α-Galactosidase in immobilized cells of Papaver somniferum L.

A poppy cell suspension culture was permeabilized by Tween 80 and immobilized by glutaraldehyde. The α-Galactosidase in these cells showed an optimum pH level at 5.2 and an optimum temperature at 70 °C. Enzyme hydrolysis was linear for 3 h, reaching 86% conversion. A very good level of storage stability was achieved when using dry catalyst and immobilized cells in 0.15 M NaCl solution (with the addition of chloramphenicol, [1-methyldodecy1)-dimethylamin-4-oxide (ATDNO), chlortetracycline hydrochloride (CLCTC)] or by freezing them in 0.15 M NaCl solution.     

Isozymes of <Emphasis Type="Italic">α</Emphasis>-amylases from newly isolated <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> CKB19: Production from immobilized cells

The aim of this study was to produce two isozymes of α-amylase by immobilization of a newly isolated soil bacterium. The bacterium was identified as Bacillus thuringiensis CKB19 on the basis of its 16S rRNA profile. Enzyme production by free cells increased linearly with cell growth up to 34 h in starch containing enriched liquid media. The active bacterial cells were immobilized in Caalginate beads, and operational stability of the entrapped cell was optimized for amylase production. Enzyme production was optimal at an alginate concentration of 2 g% (w/v), calcium chloride concentration of 1 M, and with 300 beads (each bead contained 2 × 10⁷ cells)/250 mL flask. Amylase production by the immobilized cells was about 3 times higher than free cell fermentation after 34 h of incubation. It was observed that the immobilized bacterium secreted two different amylases (Am-I and Am-II) into the culture fluid. The molecular masses of Am-I and Am-II were 59.6 and 44.7 kd, respectively, and showed optimum activity at pH 5.0 and 9.0. Both amylases showed optimum activity at 40°C and were stable at the same temperature, with losses of only 10 and 20% (for Am I and Am II, respectively) of their original activities after 24 h of incubation. Further, both amylases were salt tolerant (up to 4 M NaCl) and hydrolyzed raw starchy foods into glucose. All these characteristics make this enzyme mixture suitable for use as a digestive aid and for the improvement of digestibility of animal feed ingredients.     

The potential of immobilized bacterial α-amylase on coconut coir,a smart carrier for biocatalysts

Purified α-amylase from a soil bacterium Bacillus sp. SKB4 was immobilized on coconut coir, an inexpensive cellulosic fiber, with the cross-linking agent glutaraldehyde. The catalytic properties and stability of the immobilized enzyme were compared with those of its soluble form. The enzyme retained 97.2% of its activity and its catalytic properties were not drastically altered after immobilization. The pH optimum and stability of the immobilized enzyme were shifted towards the alkaline range compared to the free enzyme. The optimum temperature for enzymatic activity was 90°C in both forms of the enzyme. The soluble and immobilized enzyme retained 19% and 70% of original activity, respectively, after pre-incubation for 1 h at 90°C. Immobilized amylase was less susceptible to attack by heavy metal ions and showed higher K_m and V_max values than its free form. The bound enzyme showed significant activity and stability after 6 months of storage at 4°C. All of these characteristics make the new carrier system suitable for use in the bioprocess and food industries.     

Immobilization in the presence of Triton X-100: modifications in activity and thermostability of <Emphasis Type="Italic">Geobacillus thermoleovorans</Emphasis> CCR11 lipase

A partially purified lipase produced by the thermophile Geobacillus thermoleovorans CCR11 was immobilized by adsorption on porous polypropylene (Accurel EP-100) in the presence and absence of 0.1% Triton X-100. Lipase production was induced in a 2.5% high oleic safflower oil medium and the enzyme was partially purified by diafiltration (co. 500,000 Da). Immobilization conditions were established at 25 °C, pH 6, and a protein concentration of 0.9 mg/mL in the presence and absence of 0.1% Triton X-100. Immobilization increased enzyme thermostability but there was no change in neither the optimum pH nor in pH resistance irrelevant to the presence of the detergent during immobilization. Immobilization with or without Triton X-100 allowed the reuse of the lipase preparation for 11 and 8 cycles, respectively. There was a significant difference between residual activity of immobilized and soluble enzyme after 36 days of storage at 4 °C (P < 0.05). With respect to chain length specificity, the immobilized lipase showed less activity over short chain esters than the soluble lipase. The immobilized lipase showed good resistance to desorption with phosphate buffer and NaCl; minor loses with detergents were observed (less than 50% with Triton X-100 and Tween-80), but activity was completely lost with SDS. Immobilization of G. thermoleovorans CCR11 lipase in porous polypropylene is a simple and easy method to obtain a biocatalyst with increased stability, improved performance, with the possibility for re-use, and therefore an interesting potential use in commercial conditions.     

