Resolution of (±)-menthol by immobilized Candida rugosa lipase on superparamagnetic nanoparticles

Lauric acid-stabilized magnetic particles were prepared by coprecipitation in the presence of lauric acid and used for the covalent immobilization of Candida rugosa lipase via carbodiimide activation. Size analysis by transmission electron microscopy (TEM) and measurement of magnetization curves revealed that the immobilized lipase was superparamagnetic. Resolution of (±)-menthol was performed by the immobilized lipase-catalyzed enantioselective esterification with propionic anhydride. Effects of various reaction parameters, such as enzyme load, solvents, water activity, substrate concentration, reaction time and temperature, on the conversion as well as enantioselectivity were investigated. As a result, (−)-menthyl propionate with a yield higher than 96% and over 88% enantiomeric excess of products was obtained. Better conversion and enantioselectivity could be expected for the immobilized lipase-catalyzed reaction performed at 30 °C for 2.5 h with 0.2 mol/l of (±)-menthol. Hexane was found to be the most suitable solvent, and the activity as well as enantioselectivity of the immobilized lipase decreased gradually with increasing water activity. Good durability of the immobilized lipase to catalyze the resolution of (±)-menthol was also observed.     

Alcoholysis of salicornia oil using free and covalently bound lipase onto chitosan beads

Porcine pancreatic lipase was immobilized on chitosan by covalent binding and retention of its activity was examined. The activities of free and immobilized lipase were determined using olive oil as substrate. The free and immobilized enzymes showed pH 9 as optimum and retained 50% of activity after five cycles. When the substrate concentration was kept constant and enzyme concentration was varied, the K_m and V_max were observed to be 4.0 × 10⁻⁷ and 0.32, and 3.32 × 10⁻⁷ and 0.32, respectively, for free and covalently bound enzyme. This indicates that there is no possibility of conformational change during immobilization. Immobilized enzyme showed improved thermal and storage stability. Alcoholysis of salicornia oil, mediated by free and immobilized lipase, was carried out at 25 °C using methanol in hexane and acetone. Free and immobilized enzyme in hexane produced, respectively, 45% and 55% of fatty acid methyl ester after 12 h.     

Catalytic efficiency of immobilized glucose isomerase in isomerization of glucose to fructose

Glucose isomerase (GI) from Streptomycesrubiginosus was immobilized covalently onto Eupergit C 250 L made by copolymerization of N,N-methylene-bis-methacrylamide, glycidyl methacrylate, allyl glycidyl ether and methacrylamide. The catalytic efficiency of immobilized GI in isomerization of glucose to fructose was found as three fold higher than that of free GI. The residual activity of immobilized GI after 18 reuses in a batch type stirred reactor was about 85% of its initial activity. The thermal stability of immobilized GI was almost same with that of the free GI at 60 °C for 18 h preincubation time. The residual activities of immobilized GI when stored at 5 °C and 25 °C for four weeks were 72% and 69% of the initial activity, respectively. However, free GI retained 88% and 78% of its initial activity at 5 °C and 25 °C upon four weeks storage, respectively. Thus, the use of Eupergit C 250 L immobilized GI instead of free GI is suggested in enzymatic isomerization of glucose to fructose.     

Effect of Treatment with Compressed Propane on Lipases Hydrolytic Activity

The objective of this work was to assess the influence of compressed propane treatment on the hydrolytic activity of three lipases (Amano PS, Amano AY30, and a non-commercial lipase from Yarrowia lipolytica) free, resuspended, and in immobilized form. To evaluate the effect of process variables on the lipase activity, a semi-factorial experimental design with two levels and four variables was employed for free and immobilized lipases and a full 2² experimental design was carried out for lipases in solution. The residual activity was defined as the ratio of lipase activity before and after treatment with pressurized propane. For free and immobilized lipases, an enhancement in residual lipase activity in most of the experimental conditions investigated was observed. In the case of resuspended lipases, it is shown that enzyme kinetics is sensitive to treatment with compressed propane resulting in remarkable gains and losses of enzyme activity. In a general way, the results showed that the enzyme activity changes significantly depending on the enzyme, the presentation form, and the experimental conditions investigated, allowing the selection of operational conditions in terms of temperature, pressure, exposure time, and depressurization rate for advantageous application of these biocatalysts in hydrolysis reactions.     

