Recombinant Saccharomyces cerevisiae expressing P450 in artificial digestive systems: a model for biodetoxication in the human digestive environment

The use of genetically engineered microorganisms such as bacteria or yeasts as live vehicles to carry out bioconversion directly in the digestive environment is an important challenge for the development of innovative biodrugs. A system that mimics the human gastrointestinal tract was combined with a computer simulation to evaluate the survival rate and cinnamate 4-hydroxylase activity of a recombinant model of Saccharomyces cerevisiae expressing the plant P450 73A1. The yeasts showed a high level of resistance to gastric and small intestinal secretions (survival rate after 4 h of digestion, 95.6% +/- 10.1% [n = 4]) but were more sensitive to the colonic conditions (survival rate after 4 h of incubation, 35.9% +/- 2.7% [n = 3]). For the first time, the ability of recombinant S. cerevisiae to carry out a bioconversion reaction has been demonstrated throughout the gastrointestinal tract. In the gastric-small intestinal system, 41.0% +/- 5.8% (n = 3) of the ingested trans-cinnamic acid was converted into p-coumaric acid after 4 h of digestion, as well as 8.9% +/- 1.6% (n = 3) in the stomach, 13.8% +/- 3.3% (n = 3) in the duodenum, 11.8% +/- 3.4% (n = 3) in the jejunum, and 6.5% +/- 1.0% (n = 3) in the ileum. In the large intestinal system, cinnamate 4-hydroxylase activity was detected but was too weak to be quantified. These results suggest that S. cerevisiae may afford a useful host for the development of biodrugs and may provide an innovative system for the prevention or treatment of diseases that escape classical drug action. In particular, yeasts may provide a suitable vector for biodetoxication in the digestive environment.     

Recombinant Saccharomyces cerevisiae strain expressing a model cytochrome P450 in the rat digestive environment: viability and bioconversion activity

An innovative "biodrug" concept, based on the oral administration of living recombinant microorganisms, has recently emerged for the prevention or treatment of various diseases. An engineered Saccharomyces cerevisiae strain expressing plant P450 73A1 (cinnamate-4-hydroxylase [CA4H] activity) was used, and its survival and ability to convert trans-cinnamic acid (CIN) into p-coumaric acid (COU) were investigated in vivo. In rats, the recombinant yeast was resistant to gastric and small intestinal secretions but was more sensitive to the conditions found in the large intestine. After oral administration of yeast and CIN, the CA4H activity was shown in vivo, with COU being found throughout the rat's digestive tract and in its urine. The bioconversion reaction occurred very fast, with most of the COU being produced within the first 5 min. The gastrointestinal sac technique demonstrated that the recombinant yeast was able to convert CIN into COU (conversion rate ranging from 2 to 5%) in all the organs of the rat's digestive tract: stomach, duodenum, jejunum, ileum, cecum, and colon. These results promise new opportunities for the development of drug delivery systems based on engineered yeasts catalyzing a bioconversion reaction directly in the digestive tract.     

Functionalisation of allergen-loaded microspheres with wheat germ agglutinin for targeting enterocytes

In this study, we constructed particles applicable for oral immunotherapy of type I allergy by protecting allergens from digestion and supporting intestinal antigen uptake. Therefore, birch-pollen allergens were entrapped in poly(d,l-lactic-co-glycolic acid) microspheres by spray-drying rendering microspheres with a main population of 1-3microm. Microspheres were further coated with wheat germ agglutinin (WGA) to target enterocytes. Coating with WGA did not alter the surface characteristics of the microspheres as demonstrated in scanning electron microscopy. Binding of WGA was specific and could be inhibited by chitotriose to 14.7+/-6.9%. Comparable amounts of allergen were released from both particle-types with 46.3+/-1.7% and 44.5+/-2.6% during 21 days. Simulating gastric digestion in vitro, antigenicity of allergens entrapped in WGA-microspheres was preserved to 59.8+/-1.5% even after 2h. Feedings of BALB/c mice with WGA-microspheres induced higher levels of allergen-specific IgG-levels than gavages of uncoated microparticles or naked protein. Thus, we conclude that WGA-microspheres are suitable vehicles for oral delivery and mucosal targeting due to lectin-mediated bioadhesion.     

