Structured Model for Batch Culture Growth of Lactobacillus bulgaricus

Batch fermentation kinetics of Lactobacillus bulgaricus were examined in detail using the methodology of cybernetic modelling. The effect of pH and lactate ion on the activity of the enzyme β-galactosidase was simulated. Cybernetic modelling is mainly used for simulation of growth on multiple substrates. Here, it has been incorporated in a model which simulates pH effects on single-substrate batch growth. The expression of active enzyme is crucial for substrate and growth. It was seen that the effect of lactate ion on the activity of the enzyme was dependant on the system pH. A semi-empirical expression was obtained for the relative amount of active enzyme present in the organism and used to model the enzyme, biomass, substrate and lactic acid concentrations for the batch fermentation. The rate of biomass formation and product formation depended on the amount of enzyme synthesised, which in turn was dependant on the pH value of the system. The model developed simulates the effect of pH and lactate concentration on the expression and degradation of the enzyme. © 1997 SCI.     

Hydrolysis of milk lactose in a packed bed reactor system using immobilized yeast cells

BACKGROUND: Hydrolysis of lactose with β‐D‐galactosidase is one of the most promising biotechnological applications in the food industry because of its use in the production of low lactose milk products and whey hydrolysis. To overcome the problem of enzyme extraction from cells due to the intracellular nature of β‐D‐galactosidase and the poor permeability of the cell membrane to lactose, permeabilization of yeast cells was investigated. Permeabilized whole cells have been claimed to have an advantage over more pure enzyme preparations. In view of the advantages of immobilized cell systems over free cell systems, permeabilized cells were immobilized by an entrapment method in calcium alginate gel. A packed bed reactor together with this immobilized cell system has been used for hydrolysis of milk lactose in a continuous system. RESULTS: Different process parameters (temperature, substrate feed rate, biomass load and time‐course) were optimized to maximize lactose hydrolysis. The immobilized yeast cells (300 mg dry wt) resulted in 87.2% hydrolysis of milk lactose at 30 °C and flow rate 7 mL h^?1 in a packed bed reactor system. CONCLUSION: This convenient and relatively inexpensive method of immobilization, resulting in high hydrolysis potential in a continuous system, indicates that permeabilized yeast cells have the potential for the production of low lactose milk and milk products. Copyright © 2010 Society of Chemical Industry     

Effect of hydrolysis products and Mg2+, Mn2+ and Ca2+ ions on whey lactose hydrolysis and β‐galactosidase stability

BACKGROUND: Enzyme inhibition is one of the constraints of reactions catalysed by enzymes, and information is required on the inhibition mechanisms that affect the process yield. Therefore the aim of the present study was to investigate the effect of hydrolysis products and ions on the hydrolysis of lactose recovered from whey and enzyme inactivation during the reaction. The experiments were carried out in 250 mL of 25 mmol L^?1 phosphate buffer solution using β‐galactosidase from Kluyveromyces marxianus lactis in a batch reactor system. RESULTS : The degree of lactose hydrolysis (%) and the residual enzyme activity (%) in the presence and absence of lactose over time were investigated versus hydrolysate amount, glucose and galactose concentrations and Mg²⁺, Mn²⁺ and Ca²⁺ ion concentrations. The hydrolysis degree decreased with the addition of all hydrolysis products, as enzyme inhibition occurred. The residual enzyme activity increased with the addition of hydrolysate and glucose but decreased with the addition of galactose. It was observed that Mn²⁺ and Mg²⁺ ions activated the enzyme. It was also found that the hydrolysis degree was not affected by the addition of Mn²⁺ ions. On the other hand, the hydrolysis degree decreased with the addition of Ca²⁺ ions, as the enzyme was inactivated. CONCLUSION: Evaluation of the experimental data showed that both β‐galactosidase activity and lactose hydrolysis were affected by the addition of hydrolysis products and ions. Moreover, mathematical models proposed to predict the residual lactose concentration and residual enzyme activity were confirmed by the experimental results. Copyright © 2008 Society of Chemical Industry     

Amperometric Bi-enzyme based biosensor for the detection of lactose—characterization and application

Based on the glucose oxidase-β-galactosidase sequence an enzyme probe for the specific determination of lactose has been developed. β-Galactosidases from different sources have been compared, the sensor containing β-galactosidase from Curvularia inaequalis has been characterized in respect of optimal pH, enzyme loading, apparent activity and functional stability. The response of the bi-enzyme probe depends linearly on lactose concentration between 0.02 and 3.00 mmol dm^?3. The application to different milk and foodstuff samples resulted in good correlations toward enzymatic photometric (y = (0.956x-1.67) mmol dm^?3) and infrared detection (y = (1.0772x-0.3909)%). Using a measuring frequency of 100 h^?1 the serial imprecision is about 2% for diluted milk, urine, or foodstuff samples.     

