenge the intracellular reactive oxygen species (ROS) through the cascade catalysis of SOD and CAT, and thus protect the cells from ROS-induced oxidative damage. Meanwhile, the enzyme capsules could inhibit the secretion of pro-inflammatory cytokines from macrophages, thereby achieving favorable anti-inflammation effect. Oral administration of enzyme capsules could facilitate the accumulation of enzymes in the inflamed colon tissues of DSS-induced colitis mice. Moreover, the oral delivery of enzyme capsules could effectively alleviate the symptoms associated with colitis, attributing to the excellent ROS scavenging ability and the inhibition of pro-inflammatory cytokines’ level.ConclusionIn summary, our findings provided a promising approach to construct enzyme-based nano-formulations with favorable therapeutic efficacy and biocompatibility, exhibiting great potential in the treatment of gastrointestinal diseases in an oral administration manner.

Efficacy of a Viokace Pancreatic Enzyme Protocol for Clearing Occluded Enteral Feeding Tubes: A Quality Assurance Evaluation

Background: A previous retrospective study documented restored patency to 48.2% of occluded enteral feeding tubes using alkalinized Creon pancreatic enzyme capsules. In light of the low efficacy rate, the institutional enteral feeding tube clearance protocol was subsequently revised to incorporate a newly marketed non–enteric-coated Viokace pancreatic enzyme tablet, despite the lack of published data for this indication. Objective: This study aims to evaluate the effectiveness of a Viokace-based alkalinized pancreatic enzyme protocol to clear occluded enteral feeding tubes in a university health system. Methods: This retrospective, cohort quality assurance study included adult and pediatric patients receiving a Viokace-based pancreatic enzyme protocol for enteral feeding tube occlusions in a university health system during a 12-month period. The primary outcome was effectiveness in enteral tube clearance as documented in the electronic medical record. Efficacy of the new protocol was also compared with a Creon-based alkalinized solution using historical data. Results: The Viokace protocol successfully cleared 176 of the 277 (63.5%) occluded enteral feeding tubes occurring in 205 patients included in the analysis. The revised protocol was significantly more effective at clearing occluded enteral feeding tubes (P = 0.0056) than a protocol using Creon pancreatic enzyme capsules. Conclusion: According to this retrospective evaluation, an alkalinized Viokace pancreatic enzyme protocol was effective in clearing 63.5% of occluded enteral feeding tubes. This significantly higher success rate than previously documented with a Creon-based protocol supports the change in pancreatic enzyme formulations in the institutional protocol.

Clinical value of itopride combined with compound digestive enzyme capsules in treating dyspepsia in patients with chronic cardiac insufficiency

Clinical value of itopride combined with compound digestive enzyme capsules in treating dyspepsia in patients with chronic cardiac insufficiency

Production of encapsulated creatinase using yeast spores

ABSTRACTYeast spores can be used as a carrier to produce enzyme capsules. In the present study, this technique was applied to a diagnostic enzyme named creatinase. We found that a secretory form of Pseudomonas putida creatinase could be entrapped in the spore wall, and such spores were used as creatinase capsules. The activity of the encapsulated creatinase was largely improved by mild spore wall defective mutations, such as DIT1 or OSW2 deletions. The advantages of this method include the following: encapsulated and freeze-dried creatinase is produced without preparing the purified enzyme, and it exhibits resistance to environmental stresses, such as high temperature and SDS treatments. Thus, yeast spores could be applied to establish quick and easy clinical diagnostic methods.

Clinical study on the efficacy and safety of composite milk clotting enzyme capsules in children with functional dyspepsia