Soluble and immobilized phenol oxidase of the fungusMycelia sterilia IBR 35219/2: A comparative study

Phenol oxidase (EC 1.14.18.1) from the microscopic fungusMycelia sterilia IBR 35219/2 was immobilized using glutaraldehyde on macroporous silica carriers. The enzyme immobilized on amino-Silochrome SKh-2 or aminopropyl-Silochrome 350/80 exhibited maximum activity. Soluble and immobilized phenol oxidases were compared. Compared to the soluble enzyme, the activity of which was optimum at pH 5.5, immobilized phenol oxidase exhibited optimum activity under slightly more acidic conditions (pH 5.2). Immobilization considerably increased enzyme stability. Both soluble and immobilized forms of phenol oxidase fromM. sterilia IBR 35 219/2 catalyze oxidative conversion of phenolic compounds of green tea extract.     

Immobilization of urease by using chitosan–alginate and poly(acrylamide-co-acrylic acid)/κ-carrageenan supports

Jack bean urease (urea aminohydrolase, E.C. 3.5.1.5) was entrapped into chitosan–alginate polyelectrolyte complexes (C-A PEC) and poly(acrylamide-co-acrylic acid)/κ-carrageenan (P(AAm-co-AA)/carrageenan) hydrogels for the potential use in immobilization of urease, not previously reported. The effects of pH, temperature, storage stability, reuse number, and thermal stability on the free and immobilized urease were examined. For the free and immobilized urease into C-A PEC and P(AAm-co-AA)/carrageenan, the optimum pH was found to be 7.5 and 8, respectively. The optimum temperature of the free and immobilized enzymes was also observed to be 55 and 60 °C, respectively. Michaelis–Menten constant (K _m) values for both immobilized urease were also observed smaller than free enzyme. The storage stability values of immobilized enzyme systems were observed as 48 and 70%, respectively, after 70 days. In addition to this, it was observed that, after 20th use in 5 days, the retained activities for immobilized enzyme into C-A PEC and P(AAm-co-AA)/carrageenan matrixes were found as 55 and 89%, respectively. Thermal stability of the free urease was also increased by a result of immobilization.     

Immobilization of cellulase from newly isolated strain Bacillus subtilis TD6 using calcium alginate as a support material

Bacillus subtilis TD6 was isolated from Takifugu rubripes, also known as puffer fish. Cellulase from this strain was partially purified by ammonium sulphate precipitation up to 80% saturation, entrapped in calcium alginate beads, and finally characterized using CMC as the substrate. For optimization, various parameters were observed, including pH maximum, temperature maximum, sodium alginate, and calcium chloride concentration. pH maximum of the enzyme showed no changes before and after immobilization and remained stable at 6.0. The temperature maximum showed a slight increase to 60 °C. Two percent sodium alginate and a 0.15 M calcium chloride solution were the optimum conditions for acquisition of enzyme with greater stability. K (m) and V (max) values for the immobilized enzyme were slightly increased, compared with those of free enzyme, 2.9 mg/ml and 32.1 μmol/min/mL, respectively. As the purpose of immobilization, reusability and storage stability of the enzyme were also observed. Immobilized enzyme retained its activity for a longer period of time and can be reused up to four times. The storage stability of entrapped cellulase at 4 °C was found to be up to 12 days, while at 30 °C, the enzyme lost its activity within 3 days.     

Immobilization of chloroperoxidase onto highly hydrophilic polyethylene chains via bio-conjugation: Catalytic properties and stabilities

Chloroperoxidase (CPO) was covalently immobilized on poly(hydroxypropyl methacrylate-co-polyethyleneglycole-methacrylate) membranes, which were characterized, by swelling test, FT-IR spectroscopy, scanning electron microscopy, and contact angle measurement. The K_m and V_max values for free and immobilized CPO were found to be 34.6 and 47.2 μM, and 287.5 and 245.2 U/mg protein, respectively. The optimum pH for both the free and immobilized enzyme was observed at 3.0. The immobilized enzyme showed wide pH and temperature profiles. Most importantly, the increased thermal, storage and operational stability of immobilized CPO should depend on the creation of a comfortable strong hydrophilic microenvironment on the designed support to the host enzyme molecule.     