Lipase Mediated Synthesis of Low Molecular Weight Flavor Esters

Screening 27 commercial lipases showed that enzymes from Candida cylindracea, Pseudomonas fluorescens and Mucor miehei (immobilized) promoted synthesis of selected low molecular weight esters in nonaqueous systems. Maximum production after 24 hr incubation was obtained with substrate concentrations of 0.05 mol/L for isopentyl acetate, 0.2 mol/L for ethyl butyrate and 0.3 mol/L for isopentyl butyrate. Yield of butyl butryate was almost 100% at acid substrate greater than 0.2 mol/L. Substrate inhibition was observed with P. fluorescens lipase but not with C. cylindracea or M. miehei lipases, up to 1 mol/L. Hexane, octane and decane could be used as reaction media except for ethyl butyrate synthesis where hexane was the medium of choice. Poor synthesis was achieved when methylene chloride was used.     

Immobilization of pepsin on chitosan beads

In this study, chitosan beads were prepared by using a cross-linking agent and the resulting beads were employed in immobilization process. Studies on free and immobilized pepsin systems for determination of optimum temperature, optimum pH, thermal stability, pH stability, operational stability, storage stability and kinetic parameters were carried out. The optimum temperature interval for free pepsin and immobilized pepsin were 30–40 and 40–50 °C, respectively. Free and immobilized pepsin showed higher activity at pH 2.0–4.0. Apparent K_m = 12.0 g L⁻¹ haemoglobin (1.56 mM tyrosine) and V_max = 5220 μmol (mg protein min)⁻¹ values were obtained for free pepsin, while apparent K_m = 20.0 g L⁻¹ haemoglobin (2.16 mM tyrosine) and V_max = 2780 μmol (mg protein min)⁻¹ values were obtained for immobilized pepsin. Thermal stability and storage stability of immobilized pepsin were higher than that of free pepsin. Milk clotting activity was used for evaluation of the applicability of pepsin immobilization to industrial process. Optimum milk clotting temperature was found as 40 °C for free pepsin and 50 °C for immobilized pepsin.     

Effect of reaction rate on converted products from wheat germ oil by immobilized lipase ethanolysis

Monoglycerides and diglycerides containing function of wheat germ oil were to produce and to assess the influence of various conditions on the ethanolysis activities of immobilized lipases. Immobilized lipases like lipozyme (Thermonuces lanuginose immobilized on silica gel and Rhizomucor miehei immobilized on an ion exchange resin were used for enzymatic ethanolysis. Ethanolysis was carried out in different processes (non-pressured and pressured system) to compare the reaction rate and yield. For immobilized lipase, the optimal condition was found at 1.0 of ethanol mol ratio, temperature of 60°C, and lipases amount of 4% in non-pressured system. However, in pressured system, the optimal temperature was found at 50°C. The enzyme activity was changed depending on the enzyme source, reaction time, pressure, and temperature. Changing experimental parameters (temperature, ethanol mol ratio, enzyme amount, and reaction time) affecting wheat germ oil ethanolysis reaction, the optimal reaction conditions were established.     

Preparation and Properties of Rhizopus oryzae Lipase Immobilized Using an Adsorption-Crosslinking Method

The aim was to develop an enzyme immobilization method for Rhizopus oryzae lipase to improve its acidolysis activity and stability. Lipase was adsorbed into NKA-9 resin and then crosslinked with glutaraldehyde as a crosslinker. The optimum conditions obtained using the response surface methodology were as follows: 34.6 mg lipase/100 mg support, 4.1 h of adsorption time, 45°C, pH 6.8, 0.5% glutaraldehyde concentration, and 2.5 h of crosslinking time. The acidolysis activity of the immobilized lipase was 31.78%, the free lipase activity was only 11.02%. The immobilized lipase showed better performance, such as higher acidolysis activity, better pH tolerance and temperature stability, enhanced storage stability, and improved reusability.     

