Comparison of catalytic properties of free and immobilized cellobiase Novozym 188

The enzyme cellobiase from Novo was immobilized in controlled pore silica particles by covalent binding with the silane-glutaraldehyde method with protein and activity yields of 67 and 13.7%, respectively. The activity of the free enzyme (FE) and immobilized enzyme (IE) was determined with 2 g/L of cellobiose, from 40 to 75°C at pH 3.0–7.0 for FE and from 40 to 70°C at pH 2.2–7.0 for IE. At pH 4.8 the maximum specific activity for the FE and IE occurred at 65°C: 17.8 and 2.2 micromol of glucose/(min·mg of protein), respectively. For all temperatures the optimum pH observed for FE was 4.5 whereas for IE it was shifted to 3.5. The energy of activation was 11 kcal/mol for FE and 5 kcal/mol for IE at pH 4.5–5, showing apparent diffusional limitation for the latter. Thermal stability of the FE and IE was determined with 2 g/L of cellobiose (pH 4.8) at temperatures from 40 to 70°C for FE and 40 to 75°C for IE. Free cellobiase maintained its activity practically constant for 240 min at temperatures up to 55°C. The IE has shown higher stability, retaining its activity in the sametest up to 60°C. Half-life experimental results for FE were 14.1, 2.1, and 0.17 h at 60, 65, and 70°C, respectively, whereas IE at the same temperatures had half-lives of 245, 21.3, and 2.9 h. The energy of thermal deactivation was 80.6 k cal/mol for the free enzyme and 85.2 k cal/mol for the IE, suggesting stabilization by immobilization.     

Immobilization of Xylanase from <Emphasis Type="Italic">Bacillus pumilus</Emphasis> Strain MK001 and its Application in Production of Xylo-oligosaccharides

Xylanase from Bacillus pumilus strain MK001 was immobilized on different matrices following varied immobilization methods. Entrapment using gelatin (GE) (40.0%), physical adsorption on chitin (CH) (35.0%), ionic binding with Q-sepharose (Q-S) (45.0%), and covalent binding with HP-20 beads (42.0%) showed the maximum xylanase immobilization efficiency. The optimum pH of immobilized xylanase shifted up to 1.0 unit (pH 7.0) as compared to free enzyme (pH 6.0). The immobilized xylanase exhibited higher pH stability (up to 28.0%) in the alkaline pH range (7.0–10.0) as compared to free enzyme. Optimum temperature of immobilized xylanase was observed to be 8 °C higher (68.0 °C) than free enzyme (60.0 °C). The free xylanase retained 50.0% activity, whereas xylanase immobilized on HP-20, Q-S, CH, and GE retained 68.0, 64.0, 58.0, and 57.0% residual activity, respectively, after 3 h of incubation at 80.0 °C. The immobilized xylanase registered marginal increase and decrease in K _m and V _max values, respectively, as compared to free enzyme. The immobilized xylanase retained up to 70.0% of its initial hydrolysis activity after seven enzyme reaction cycles. The immobilized xylanase was found to produce higher levels of high-quality xylo-oligosaccharides from birchwood xylan, indicating its potential in the nutraceutical industry.     

Design and Properties of an Immobilization Enzyme System for Inulin Conversion

A commercial inulinase could convert inulin into fructose, which was optimized to be entrapped in the calcium alginate-gelatin beads with the immobilization yield of 86% for free inulinase activities. The optimum pH values and temperatures were 4.5 and 40 °C for the free enzyme and 5.0–5.5 and 45–50 °C for the immobilized enzyme. The kinetic parameters of V _max and K _m were 5.24 μmol/min and 57.6 mg/mL for the free inulinase and 4.32 μmol/min and 65.8 mg/mL for the immobilized inulinase, respectively. The immobilized enzyme retained 80% of its initial activities at 45 °C for 4 days, which could exhibit better thermal stability. The reuse of immobilized inulinase throughout the continuous batch operations was explored, which had better reusability of the immobilized biocatalyst. At the same time, the stability of immobilized enzyme in the continuous packed-bed bioreactor was estimated, which showed the better results and had its potential scale-up fructose production for inulin conversion.     

