Crystal structures of D-tagatose 3-epimerase from Pseudomonas cichorii and its complexes with D-tagatose and D-fructose

Pseudomonas cichoriiid-tagatose 3-epimerase (P. cichoriid-TE) can efficiently catalyze the epimerization of not only d-tagatose to d-sorbose, but also d-fructose to d-psicose, and is used for the production of d-psicose from d-fructose. The crystal structures of P. cichoriid-TE alone and in complexes with d-tagatose and d-fructose were determined at resolutions of 1.79, 2.28, and 2.06 Å, respectively. A subunit of P. cichoriid-TE adopts a (β/α)₈ barrel structure, and a metal ion (Mn²⁺) found in the active site is coordinated by Glu152, Asp185, His211, and Glu246 at the end of the β-barrel. P. cichoriid-TE forms a stable dimer to give a favorable accessible surface for substrate binding on the front side of the dimer. The simulated omit map indicates that O2 and O3 of d-tagatose and/or d-fructose coordinate Mn²⁺, and that C3-O3 is located between carboxyl groups of Glu152 and Glu246, supporting the previously proposed mechanism of deprotonation/protonation at C3 by two Glu residues. Although the electron density is poor at the 4-, 5-, and 6-positions of the substrates, substrate-enzyme interactions can be deduced from the significant electron density at O6. The O6 possibly interacts with Cys66 via hydrogen bonding, whereas O4 and O5 in d-tagatose and O4 in d-fructose do not undergo hydrogen bonding to the enzyme and are in a hydrophobic environment created by Phe7, Trp15, Trp113, and Phe248. Due to the lack of specific interactions between the enzyme and its substrates at the 4- and 5-positions, P. cichoriid-TE loosely recognizes substrates in this region, allowing it to efficiently catalyze the epimerization of d-tagatose and d-fructose (C4 epimer of d-tagatose) as well. Furthermore, a C3-O3 proton-exchange mechanism for P. cichoriid-TE is suggested by X-ray structural analysis, providing a clear explanation for the regulation of the ionization state of Glu152 and Glu246.     

Immobilization of α-galactosidase on galactose-containing polymeric beads

Industrial application of α-galactosidase requires efficient methods to immobilize the enzyme, yielding a biocatalyst with high activity and stability compared to free enzyme. An α-galactosidase from tomato fruit was immobilized on galactose-containing polymeric beads. The immobilized enzyme exhibited an activity of 0.62 U/g of support and activity yield of 46%. The optimum pH and temperature for the activity of both free and immobilized enzymes were found as pH 4.0 and 37 °C, respectively. Immobilized α-galactosidase was more stable than free enzyme in the range of pH 4.0–6.0 and more than 85% of the initial activity was recovered. The decrease in reaction rate of the immobilized enzyme at temperatures above 37 °C was much slower than that of the free counterpart. The immobilized enzyme shows 53% activity at 60 °C while free enzyme decreases 33% at the same temperature. The immobilized enzyme retained 50% of its initial activity after 17 cycles of reuse at 37 °C. Under same storage conditions, the free enzyme lost about 71% of its initial activity over a period of 7 months, whereas the immobilized enzyme lost about only 47% of its initial activity over the same period. Operational stability of the immobilized enzyme was also studied and the operational half-life (t_1/2 was determined as 6.72 h for p-nitrophenyl α-d-galactopyranoside (PNPG) as substrate. The kinetic parameters were determined by using PNPG as substrate. The K_m and V_max values were measured as 1.07 mM and 0.01 U/mg for free enzyme and 0.89 mM and 0.1 U/mg for immobilized enzyme, respectively. The synthesis of the galactose-containing polymeric beads and the enzyme immobilization procedure are very simple and also easy to carry out.     