Biochemical characterisation and application of lipases produced by Aspergillus sp. on solid‐state fermentation using three substrates

Lipase from Aspergillus sp. obtained by solid‐state fermentation (SSF) on wheat bran (LWB), soybean bran (LSB) and soybean bran combined with sugarcane bagasse (LSBBC) were 67.5, 58 and 57.3 U of crude lipase per gram substrate, respectively. The optimum pH of activity and stability of the LWB was between 8 and 9, and the optimum temperature of activity and stability was 50 °C and up to 60 °C, respectively. The LSB and LSBBC showed two peaks of optimum pH (4 and 6) and optimal values of temperature and stability at 50 °C. The LSB was stable in the pH range of 6–7, while LSBBC in the range of pH 4–7. All the enzymes show activities on p‐nitrophenyl esters (butyrate, laurate and palmitate). LWB stood out either on the hydrolysis of sunflower oil, presenting 66.1% of the activity over commercial lipase and on the esterification of oleic acid and ethanol, surpassing the activities of the commercial lipases studied. The thin layer chromatography showed that LWB and LSB have produced ethyl esters from corn oil, while LWB produced it from sunflower oil.     

Production of n − 3 polyunsaturated fatty acid concentrate from sardine oil by lipase-catalyzed hydrolysis

The production of n − 3 polyunsaturated fatty acids (n − 3 PUFAs) concentrate from oil extracted from Pacific sardines (Sardinops sagax) was studied using lipase-catalyzed hydrolysis. Commercially available microbial lipases, from Candida Rugosa (CR), Candida cylindracea (CC), Mucor javanicus (MJ), and Aspergillus niger (AN) were used for enzymatic hydrolyses with extracted sardine oil, run at 37 °C with constant stirring for 1.5, 3, 6, and 9 h. Fatty acid composition analysis by gas chromatography showed that the refined unhydrolyzed oil contained 26.86% of eicosapentaenoic acid (EPA) and 13.62% of docosahexaenoic acid (DHA) (wt/wt%). CR lipase was the most effective in concentrating n − 3 PUFA. Hydrolysis with 250 U CR lipase increased EPA concentration to a relatively constant level of 33.74% after 1.5 h. DHA levels were also significantly increased from 13.62% to 29.94% with 500 U after 9 h. Compared to CR and CC lipases, MJ and AN lipases resulted in low n − 3 PUFA concentration. Triacylglycerol levels decreased significantly as reaction time progressed.     

Purification and partial characterization of psychrotrophic Serratia marcescens lipase

Serratia marcescens isolated from raw milk was found to produce extracellular lipase. The growth of this organism could contribute to flavor defects in milk and dairy products. Serratia marcescens was streaked onto spirit blue agar medium, and lipolytic activity was detected after 6 h at 30 degrees C and after 12 h at 6 degrees C. The extracellular crude lipase was collected after inoculation of the organism into nutrient broth and then into skim milk. The crude lipase was purified to homogeneity by ion-exchange chromatography and gel filtration. The purified lipase had a final recovered activity of 45.42%. Its molecular mass was estimated by SDS-PAGE assay to be 52 kDa. The purified lipase was characterized; the optimum pH was likely between 8 and 9 and showed about 70% of its activity at pH 6.6. The enzyme was very stable at pH 8 and lost about 30% of its activity after holding for 24 h at 4 degrees C in buffer of pH 6.6. The optimum temperature was observed at 37 degrees C and exhibited high activity at 5 degrees C. The thermal inactivation of S. marcescens lipase was more obvious at 80 degrees C; it retained about 15% of its original activity at 80 degrees C and was completely inactivated after heating at 90 degrees C for 5 min. Under optimum conditions, activity of the enzyme was maximum after 6 min. The Michaelis-Menten constant was 1.35 mM on tributyrin. The enzyme was inhibited by a concentration more than 6.25mM. Purified lipase was not as heat-stable as other lipases from psychrotrophs, but it retained high activity at 5 degrees C. At pH 6.6, the pH of milk, purified lipase showed some activity and stability. Also, the organism demonstrated lipolytic activity at 6 degrees C after 12 h. Therefore, S. marcescens and its lipase were considered to cause flavor impairment during cold storage of milk and dairy products.     