Covalent Immobilization of α-Galactosidase from <Emphasis Type="Italic">Penicillium griseoroseum</Emphasis> and its Application in Oligosaccharides Hydrolysis

Partially purified α-Galactosidase from Penicillium griseoroseum was immobilized onto modified silica using glutaraldehyde linkages. The effective activity of immobilized enzyme was 33%. Free and immobilized α-galactosidase showed optimal activity at 45 °C and pH values of 5 and 4, respectively. Immobilized α-galactosidase was more stable at higher temperatures and pH values. Immobilized α-galactosidase from P. griseoroseum maintained 100% activity after 24 h of incubation at 40 °C, while free enzyme showed only 32% activity under the same incubation conditions. Defatted soybean flour was treated with free and immobilized α-galactosidase in batch reactors. After 8 h of incubation, stachyose was completely hydrolyzed in both treatments. After 8 h of incubation, 39% and 70% of raffinose was hydrolyzed with free and immobilized α-galactosidase respectively. Immobilized α-galactosidase was reutilized eight times without any decrease in its activity.     

Physical and Covalent Immobilization of <Emphasis Type="Italic">Lipase</Emphasis> onto Amine Groups Bearing Thiol-Ene Photocured Coatings

In this study, amine groups containing thiol-ene photocurable coating material for lipase immobilization were prepared. Lipase (EC 3.1.1.3) from Candida rugosa was immobilized onto the photocured coatings by physical adsorption and glutaraldehyde-activated covalent bonding methods, respectively. The catalytic efficiency of the immobilized and free enzymes was determined for the hydrolysis of p-nitrophenyl palmitate and also for the synthesis of p-nitrophenyl linoleate. The storage stability and the reusability of the immobilized enzyme and the effect of temperature and pH on the catalytic activities were also investigated. The optimum pH for free lipase and physically immobilized lipase was determined as 7.0, while it was found as 7.5 for the covalent immobilization. After immobilization, the optimum temperature increased from 37 °C (free lipase) to 50–55 °C. In the end of 15 repeated cycles, covalently bounded enzyme retained 60 and 70 % of its initial activities for hydrolytic and synthetic assays, respectively. While the physically bounded enzyme retained only 56 % of its hydrolytic activity and 67 % of its synthetic activity in the same cycle period. In the case of hydrolysis V _max values slightly decreased after immobilization. For synthetic assay, the V _max value for the covalently immobilized lipase was found as same as free lipase while it decreased dramatically for the physically immobilized lipase. Physically immobilized enzyme was found to be superior over covalent bonding in terms of enzyme loading capacity and optimum temperature and exhibited comparable re-use values and storage stability. Thus, a fast, easy, and less laborious method for lipase immobilization was developed.     

Immobilization of Horseradish Peroxidase on Nonwoven Polyester Fabric Coated with Chitosan

The immobilization of horseradish peroxidase (HRP) on composite membrane has been investigated. This membrane was prepared by coating nonwoven polyester fabric with chitosan glutamate in the presence of glutraldehyde as a crosslinking agent. The physico-chemical properties of soluble and immobilized HRP were evaluated. The soluble HRP lost 90% of its activity after 4 weeks of storage at 4°C, whereas the immobilized enzyme retained 85% of its original activity at the same time. A reusability study of immobilized HRP showed that the enzyme retained 54% of its activity after 10 cycles of reuse. Soluble and immobilized HRP showed the same pH optima at pH 5.5. The immobilized enzyme had significant stability at different pH values, where it had maximum stability at pH 3.0 and 6.0. The kinetic properties indicated that the immobilized enzyme had more affinity toward substrates than soluble enzyme. The soluble and immobilized enzymes had temperature optima at 30 and 40°C and were stable up to 40 and 50°C, respectively. The stability of HRP against metal ion inactivation was improved after immobilization. Immobilized HRP exhibited high resistance to proteolysis by trypsin. The immobilized HRP was more resistant to inactivation induced by urea, Triton X-100, and organic solvents compared to its soluble counterpart. The immobilized HRP showed very high yield of immobilization and markedly high stabilization against several forms of denaturants that offer potential for several applications.     