Synthesis of glyceryl ferulate by immobilized ferulic acid esterase

Glyceryl ferulate was synthesized by the condensation of ferulic acid with glycerol using Pectinase PL “Amano” from Aspergillus niger, which contained ferulic acid esterase, to improve the water-solubility of ferulic acid. The optimum reaction medium was glycerol/0.1 M acetate buffer, pH 4.0, (98:2 v/v). The enzyme immobilized onto Chitopearl BCW3003 exhibited the highest activity among the those immobilized onto various kinds of Chitopearl BCW resins. The optimum temperature for the immobilized enzyme was 50°C, and it could be reused at least five times without a significant loss in activity for the synthesis of glyceryl ferulate in batch reaction. Storage of the reaction mixture at 25°C improved the molar fraction of glyceryl ferulate relative to the dissolved ferulic residues.     

Immobilization and characterization of cellulase on concanavalin A (Con A)-layered calcium alginate beads

Cellulase has been immobilized on hybrid concanavalin A (Con A)-layered calcium alginate–starch beads. Immobilized cellulase retained about 82% of its activity. Con A was extracted from jack bean and the obtained crude protein was characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. The immobilized beads showed high mechanical and storage stability; immobilized cellulase retained 100% and 85% activity at 4°C and 30°C, respectively, over one month. The immobilized cellulase retained about 70% of its activity after five cycles of use. The immobilized cellulase retained 70% activity after 120-min exposure to 60°C, whereas the soluble form only retained about 20%, showing that immobilization improved thermal stability. Surface morphology and elemental analysis of immobilized cellulase were examined using scanning electron microscope equipped with energy-dispersive X-ray. Based on the enzyme stability and reuse, this method of immobilization is both convenient and cheap.     

Covalent immobilization of ω-transaminase from Vibrio fluvialis JS17 on chitosan beads

Chitosan beads were prepared by emulsion method and used for the immobilization of ω-transaminase of Vibrio fluvialis. The yield of enzyme immobilization (54.3%) and its residual activity (17.8%) were higher than those obtained with other commercial beads. ω-Transaminase was effectively immobilized on the chitosan beads at pH 6.0. The optimal pH of the immobilized enzyme was pH 9.0, which is the same as that of the free enzyme. The immobilized enzyme on chitosan beads retained ca. 77% of its conversion after five consecutive reactions with the 25 mM substrate, while the immobilized enzyme on Eupergit^® C retained 12%. Also, the immobilized ω-transaminase on chitosan bead retained 70% of initial activity when it's stored at 4 °C for 3.5 weeks. Addition of the co-factor, pyridoxal 5-phosphate (PLP), was needed to maintain the stability of the immobilized ω-transaminase.     

Repeated batch production of agar-oligosaccharides from agarose by an amberlite IRA-900 immobilized agarase system

The production of agar-oligosaccharides from agarose by free and immobilized agarase, obtained from a Pseudomonas aeruginosa AG LSL-11 was investigated and the activity, longevity and the operational stability of immobilized enzyme was compared with that of the free enzyme. The agar hydrolyzed products of free enzyme and immobilized enzyme were neoagarobiose, neoagarotetraose and neoagarohexaose as evidenced by LC-MS analysis. The immobilization of agarase was confirmed by SEM and also by the enzymatic transformation of agarose into agaroligosaccharides. The free agarase showed maximum activity at 40°C, whereas it’s immobilized counterpart showed maximum activity at 45oC, however, the optimum pH for both systems remained unchanged (pH 8.0). The relative activities of free agarase at pH 9.0 and 10.0 were 90 and 74%, respectively, whereas, the corresponding activities of the immobilized system were determined to be 97 and 90%. The stabilities of free agarase at pH 9.0 and 10.0 were 80 and 60% respectively, but for the immobilized system the respective residual activities were estimated to be 97 and 85%. Immobilized agarase appears to be more tolerant to high temperatures in terms of its activity and stability as it is compared to that of the free enzyme which retained 74 and 50.84% of relative activity at 55 and 60°C while, free agarase retained only 40 and 16.79% of its original activity. Furthermore, the immobilized agarase could be reused in batches efficiently for eight cycles, and could be stored for 3 months at 4°C as wet beads and for more than 6 months as dry beads.     