Properties of Rhodotorula gracilis D-amino acid oxidase immobilized on magnetic beads through his-tag

D-amino acid oxidase catalyzes one of the key steps in the production of semisynthetic cephalosporins. We expressed and purified recombinant Rhodotorula gracilis D-amino acid oxidase with C-terminal his-tags. This engineered enzyme was immobilized onto Ni(2+)-chelated nitrilotriacetic acid magnetic beads through the interaction between his-tag and Ni(2+). The kinetic constants, storage properties, and the reusability of the immobilized d-amino acid oxidase were determined. The effects of temperature, pH, and hydrogen peroxide on the activity of immobilized d-amino acid oxidase were also studied. The highest activity recovery was 75%. Thermal stability was improved after immobilization; the relative activity of the immobilized enzyme was 56% whereas the free enzyme was completely inactivated after incubation at 50 degrees C for 1 h. In the presence of 10 mM hydrogen peroxide, the immobilized enzyme did not show a rapid loss of activity during the first 2 h of incubation, which was observed in the case of the free enzyme; the residual activity of the immobilized enzyme after 9 h was 72% compared with 22% of the free form. The long-term storage stability was improved; the residual activity of the immobilized enzyme was 74% compared with 20% of the free enzyme when stored at room temperature for 10 d. The immobilized form retained 37% of its initial activity after 20 consecutive reaction cycles.     

Enzymatic synthesis of structured lipids using a novel cold-active lipase from Pichia lynferdii NRRL Y-7723

Structured lipids (SL) were synthesized by the acidolysis of borage oil with caprylic acid using lipases. Six commercial lipases from different sources and a novel lipase from Pichia lynferdii NRRL Y-7723 were screened for their acidolysis activities and Lipozyme RM IM and NRRL Y-7723 lipase were selected to synthesize symmetrical SL since recently NRRL Y-7723 lipase was identified as a novel cold-active lipase. Both lipases showed 1,3-regiospecifity toward the glycerol backbone of borage oil. The effects of enzyme loading and temperature on caprylic acid incorporation into the borage oil were investigated. For Lipozyme RM IM and NRRL Y-7723 lipase, the incorporation of caprylic acid increased as enzyme loading increased up to 4% of total weight of the substrate, but significant increases were not observed when enzyme loading was further increased. The activity of NRRL Y-7723 lipase was higher than that of Lipozyme RM IM in the temperature range between 10 and 20 °C.     

Kinetic Properties and Enantioselectivity of The Lipases Produced by Four Aspergillus Species

Four high lipase-producing Aspergillus species, selected in our laboratory, were compared in terms of their stability and reactivity for enantioselective esterification between (R, S)-2-octanol with octanoic acid in n-hexane. We determined the pH and temperature reactions dependences of lipases activities, and we found that these enzymes exhibited various pH sensitivities. The optimum pH observed for Aspergillus terreus lipase was 5.5, for A. niger and A. oryzae lipases in the range of 6.0 to 6.5 and pH 7.0 for A. flavus lipase. Good stability was observed at pH ranging from 5.0 to 8.5 after 24 hours at 40° C, and the optimum activity was observed at 35-40° C for all lipases tested. The lipases from A. terreus and A. niger were highly thermostable, retaining 60% and 50% activity at 60° C after 1 hour, respectively. The lipases from A. niger and A. terreus lipases provided the best results in terms of enantioselectivity (E) in the esterification of (R, S)-2-octanol with octanoic acid in n-hexane (E = 4.9 and E = 4.5, respectively). These properties make these lipases good candidates for biocatalysis in organic media.     

