Evaluation of a silica-coated magnetic nanoparticle for the immobilization of a His-tagged lipase

Magnetic particles of size 10 nm have been coated with silica to a mean diameter of 40 nm and charged with Cu²⁺ ions via a multidentate ligand, iminodiacetic acid (IDA), for the immobilization of His-tagged Bacillus stearothermopilus L1 lipase. Microporous (average pore diameter of 60 Å) silica gel with a mean particle diameter of 115 µm has been used as a comparative support material. The molar ratio of Cu²⁺ to IDA was found to be 1:1.14 and 1:1.99 in the silica gel and the silica-coated magnetic nanoparticles (SiMNs), respectively. The specific activity of the immobilized enzyme was found to conform to the following order: Cu²⁺-charged SiMN>SiMN>Cu²⁺-charged silica gel>silica gel. When it was immobilized on the Cu²⁺-charged SiMNs, over 70% of the initial activity of the lipase remained after it had been reused five times. However, only 20% of the initial activity remained after the enzyme immobilized on the Cu²⁺-charged silica gel had been reused five times. For the enzyme immobilized on supports without Cu²⁺ cations, all activity was lost after threefold reuse. The differences in the specific activities and the efficiencies of reuse of the enzymes immobilized on the various support materials are discussed in terms of immobilization mechanisms (physical adsorption vs. coordination bonding), mass transfer of a substrate and a product of the enzyme reaction, and the status of the Cu (Cu bound to the IDA on the silica layer vs. Cu directly adsorbed on the silica layer).     

Fabrication of an organic–inorganic nanocomposite carrier for enzyme immobilization based on metal–organic coordination

An organic–inorganic nanocomposite which combined mesoporous silica SBA-15 and chitosan using a carboxyl functionalized ionic liquid as the bridging agent (SBA@CS) was successfully fabricated, and was used to immobilize porcine pancreas lipase (PPL) by physical adsorption, cross-linking and metal–organic coordination, respectively. The as-prepared carriers were characterized by scanning electron microscopy, Fourier transform infrared and energy-dispersive X-ray spectroscopy. Compared with immobilization onto the pure mesoporous silicon material SBA-15, all the batches of PPL immobilized onto organic–inorganic nanocomposites showed higher activity, improved stability and reusability as well as better resistance to pH and temperature changes. Among the immobilized PPLs, immobilization based on Co²⁺ coordination (SBA@CS-Co-PPL) produced the best enzymatic properties. The maximum immobilization efficiency and specific activity of 79.6% and 1975.8 U g⁻¹ were obtained with SBA@CS-Co, separately. More importantly, the activity of immobilized enzyme can still maintain 84.0% after 10 times of reuse. These results demonstrated that thus prepared organic–inorganic nanocomposite could be an ideal carrier for enzyme immobilization by metal–organic coordination.     

Comparison of physical and covalent immobilization of lipase from Candida antarctica on polyamine microspheres of alkylamine matrix

Abstract

Polyamine microspheres (PA-M) prepared using polyethyleneimine as matrix were used for the immobilization of Candida antarctica lipase. The isoelectric point of PA-M is 10.6, and the hydrophobicity of PA-M was indicated using naphthalene. Optimization of conditions showed that the maximal loading of lipase on PA-M reached 230.2 mg g^{? 1} at pH 9.0 and 35°C. An increased buffer concentration had no effect on the activity of lipase but decreased the amount of lipase adsorbed. Simulation with Langmuir and Freundlich isotherms demonstrated that the adsorption of lipase on PA-M was thermodynamically favorable. Covalent crosslinking of the lipase adsorbed extended the pH range and increased the optimal temperature of the lipase activity. The physically adsorbed lipase (P-lipase) and the covalently immobilized derivative (C-lipase) retained more than 75% and 85% of their initial activity, respectively, after 10 cycles of usage. The half-lives of P-lipase and C-lipase at 50°C were 15.70 and 27.67 times higher than that of the free enzyme, respectively. Compared to P-lipase, covalent immobilization obviously reduced the catalytic efficiency and activation energy of the enzyme.     