Immobilization of the cross-linked para-nitrobenzyl esterase of Bacillus subtilis aggregates onto magnetic beads

The para-nitrobenzyl esterase (PNBE), which was encoded by pnbA gene from Bacillus subtilis, was immobilized on amino-functionalized magnetic supports as cross-linked enzyme aggregates (CLEA). The maximum amount of PNBE-CLEA immobilized on the magnetic beads using glutaraldehyde as a coupling agent was 31.4 mg/g of beads with a 78% activity recovery after the immobilization. The performance of immobilized PNBE-CLEA was evaluated under various conditions. As compared to its free form, the optimal pH and temperature of PNBE-CLEA were 1 unit (pH 8.0) and 5 °C higher (45 °C), respectively. Under different temperature settings, the residual enzyme activity was highest for the PNBE-CLEA, followed by covalently fixed PNBE without further cross-linking and the free PNBE. During 40 days of storage pried, the PNBE-CLEA maintained more than 90% of its initial activity while the free PNBE maintained about 60% under the same condition. PNBE-CLEA also retained more than 80% activity after 30 reuses with 30 min of each reaction time, indicating stable reusability under aqueous medium.     

Improving of Catalase Stability Properties by Encapsulation in Alginate/Fe3O4 Magnetic Composite Beads for Enzymatic Removal of H2O2

The aim of this study was enhancing of stability properties of catalase enzyme by encapsulation in alginate/nanomagnetic beads. Amounts of carrier (10–100 mg) and enzyme concentrations (0.25–1.5 mg/mL) were analyzed to optimize immobilization conditions. Also, the optimum temperature (25–50°C), optimum pH (3.0–8.0), kinetic parameters, thermal stability (20–70°C), pH stability (4.0–9.0) operational stability (0–390 min), and reusability were investigated for characterization of the immobilized catalase system. The optimum pH levels of both free and immobilized catalase were 7.0. At the thermal stability studies, the magnetic catalase beads protected 90% activity, while free catalase maintained only 10% activity at 70°C. The thermal profile of magnetic catalase beads was spread over a large area. Similarly, this system indicated the improving of the pH stability. The reusability, which is especially important for industrial applications, was also determined. Thus, the activity analysis was done 50 times in succession. Catalase encapsulated magnetic alginate beads protected 83% activity after 50 cycles.     

Immobilization of levan fructotransferase for the production of di-fructose anhydride from levan

Levan fructotransferase (LFTase) from Arthrobacter ureafaciens K2032 was immobilized on various carriers of which Chitopearl BCW2501 beads showed the higher activity of 320 U g^–1 for the formation of di-fructose anhydride compounds. The immobilized enzyme retained about 60% of its initial activity after being used for 20 cycles.     

Immobilization and characterization of lipase from an indigenous Bacillus aryabhattai SE3-PB isolated from lipid-rich wastewater

Abstract

Extracellular lipase from an indigenous Bacillus aryabhattai SE3-PB was immobilized in alginate beads by entrapment method. After optimization of immobilization conditions, maximum immobilization efficiencies of 77%?±?1.53% and 75.99%?±?3.49% were recorded at optimum concentrations of 2% (w/v) sodium alginate and 0.2?M calcium chloride, respectively, for the entrapped enzyme. Biochemical properties of both free and immobilized lipase revealed no change in the optimum temperature and pH of both enzyme preparations, with maximum activity attained at 60?°C and 9.5, respectively. In comparison to free lipase, the immobilized enzyme exhibited improved stability over the studied pH range (8.5–9.5) and temperature (55–65?°C) when incubated for 3?h. Furthermore, the immobilized lipase showed enhanced enzyme-substrate affinity and higher catalytic efficiency when compared to soluble enzyme. The entrapped enzyme was also found to be more stable, retaining 61.51% and 49.44% of its original activity after being stored for 30 days at 4?°C and 25?°C, respectively. In addition, the insolubilized enzyme exhibited good reusability with 18.46% relative activity after being repeatedly used for six times. These findings suggest the efficient and sustainable use of the developed immobilized lipase for various biotechnological applications.     