A new method for immobilization of acetylcholinesterase

A new method for immobilization of acetylcholinesterase (AChE) to alginate gel beads by activating the carbonyl groups of alginate using carbodiimide coupling agent has been successfully developed. Maximum reaction rate (V _max) and Michaelis–Menten constant (K _m) were determined for the free and binary immobilized enzyme. The effects of pH, temperature, storage stability, reuse number and thermal stability on the free and immobilized AChE were also investigated. For the free and binary immobilized enzyme on the Ca–alginate gel beads, optimum pH values were found to be 7 and 8, respectively. Optimum temperatures for the free and immobilized enzyme were observed to be 30 and 35 °C, respectively. Upon 60 days of storage the preserved activity of free and immobilized enzyme were found as 4 and 68%, respectively. In addition, reuse number, and thermal stability of the free AChE were increased by as a result of binary immobilization.     

Immobilized preparation of cold-adapted and halotolerant Antarctic beta-galactosidase as a highly stable catalyst in lactose hydrolysis

A cold-active beta-galactosidase of Antarctic marine bacterium Pseudoalteromonas sp. 22b was synthesized by an Escherichia coli transformant harboring its gene and immobilized on glutaraldehyde-treated chitosan beads. Unlike the soluble enzyme the immobilized preparation was not inhibited by glucose, its apparent optimum temperature for activity was 10 degrees C higher (50 vs. 40 degrees C, respectively), optimum pH range was wider (pH 6-9 and 6-8, respectively) and stability at 50 degrees C was increased whilst its pH-stability remained unchanged. Soluble and immobilized preparations of Antarctic beta-galactosidase were active and stable in a broad range of NaCl concentrations (up to 3 M) and affected neither by calcium ions nor by galactose. The activity of immobilized beta-galactosidase was maintained for at least 40 days of continuous lactose hydrolysis at 15 degrees C and its shelf life at 4 degrees C exceeded 12 months. Lactose content in milk was reduced by more than 90% over a temperature range of 4-30 degrees C in continuous and batch systems employing the immobilized enzyme.     

Immobilization of adenosine deaminase onto agarose and casein

In the present study adenosine deaminase (ADA) was immobilized onto two different polymeric materials, agarose and casein. The factors affecting the amount of enzyme attachment onto the polymeric supports such as incubation time were investigated. The maximum amount of enzyme immobilized onto different polymeric supports occurred at incubation pH value 7.5 and ADA concentration 42 units/g and the incubation time needed for the maximum amount of enzyme attachment to the polymeric supports was found to be 8 h. Some phsicochemical properties of the free and immobilized ADA such as operational stability, optimum temperature and thermal stability, pH optimum and stability, storage stability, and the effect of gamma-radiation were studied. The operational stability of the free and immobilized enzyme showed that the enzyme immobilized by a cross-linking technique using gultaric dialdehyde showed poor durability and the relative activity decreased sharply due to the leakage after repeated washing, while the enzymes immobilized by covalent bonds to the carriers showed a slight decrease in most cases in the relative activity (around 20%) after being used 10 times. Storage for 4-6 months, showed that the free enzyme lost its activity, while the immobilized enzyme showed the opposite behavior. Subjecting the immobilized enzyme to a dose of gamma radiation of 0.5-10 Mrad showed complete loss in the activity of the free enzyme at a dose of 5 Mrad, while the immobilized enzymes showed relatively high resistance to gamma radiation up to a dose of 5 Mrad.     

Thermo-sensitive amphiphilic block copolymer poly (styrene-<Emphasis Type="Italic">b</Emphasis>-N-isopropylacrylamide) with switchable catalytic activity immobilizing pectinase

Pectinase was immobilized onto thermo-sensitive amphiphilic block copolymers poly(styrene-b-Nisopropylacrylamide) PS-b-poly(N-isopropylacrylamide) (PNIPAM) by covalent attachment. Biochemical studies have found that the stability of the PS-b-PNIPAM support is not impeded by the bound proteins despite that up to 242.5 mg of enzyme is immobilized per gram of carrier particles. The immobilized enzyme retained nearly 65% of its initial activity over 30 days, and the optimum temperature and pH also increased to the range of 60 ∼ 70°C and 4.0 ∼ 6.0, respectively. The immobilized enzyme also exhibited great operational stability, and more than 60% residual activity was observed in the immobilized enzyme after 10 batch reactions. Moreover, the lower critical solution temperature of the PS-b-PNIPAM support could be switched on or off by a small change in solution temperature. Thus, the immobilized pectinase could be recovered and showed durable activity during the recycle process.     