Objective To evaluate the efficacy and safety of composite milk clotting enzyme capsules in children with functional dyspepsia. Methods A multicenter, randomized, open-label clinical trial was carried out in 4 Guangzhou hospitals during Feb.2012 to Mar.2013.Children with functional dyspepsia on basis of Rome Ⅲ criteria were enrolled and treated with composite milk clotting enzyme capsules.Total symptom score and drug-related adverse reactions were evaluated after one-week and two-week therapy.The treatment outcome were divided into very effective, fairly effective and ineffective according to the symptom score changes.Superiority test was done for effectiveness of the treatment based on efficacy difference by 10% regarded as superiority. Results Two hundred and one children were enrolled.One hundred and ninety-six children completed the study as planned.After two-week therapy, better effective rate and total effective rate were 68.88% and 87.76%, higher than those of one-week therapy(27.04%, 76.02%)(U=2.935, P<0.05). The lower limits of 95% confidence interval of difference in symptoms improve rate, better efficacy rate and symptom score decrease rate between two-week and one-week therapy were more than 10%, and two-week therapy was superior to one-week therapy.No significant drug-related adverse reaction was found during the study. Conclusions Composite milk clotting enzyme capsules can improve functional dyspepsia symptoms significantly.The effectiveness of two-week of treatment is better than that of one-week treatment.No obvious drug-related adverse reaction is observed. Key words: Functional dyspepsia; Child; Composite milk clotting enzyme capsules; Fisher

Isolation and characterization of a thermotolerant ene reductase from Geobacillus sp. 30 and its heterologous expression in Rhodococcus opacus.

Rhodococcus opacus B-4 cells are adhesive to and even dispersible in water-immiscible hydrocarbons owing to their highly lipophilic nature. In this study, we focused on the high operational stability of thermophilic enzymes and applied them to a biocatalytic conversion in an organic reaction medium using R. opacus B-4 as a lipophilic capsule of enzymes to deliver them into the organic medium. A novel thermo- and organic-solvent-tolerant ene reductase, which can catalyze the enantioselective reduction of ketoisophorone to (6R)-levodione, was isolated from Geobacillus sp. 30, and the gene encoding the enzyme was heterologously expressed in R. opacus B-4. Another thermophilic enzyme which catalyzes NAD(+)-dependent dehydrogenation of cyclohexanol was identified from the gene-expression library of Thermus thermophilus and the gene was coexpressed in R. opacus B-4 for cofactor regeneration. While the recombinant cells were not viable in the mixture due to high reaction temperature, 634mM of (6R)-levodione could be produced with an enantiopurity of 89.2% ee by directly mixing the wet cells of the recombinant R. opacus with a mixture of ketoisophorone and cyclohexanol at 50°C. The conversion rate observed with the heat-killed recombinant cells was considerably higher than that obtained with a cell-free enzyme solution, demonstrating that the accessibility between the substrates and enzymes could be improved by employing R. opacus cells as a lipophilic enzyme capsule. These results imply that a combination of thermophilic enzymes and lipophilic cells can be a promising approach for the biocatalytic production of water-insoluble chemicals.

Self-Assembled Enzyme Capsules in Ionic Liquid [BMIM][BF4] as Templating Nanoreactors for Hollow Silica Nanocontainers

Most of the self-assembly studies have hitherto explored the aqueous media as fluid phase for self-assembly of amphiphilic biomacromolecules, wherein architectural modification of biomolecules is generally a prerequisite for self-assembly of modified biomolecules. We demonstrate for the first time that ionic liquids can act as nonaqueous designer solvents to self-assemble amphiphilic biomacromolecules without requiring their prior modification. To this end, we show that enzyme (phytase) molecules self-assembled in the presence of an appropriate ionic liquid, resulting in the formation of enzyme capsules. Phytase capsules synthesized using this approach were further used as templating nanoreactors for the synthesis of enzyme-containing hollow silica nanocontainers. In situ immobilized phytase enzyme in the silica nanocontainers, when subjected to enzyme-reusability application, establishes them as excellent reusable biocatalysts.

Enteric‐Coated Pancreatic Enzyme with Bicarbonate Is Equal to Standard Enteric‐Coated Enzyme in Treating Malabsorption in Cystic Fibrosis