A novel aspartic protease from the viscera of Sardinelle (Sardinella aurita): Purification and characterisation

A novel aspartic protease was extracted from the defatted viscera of sardinelle (Sardinella aurita) and purified, with a 9.5-fold increase in specific activity and 23.3% recovery. The molecular weight of the purified enzyme was estimated to be 17 kDa by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE). The purified enzyme appeared as a single band on native-PAGE. The optimum pH and temperature for protease activity were around 3.0 and 40 °C, respectively. The enzyme showed pH stability between 2.0 and 5.0 and retained more than 50% of its activity after heating for 30 min at 50 °C. The enzyme lost 90% of its activity after incubation with pepstatin A at room temperature, but was not inhibited by soybean trypsin inhibitor or phenylmethylsulfonyl fluoride. Its K_m value was determined to be 0.73 × 10⁻⁴ M using haemoglobin as a substrate. The N-terminal 12 amino acid sequence of the purified acidic protease was R V I I E D X D Q F C T. This sequence showed low homology with aspartic peptidases of several other species of fish, suggesting that the enzyme is a new aspartic protease.     

Kinetic Properties and Enantioselectivity of The Lipases Produced by Four Aspergillus Species

Four high lipase-producing Aspergillus species, selected in our laboratory, were compared in terms of their stability and reactivity for enantioselective esterification between (R, S)-2-octanol with octanoic acid in n-hexane. We determined the pH and temperature reactions dependences of lipases activities, and we found that these enzymes exhibited various pH sensitivities. The optimum pH observed for Aspergillus terreus lipase was 5.5, for A. niger and A. oryzae lipases in the range of 6.0 to 6.5 and pH 7.0 for A. flavus lipase. Good stability was observed at pH ranging from 5.0 to 8.5 after 24 hours at 40° C, and the optimum activity was observed at 35–40° C for all lipases tested. The lipases from A. terreus and A. niger were highly thermostable, retaining 60% and 50% activity at 60° C after 1 hour, respectively. The lipases from A. niger and A. terreus lipases provided the best results in terms of enantioselectivity (E) in the esterification of (R, S)-2-octanol with octanoic acid in n-hexane (E = 4.9 and E = 4.5, respectively). These properties make these lipases good candidates for biocatalysis in organic media.     

Comparison of lipase-catalyzed enantioselective esterification of (±)-menthol in ionic liquids and organic solvents

Enantioselective esterification of (±)-menthol was studied using Candida rugosa lipase (CRL) in ionic liquids (1-butyl-3-methyl-imidazolium hexafluorophosphate ([BMIM][PF₆]) and 1-butyl-3-methyl-imidazolium tetraflouroborate) and organic solvents of different hydrophobicities. Propionic anhydride was employed as an acylating agent. Because the enzyme showed comparable conversion yield and enantioselectivity in [BMIM][PF₆] and hexane in a 24-h reaction, more work focused on these two reaction media. Comparison of the activity, stability and enantioselectivity of CRL was carried out by examining the effects of the mole ratio of substrates, temperature, incubation time and enzyme recycling. It was found that temperature control was more crucial in the ionic liquid than in hexane to reach high conversion and enantioselectivity. The ionic liquid system showed an advantage of using less acid anhydride to achieve higher (±)-menthol conversion yield and better enantioselectivity. Moreover, during an incubation of 4–60 days in the ionic liquid, CRL activity was 2.5 times higher than its initial value, while that in hexane decreased to less than 60% in 2 days. In addition, the enzyme showed potentiality of recycled use in the ionic liquid. These advantages of the ionic liquid suggest that it would be used as a green alternative to organic solvents for the enantioselective esterification of (±)-menthol.     