Production of 2-O-α-glucopyranosyl l-ascorbic acid from ascorbic acid and β-cyclodextrin using immobilized cyclodextrin glycosyltransferase

Cyclodextrin glycosyltransferase (CGTase) isolated and purified from Paenibacillus sp. A11 was immobilized on various carriers by covalent linkage using bifunctional agent glutaraldehyde. Among tested carriers, alumina proved to be the best carrier for immobilization. The effects of several parameters on the activation of the support and on the immobilization of enzyme were optimized. The best preparation of immobilized CGTase retained 31.2% of its original activity. After immobilization, the enzymatic properties were investigated and compared with those of the free enzyme. The optimum pH of the immobilized CGTase was shifted from 6.0 to 7.0 whereas optimum temperature remained unaltered (60°C). Free and immobilized CGTase showed similar pH stability profile but the thermal stability of the immobilized CGTase was 20% higher. Kinetic data (K _M and V _max) for the free and immobilized enzymes were determined from the rate of β-CD formation and it was found that the immobilized form had higher K _M and lower V _max. The immobilized CGTase also exhibited higher stability when stored at both 4°C and 25°C for 2 months. The enzyme immobilized on alumina was further used in a batch production of 2-O-α-glucopyranosyl-l-ascorbic acid (AA-2G) from ascorbic acid and β-cyclodextrin. The yield of AA-2G was 2.92% and the immobilized CGTase retained its activity up to 74.4% of the initial catalytic activity after being used for 3 cycles. The immobilized CGTase would have a promising application in the production of various transglycosylated compounds and in the production of cyclodextrin by the hydrolysis of starch.     

Inulin Hydrolysis by Immobilized Inulinase on Functionalized Magnetic Nanoparticles Using Soy Protein Isolate and Bovine Serum Albumin

Inulin hydrolysis was performed by inulinase from Aspergillus niger covalently immobilized on magnetite nanoparticles (Fe₃O₄) covered with soy protein isolate (Fe₃O₄/SPI) functionalized by bovine serum albumin (Fe₃O₄/SPI/BSA) nanoparticles as a new bio‐functional carrier. The specific activity and protein content of the immobilized enzyme were 25.99 U/mg and 3.52 mg/mL, respectively, with 80% enzyme loading. The immobilized inulinase showed maximum activity at 45 °C, which is 5 °C higher than the optimum temperature of the free enzyme. Also, the optimum pH of the immobilized enzyme shifted from 6 to 5.5, which is more acidic compared to that of the free enzyme. The K_m value of immobilized inulinase decreased to 2.03 mg/mL. Thermal stability increased considerably at 65 and 75 °C, and a 5.13‐fold rise was detected in the enzyme half‐life at 75 °C after immobilization. Moreover, 80% of initial activity of immobilized inulinase remained after 10 cycles of hydrolysis.     

Chemical physics: The standing of a mature discipline

Background

The use of immobilized enzymes for catalyzing various biotransformations is now a widely used approach. In recent years, cross-linked enzyme aggregates (CLEAs) have emerged as a novel and versatile biocatalyst design. The present work deals with the preparation of a CLEA from a commercial preparation, Pectinex? Ultra SP-L, which contains pectinase, xylanase and cellulase activities. The CLEA obtained could be used for any of the enzyme activities. The CLEA was characterized in terms of kinetic parameters, thermal stability and reusability in the context of all the three enzyme activities.