Gastric acid secretion in aquaporin-4 knockout mice

The aquaporin-4 (AQP4) water channel has been proposed to play a role in gastric acid secretion. Immunocytochemistry using anti-AQP4 antibodies showed strong AQP4 protein expression at the basolateral membrane of gastric parietal cells in wild-type (+/+) mice. AQP4 involvement in gastric acid secretion was studied using transgenic null (-/-) mice deficient in AQP4 protein. -/- Mice had grossly normal growth and appearance and showed no differences in gastric morphology by light microscopy. Gastric acid secretion was measured in anesthetized mice in which the stomach was luminally perfused (0. 3 ml/min) with 0.9% NaCl containing [(14)C]polyethylene glycol ([(14)C]PEG) as a volume marker. Collected effluent was assayed for titratable acid content and [(14)C]PEG radioactivity. After 45-min baseline perfusion, acid secretion was stimulated by pentagastrin (200 microg. kg(-1). h(-1) iv) for 1 h or histamine (0.23 mg/kg iv) + intraluminal carbachol (20 mg/l). Baseline gastric acid secretion (means +/- SE, n = 25) was 0.06 +/- 0.03 and 0.03 +/- 0.02 microeq/15 min in +/+ and -/- mice, respectively. Pentagastrin-stimulated acid secretion was 0.59 +/- 0.14 and 0.70 +/- 0.15 microeq/15 min in +/+ and -/- mice, respectively. Histamine plus carbachol-stimulated acid secretion was 7.0 +/- 1.9 and 8.0 +/- 1.8 microeq/15 min in +/+ and -/- mice, respectively. In addition, AQP4 deletion did not affect gastric fluid secretion, gastric pH, or fasting serum gastrin concentrations. These results provide direct evidence against a role of AQP4 in gastric acid secretion.     

Living recombinant Saccharomyces cerevisiae secreting proteins or peptides as a new drug delivery system in the gut

New strategies to prevent or treat diseases have been focusing on innovative approaches, such as the oral administration of living recombinant micro-organisms delivering active compounds in the digestive environment. The survival rate and the ability of two recombinant Saccharomyces cerevisiae strains (WppV(5)H(6) and WppGSTV(5)H(6)) to initiate the synthesis and secrete either a model peptide (peptide-V(5)H(6), MW: 5.6 kDa) or a model protein (glutathione-S-transferase-V(5)H(6), MW: 31.5 kDa) were studied in a gastric-small intestinal system simulating human digestive conditions. The WppV(5)H(6) and WppGSTV(5)H(6) strains respectively showed 83.1%+/-9.6 (n=3) and 95.3%+/-22.7 (n=4) survival rates in the model upper digestive tract after 270 min of digestion. The secretion products were detected as early as 90 min after the yeast intake/gene induction in each compartment of the in vitro system, but mostly in the jejunum and ileum. The GST-V(5)H(6) concentrations in the digestive medium reached 15 ng ml(-1), close to values measured in batch cultures. These results open up new opportunities for the set up of drug delivery systems based on engineered yeasts secreting compounds directly in the digestive tract. The main potential medical applications include the development of oral vaccines, the correction of metabolic disorders and the in situ production of biological mediators.     

The digestion rate of protein is an independent regulating factor of postprandial protein retention

To evaluate the importance of protein digestion rate on protein deposition, we characterized leucine kinetics after ingestion of "protein" meals of identical amino acid composition and nitrogen contents but of different digestion rates. Four groups of five or six young men received an L-[1-13C]leucine infusion and one of the following 30-g protein meals: a single meal of slowly digested casein (CAS), a single meal of free amino acid mimicking casein composition (AA), a single meal of rapidly digested whey proteins (WP), or repeated meals of whey proteins (RPT-WP) mimicking slow digestion rate. Comparisons were made between "fast" (AA, WP) and "slow" (CAS, RPT-WP) meals of identical amino acid composition (AA vs. CAS, and WP vs. RPT-WP). The fast meals induced a strong, rapid, and transient increase of aminoacidemia, leucine flux, and oxidation. After slow meals, these parameters increased moderately but durably. Postprandial leucine balance over 7 h was higher after the slow than after the fast meals (CAS: 38 +/- 13 vs. AA: -12 +/- 11, P < 0.01; RPT-WP: 87 +/- 25 vs. WP: 6 +/- 19 micromol/kg, P < 0.05). Protein digestion rate is an independent factor modulating postprandial protein deposition.     