Immobilization of thermostable α-galactosidase from Pycnoporus cinnabarinus on chitosan beads and its application to the hydrolysis of raffinose in beet sugar molasses

α-Galactosidase (EC 3.2.1.22) from Pycnoporus cinnabarinus was immobilized on chitosan beads, BCW 1000, and crosslinked chitosan beads, BCW 3000 and 3500, of three different sizes, which were untreated or previously treated with glutaraldehyde. The activity yields of the immobilized enzymes were between 25 to 45%, except for glutaraldehyde-untreated B BCW 1000. Leakage of the enzyme with increasing ionic strength was observed in glutaraldehyde-untreated BCW 1000 and 3000. The α-galactosidases immobilized on glutaraldehyde-treated BCW 3000 and 3500 were active at pH 3–6 and at 70–80°C, and stable between pH 3 and 9, and below 70°C. The immobilized α-galactosidase was continuously used for 30 days to hydrolyze raffinose in beet sugar molases.     

Immobilized CALB Catalyzed Transesterification of Soybean Oil and Phytosterol

Candida antarctica lipase B (CALB) was immobilized on Fe₃O₄/SiO_x-g-P(GMA) polymer carrier to catalyzed the transesterification of soybean oil and phytosterol. The enzyme loading of the obtained particles was 98.7 mg/g supports and the enzyme activity was 1226.5 U/g. The average particle size was 100.5?±?1.30 nm and the magnetization was 15.80 emu/g. The immobilized enzyme showed higher activities at a wider range of pH and temperatures. Its optimum reaction temperature was up to 50 °C; increased by 5 °C compared to the free enzyme. The obtained magnetic immobilized Fe₃O₄/SiO_x-g-P(GMA) lipase was nanoscale. First-grade soybean oils were used as a substrate. System pH was adjusted to 7.0. The optimal reaction temperature was 50 °C and the reaction time was 3 h. The phytosterol concentration of 5% and immobilized CALB of 2% were obtained. The conversion rate of transesterification reaction between soybean oil and phytosterol was 86.2%. The use of magnets can quickly separate the immobilized enzymes from the substrates. The relative activity of the immobilized enzymes was 83.0% when reused seven times. The prepared immobilized CALB can improve efficiently enzyme activity and reutilization.     

Inversion of sucrose with β-d-fructofuranosidase (invertase) immobilized on bead DEAHP-cellulose: Batch process

The inversion of sucrose with β-d-fructofuranosidase (EC 3.2.1.26) immobilized by an ionic bond on bead cellulose containing weak basic N,N-diethylamino-2-hydroxypropyl groups has been investigated. The immobilized enzyme is strongly bound at an ionic strength up to 0.1 M in the pH range 3–6. The amount adsorbed is proportional to porosity and to the exchange capacity of the ion exchange cellulose, reaching values up to 200 mg/g dry carrier, with an activity in 10% sucrose solution at 30°C, pH 5, >8000 μmol min^?1 g^?1. The inversion of sucrose with immobilized β-d-fructofuranosidase was carried out in a stirred reactor. The dependence of activity on pH (3–7), temperature (0–70°C) and concentration of the substrate (2–64 wt%) were determined, and the inversion was compared with that obtained using non-immobilized enzyme under similar conditions. The rate of inversion at low substrate concentration (2–19 wt%) was described by Michaelis-Menten kinetics.     