Results

Complete precipitation of the three enzyme activities was obtained with n-propanol. When resulting precipitates were subjected to cross-linking with 5 mM glutaraldehyde, the three activities initially present (pectinase, xylanase and cellulase) were completely retained after cross-linking. The V_max/K_m values were increased from 11, 75 and 16 to 14, 80 and 19 in case of pectinase, xylanase and cellulase activities respectively. The thermal stability was studied at 50°C, 60°C and 70°C for pectinase, xylanase and cellulase respectively. Half-lives were improved from 17, 22 and 32 minutes to 180, 82 and 91 minutes for pectinase, xylanase and cellulase respectively. All three of the enzymes in CLEA could be reused three times without any loss of activity.

Conclusion

A single multipurpose biocatalyst has been designed which can be used for carrying out three different and independent reactions; 1) hydrolysis of pectin, 2) hydrolysis of xylan and 3) hydrolysis of cellulose. The preparation is more stable at higher temperatures as compared to the free enzymes.     

Production of l-DOPA by tyrosinase immobilized on modified polystyrene

Mushroom tyrosinase was immobilized on modified polystyrene—polyaminostyrene (PSNH) and polymethylchloridestyrene (PSCL)—to produce l-DOPA from l-tyrosine. Glutaraldehyde was used as an activating agent for the PSNH to immobilize the tyrosinase and 10% (w/v) glutaraldehyde was optimal in conferring the highest specific activity (11.96 U/g) to the PSNH. Methylchloride on the PSCL was directly linked with the tyrosinase, and 1.5 mmol of Cl/g was optimal in attaining the specific activity of 17.0 U/g. The temperature and optimal acidity were, respectively, 60°C and pH 5.5 for the PSNH, and 70°C and pH 3.0 for the PSCL. In a 50-mL batch reactor working over 36 h, the l-DOPA production rate at 30°C was 1.44 mg/(L·h) for the PSNH and 2.33 mg/(L·h) for the PSCL. The production rate over 36 h was 3.86 mg/(L·h) for the PSNH at 60°C and 5.54 mg/(L·h) for the PSCL at 70°C. Both of the immobilized enzymes showed a remarkable stability with almost no change in activity after being stored wet. The operational stability study indicated a 22.4% reduction in l-DOPA production for the PSNH and an 8.63% reduction for the PSCL over seven runs (each run was for 144h at 30°C) when the immobilized enzymes were used under turnover conditions. The immobilized tyrosinase was more stable on the PSCL than on the PSNH.     

Lactose Hydrolysis by β-Galactosidase Covalently Immobilized to Thermally Stable Biopolymers

Lactose has been hydrolyzed using covalently immobilized β-galactosidase on thermally stable carrageenan coated with chitosan (hydrogel). The hydrogel’s mode of interaction was proven by Fourier transform infrared spectroscopy, differential scanning calorimetry (DSC), and Schiff’s base formation. The DSC thermogram proved the formation of a strong polyelectrolyte complex between carrageenan and chitosan followed by glutaraldehyde as they formed one single peak. The modification of carrageenan improved the gel’s thermal stability in solutions from 35 °C to 95 °C. The hydrogel has been proven to be efficient for β-galactosidase immobilization where 11 U/g wet gel was immobilized with 50% enzyme loading capacity. Activity and stability of free and immobilized β-galactosidase towards pH and temperature showed marked shifts in their optimum pH from 4.5–5 to 5–5.5 and temperature from 50 °C to 45–55 °C after immobilization, which reveals higher catalytic activity and reasonable stability at wider pHs and temperatures. The apparent K _m of the immobilized enzyme increased from 13.2 to 125 mM, whereas the V _max increased from 3.2 to 6.6 μmol/min compared to the free enzyme, respectively. The free and immobilized enzymes showed lactose conversion of 87% and 70% at 7 h, respectively. The operational stability showed 97% retention of the enzyme activity after 15 uses, which demonstrates that the covalently immobilized enzyme is unlikely to leach. The new carrier could be suitable for immobilization of other industrial enzymes.     