Β-Galactosidase in immobilized cells ofPapaver somniferum

Cell suspension culture of poppy was permeabilized by Tween 80 and immobilized by glutaraldehyde. Β-Galactosidase showed optimum pH 4.8 and temperature of 50 ‡C. The enzyme hydrolysis was linear during 3 h reaching 68 % conversion. The cells characterized by high Β-galactosidase activity and stability in long-term storage showed convenient physico-mechanical properties.     

Optimization of culture conditions for the production of halothermophilic protease from halophilic bacterium <Emphasis Type="Italic">Chromohalobacter</Emphasis> sp. TVSP101

An extremely halophilic Chromohalobacter sp. TVSP101 was isolated from solar salterns and screened for the production of extracellular halothermophilic protease. Identification of the bacterium was done based upon biochemical tests and the 16S rRNA sequence. The partially purified enzyme displayed maximum activity at pH 8 and required 4.5 M of NaCl for optimum proteolytic activity. In addition, this enzyme was thermophilic and active in broad range of temperature 60–80°C with 80°C as optimum. The Chromohalobacter sp. required 4 M NaCl for its optimum growth and protease secretion and no growth was observed below 1 M of NaCl. The initial pH of the medium for growth and enzyme production was in the range 7.0–8.0 with optimum at pH 7.2. Various cations at 1 mM concentration in the growth medium had no significant effect in enhancing the growth and enzyme production but 0.5 M MgCl₂ concentration enhanced enzyme production. Casein or skim milk powder 1% (w/v) along with 1% peptone proved to be the best nitrogen sources for maximum biomass and enzyme production. The carbon sources glucose and glycerol repressed the protease secretion. Immobilization of whole cells in absence of NaCl proved to be useful for continuous production of halophilic protease.     

Trichloroethylene degradation by cells of a methane-utilizing bacterium,Methylocystis sp. M,immobilized in calcium alginate

When Methylocystis sp. M cells were immobilized in calcium alginate, the resulting cell beads showed optimum trichloroethylene (TCE) degradation activity at pH 7.0 and 35°C. In comparison with free cells, the immobilized cells were more stable at low pH, and to some extent, at higher temperatures. Studies on the kinetics and the influence of cell density suggest that oxygen permeation was a rate-limiting step. Investigation of the storage stability and the optimum concentration of dissolved oxygen revealed that the TCE degradability was greater under anaerobic than aerobic conditions. Although a toxic effect caused by TCE was observed, methane seemed to restore activity, suggesting that the development of a two-step reactor system might be advantageous. The finding that the immobilized cells showed TCE degradation activity in actual groundwater suggests that TCE bioremediation could be achieved through the use of bioreactors with such cells.     

Covalent immobilization of alkaline proteinase on amino-functionalized magnetic nanoparticles and application in soy protein hydrolysis

Magnetic nanoparticles (MNPs) were synthesized and surface modified with (3-Aminopropyl)triethoxysilane (APTES). The alkaline proteinase (AP) was covalently immobilized on the APTES-modified MNPs through glutaraldehyde linkage. The resulting AP-loaded MNPs have an average size of 84 nm in aqueous solution, and a magnetization of 40 emu/g, endowing the immobilized enzyme with excellent magnetic responsively and dispersity. The maximum amount of AP and catalytic activity immobilized 1.0 mg MNPs was 120 μg and 25.3 units, respectively. Immobilized AP showed maximum activity at pH 10.0 and 50°C. Compared with free enzyme, the immobilized AP exhibited better storage stability. Moreover, immobilized AP can be reused 10 times and still maintained about 50% of its initial activity. The degree of hydrolysis of soy protein hydrolysates for immobilized AP could reach 19.0%, which was closer to the value of free enzyme. The molecular weight (M.W.) analysis showed that the soy protein was hydrolyzed successfully into small peptides of two main fractions with an average M.W. of 742 and 2126 Da. This study indicated that the immobilized AP could be used to hydrolyze continuously soy protein for potential industry application. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 35: e2756, 2019.     