To compare the efficacy of an enteric-coated buffered pancreatic enzyme (EC buffered PE) containing 1.5 mEq of bicarbonate per capsule with a conventional enteric-coated enzyme (EC-PE) capsule in cystic fibrosis patients with signs or symptoms of moderate to severe malabsorption. In a double-blind crossover study, subjects were randomly assigned to two consecutive, 2-week phases using an EC buffered PE product and conventional EC-PE product. Seventy-two hour stool collections from each phase were analyzed for energy, fat, and nitrogen content and expressed as percent of estimated intake. Twenty-one patients with cystic fibrosis and pancreatic insufficiency (14 female, mean age 20.6 +/- 11.5 years, range 8.8-41.9) completed the study. There was no significant difference in percent malabsorption of energy (19.4% vs. 19.0%), fat (20.7% vs. 20.2%), or nitrogen (10.4% vs. 10.7%) between the EC buffered PE product and the conventional EC-PE product. However, patients taking the EC buffered PE product received less enzyme based on actual enzyme activity measured in vitro (3,468 +/- 1,434 U lipase/g fat vs. 3,978 +/- 1,474 U lipase/g fat, P < 0.02). In the doses used, nutrient absorption of patients taking EC buffered PE preparation offers no advantage over a conventional EC-PE preparation.

Clinical Quiz

A 17-month-old boy with cystic fibrosis presented with a 2-month history of bloody diarrhea and poor weight gain. He had no previous history of meconium ileus, constipation, distal intestinal obstruction syndrome or abdominal surgery. The patient's mother reported giving the child six capsules of pancreatic enzymes (Ultrase, 4500 units lipase/capsule) with each breastfeeding session. Stool culture was negative for any pathogens; stool Clostridium difficile toxin was negative. Colonoscopy was performed showing edema, erythema and friability; a stricture was noted in the distal ascending colon in proximity of the hepatic flexure. Pinch biopsies of the colonic mucosa showed an increase in capillaries and extravasation of erythrocytes into the lamina propria but no evidence of colitis. A barium enema was done (Fig. 1).FIG. 1: Barium enema showing a featureless colon with retraction and narrowing of the proximal colon (arrow). There is flow of contrast material into the terminal ileum and appendix (*).What is the diagnosis? Crohn disease. Distal intestinal obstruction syndrome. Fibrosing colonopathy. Stricture from necrotizing enterocolitis. Adenocarcinoma. ANSWER/DISCUSSION The diagnosis is C, fibrosing colonopathy. Medical management was instituted with elemental formula, decreasing pancreatic enzymes, and eventually nil per os and total parenteral nutrition. Symptoms did not abate and the patient was taken to surgery, where 9 cm of fibrotic ascending colon was resected (Fig. 2). Histologic findings were consistent with fibrosing colonopathy, including submucosal and serosal fibrosis with a few inflammatory cells present; there was no evidence of cryptitis or granuloma formation (Fig. 3).FIG. 2: Surgical specimen showing the thickened mucosa and narrowed lumen of the proximal colon.FIG. 3: Histology shows an expanded submucosal layer with severe fibrosis. A, hematoxylin and eosin stain, original magnification ×40. The fibrosis is highlighted with a trichrome stain that stains collagen blue. B, Masson trichrome stain, original magnification ×40.Submucosal inflammation and fibrosis leading to colonic stricture and subsequent bowel obstruction characterize fibrosing colonopathy (1,2). The initial description of this disorder was temporally related to the introduction of high-strength pancreatic enzyme supplement (up to 20,000 U of lipase/capsule) in children with cystic fibrosis. Case-controlled studies confirmed a higher risk of developing fibrosing colonopathy with the use of large amounts of high-strength pancreatic enzyme supplements (3,4). Subsequently, a consensus statement was issued recommending limiting enzyme supplementation to <2500 units lipase/kg/meal and <10,000 units lipase/kg/day (3,4). The patient described here was receiving the recommended enzyme supplementation per meal; however, he was breastfeeding 10 to 12 times per day. The mother diligently gave replacement enzymes with each feed to optimize growth, resulting in a total daily dose of 27,000 to 32,000 units lipase/kg/day. The exact pathogenic mechanism for the development of fibrosing colonopathy is unclear, but proposed contributors to the development of fibrosing colonopathy include abnormal intestinal permeability, genetic predisposition and resins in the enteric coating of the pancreatic enzyme capsules (1,2). Radiological modalities that are useful in the investigation of colonic inflammation in patients with cystic fibrosis include barium enema and abdominal ultrasound. Characteristic barium enema findings in fibrosing colonopathy include stricture, abnormal haustra, and longitudinal shortening of the colon, beginning in the right colon and sometimes involving the distal colon without skip areas (5). Abdominal ultrasound has also been used to evaluate intestinal wall thickness as a sign of inflammation of preinflammatory processes in the setting of cystic fibrosis (6). Diagnosis of fibrosing colonopathy is based on histologic findings. A full-thickness biopsy is required because endoscopic pinch biopsies are too superficial to show the extensive submucosal fibrosis. The natural history of fibrosing colonopathy is difficult to ascertain. Although less severe subclinical cases may exist, most reported cases have involved advanced disease with stricture formation. It is unlikely that the degree of fibrosis present in symptomatic cases will respond to medical therapy. The patient we describe had a stricture that was treated conservatively with medication, bowel rest and nutritional support for 6 months without any improvement in symptoms. Surgical resection of the stenotic area is the best option for advanced disease. Fibrosing colonopathy does not recur after surgery and decreasing the pancreatic enzyme supplementation (7).