A feasible enzymatic process for D-tagatose production by an immobilized thermostable L-arabinose isomerase in a packed-bed bioreactor

To develop a feasible enzymatic process for d-tagatose production, a thermostable l-arabinose isomerase, Gali152, was immobilized in alginate, and the galactose isomerization reaction conditions were optimized. The pH and temperature for the maximal galactose isomerization reaction were pH 8.0 and 65 degrees C in the immobilized enzyme system and pH 7.5 and 60 degrees C in the free enzyme system. The presence of manganese ion enhanced galactose isomerization to tagatose in both the free and immobilized enzyme systems. The immobilized enzyme was more stable than the free enzyme at the same pH and temperature. Under stable conditions of pH 8.0 and 60 degrees C, the immobilized enzyme produced 58 g/L of tagatose from 100 g/L galactose in 90 h by batch reaction, whereas the free enzyme produced 37 g/L tagatose due to its lower stability. A packed-bed bioreactor with immobilized Gali152 in alginate beads produced 50 g/L tagatose from 100 g/L galactose in 168 h, with a productivity of 13.3 (g of tagatose)/(L-reactor.h) in continuous mode. The bioreactor produced 230 g/L tagatose from 500 g/L galactose in continuous recycling mode, with a productivity of 9.6 g/(L.h) and a conversion yield of 46%.     

Immobilized Sclerotinia sclerotiorum invertase to produce invert sugar syrup from industrial beet molasses by product

The fungus Sclerotinia sclerotiorum produces invertase activity during cultivation on many agroindustrial residues. The molasses induced invertase was purified by DEAE-cellulose chromatography. The molecular mass of the purified enzyme was estimated at 48 kDa. Optimal temperature was determined at 60 °C and thermal stability up to 65 °C. The enzyme was stable between pH 2.0 and 8.0; optimum pH was about 5.5. Apparent K_m and V_max for sucrose were estimated to be respectively 5.8 mM and 0.11 μmol/min. The invertase was activated by β-mercaptoethanol. Free enzyme exhibited 80 % of its original activity after two month’s storage at 4 °C and 50 % after 1 week at 25 °C. In order to investigate an industrial application, the enzyme was immobilized on alginate and examined for invert sugar production by molasses hydrolysis in a continuous bioreactor. The yield of immobilized invertase was about 78 % and the activity yield was 59 %. Interestingly the immobilized enzyme hydrolyzed beet molasses consuming nearly all sucrose. It retained all of its initial activity after being used for 4 cycles and about 65 % at the sixth cycle. Regarding productivity; 20 g/l of molasses by-product gave the best invert sugar production 46.21 g/day/100 g substrate related to optimal sucrose conversion of 41.6 %.     

Immobilization of phytase on epoxy-activated Sepabead EC-EP for the hydrolysis of soymilk phytate

In this work, an active phytase concentrated extract from soybean sprout was immobilized on a polymethacrylate-based polymer Sepabead EC-EP which is activated with epoxy groups. The immobilized enzyme exhibited an activity of 0.1 U/g of carrier and activity yield of 64.7%. The optimum temperature and pH for the activity of both free and immobilized enzymes were found as 60 °C and pH 5.0, respectively. The immobilized enzyme was more stable than free enzyme in the range of pH 3.0–8.0 and more than 70% of the original activity was recovered. Both the enzymes completely retained nearly about 84% of their original activity at 65 °C. The K_m and V_max values were measured as 5 mM and 0.63 U/mg for free enzyme and 12.5 mM and 0.71 U/mg for immobilized enzyme, respectively. Free and immobilized soybean sprout phytase enzymes were also used in the biodegradation of soymilk phytate. The immobilized enzyme hydrolysed 92.5% of soymilk phytate in 7 h at 60 °C, as compared with 98% hydrolysis observed for the native enzyme over the same period of time. The immobilization procedure on Sepabead EC-EP is very cheap and also easy to carry out, and the features of the immobilized enzyme are very attractive that the potential for practical application is considerable.     