Combined bioreaction and separation in a simulated counter-current chromatographic bioreactor–separator for the hydrolysis of lactose

The hydrolysis of lactose using the enzyme lactase from Aspergillus oryzae and the simultaneous separation of the products galactose and glucose was examined in a Simulated Counter-Current Chromatographic Bioreactor–Separator (SCCR-S) system. The system was operated in continuous and batch modes and the results obtained used to compare the performance in both modes. Mathematical modelling and computer simulation of the system operating in the continuous mode are reported.     

Immobilisation of β-galactosidase onto gelatin by glutaraldehyde and chromium(III) acetate

β-Galactosidase (EC 3.2.1.23) was immobilised onto a gelatin carrier system by crosslinking. Glutaraldehyde and chromium(III) acetate were used as crosslinking agents. The effect of pH on enzyme activity, kinetic parameters and reusability of the immobilised enzyme were investigated. To obtain a better relative activity, a method was developed and further modified to increase the surface area of the support system by using polyacrylamide. Relative activity enhancement of 68% was achieved in the gelatin–glutaraldehyde system.     

Microbial production,immobilization and applications of β‐D‐galactosidase

β‐D ‐Galactosidase (β‐D ‐galactoside galactohydrolase, E.C. 3.2.1.23), most commonly known as lactase, is one of the most important enzymes used in food processing, which catalyses the hydrolysis of lactose to its constituent monosaccharides, glucose and galactose. The enzyme has been isolated and purified from a wide range of microorganisms but most commonly used β‐D ‐galactosidases are derived from yeasts and fungal sources. The major difference between yeast and fungal enzyme is the optimum pH for lactose hydrolysis. The application of β‐D ‐galactosidase for lactose hydrolysis in milk and whey offers nutritional, technological and environmental applications to human life. In this review, the main emphasis has been given to elaborate the various techniques used in recent times for the production, purification, immobilization and applications of β‐D ‐galactosidase. Copyright © 2006 Society of Chemical Industry     

Antarctic Rahnella inusitata: A Producer of Cold-Stable β-Galactosidase Enzymes

There has been a recent increase in the exploration of cold-active β-galactosidases, as it offers new alternatives for the dairy industry, mainly in response to the current needs of lactose-intolerant consumers. Since extremophilic microbial compounds might have unique physical and chemical properties, this research aimed to study the capacity of Antarctic bacterial strains to produce cold-active β-galactosidases. A screening revealed 81 out of 304 strains with β-galactosidase activity. The strain Se8.10.12 showed the highest enzymatic activity. Morphological, biochemical, and molecular characterization based on whole-genome sequencing confirmed it as the first Rahnella inusitata isolate from the Antarctic, which retained 41–62% of its β-galactosidase activity in the cold (4 °C–15 °C). Three β-galactosidases genes were found in the R. inusitata genome, which belong to the glycoside hydrolase families GH2 (LacZ and EbgA) and GH42 (BglY). Based on molecular docking, some of these enzymes exhibited higher lactose predicted affinity than the commercial control enzyme from Aspergillus oryzae. Hence, this work reports a new Rahnella inusitata strain from the Antarctic continent as a prominent cold-active β-galactosidase producer.     

Enzyme hydrolysis of grafted amylopectin

Previous methods of proof of grafting are based on separation of homopolymers from crude reaction products and further characterization of extracted component. This article reports the proof of grafting by a combined use of viscometry and enzyme hydrolysis that, to our knowledge, has not been reported so far. Two series of graft copolymers of amylopectin with polyacrylamide were synthesized using ceric ion-induced redox initiation technique. In the first series, a variation of ceric ion concentration at fixed acrylamide concentration and in the second series, a variation of acrylamide concentration at fixed ceric ion concentration were undertaken to effect a variation in the number and length of polyacrylamide chains. Qualitatively, it has been observed that there may be some homopolymers formed at a very high acrylamide concentration. The products may at best be a mixture of graft copolymer and homopolymer, but it contradicts the view that the products are purely physical mixtures of polysaccharide and polyacrylamide. © 1998 John Wiley & Sons, Inc. J Appl Polym Sci 70: 2627–2633, 1998     

β‐Galactosidase immobilization into poly(hydroxyethyl methacrylate) membrane and performance in a continuous system

The activity of β‐galactosidase immobilized into a poly(2‐hydroxyethyl methacrylate) (pHEMA) membrane increased from 1.5 to 10.8 U/g pHEMA upon increase in enzyme loading. The K_m values for the free and the entrapped enzyme were found to be 0.26 and 0.81 mM, respectively. The optimum reaction temperatures for the free and the entrapped β‐galactosidase were both found to be 50°C. Similarly, the optimum reaction pH was 7.5 for both the free and the entrapped enzyme. The immobilized β‐galactosidase was characterized in a continuous system during lactose hydrolysis and the operational inactivation rate constant (k_iop) of the entrapped enzyme was found to be 3.1 × 10⁻⁵ min⁻¹. © 1999 John Wiley & Sons, Inc. J Appl Polym Sci 72: 1367–1373, 1999     