Magnetic nanoparticles supported ionic liquids for lipase immobilization: Enzyme activity in catalyzing esterification

Candida rugosa lipase was immobilized on magnetic nanoparticles supported ionic liquids having different cation chain length (C₁, C₄ and C₈) and anions (Cl⁻, BF₄⁻ and PF₆⁻). Magnetic nanoparticles supported ionic liquids were obtained by covalent bonding of ionic liquids–silane on magnetic silica nanoparticles. The particles are superparamagnetic with diameter of about 55 nm. Large amount of lipase (63.89 mg/(100 mg carrier)) was loaded on the support through ionic adsorption. Activity of the immobilized lipase was examined by the catalysis of esterification between oleic acid and butanol. The activity of bound lipase was 118.3% compared to that of the native lipase. Immobilized lipase maintained 60% of its initial activity even when the temperature was up to 80 °C. In addition, immobilized lipase retained 60% of its initial activity after 8 repeated batches reaction, while no activity was detected after 6 cycles for the free enzyme.     

表面改性SBA-15材料固载猪胰脂肪酶

采用试剂y-氯丙基三乙氧基硅烷（cvrEs）对介孔硅材料SBA-15进行表面改性,并通过红外图谱（FT-IR）和N2吸附脱附等温图（BET）对其进行表征。结果表明：改性前原材料的比表面积为460．9m2／g,改性后材料比表面积提高到512．0m2／g。利用改性前和改性后的SBA-15对猪胰脂肪酶进行固载实验,并对实验结果进行比较,发现改性后的SBA-15在脂肪酶活性、pH环境适应性、热耐受性和可操作性都优于改性前的SBA-15,在最优条件下的酶活力提高超过60％。     

Crosslinked enzyme aggregates in hierarchically-ordered mesoporous silica: a simple and effective method for enzyme stabilization

alpha-chymotrypsin (CT) and lipase (LP) were immobilized in hierarchically-ordered mesocellular mesoporous silica (HMMS) in a simple but effective way for the enzyme stabilization, which was achieved by the enzyme adsorption followed by glutaraldehyde (GA) crosslinking. This resulted in the formation of nanometer scale crosslinked enzyme aggregates (CLEAs) entrapped in the mesocellular pores of HMMS (37 nm), which did not leach out of HMMS through narrow mesoporous channels (13 nm). CLEA of alpha-chymotrypsin (CLEA-CT) in HMMS showed a high enzyme loading capacity and significantly increased enzyme stability. No activity decrease of CLEA-CT was observed for 2 weeks under even rigorously shaking condition, while adsorbed CT in HMMS and free CT showed a rapid inactivation due to the enzyme leaching and presumably autolysis, respectively. With the CLEA-CT in HMMS, however, there was no tryptic digestion observed suggesting that the CLEA-CT is not susceptible to autolysis. Moreover, CLEA of lipase (CLEA-LP) in HMMS retained 30% specific activity of free lipase with greatly enhanced stability. This work demonstrates that HMMS can be efficiently employed as host materials for enzyme immobilization leading to highly enhanced stability of the immobilized enzymes with high enzyme loading and activity.     

Design of β‐galactosidase/silica biocatalysts: Impact of the enzyme properties and immobilization pathways on their catalytic performance

The use of heterogeneous biocatalysis in industrial applications is advantageous and the enzyme stability improvement is a continuous challenge. Therefore, we designed β‐galactosidase heterogeneous biocatalysts by immobilization, involving the support synthesis and enzyme selection (from Bacillus circulans, Kluyveromyces lactis, and Aspergillus oryzae). The underivatized, tailored, macro‐mesoporous silica exhibited high surface area, offered high enzyme immobilization yields and activity. Its chemical activation with glyoxyl groups bound the enzyme covalently, which suppressed lixiviation and conferred higher pH and thermal stability (120‐fold than for the soluble enzyme), without observable reduction of activity/stability due to the presence of silica. The best balance between the immobilization yield (68%), activity (48%), and stability was achieved for Bacillus circulans β‐galactosidase immobilized on glyoxyl‐activated silica, without using stabilizing agents or modifying the enzyme. The enzyme stabilization after immobilization in glyoxyl‐activated silica was similar to that observed in macroporous agarose‐glyoxyl support, with the reported microbiological and mechanical advantages of inorganic supports. The whey lactolysis at pH 6.0 and 25°C by using this catalyst (1 mg ml^?1, 290 UI g^?1) was still 90%, even after 10 cycles of 10 min, in batch process but it could be also implemented on continuous processes at industrial level with similar results.     