Studies on the activity and stability of immobilized horseradish peroxidase on poly(ethylene terephthalate) grafted acrylamide fiber

Having been activated with glutaraldehyde, modified poly(ethylene terephthalate) grafted acrylamide fiber was used for the immobilization of horseradish peroxidase (HRP). Both the free HRP and the immobilized HRP were characterized by determining the activity profile as a function of pH, temperature, thermal stability, effect of organic solvent and storage stability. The optimum pH values of the enzyme activity were found as 8 and 7 for the free HRP and the immobilized HRP respectively. The temperature profile of the free HRP and the immobilized HRP revealed a similar behaviour, although the immobilized HRP exhibited higher relative activity in the range from 50 to 60 °C. The immobilized HRP showed higher storage stability than the free HRP.     

Purification and stability characteristics of an alkaline serine protease from a newly isolated Haloalkaliphilic bacterium sp. AH-6

An alkaline protease secreting Haloalkaliphilic bacterium (Gene bank accession number EU118361) was isolated from the Saurashtra Coast in Western India. The alkaline protease was purified by a single step chromatography on phenyl sepharose 6 FF with 28% yield. The molecular mass was 40 kDa as judged by SDS-PAGE. The enzyme displayed catalysis and stability over pH 8–13, optimally at 9–11. It was stable with 0–4 M NaCl and required 150 mM NaCl for optimum catalysis at 37 °C; however, the salt requirement for optimal catalysis increased with temperature. While crude enzyme was active at 25–80 °C (optimum at 50 °C), the purified enzyme had temperature optimum at 37 °C, which shifted to 80 °C in the presence of 2 M NaCl. The NaCl not only shifted the temperature profile but also enhanced the substrate affinity of the enzyme as reflected by the increase in the catalytic constant (K _cat). The enzyme was also calcium dependent and with 2 mM Ca⁺², the activity reached to maximum at 50 °C. The crude enzyme was highly thermostable (37–90 °C); however, the purified enzyme lost its stability above 50 °C and its half life was enhanced by 30 and sevenfold at 60 °C with 1 M NaCl and 50 mM Ca⁺², respectively. The activity of the enzyme was inhibited by PMSF, indicating its serine type. While the activity was slightly enhanced by Tween-80 (0.2%) and Triton X-100 (0.05%), it marginally decreased with SDS. In addition, the enzyme was highly stable with oxidizing-reducing agents and commercial detergents and was affected by metal ions to varying extent. The study assumes significance due to the enzyme stability under the dual extremities of pH and salt coupled with moderate thermal tolerance. Besides, the facts emerged on the enzyme stability would add to the limited information on this enzyme from Haloalkaliphilic bacteria.     

Immobilization of Kluyveromyces marxianus cells containing inulinase activity in open pore gelatin matrix: 1. Preparation and enzymatic properties

Kluyveromyces marxianus cells with inulinase (2,1-β-d-fructan fructanohydrolase, EC 3.2.1.7) activity have been immobilized in open pore gelatin pellets with retention of > 90% of the original activity. The open pore gelatin pellets with entrapped yeast cells were obtained by selective leaching out of calcium alginate from the composite matrix, followed by crosslinking with glutaraldehyde. Enzymatic properties of the gelatin-entrapped cells were studied and compared with those of the free cells. The immobilization procedure did not alter the optimum pH of the enzymatic preparation; the optimum for both free and immobilized cells was pH 6.0. The optimum temperature of inulin hydrolysis was 10°C higher for immobilized cells. Activation energies for the reaction with the free and immobilized cells were calculated to be 6.35 and 2.26 kcal mol^?1, respectively. K_m values were 8 mM inulin for the free cells and 9.52 mM for the immobilized cells. The thermal stability of the enzyme was improved by immobilization. Free and immobilized cells showed fairly stable activities between pH 4 and 7, but free cell inulinase was more labile at pH values below 4 and above 7 compared to the immobilized form. There was no loss of enzyme activity of the immobilized cells on storage at 4°C for 30 days. Over the same period at room temperature only 6% of the original activity was lost.     

Properties of fibre-entrapped aspartase

Aspartase (l-aspartate ammonia lyase, EC 4.3.1.1) was extracted and purified from Escherichia intermedia cells. The enzyme was entrapped in cellulose triacetate porous fibres and the properties of the immobilized enzyme compared with those of the free enzyme. Similar behaviour was observed with regard to optimum pH, temperature, heat stability and kinetic constants. The stability of the entrapped enzyme was tested under operating conditions in a series of batch reactions. Good results were obtained for both the stability and the efficiency of the immobilized enzyme. The potential use of aspartase fibres for the production of l-aspartic acid is discussed.