Effect of skim milk-alginate beads on survival rate of bifidobacteria

In this study, an attempt was made to increase the survival rate of bifidobacteria entrapped in alginate in the gastrointestinal tract, and to investigate the potential industrial applications, for example lyophilized capsules and yogurt. First, the protective effect of various food additives on bifidobacterial survivability was determined after exposure to simulated gastric juices and bile salts. The additives used in this study were skim milk (SM), poly dextrose (PD), soy fiber (SF), yeast extract (YE), chitosan (CS), κ-carageenan (κ-C) and whey, which were added at 0.6% concentration (w/v) to 3% alginate-bifidobacterial solution. In the simulated gastric juices and bile salts, the protective effect of 0.6% skim milk-3% alginate (SM-A) beads on the survival rate of bifidobacteria proved to be higher than the other additives. Second, the hydrogen ion permeation was detected through SM-A vessel without bifidobacterial cells at different SM concentrations (0.2%, 0.4%, 0.6%, 0.8%, and 1.0%). There were no differences in terms of the pH decrease in SM-A vessels at 0.6%, 0.8%, and 1.0% (w/v) SM concentrations. The survival rate of bifidobacteria in SM-A beads would appear to be related to the SM buffering capacity against hydrogen ions and its tendency to reduce the pore size of bead. In this experiment, the survival rate of bifidobacteria entrapped in beads containing 0.6% SM showed the highest viability after exposure to simulated gastric juices for 3 h, thereby indicating that 0.6% SM is the optimum concentration for 3% alginate bead preparation. Third, the effect of SM-A beads on the freeze-drying and yogurt storage for 10 days was investigated. SM-A beads were found to be more efficient for freeze drying and yogurt storage than untrapped cells and the alginate bead. Consequently, the survival rate of bifidobacteria entrapped in SM-A beads was increased in simulated gastric juices, bile salts and probiotic products such as lyophilized capsules and yogurt, SM-A beads can be expected to produce high value probiotic products.     

Cloning and expression of human UDP-glucuronosyltransferase 1A4 in Bac-to-Bac system

UDP-glucuronosyltransferases (UGTs) catalyze the transfer of glucuronic acid from uridine diphosphate-glucuronic acid (UDP-GA) to compounds with amine, hydroxyl, and carboxylic acid moieties. N-glucuronidation is an important pathway for elimination of many tertiary amine therapeutic agents used in humans. UGT1A4 has been reported to be specific for glucuronidating primary, secondary, and tertiary amines, forming N-glucuronides. To further investigate the drugs metabolized by UGT1A4, the Bac-to-Bac expression system was used to express the recombinant UGT1A4 with His-tag on the C-terminal. The His-tagged recombinant UGT1A4 expressed in Spodoptera frugiperda (Sf9) cells were detected using anti-His antibody and the molecular weight of the recombinant protein was approximately 55kDa. The enzyme activity towards imipramine in cell homogenate protein was found to be 83.14+/-15pmol/min/mg protein (n=3) with 0.5mM imipramine by HPLC, but was not detectable in blank Sf9 cells. It paved the way for the further studies for drug glucuronidation by UGT1A4. The purification of the UGT1A4 can be done by Ni-resin. This is helpful to do research on the structure of the UFT1A4.     

Phospholipase A2 activities with a plasmalogen substrate in brain and in neural tumor cells: a sensitive and specific assay using pyrenesulfonyl-labeled plasmenylethanolamine

We have developed a new assay method for phospholipase A2 (EC 3.1.1.4.), towards ethanolamine plasmalogen using pyrenesulfonyl-labeled plasmenylethanolamine as the substrate. This procedure is sensitive to about 3 pmol/ml per min and is absolutely specific for plasmalogen. In this method, the product of phospholipase A2, pyrenesulfonyl-labeled lysoplasmalogen, is hydrolyzed to aldehyde and labeled glycerophosphoethanolamine with hydrochloric acid exposure, and after TLC separation, the pyrenesulfonyl-glycerophosphoethanolamine is quantitated spectrofluorometrically. The excitation and emission wave lengths were 340 and 376 nm, respectively. The activity of bovine brain homogenate was 44.1 +/- 6.47 pmol/min per mg protein (n = 3). Among bovine brain subcellular fractions, the distribution and specific activity of the enzymes were highest in cytosol (38.7 +/- 1.58% and 102.6 +/- 16.2 pmol/min per mg protein, n = 3). The activities of neural tumor cells, PC12 pheochromocytoma, Neuro2A and SKNSH neuroblastoma and U1242MG glioblastoma, were 34.4 +/- 6.83 (n = 5), 7.05 +/- 0.97 (n = 4), 5.25 +/- 1.69 (n = 5), and 9.68 +/- 1.35 (n = 4), pmol/min per mg protein (M +/- S.E.M.), respectively.     