Young adults with cystic fibrosis are shorter than healthy peers because their parents are also short

The aim of this study was to evaluate whether cysticfibrosis (CF) patients achieve a final height (FH) similarto the healthy population and/or appropriate for theirgenetic potential.FHs of the patients were compared with both therespective target heights (THs) and the FHs of age-matched healthy adults living in the same geographicareas, who served as controls. Patients’ THs were alsocompared with controls. Heights of both patients andtheir parents were measured with the same portableHarpenden stadiometers and by the same personsaccording to a standardised technique [4]. Definition ofFH in the CF series was based on two criteria: bone ageof at least 18 (boys) or 17 years (girls) and completepubertal development. TH was calculated by father’sheight + mother’s height ± 13 / 2.Patients were genotyped by screening for the ninedifferent mutations affecting the CFTR gene repre-senting more than 80% of CF carriers in the WesternEuropean population [5].The control population consisted of 371 young adultsaged 18–20 years (130 males, 241 females) covering thetwo regions of origin of the CF patients admitted to thisstudy, namely Sicily and Calabria. Auxological data ofcontrols were collected to draw up Italian growth chartsfor height, weight and body mass index (BMI) [3].Heights were assessed with the same portable Harpen-den stadiometers, by the same persons who measuredthe CF patients and their parents and according to thesame technique [4].In boys with CF, the average FH was comparable toboth parental height and TH (Fig.1). When comparedwith control stature, the patients’ FH was lower (Fig. 1).In girls with CF, the mean FH was very close to bothmaternal stature and TH (Fig. 2). When compared tocontrol adult height, however, the patients’ FH waslower (Fig. 2).In both sexes, TH was lower than control FH due tothe fact that both fathers and mothers of CF patientswere shorter than controls of the respective sex. On thecontrary, when compared with their sons and daughters,patient parents had a superimposable stature (Fig. 1 andFig. 2).In the overall CF series, patient FH was positivelyrelated to TH (r =0.55; P <0.001). A positive re-lationship was also found between patients’ FHs andforced expiratory volume in 1 s (r =0.25; P <0.05). Norelationship was found between patient FH and eitherBMI, pancreatic enzyme capsule intake, age at diagnosisor genotype. In CF patients, the BMI was lower thanthat of controls (19.8±1.8 vs. 22.4±1.9; P <0.0005).Our data have shown that even nowadays, CFpatients achieve a FH which remains slightly below thatof controls, despite clinical impressions and demon-strated improvements during the last 20 years [1, 2,6].Patient FHs were mainly conditioned by parental stat-ures, whereas the prognostic role of other factors wasless important. Our results might suggest the hypothesisof abnormal parental height in CF secondary to carryingthe CFTR gene, although the mechanism responsiblefor this auxological impairment is not clear.We conclude that FH in CF is lower than thatrecorded in healthy young adults living in the samegeographic regions; patient FH is superimposable tothat of their parents and strongly related to it; TH of CFpatients is lower than control stature and FH in CF ismainly conditioned by TH, at least in a selected popu-lation consisting of patients who are able to achieve 20years of age.