Enzymatic Decolorization of Anthraquinone and Diazo Dyes Using Horseradish Peroxidase Enzyme Immobilized onto Various Polysulfone Supports

In this study, covalent immobilization of the horseradish peroxidase (HRP) onto various polysulfone supports was investigated. For this purpose, different polysulfones were methacrylated with methacryloyl chloride, and then, nonwoven fabric samples were coated by using solutions of these methacrylated polysulfones. Finally, support materials were immersed into aquatic solution of HRP enzyme for covalent immobilization. Structural analysis of enzyme immobilization onto various polysulfones was confirmed with Fourier transform infrared spectroscopy, atomic force microscopy, and proton nuclear magnetic resonance spectroscopy. Decolorization of textile diazo (Acid Black 1) and anthraquinone (Reactive Blue 19) dyes was investigated by UV–visible spectrophotometer. Covalently immobilized enzyme has been used seven times in freshly prepared dye solutions through 63 days. Dye decolorization performance of the immobilized systems was observed that still remained high (70 %) after reusing three times. Enzyme activities of immobilized systems were determined and compared to free enzyme activity at different conditions (pH, temperature, thermal stability, storage stability). Enzyme activities of immobilized systems and free enzyme were also investigated at the different temperatures and effects of temperature and thermal resistance for different incubation time at 50 °C. In addition, storage activity of free and immobilized enzymes was determined at 4 °C at different incubation days.     

Highly Efficient Method Towards In Situ Immobilization of Invertase Using Cryogelation

A novel method was developed for the immobilization of Saccharomyces cerevisiae invertase within supermacroporous polyacrylamide cryogel and was used to produce invert sugar. First, the cross-linking of invertase with soluble polyglutaraldehyde (PGA) was carried out prior to immobilization in order to increase the bulkiness of invertase and thus preventing the leakage of the cross-linked enzyme after immobilization by entrapment. And then, in situ immobilization of PGA cross-linked invertase within cryogel synthesis was achieved by free radical polymerization in semi-frozen state. The method resulted in 100 % immobilization and 74 % activity yields. The immobilized invertase retained all the initial activity for 30 days and 30 batch reactions. Immobilization had no effect on optimum temperature and it was 60 °C for both free and immobilized enzyme. However, optimum pH was affected upon immobilization. Optimum pH values for free and immobilized enzyme were 4.5 and 5.0, respectively. The immobilized enzyme was more stable than the free enzyme at high pH and temperatures. The kinetic parameters for free and immobilized invertase were also determined. The newly developed method is simple yet effective and could be used for the immobilization of some other enzymes and microorganisms.     

Bamboo (<Emphasis Type="Italic">Phyllostachys pubescens</Emphasis>) as a Natural Support for Neutral Protease Immobilization

Lignin polymers in bamboo (Phyllostachys pubescens) were decomposed into polyphenols at high temperatures and oxidized for the introduction of quinone groups from peroxidase extracted from bamboo shoots and catalysis of UV. According to the results of FT-IR spectra analysis, neutral proteases (NPs) can be immobilized on the oxidized lignin by covalent bonding formed by amine group and quinone group. The optimum condition for the immobilization of NPs on the bamboo bar was obtained at pH 7.0, 40 °C, and duration of 4 h; the amount of immobilized enzyme was up to 5 mg g^?1 bamboo bar. The optimal pH for both free NP (FNP) and INP was approximately 7.0, and the maximum activity of INP was determined at 60 °C, whereas FNP presented maximum activity at 50 °C. The K_m values of INP and FNP were determined as 0.773 and 0.843 mg ml^?1, respectively; INP showed a lower K_m value and V_max, than FNP, which demonstrated that INP presented higher affinity to substrate. Compared to FNP, INP showed broader thermal and storage stability under the same trial condition. With respect to cost, INP presented considerable recycling efficiency for up to six consecutive cycles.     