Immobilization and activity of Rhizomucor miehei lipase. Effect of the matrix properties prepared from nonionic fluorinated surfactants

We report the immobilization of Rhizomucor miehei lipase (RmL) onto mesoporous silica materials, in particular the investigations concerning the effects of the level of silica condensation and of the pore size on the enzyme activity. The efficiency of the immobilization was revealed by FTIR spectroscopy. Infrared was also used to determine the quantity of adsorbed enzyme. Immobilization efficiency increased when the RmL concentration in the buffer solution was changed from 2 to 10 mg/mL. Nevertheless, while upon enzyme immobilization the mesopore ordering was sustained for the support recovered after hydrothermal treatment at 100 °C, a structure collapse occurred for the one prepared at 80 °C. The difference in behavior is attributed to the lower hydrothermal stability of this material, which reflects the lower level of silica condensation. The enzyme-containing mesostructured silica was effectively used to catalyze the model esterification reaction of lauric acid with 1-propanol, as the immobilized lipase retained its catalytic activity. A linear relationship was observed between the reaction rate and the amount of catalyst. RmL immobilized on mesoporous materials presented a satisfactory reusability, while the remaining activity of RmL after 4 months of storage was 47% of the initial one.     

Immobilisation of lipases by adsorption and deposition: high protein loading gives lower water activity optimum

Two different immobilisation techniques for lipases were investigated: adsorption on to Accurel EP-100 and deposition on to Celite. The specific activities were in the same order of magnitude, 2.9 (mol min^–1 mg protein) when Celite was used as support and 2.3 (mol min^–1 mg^–1 protein) when Accurel EP-100 was used as support, even if the amount of lipase loaded differed by 2 orders of magnitude. Immobilisation on Accurel EP-100 was the preferred technique since 40–100 times more protein can be loaded/per g carrier, thus yielding a more active catalyst. The water activity profiles in lipase catalysed esterification were influenced by the amount of protein adsorbed to Accurel EP-100. Higher protein loading (40 mg g^–1) resulted in a bell-shaped water activity profile with highest specific activity (6.1 mol min^–1 mg^–1 protein) at a _w=0.11, while an enzyme preparation with low protein loading (4 mg g^–1) showed highest specific activity at a _w=0.75.     

Immobilization of lipase on methyl-modified silica aerogels by physical adsorption

In this work, methyl-modified silica aerogels, a new kind of macro-porous material with high porosity, were used as carriers to immobilize lipase by adsorption. SEM, TEM, nitrogen adsorption device, and thermogravimetric analysis were used to characterize the properties of modified aerogels. The surface area was 395.6 m(2)/g, and the average pore diameter was 68.72 nm. The contact angle of aerogel particles increased from 20.9 degrees to 99.2 degrees after methyl modification. Reaction characteristics of the material after enzyme loading were also discussed. The results showed that adsorption capacity could reach 67.42 mg/g; and the maximal enzyme activity was 19.87 micromol min(-1)mg(-1) (protein), and activity retention could reach 56.44%. It is worth mentioning that the amount of modified aerogels added had significant effects on the diameter of droplets and the mass transfer behavior of substrates in the reaction emulsion. Online microscope was used to visualize the droplets in the emulsion, where the aerogel particles were observed locating at the interface of oil and water. The average diameter of droplets reached the minimum when 0.06 g of modified aerogels was added into the reaction emulsion which contained 10 ml of oil and 10 ml of phosphate buffer solution. The phenomenon was resulted from the wettability of methyl-modified silica aerogels.     

Enhancing performance of lipase immobilized on methyl-modified silica aerogels at the adsorption and catalysis processes: Effect of cosolvents

Hydrophobic silica aerogels modified with methyl group were applied as support to immobilize Candida rugosa lipase (CRL). At the adsorption process, different alcohols were used to intensify the immobilization of CRL. The results showed that n-butanol wetting the hydrophobic support prior to contacting with enzyme solution could promote lipase activity, but the adsorption quantity onto the support decreased. Based on this, a novel immobilization method was proposed: the support contacted with enzyme solution without any alcohols, and then the immobilized enzymes were activated by 90% (V) n-butanol solution. The experimental results showed that this method could keep high adsorption quantity (413.0 mg protein/g support) and increase the lipase specific activity by more than 50%. To improve the stability of immobilized lipase, the support after adsorption was contacted with n-octane to form an oil layer covering the immobilized lipases, thus the leakage can be decreased from over 30–4% within 24 h. By utilizing proper cosolvents, a high enzyme activity and loading capacity as well as little loss of lipase was achieved without covalent linkage between the lipase and the support. This is known to be an excellent result for immobilization achieved by physical adsorption only.     