Acid hydrolysis of glycosidic bonds in oat β‐glucan and development of a structured kinetic model

Homogeneous acid‐catalyzed hydrolysis of oat β‐glucan, which contains β‐(1,4) and β‐(1,3) glycosidic bonds in a nonrandom order, was studied at 353 K using HCl and H₂SO₄. A new structured kinetic model was developed that takes into account the difference in the reactivity of β‐(1,4) and β‐(1,3) glycosidic bonds as well as their positions in the polysaccharide chain. To minimize the correlation of adjustable parameters in the new model, the reactivities of these bonds were studied independently (T = 313…363 K; c_H+ = 0.1…2 mol/L) using cellobiose and laminaribiose. The difference in kinetic parameters (e.g., T = 338 K: k_β‐(1,4) = 0.693 × 10^?3 L/mol/min, k_β‐(1,3) = 1.027 × 10^?3 L/mol/min) was found to be statistically significant (P < 0.0001), which emphasizes the need for the structured model for oat β‐glucan hydrolysis. The simulation of β‐glucan hydrolysis with the new model was in good agreement with the experimental data and shows improvement over existing nonstructured models. © 2018 American Institute of Chemical Engineers AIChE J, 64: 2570–2580, 2018     

Simulation of batch and continuous reactors with co-immobilized yeast and β-galactosidase

A reaction-diffusion model was used to simulate a co-immobilized system utilizing the numerical method of orthogonal collocation. The production of ethanol from deproteinized whey using β-galactosidase co-immobilized with Saccharomyces cerevisiae in calcium alginate gel beads was chosen as a model system. Calculated concentrations of lactose, glucose, galactose and ethanol were compared with experimental data for a batch reactor and a continuous horizontal packed-bed reactor. The mathematical model has been used to analyse the influence of internal and external mass transfer for the continuous reactor. The external mass transfer was shown to be of minor importance. The introduction of baffles decreased the backmixing in the horizontal packed-bed reactor. Internal mass transfer was found to be the main cause of the reduction in the apparent reaction rate. Thus, much of the expected increase in reaction rate is diminished by mass transfer hindrance when the cell concentration is increased.     

Enzymatic hydrolysis of dissolved corn stalk hemicelluloses: reaction kinetics and modeling

The enzymatic hydrolysis of hemicelluloses as a filtrate originating from aqueous/steam pretreated corn stalks was carried out using commercial enzyme systems of several activities composed of cellulases and β‐D ‐xylosidases from Aspergillus niger. The hydrolysis was conducted using free enzymes in aqueous substrate solution at a temperature of 30 °C and a pH of 5. Saccharification corresponding to 90% of potential simple sugar release was obtained after 10 h using 0.12 activity units (U) of Cellulases_1 complex per mg of dissolved solids present in the filtrate. Synergetic action of Cellulases_1 enzyme complex and β‐D ‐xylosidases proved to be effective for the hydrolysis of plant hemicelluloses. A lumped model based on the Michaelis–Menten approach successfully described the fate of the lumped variables describing the hydrolysis of the overall kinetics of corn stalk hemicelluloses. The maximum saccharification rate evolved with the cellulases concentration as . This overall and pseudokinetic tendency was comparable to those reported in the literature for more simple systems employing a defined substrate and a pure hydrolytic enzyme. The n‐value was found to be in the range of 0.1–0.9 depending on the substrate lump involved in the reaction system. Copyright © 2003 Society of Chemical Industry     

超声催化水解法制邻氯苯甲醛的影响因素

使用超声波促进邻氯苄叉二氯的水解,研究各反应条件对反应速度的影响。实验结果表明超声水解反应速率大大高于普通反应速度,24min内声化学反应完成了97.7%,而普通化学反应仅完成了32.6%,声化学反应比普通反应快3倍。反应的影响因素有温度、声强、催化剂质量分数、催化剂A、B摩尔比、氮气通入速度等,最佳值分别为110℃、4.03W/cm2、4%、2∶1、60mL/min。影响因子以温度为最大,声强、催化剂质量分数、氮气流量依次递降。     

Pressure Cycling to Enhance an Immobilized Enzymic Reaction by Enzyme Entrapment in a Pressure-Sensitive Gel

The enzyme β-galactosidase was entrapped in poly(N-isopropylacrylamide) gel. The gel swelled or collapsed sharply upon the rise or fall of environmental pressure. The gel beads, prepared by reverse suspension polymerization, were placed in a stirred batch reactor filled with a solution of the substrateo-nitrophenol β-D -galactopyranoside. Operations under constant or cycling pressure were performed. A considerable increase in conversion rate was observed under pressure cycling operation, compared with isobaric operations at either atmospheric or high pressure. This result could be explained by the enhancement of mass transfer inside the gel beads during the pressure cycling operation.     