Whey protein coating bead improves the survival of the probiotic Lactobacillus rhamnosus CRL 1505 to low pH

Aims: To evaluate the efficacy of a novel microencapsulation procedure using whey protein and pectin to improve the survival rate of Lactobacillus rhamnosus CRL 1505 to low pH and bile. Methods and Results: Lactobacillus rhamnosus CRL 1505 was encapsulated by ionotropic gelation using pectin (PE) and pectin–whey protein (PE–WP). Both types of beads (MC_PE/WP and MC_PE–WP/WP) were covered with a layer of whey protein by complex coacervation. The noncapsulated lactobacilli were not sensitive to bile salts but to acid. Both microparticles protected Lact. rhamnosus CRL 1505 at pH 2·0, but only MC_PE/WP was effective at pH 1·2. Conclusions: The combination of ionotropic gelation and complex coacervation techniques is efficient to obtain microcapsules of pectin covered with whey proteins. The MC_PE/WP beads were more stable than the MC_PE–WP/WP beads in simulated gastric conditions, thus offering better protection to Lact. rhamnosus CRL 1505 at low pH. Significance and Impact of the Study: Pectin beads with a whey protein layer (MC_PE/WP) could be used as probiotic carrier in functional foods of low pH (e.g. apple juice), thus protecting Lact. rhamnosus CRL 1505 against the stressful conditions of the gastric tract.     

Ghrelin gastrokinetic action in patients with neurogenic gastroparesis

Ghrelin has been shown to accelerate gastric emptying in animals where its effect appeared mediated through the vagus nerve. We aimed to verify the gastrokinetic capacity of ghrelin in human. Patients with gastroparesis attributed to a neural dysregulation by diabetes (n = 5) or surgical vagotomy (n = 1) were evaluated. The emptying of a test meal (420 kcal) was determined by the C13 octanoic acid breath test. Saline or synthetic ghrelin 1-4 microg/kg were given in 1 min bolus at the end of the meal. T-lag and T-1/2 were shorter during ghrelin than during saline administration [33 +/- 5 min versus 65 +/- 14 min (p < 0.01) and 119 +/- 6 min versus 173 +/- 38 min (p < 0.001)]. Ghrelin injection therefore accelerated gastric emptying of a meal in humans even in presence of a deficient gastric innervation.     

Lingual and gastric lipases: species differences in the origin of prepancreatic digestive lipases and in the localization of gastric lipase

The source of the lipase(s) acting in the stomach was investigated in five animal species: rat, mouse (rodents), rabbit (lagomorphs), guinea pig (caviidae), baboon and human (primates). The activity of lingual and gastric lipases was quantitated in homogenates of lingual serous glands and of gastric mucosa, respectively, by the hydrolysis of tri[3H]oleylglycerol and is expressed in units/g (1 U = 1 mumol [3H]oleic acid released/min) per g tissue wet weight, mean +/- S.E. There were marked differences in the activity level of lingual and gastric lipases among species: mouse and rat had high levels of lingual lipase activity (250 +/- 20 and 824 +/- 224 U/g) and only traces of gastric lipase activity (4.5 +/- 0.9 and 0.04 U/g, respectively), whereas rabbit and guinea pig had no lingual lipase activity and only gastric lipase activity (78 +/- 48 and 27 +/- 7.4 U/g, respectively). In the baboon and human, gastric lipase was the predominant enzyme (109 +/- 20 U/g and 118 +/- 8.8 U/g, respectively), whereas lingual lipase activity was present in trace amounts only (0.04 U/g and 0.3 U/g, respectively). In addition to species differences in the origin of the preduodenal lipases, there were also species differences in the distribution of gastric lipase in the stomach. Thus, while in the rabbit, gastric lipase was localized exclusively in the cardia and body of the stomach, it was diffusely distributed in the entire stomach of the guinea pig and baboon. A comparison between the level of activity of lipase and pepsin (the two chief digestive enzymes secreted by the stomach), showed differences in their localization in the species studied. The difference in source (tongue vs. stomach) and site (cardia-body vs. entire stomach) of lipase secretion must be taken into account in future studies of these digestive enzymes. Although the exact contribution of lingual and gastric lipases individually to fat digestion in species which contain both enzymes cannot yet be evaluated, the markedly higher levels of gastric lipase activity in the baboon and human suggests that, in primates, gastric lipase is probably the major non-pancreatic digestive lipase.     