Kinetics of solvent-free geranyl acetate synthesis by Rhizopus oligosporus NRRL 5905 lipase immobilized on to cross-linked silica

The objective of the present work was to study the kinetics of the solvent-free synthesis of geranyl acetate by a novel lipase (activity 60 U g^?1) made by immobilization of lipase from Rhizopus oligosporous NRRL 5905 on to cross-linked silica gel. Transesterification was performed with vinyl acetate as the acyl donor. Vinyl acetate was used in large excess compared to geraniol, which made the reaction pseudo first order with respect to geraniol and the reaction rate followed Michaelis–Menten kinetics for a single substrate. To obtain the highest yield for geranyl acetate, various relevant physical parameters such as shaking speed, reaction time, enzyme concentration, initial water amount and reaction temperature that influence the activity of lipase were investigated. A maximum molar conversion of 67% was achieved after 48 h of reaction at 30°C, at an enzyme concentration of 25% w/v of reaction mixture. Substrate conversion remained constant for five successive cycles; thereafter the conversion dropped by only 11%. Using a pseudo first-order kinetic model for geranyl acetate synthesis in the absence of organic solvents, apparent K_m and V_max values were evaluated as 60 mM and 141 µmol g^?1 h^?1, respectively.     

Activity and adsorption of lipase from Nigella sativa seeds on Celite at different pH values

Lipase from Nigella sativa seeds was immobilized by adsorption on Celite 535 from phosphate buffer solutions varying pH values of 5.0–8.0 at 25?°C. Langmuir isotherms described the adsorption equilibria well for lipase adsorption at all pH range. The saturation capacity for adsorption of lipase increased from 14.5 to 24.3 mg g^?1 Celite as the adsorption pH was reduced from 8 to 5, but the adsorption equilibrium constant remained constant and was determined to be 1.92 × 10⁵ M^?1. The adsorbed enzymes showed different activity values depending on the pH of the adsorption medium. The immobilized enzymes prepared at pH 6 displayed the highest activity values.     

The inhibitory kinetics and mechanism of glycolic acid on lipase

Abstract

Obesity is prone to cause a variety of chronic metabolic diseases, and it has aroused people’s attention that the rapid increase in the global population of obese people in the past years. As a kind of weight-loss drug acting in the intestine, lipase inhibitor does not enter the bloodstream without producing central nervous side effects. Because they do not affect the metabolism system, lipase inhibitors and obesity have become one of the hot spots in recent years. Glycolic acid is a new substrate analog inhibitor with the value of the semi-inhibitory concentration of lipase is estimated to be 17.29?±?0.14?mM. Using the plots of Lineweaver-Burk, the inhibition mechanism of lipase by glycolic acid was reversible and the inhibition type belongs to competitive inhibition with a K_I value of 19.61?±?0.26?mM. The inhibitory kinetics assay showed that the microscopic velocity constant k₊₀ of inhibition kinetics is 1.79?×?10^?3?mM^?1s^?1, and k_?0 is 0.73?×?10^?3 s^?1. The results of UV full-wavelength scanning on product cumulative, fluorescence quenching and molecular simulation also indicated that glycolic acid and substrate competitive with lipase by binding to Lys137. Thereby glycolic acid inhibiting the oxidation-catalyzed reaction and reducing the product of the enzyme and substrate. This adds a new direction for the search for lipase inhibitors and provides new ideas about the development of anti-obesity drugs.

Communicated by Ramaswamy H. Sarma     

Immobilization of Lipase from Rhizopus Niveus: A Way to Enhance its Synthetic Activity in Organic Solvent

Lipase (EC 3.1.1.3) from Rhizopus niveus was immobilized by physical adsorption on various carriers, including different types of Celite, Spherosil and Duolite. After the enzyme immobilization, the recovered hydrolytic and synthetic activities on the different carriers were then determined. The results showed that the highest synthetic activity was obtained when Duolite XAD 761 was used as the carrier. However the recovered hydrolytic activity after the immobilization on this resin was relatively low although this carrier showed the best protein loading capacity. The highest recovered hydrolytic activity was observed when the lipase was immobilized on Celite Hyflo-Supercel using an immobilization buffer adjusted to pH 4. The comparison of the free and immobilized lipase specific activities suggest that the immobilization on Celite Hyflo-Supercel, Spherosil XOA 200 and silica has enhanced the lipase hydrolytic activity. On the other hand, the use of the lipase immobilized on Duolite XAD 761 as biocatalyst of synthetic reaction, compared to that of the free enzyme, allows the reaction initial velocity to be increased 12.2-fold. In addition, the synthetic activity of the lipase immobilized on Duolite XAD 761 was shown to be maximum at a water activity in the range of 0.32-0.52.     