Combined effects of freezing rate, storage temperature and time on bread dough and baking properties

This study compares the effects of freezing temperature and rate as well as storage temperature and time on the quality of frozen dough. Yeasted bread dough was frozen using four freezing rates (19-69 °C/h), then stored at −10, −20, −30, or −35 °C for up to 180 days. Dough strength diminished with longer storage time and higher storage temperatures. Cryo-SEM showed that dough stored at −30 and −35 °C had the least damaged gluten network. NMR studies showed that more rapidly frozen dough, and that stored at lower temperatures had lower transverse relaxation (T₂) times (9-10 ms). However, dough stored at −20 °C displayed the highest yeast activity among samples. Bread loaf volume decreased with storage time, and bread made from dough stored at −20 °C showed the highest loaf volume. Breads produced from −30 and −35 °C stored dough displayed less change in the texture profile during storage as well as less change in T₂ values. Response surface analysis showed that optimal properties occurred at freezing rates of around 19-41 °C/h and storage temperatures of −15 to −20 °C.     

Short communication: Effect of active food packaging materials on fluid milk quality and shelf life

Active packaging, in which active agents are embedded into or on the surface of food packaging materials, can enhance the nutritive value, economics, and stability of food, as well as enable in-package processing. In one embodiment of active food packaging, lactase was covalently immobilized onto packaging films for in-package lactose hydrolysis. In prior work, lactase was covalently bound to low-density polyethylene using polyethyleneimine and glutaraldehyde cross-linkers to form the packaging film. Because of the potential contaminants of proteases, lipases, and spoilage organisms in typical enzyme preparations, the goal of the current work was to determine the effect of immobilized-lactase active packaging technology on unanticipated side effects, such as shortened shelf-life and reduced product quality. Results suggested no evidence of lipase or protease activity on the active packaging films, indicating that such active packaging films could enable in-package lactose hydrolysis without adversely affecting product quality in terms of dairy protein or lipid stability. Storage stability studies indicated that lactase did not migrate from the film over a 49-d period, and that dry storage resulted in 13.41% retained activity, whereas wet storage conditions enabled retention of 62.52% activity. Results of a standard plate count indicated that the film modification reagents introduced minor microbial contamination; however, the microbial population remained under the 20,000 cfu/mL limit through the manufacturer’s suggested 14-d storage period for all film samples. This suggests that commercially produced immobilized lactase active packaging should use purified cross-linkers and enzymes. Characterization of unanticipated effects of active packaging on food quality reported here is important in demonstrating the commercial potential of such technologies.     

Effect of membranes with various hydrophobic/hydrophilic properties on lipase immobilized activity and stability

In this study, three membranes: regenerated cellulose (RC), glass fiber (GF) and polyvinylidene fluoride (PVDF), were grafted with 1,4-diaminobutane (DA) and activated with glutaraldehyde (GA) for lipase covalent immobilization. The efficiencies of lipases immobilized on these membranes with different hydrophobic/hydrophilic properties were compared. The lipase immobilized on hydrophobic PVDF-DA-GA membrane exhibited more than an 11-fold increase in activity compared to its immobilization on a hydrophilic RC-DA-GA membrane. The relationship between surface hydrophobicity and immobilized efficiencies was investigated using hydrophobic/hydrophilic GF membranes which were prepared by grafting a different ratio of n-butylamine/1,4-diaminobutane (BA/DA). The immobilized lipase activity on the GF membrane increased with the increased BA/DA ratio. This means that lipase activity was exhibited more on the hydrophobic surface. Moreover, the modified PVDF-DA membrane was grafted with GA, epichlorohydrin (EPI) and cyanuric chloride (CC), respectively. The lipase immobilized on the PVDF-DA-EPI membrane displayed the highest specific activity compared to other membranes. This immobilized lipase exhibited more significant stability on pH, thermal, reuse, and storage than did the free enzyme. The results exhibited that the EPI modified PVDF is a promising support for lipase immobilization.