Application of encapsulated enzymes to accelerate cheese ripening

Abstract The suitability of gellan, κ-carrageenan and a high-melting-fat-fraction of milk fat (HMFF) to encapsulate protease enzymes (Flavourzyme) and impact in accelerating Cheddar cheese ripening were studied. The rates of enzyme entrapment were 48.2%, 55.6%, and 38.9% for gellan, κ-carrageenan and HMFF, respectively. The enzyme capsules were incorporated into milk during cheese manufacture. The moisture content of cheeses with added gum capsules was higher than control cheeses. Casein (β) degradation was monitored by High-Performance Capillary Electrophoresis. All cheeses treated with encapsulated enzyme showed higher rates of proteolysis than the control cheese throughout the ripening period. The rate of proteolysis was greater with cheeses made incorporating κ-carrageenan capsules containing protease. Cheese texture and sensory quality were not significantly influenced by the type of encapsulating material (gum or milk fat). Differences in textural and sensory quality between treated and control cheeses were consistent with release of protease enzymes from capsules.

DEGRADATION OF UREMIC TOXINS WITH FREE AND ENCAPSULATED ENZYMES

Oral delivery of encapsulated bioactive agents (eg. cells or enzymes) for metabolic degradation of urea, uric acid and creatinine represents a novel approach to treat uremia We report here on an in vitro evaluation demonstrating preliminary feasibility of this new therapy format. Three enzymes (Urease from jack beans, Uricase from Arthrobacter globiformis, Creatininase from Flavobacterium (Sigma)) were simultaneously encapsulated in alginate, forming a combination enzyme capsule; ultimately these capsules will be administered orally and act on uremic toxins which freely diffuse into the gut. A test solution consisting of metabolites at similar levels to those found in the blood of kidney failure patients was prepared: urea (100 mg/dl), uric acid (10 mg/dl), and creatinine (10 mg/dl) and we measured the reduction of the metabolites by assay.TableOur results show no real difference between free and encapsulated enzyme trials. Therefore, the enzymes are active within the capsule and the data suggests that mass transfer was not limiting. A twenty-four hour period or Gl transit time, appears adequate for these ratios of metabolite to enzyme. Continued evaluations to confirm that this approach could supplement and improve existing renal therapy treatment are underway.

Comparison of High‐Dose and Standard‐Dose Pancreatic Enzyme Capsules in Children with Cystic Fibrosis

Objective: To compare the efficacy of a high‐dose, pancreatic enzyme supplement Creon Forte to that of a standard pancreatic enzyme supplement Cotazym S Forte in a group of children with cystic fibrosis.Methodology: 18 children with cystic fibrosis were enrolled in a 4‐week open‐labelled, randomised, twoperiod, crossover study in which equal doses of lipase were provided by either Creon Forte or Cotazym S Forte. Efficacy was determined by a 4‐day faecal fat study at the end of each 2‐week period. Patients were also asked to record daily the degree of abdominal symptoms including abdominal pain, abdominal bloating, diarrhoea, constipation, nausea, vomiting and anorexia.Results: No significant differences were found between the two enzyme preparations as assessed by fat absorption studies when equivalent doses of lipase were used. The mean fat absorption for Creon Forte was 89% and 91% for Cotazym S Forte (p &gt; 0.05). This allowed a 2.5‐fold reduction in the number of capsules taken per day with Creon Forte compared to Cotazym S Forte. Scoring of abdominal symptoms were not significantly different between the two medications. The 14 patients who completed the trial preferred Creon Forte.Conclusions: Creon Forte is as effective as Cotazym S Forte in improving fat absorption in children with cystic fibrosis when equivalent doses of lipase are used with a lower number of capsules.

High Strength Pancreatic Exocrine Enzyme Capsules Associated with Colonic Strictures in Patients with Cystic Fibrosis

High Strength Pancreatic Exocrine Enzyme Capsules Associated with Colonic Strictures in Patients with Cystic Fibrosis

Impact of lactation on women with cystic fibrosis and their infants: A review of five cases