Effect of the Presence of Surfactants and Immobilization Conditions on Catalysts’ Properties of <Emphasis Type="Italic">Rhizomucor miehei</Emphasis> Lipase onto Chitosan

Lipase from Rhizomucor miehei (RML) was immobilized onto chitosan support in the presence of some surfactants added at low levels using two different strategies. In the first approach, the enzyme was immobilized in the presence of surfactants on chitosan supports previously functionalized with glutaraldehyde. In the second one, after prior enzyme adsorption on chitosan beads in the presence of surfactants, the complex chitosan beads-enzyme was then cross-linked with glutaraldehyde. The effects of surfactant concentrations on the activities of free and immobilized RML were evaluated. Hexadecyltrimethylammonium bromide (CTAB) promoted an inhibition of enzyme activity while the nonionic surfactant Triton X-100 caused a slight increase in the catalytic activity of the free enzyme and the derivatives produced in both methods of immobilization. The best derivatives were achieved when the lipase was firstly adsorbed on chitosan beads at 4 °C for 1 h, 220 rpm followed by cross-link the complex chitosan beads-enzyme with glutaraldehyde 0.6% v.v^?1 at pH 7. The derivatives obtained under these conditions showed high catalytic activity and excellent thermal stability at 60° and 37 °C. The best derivative was also evaluated in the synthesis of two flavor esters namely methyl and ethyl butyrate. At non-optimized conditions, the maximum conversion yield for methyl butyrate was 89%, and for ethyl butyrate, the esterification yield was 92%. The results for both esterifications were similar to those obtained when the commercial enzyme Lipozyme® and free enzyme were used in the same reaction conditions and higher than the one achieved in the absence of the selected surfactant.     

Characterization of Sol-gel bioencapsulates for ester hydrolysis and synthesis

Candida rugosa lipase was entrapped in silica sol-gel particles prepared by hydrolysis of methyltrimethoxysilane and assayed by p-nitrophenyl palmitate hydrolysis, as a function of pH and temperature, giving pH optima of 7.8 (free enzyme) and 5.0–8.0 (immobilized enzyme). The optimum temperature for the immobilized enzyme (50–55°C) was 19°C higher than for the free enzyme. Thermal, operational, and storage stability were determined with n-butanol and bytyric acid, giving at 45°C a half-life 2.7 times greater for the immobilized enzyme; storage time was 21 d at room temperature. For ester synthesis, the optimum temperature was 47°C, and high esterification conversions were obtained under repeated batch cycles (half-life of 138 h).     

Immobilization of a Recombinant Esterase from <Emphasis Type="Italic">Lactobacillus plantarum</Emphasis> on Polypropylene Accurel MP1000

A recombinant esterase from Lactobacillus plantarum was immobilized on hydrophobic support polypropylene Accurel MP1000 by adsorption. Adsorption efficiency was 83%, and the immobilized protein was 12.4 mg/g of support. Esterase activity was determined using p-nitrophenyl butyrate as substrate, and highest activities were observed at 50 °C for immobilized enzyme and 30 °C for free enzyme extract. Concerning thermal stability, after enzyme incubation at 80 °C for 30 min, immobilized and free enzyme retained 91% and 56% of initial activity, respectively. Immobilized enzyme presented lower V _max and higher K _m than free enzyme. Protein was not released from the support, and esterase activity increased after 3 cycles of reuse.     