The Effect of Gel Microstructure on Simulated Gastric Digestion of Protein Gels

The objective of this study was to analyse the impact of the gel structure obtained by different heat-induced temperatures on the in vitro gastric digestibility at pH 2. To achieve this, gels were prepared from soy protein, pea protein, albumin from chicken egg white and whey protein isolate at varying temperatures (90, 120 and 140 °C) for 30 min. Gels were characterised prior to digestion via microstructure and SDS-PAGE analysis. Subsequently, the gastric digestion process was followed via the protein hydrolysis and HPSEC analysis up to 180 min. Peptides of different sizes (<5 kDa) were gradually formed during the digestion. Our results showed that gels induced at 140 °C were digested faster. The protein source and gelation temperature had great influence on the in vitro gastric protein digestibility.     

The distinctiveness of ATP:citrate lyase from Aspergillus nidulans

ATP:citrate lyase (ACL), an important enzyme in lipid synthesis, has been purified from Aspergillus nidulans to a specific activity of 19.6 micromol min(-1) mg(-1), almost twice that of any other purified ACL and shown to be distinct from any previously purified ACL. The enzyme is a 371+/-31 kDa hexamer of 3 alpha, 3 beta proteins, unlike the 4 alpha tetramer found in rats or yeasts. The molecular weights of the alpha and beta protein subunits were determined by SDS-PAGE to be 70 and 55 kDa.ACL in A. nidulans (unlike Aspergillus niger) appears to be regulated by the carbon source present in the media. In crude extracts, it was found at high activity (88 micromol min(-1) mg protein(-1)) in glucose-grown cells but only at low activity (10 micromol min(-1) mg protein(-1)) in acetate-grown cells.     

Method to concentrate protein solutions based on dialysis-freezing-centrifugation: enzyme applications

One of the recurrent methodological problems in preparative biochemical work is the concentration of dilute protein solutions, including culture supernatants resulting from biotechnological processes. A procedure was developed to concentrate enzymes by a novel cryoconcentration system. This approach includes a new device that facilitates the sample freezing and the subsequent solute elution from the frozen matrix by centrifugation. The optimal centrifugation conditions for this cryoconcentration system were obtained using whey protein solution as a model. The procedure was applied to concentrate dilute solutions of commercial pectinase, measuring the endopolygalacturonase (EPG) activity of this enzyme in the concentrate by a method based on the on-line torque measurement, and of recombinant fructan:fructan 1-fructosyltransferase (1-FFT) protein of Pichia pastoris from a culture in a bioreactor, as an expression system. The optimal centrifugation speed, time, and temperature were 6150 g, 20 min, and 4 °C, respectively. The concentration factors for the dilute protein solutions were 9.2-, 11.2-, and 17.1-fold for 1-FFT, whey, and commercial pectinase, respectively. Recoveries ranged from 87% to 93%. The procedure allowed concentrating proteins efficiently without affecting their enzymatic activity.     

Assessment of antral grinding of a model solid meal with echo-planar imaging

Mathematical modeling of how physical factors alter gastric emptying is limited by lack of precise measures of the forces exerted on gastric contents. We have produced agar gel beads (diameter 1.27 cm) with a range of fracture strengths (0.15-0.90 N) and assessed their breakdown by measuring their half-residence time (RT(1/2)) using magnetic resonance imaging. Beads were ingested either with a high (HV)- or low (LV)-viscosity liquid nutrient meal. With the LV meal, RT(1/2) was similar for bead strengths ranging from 0.15 to 0.65 N but increased from 22 +/- 2 min (bead strength <0.65 N) to 65 +/- 12 min for bead strengths >0.65 N. With the HV meal, emptying of the harder beads was accelerated. The sense of fullness after ingesting the LV meal correlated linearly (correlation coefficient = 0.99) with gastric volume and was independently increased by the harder beads, which were associated with an increased antral diameter. We conclude that the maximum force exerted by the gastric antrum is close to 0.65 N and that gastric sieving is impaired by HV meals.     