Objective To evaluate the impact of lactation on the health status of women with cystic fibrosis and their infants. Design Data were acquired through a retrospective review of pulmonary, obstetric, and pediatric medical records. Setting A medical school-based Pediatric Cystic Fibrosis Center in Philadelphia, Pa. Subjects Five women with cystic fibrosis and their respective infants. Main outcome measures For the women, measures of pregravid, gravid, and postpartum height, weight, pulmonary function test results, and energy intake were determined. For the infants, birth Apgar scores were obtained, as were measures of growth and development. Statistical analyses performed Means and standard deviations were calculated. Data were compared with published standardized references. Results Mean maternal age at conception was 25.4±4.1 years. Mean pregravid weight was 60.0±10.7 kg of standard body weight for height (range = 50.8 to 76.7 kg; median = 58 kg). Two women consumed up to two enteric-coated supplemental enzyme capsules per meal, three women consumed none. Pulmonary disease was mild in four of the five women. Mean weight gain during pregnancy was 10.2±7.4 kg. Mean gestation was 37.4±1.5 weeks; mean birth weight was 3.0±0.5 kg. Sweat tests performed on all infants were negative. Duration of breast-feeding was 3 to 30 weeks. Four of the five infants maintained appropriate growth velocity during breast-feeding. Mean maternal energy intake during lactation was above the Recommended Dietary Allowance for lactating women. Four of the five women were at or above their standard body weight during lactation. Conclusions/applications We conclude that women with the clinical diagnosis of mild cystic fibrosis disease can maintain a normal pregnancy with appropriate weight gain and can deliver infants of normal weight for gestational age. During lactation, women with mild cystic fibrosis disease can maintain their own weight and support growth in healthy infants. The dietitian needs to be an integral member of the health care team in assessing, monitoring and managing, women with cystic fibrosis during their child-bearing years.

Serum immunoglobulin G directed against porcine trypsin in the serum of cystic fibrosis children receiving porcine pancreatic enzyme supplements

Cystic fibrosis (CF) patients require pancreatic enzyme replacement therapy to correct pancreatic insufficiency. These enzymes are derived from porcine pancreas and are known to be antigenic. To determine the possible clinical consequences, a specific ELISA was developed to detect IgG antibody directed against porcine trypsin (PTAb) in the sera of CF patients. The assay was used to evaluate the occurrence of PTAb in a cross sectional study of 103 CF patients in relation to the introduction of porcine enzyme therapy, clinical status and genotype. Antibodies against porcine trypsin were detected in the sera of 63% of patients unrelated to the age of commencement or the duration of enzyme therapy. No differences were observed in the clinical status of CF patients who had developed PTAb (n = 65) and those who had no detectable PTAb (n = 38) as determined from: the current prescribed dose of porcine pancreatic enzyme capsules; Z scores for height and weight; and respiratory function tests. It is suggested that the PTAb commonly found in the sera of CF patients are of doubtful clinical significance but the prospect of PTAb contributing to immune complex disease should be examined further.

Endocrine and exocrine pancreatic function and the ΔF508 mutation in cystic fibrosis

The relationship between the cystic fibrosis (CF) genotype and endocrine and exocrine pancreatic function was studied in 215 CF patients. In the 211 patients with the delta F508 mutation, endocrine pancreatic function (oral glucose tolerance; WHO criteria) was normal in 72.5%, impaired in 12.3%, and diabetic in 15.2% of the patients, with no difference between CF patients homozygous (N = 163, median age 15 years, range 2-40) or heterozygous (N = 48, 18 years, 3-40; age difference not significant) for the delta F508 mutation. Exocrine pancreatic sufficiency (no need for pancreatic enzyme substitution) was found in 0.6% of the patients homozygous for the delta F508 mutation and in 10.4% of the heterozygotes (p less than 0.01). Homozygous patients with pancreatic insufficiency took more pancreatic enzyme capsules (median 42 per day, range 0-192) than the heterozygotes (29 per day, 0-300; p less than 0.001). The four patients (1.9%) without the delta F508 mutation had normal glucose tolerance but exocrine pancreatic insufficiency. In conclusion, the major mutation genotype in CF (delta F508) affects the severity of the exocrine pancreatic insufficiency, whereas endocrine pancreatic function is unrelated to this genotype.