Ultrasound‐activated Knoevenagel condensation of malononitrile with carbonylic compounds catalysed by alkaline‐doped saponites

α,β‐Unsaturated nitriles have been synthesized by Knoevenagel condensation of a carbonylic compound with malononitrile, assisted by sonochemical irradiation. Two alkaline‐promoted clays (Li⁺‐ and Cs⁺‐exchanged saponites) have been employed as catalysts. The influence of the carbonylic compound (benzaldehyde or cyclohexanone) and the use of a solvent on the catalytic activity have been studied. Remarkable increase in the conversion values has been found when the reaction is activated by ultrasound, as compared with the thermal activation. In this green, solvent‐free procedure, α,β‐unsaturated nitriles have been produced in very high yields (97%) when the Cs⁺‐saponite is used as catalyst. Copyright © 2004 Society of Chemical Industry     

Comparison of β-galactosidase immobilization by entrapment in and adsorption on poly(2-hydroxyethylmethacrylate) membranes

β-Galactosidase was immobilized in/on poly(2-hydroxyethyl methacrylate) (pHEMA) membranes by two different methods: adsorption on Cibacron F3GA derivatized pHEMA membranes (pHEMA-CB), and entrapment in the bulk of the pHEMA membranes. The maximum β-galactosidase adsorption on pHEMA-CB membranes was obtained as 95·6μgcm^-2 in 2·0mgcm^-3 enzyme solution. The adsorption phenomena appeared to follow a typical Langmuir isotherm. In the entrapment, an increase in β-galactosidase loading resulted in a consistent increase in membrane activity from 3·3×10^-2 to 17·8×10^-2Ucm^-2 pHEMA membranes. The K_m values for both immobilized β-galactosidase (adsorbed 0·32mM and entrapped 0·81mM ) were higher than that of the free enzyme (0·26mM ). The optimum reaction temperature of the adsorbed enzyme was 5°C higher than that of both the free and the entrapped enzyme. The optimum reaction pH was 7·5 for free and both immobilized preparations. After 15 successive uses the retained activity of the adsorbed and the entrapped enzymes was 80% and 95%, respectively. The storage stability of the enzyme was found to increase upon immobilization. ©1997 SCI     

Preparation and characteristics of β-chitin and poly(vinyl alcohol) blend

Mechanical properties of blend films of β-chitin and poly(vinyl alcohol) (PVA) in dry and wet states were improved compared to those of homopolymers. Fourier transformed infra-red (FTi.r.) spectra of the blend showed the transition of hydroxyl and carbonyl stretching bands upon blending. The subtraction infra-red spectra of blend 70/30 indicated that the blend showed an intermolecular interaction and the reduction of crystallinity compared with those of pure β-chitin. Wide angle X-ray diffraction (WAXD) patterns of the blend also exhibited the loss of crystallinity of β-chitin and PVA upon blending. The blends, however, showed a mixed-crystal structure. Dielectric analysis of the blend showed the transition of α and β relaxation peaks of β-chitin upon blending, appeared at 182° and 97°, respectively. For blends, the temperature of the maximum loss (T_max) of β-chitin appeared at around 182° shifted to a lower temperature region. Differential scanning calorimetric analysis of the blend also showed the transition of melting endotherms of the blend. Transmission electron microscope (TEM) studies of blends using ruthenium tetraoxide as a staining agent were examined to reveal the micro-structure and miscibility of the blends. The TEM micrograph of blend 70/30 shows some microseparations, but it is still believed to be miscible in the blends.     

Photosensitive polymers: Synthesis,characterization, and photocrosslinking properties of polymers with pendant α,β-unsaturated ketone moiety

Acrylate and methacrylate monomers with the photodimerizable α,β-unsaturated ketone moiety, such as 4-cinnamoylphenyl, 4-(4-methoxycinnamoyl)phenyl, 4-(4-nitrocinnamoyl)phenyl, or 4-(4-chlorocinnamoyl)phenyl, were prepared and homopolymerized using benzoyl peroxide as the initiator at 70°C in methyl ethyl ketone. The poly(meth)acrylates were characterized by ultraviolet, infrared, ¹H-nuclear magnetic resonance (NMR), and ¹³C-NMR spectra, and gel permeation chromatography. Their thermal properties were studied by thermogravimetric analyses in air and nitrogen, and differential scanning calorimetry. The photocrosslinking properties of the polymers were investigated as thin films and in solution in the presence and absence of sensitizer. © 1998 John Wiley & Sons, Inc. J Appl Polym Sci 70: 2111–2120, 1998