Immobilization of Mucor javanicus lipase by entrapping in alginate-silica hybrid gel beads with simultaneous cross-linking with glutaraldehyde

Mucor javanicus lipase was entrapped in alginate-silica hybrid gel beads with or without simultaneous cross-linking with glutaraldehyde. The activity and recovery of activity on immobilization of the enzyme entrapped in the hybrid beads were 1.4 and 1.7 times higher than those of the enzyme entrapped in the simple alginate beads. Entrapment with simultaneous cross-linking in the hybrid beads further improved the enzyme activity (1.6 times) and activity recovery (1.7 times) compared to those of the enzyme entrapped in the hybrid beads without simultaneous cross-linking. The leakage of the enzyme entrapped in the hybrid beads with simultaneous cross-linking was only 50% that of the enzyme entrapped in the simple alginate beads.     

Phosphatase activity in rat adipocytes: effects of insulin and insulin resistance

Insulin regulates the activity of both protein kinases and phosphatases. Little is known concerning the subcellular effects of insulin on phosphatase activity and how it is affected by insulin resistance. The purpose of this study was to determine insulin-stimulated subcellular changes in phosphatase activity and how they are affected by insulin resistance. We used an in vitro fatty acid (palmitate) induced insulin resistance model, differential centrifugation to fractionate rat adipocytes, and a malachite green phosphatase assay using peptide substrates to measure enzyme activity. Overall, insulin alone had no effect on adipocyte tyrosine phosphatase activity; however, subcellularly, insulin increased plasma membrane adipocyte tyrosine phosphatase activity 78 +/- 26% (n = 4, P < 0.007), and decreased high-density microsome adipocyte tyrosine phosphatase activity 42 +/- 13% (n = 4, P < 0.005). Although insulin resistance induced specific changes in basal tyrosine phosphatase activity, insulin-stimulated changes were not significantly altered by insulin resistance. Insulin-stimulated overall serine/threonine phosphatase activity by 16 +/- 5% (n = 4, P < 0.005), which was blocked in insulin resistance. Subcellularly, insulin increased plasma membrane and crude nuclear fraction serine/threonine phosphatase activities by 59 +/- 19% (n = 4, P < 0. 005) and 21 +/- 7% (n = 4, P < 0.007), respectively. This increase in plasma membrane fractions was inhibited 23 +/- 7% (n = 4, P < 0. 05) by palmitate. Furthermore, insulin increased cytosolic protein phosphatase-1 (PP-1) activity 160 +/- 50% (n = 3, P < 0.015), and palmitate did not significantly reduce this activity. However, palmitate did reduce insulin-treated low-density microsome protein phosphatase-1 activity by 28 +/- 6% (n = 3, P < 0.04). Insulin completely inhibited protein phosphatase-2A activity in the cytosol and increased crude nuclear fraction protein phosphatase-2A activity 70 +/- 29% (n = 3, P < 0.038). Thus, the major effects of insulin on phosphatase activity in adipocytes are to increase plasma membrane tyrosine and serine/threonine phosphatase, crude nuclear fraction protein phosphatase-2A, and cytosolic protein phosphatase-1 activities, while inhibiting cytosolic protein phosphatase-2A. Insulin resistance was characterized by reduced insulin-stimulated serine/threonine phosphatase activity in the plasma membrane and low-density microsomes. Specific changes in phosphatase activity may be related to the development of insulin resistance.     

Immobilization of Mucor javanicus lipase by entrapping in alginate-silica hybrid gel beads with simultaneous cross-linking with glutaraldehyde

Mucor javanicus lipase was entrapped in alginate-silica hybrid gel beads with or without simultaneous cross-linking with glutaraldehyde. The activity and recovery of activity on immobilization of the enzyme entrapped in the hybrid beads were 1.4 and 1.7 times higher than those of the enzyme entrapped in the simple alginate beads. Entrapment with simultaneous cross-linking in the hybrid beads further improved the enzyme activity (1.6 times) and activity recovery (1.7 times) compared to those of the enzyme entrapped in the hybrid beads without simultaneous cross-linking. The leakage of the enzyme entrapped in the hybrid beads with simultaneous cross-linking was only 50% that of the enzyme entrapped in the simple alginate beads.