Preparation, properties and uses of polymer‐enzyme conjugates

AbstractEnzymes can be immobilized by gel entrapment, by microencapsulation, by physical or ionic adsorption, by covalent binding to inorganic or organic carriers, or by whole cell immobilization. Of particular interest is the large number of chemical reactions developed for the covalent binding of enzymes via their nonessential functional groups to inorganic carriers such as glass, ceramics and iron, to natural polymers such as cellulose and Sepharose, and to synthetic polymers such as nylon, polyacrylamide, and other vinyl polymers and copolymers possessing reactive chemical groups. The stability of certain enzymes is markedly increased on their immobilization. It was thus possible to transform the biologically active polymer derivatives into active enzyme beads, enzyme capsules, enzyme columns and enzyme membranes and these enabled the construction of enzyme reactors such as the batch‐stirred tank reactors, the continuous packed bed reactors, and fluidized bed reactors. So far mainly immobilized hydralases and isomerases are being used in industry on a large scale. It seems likely, however, that once adequate techniques become available for cofactor recycling, the use of immobilized enzymes will be extended to other organic reactions, particularly those involving stereospecific synthesis of simple or complex organic molecules. Among the industrial processes in which immobilized enzymes are being used, it is worth mentioning the industrial‐scale continuous production of fructose enriched syrup from glucose by immobilized glucose‐isomerase, the batch process for the production of 6‐aminopenicillanic acid (6‐APA) from penicillin G with the aid of immobilized penicillin amidase; the production of aspartame from aspartic acid and phenylalanine by immobilized thermoase; the large scale production of optically active amino acids with immobilized amino acid acylase; and the large scale production and application of immobilized lactase for the hydrolysis of lactose. The recently developed process for acrylamide production using immobilized nitrilase containing microbial cells should also be referred to. The successful use of an NAD‐polyethylene glycol conjugate (NAD‐PEG) as a nondialyzable water‐soluble coenzyme derivative in the enzymic synthesis of leucine from α‐ketoisocaproic acid and ammonia, in a membrane‐enclosed reactor containing L‐leucine dehydrogenase, NAD‐PEG, formate and formate dehydrogenase, illustrates the new possibilities opened up by making use of cofactor‐polymer conjugates. The use of enzyme‐polymer conjugates in analytical and clinical is also illustrated.

Stabilization of cytosine deaminase from bakers' yeast by immobilization.

Cytosine deaminase (EC 3.5.4.1) from bakers’ yeast, which is labile to heat, the half-life being about 30 min at 37°C, was stabilized through immobilization by various techniques. Our purpose was to prepare implantable enzyme capsules for long-term use in cancer chemotherapy [J. Biotechnol., 2, 13 (1985)]. When the enzyme was immobilized on Eupergit C, a commercial epoxy-acrylic resin, the highest yield of apparent enzymic activity was about 80% of the starting activity, and the enzymic activity decayed exponentially at 37°C over a few weeks, the half-life being about 10 days. With lower yields, other commercial adsorbents resulted in good stability; with yields of 20~67 and 21 %, and half-lives of 15 and 24 days for Formyl-Cellulofine and CNBr-activated Sepharose 4B, respectively.

Implantable enzyme capsules for cancer chemotherapy from bakers' yeast cytosine deaminase immobilized on epoxy-acrylic resin and urethane prepolymer.

For trial use in the local chemotherapy of cancer by a combination of cytosine deaminase (EC 3.5.4.1) and 5-fluorocytosine (J. Biotechnol., (1985), 2, 13-21), 40 U of partially purified cytosine deaminase was obtained from 500 g of commercial compressed bakers' yeast. The enzyme, which is unstable, was immobilized to stabilize it by the use of commercial epoxy-acrylic beads (Eupergit C). The immobilized enzyme was made into enzyme capsules with cellulose tubing for dialysis to encapsulate it or urethane polymer to entrap it, which materials are biocompatible. The activity of the intact cellulose capsules thus made was 0.4% that of the immobilized enzyme inside. The enzyme capsules also were stable. Ten days after the cellulose capsules were implanted in rats, 25% of the starting activity remained. When the polyurethane capsules were tested in vitro for 9 mo for thermostability at 37 degrees C, the activity decreased rapidly (with a half-life of 28 d) during the first 4 mo, and then slowly (half-life, about 100 d) during the next 5 mo. A calculation to transform the biphasic decline into a sum of the exponential decline of two components of enzymic activities with different strengths and half-lives showed that the larger half-life was 5 mo.