Continuous Production of Isomalto-oligosaccharides by Thermo-inactivated Cells of <Emphasis Type="Italic">Aspergillus niger</Emphasis> J2 with Coarse Perlite as an Immobilizing Material

The coarse perlite 40–80 mesh was selected as an immobilizing material and put into a packed bed reactor (PBR) to continuously convert maltose to isomalto-oligosaccharides (IMOs). The PBR was prepared by mixing the thermo-inactivated cells (TIC) from Aspergillus niger J2 strain with the coarse perlite, then the mixture was put into an overpressure-resistant column. Compared with diatomite 40–80 mesh and thin perlite 80–120 mesh in PBR, coarse perlite was chosen as the best filtration aid, when the ratio of coarse perlite versus TIC was 1:1. The thermal and pH stability of the free and immobilized TIC and the optimum conditions for the transglycosylation reactions were determined. The results show that approximately 75 and 82% and 87 and 91% of α-glucosidase activity were reserved for free and immobilized TIC at temperatures from 30 to 60 °C and pH from 3.00 to 7.00 for 12 h, respectively. With 30% malt syrup under the conditions of 50 °C and pH 4.00, a mini-scale packed bed reactor (Mi-PBR) and medium-scale packed bed reactor (Me-PBR) could continuously produce IMO over 25 and 34 days with the yield of effective IMO (eIMO) ≥?35% and total IMO (tIMO) ≥?50%, respectively. The strategy of mixing the coarse perlite with TIC in PBR is a novel approach to continuously produce IMO and has great application potential in industry.     

Covalent Immobilization of β-Glucosidase on Magnetic Particles for Lignocellulose Hydrolysis

β-Glucosidase hydrolyzes cellobiose to glucose and is an important enzyme in the consortium used for hydrolysis of cellulosic and lignocellulosic feedstocks. In the present work, β-glucosidase was covalently immobilized on non-porous magnetic particles to enable re-use of the enzyme. It was found that particles activated with cyanuric chloride and polyglutaraldehyde gave the highest bead-related immobilized enzyme activity when tested with p-nitrophenyl-β-D-glucopyranoside (104.7 and 82.2 U/g particles, respectively). Furthermore, the purified β-glucosidase preparation from Megazyme gave higher bead-related enzyme activities compared to Novozym 188 (79.0 and 9.8 U/g particles, respectively). A significant improvement in thermal stability was observed for immobilized enzyme compared to free enzyme; after 5 h (at 65 °C), 36 % of activity remained for the former, while there was no activity in the latter. The performance and recyclability of immobilized β-glucosidase on more complex substrate (pretreated spruce) was also studied. It was shown that adding immobilized β-glucosidase (16 U/g dry matter) to free cellulases (8 FPU/g dry matter) increased the hydrolysis yield of pretreated spruce from ca. 44 % to ca. 65 %. In addition, it was possible to re-use the immobilized β-glucosidase in the spruce and retain activity for at least four cycles. The immobilized enzyme thus shows promise for lignocellulose hydrolysis.     

Screening and Immobilization <Emphasis Type="Italic">Burkholderia</Emphasis> sp. GXU56 Lipase for Enantioselective Resolution of (<Emphasis Type="Italic">R</Emphasis>,<Emphasis Type="Italic">S</Emphasis>)-Methyl Mandelate

Microorganisms producing lipase were isolated from soil and sewage samples and screened for enantioselective resolution of (R,S)-methyl mandelate to (R)-mandelic acid. A strain designated as GXU56 was obtained and identified as Burkholderia sp. Preparing immobilized GXU56 lipase by simple adsorption on octyl sepharose CL-4B, the optimum temperature was shifted from 40 °C (free lipase) to 50 °C (immobilized lipase), and the optimum pH was shifted from 8.0 (free lipase) to 7.2 (immobilized lipase). The immobilized enzyme displayed excellent stability in the pH range of 5.0–8.0, at the temperatures below 50 °C and in organic solvents compared with free enzyme. Enantioselectivity ratio for (R)-mandelic acid (E) was dramatically improved from 29.2 to more than 300 by applying immobilized lipase in the resolution of (R,S)-methyl mandelate. After five cycles of use of immobilized lipase, conversion and enantiomeric excess of (R)-mandelic acid were 34.5% and 98.5%, respectively, with enantioselectivity ratio for (R)-mandelic acid (E) of 230. Thus, octyl-sepharose-immobilized GXU56 lipase can be used as a bio-resolution reagent for producing (R)-mandelic acid.