Immobilization and stabilization of d-hydantoinase from Vigna angularis and its use in the production of N-carbamoyl-d-phenylglycine. Improvement of the reaction yield by allowing chemical racemization of the substrate

This work reports the immobilization of a multimeric d-hydantoinase (DHTase) from Vigna angularis (E.C. 3.5.2.2.) on agarose beads activated with glyoxyl groups aiming to improve its stability via multipoint covalent attachment. The final reduction with sodium borohydride resulted in a drop in enzyme activity that could be decreased by adding Zn²⁺ or Mg²⁺. The optimal preparation with high activity (58 % recovered activity) and stability (around 86-fold more stable than the free enzyme) was obtained by DHTase immobilization on glyoxyl agarose for 24 h at 25 °C and pH 10.05, and a borohydride reduction step in the presence of 10 mM Zn²⁺ (DHTase-Glx). The enzyme was almost fully immobilized on glyoxyl agarose (19.8 mg/g of support) when offering 20 mg/g. This immobilized biocatalyst was used to catalyze the hydrolysis of d,l-phenylhydantoin under substrate racemization conditions, which produced 99 % of N-carbamoyl-d-phenylglycine after 9 h reaction.     

Physico-Chemical Characterization of Watermelon Urease upon Immobilization in Alginate Beads

Watermelon (Citrullus vulgaris) urease was immobilized in 3.5% alginate leading to 72% immobilization. There was no leaching of the enzyme over a period of 15 days at 4°C. It continued to hydrolyse urea at a faster rate upto 90 min of incubation. The immobilized urease exhibited a shift of apparent pH optimum by one unit towards acidic side (from pH 8.0 to 7.0). The K_m was found to be 13.3 mM; 1.17 times higher than the soluble enzyme (11.4 mM). The beads were fairly stable upto 50°C and exhibited activity even at ?10°C. The enzyme was significantly activated by ME and it exhibited two peaks of activation; one at lower concentration and another at higher concentration. Time-dependent ureolysis in presence of ME progressed at a much elevated rate. Unlike soluble enzyme, which was inhibited at 200 mM urea, the immobilized enzyme was inhibited at 600 mM of urea and above, and about 47% activity was retained at 2000 mM urea. Moreover, the inhibition caused by high urea concentration was partially abolished by ME. The significance of the observations is discussed.     

Continuous chitosan hydrolyzate production by immobilized chitosanolytic enzyme from Enterobacter sp. G-1.

Chitosanolytic enzymes from Enterobacter sp. G-1 were immobilized on various carriers to continuously hydrolyze chitosan. Four different carriers were tested: FE-3901 (strong basic anion exchange resin, ionic binding), glutaraldehyde-treated FE-4612 (weak basic anion exchange resin, cross-linking), Chitopearl (chitosan beads), and alginate calcium. Glutaraldehyde-treated FE-4612 and Chitopearl immobilized more protein than the others. The enzyme immobilized on FE-3901 had the greatest activity. The activity of enzyme immobilized on FE-3901 decreased rapidly when exposed to a continuous flow of 1% chitosan. The enzyme immobilized with Chitopearl retained more than 50% of its original activity after 17 days, and the activity was fully restored by re-immobilization.     

Stabilization of immobilized cathepsin L in non-aqueous medium

A lysosomal cysteine protease cathepsin L (3.4.22.15) purified from goat brain has been immobilized in calcium alginate beads in the presence of BSA through entrapment. Most favorable conditions for the entrapment were standardized as 3.0%(w/v) alginate and 1.5%(w/v) calcium chloride. Comparing the properties of free and immobilized enzyme using Z-Phe-Arg-4mβNA as chromogenic substrate, it was found that the immobilized enzyme could retain～70% of the original activity after five successive batch reactions. Vis-à-vis the free enzyme, immobilization conferred high stability to the enzyme both in the acidic and alkaline range, the enzyme lost no activity up to 60°C (Temperature stability for free enzyme is only up to 50°C). The pH optima for the enzyme shifted from 6.2 to 6.6 on entrapment. The increase in activity and stability of the enzyme in immobilized form even in the presence of high concentration of DMSO and ethanol is surprising and may make it useful for catalyzing organic reactions like trans-esterification and trans-amidation.