Sulfur‐Embedded Activated Multichannel Carbon Nanofiber Composites for Long‐Life,High‐Rate Lithium–Sulfur Batteries

Despite the 3–5 fold higher energy density than the conventional Li‐ion cells at a lower cost, commercialization of Li–S batteries is hindered by the insulating nature of sulfur and the dissolution of intermediate polysulfides (Li₂S _X , 4 < X ≤ 8) into the electrolyte. The authors demonstrate here multichannel carbon nanofibers that are composed of parallel mesoporous channels connected with micropores as sulfur containment. In addition, hydroxyl functional groups are formed on the carbon surface through a chemical activation to enhance the interaction between sulfur and carbon. In the sulfur embedded composite, the mesoporous multichannel enhances the active material utilization and sulfur loading, while the micropores act as a reaction chamber for sulfur component and trap site for polysulfide with the assistance of the functional groups. This sulfur–carbon composite electrode with 2.2 mg cm^?2 sulfur displays excellent performance with high rate capability (initial capacity of 1351 mA h g^?1 at C/5 rate and 847 mA h g^?1 at 5C rate), maintaining 920 mA h g^?1 even after 300 cycles (a decay of 0.07% per cycle). Furthermore, a stable reversible capacity of as high as ≈1100 mA h g^?1 is realized with a higher sulfur loading of 4.6 mg cm^?2.     

Hormonal control of lipase activity in oilseed rape germinants

In oilseeds, storage lipids provide the respiratory fuel for seedling growth. The enzyme responsible for their initial hydrolysis is lipase (triacylglycerol acylhydrolase; EC 3.1.1.3). We investigated the possibility that lipase is regulated by gibberellins (GAs). In four oilseed rape cultivars of Brassica napus and B. rapa, seed imbibition in 10^?6 to 10^?3M GA₃ increased lipase activity 1.5- to 7-fold over control levels. Conversely, imbibition in 10^?7 to 10^?5M abscisic acid or 10^?6 to 10^?4M paclobutrazol, an inhibitor of GA biosynthesis, markedly decreased lipase activity. While lipase activity in B. napus cv. Parkland increased during the first 5 days following imbibition, concentrations of endogenous GA₁, GA₈ and GA₁₉ (as measured by GC-selected ion monitoring using [²H₂]GA internal standards) were relatively constant and GA₂₀, a precursor of GA₁, decreased. Levels of endogenous GA₃ were apparently variable. Thus, lipase activity was not correlated with GA₁ concentration, but the inverse correlation with GA₂₀ concentration suggests that GA turnover could be positively correlated with lipase activity. Lipase activity was also examined in three genotypes of rapid cycling B. rapa that vary in endogenous GA content: rosette, a GA-deficient dwarf, a normal line and elongated internode, a tall mutant with high GA content. The three genotypes showed similar patterns of lipase activity during the first 4 days following imbibition and the subcellular distribution of lipase activity was also similar in the three genotypes. Although GA may be involved in the regulation of lipase in oilseed rape germinants, it is not the sole regulatory factor.     

Luminescent thermometer based on Eu3+/Tb3+‐organic‐functionalized mesoporous silica

In this work we investigate a mesoporous silica (MS) decorated with dipyridyl‐pyridazine (dppz) ligands and further grafted with a mixture of Eu³⁺/Tb³⁺ ions (28.45%:71.55%), which was investigated as a potential thermometer in the 10–360 K temperature range. The MS material was prepared employing a hetero Diels–Alder reaction: 3,6‐di(2‐pyridyl)‐1,2,4,5‐tetrazine was reacted with the double bonds of vinyl‐silica (vSilica) followed by an oxidation procedure. We explore using the dppz‐vSilica material to obtain visible emitting luminescent materials and for obtaining a luminescent thermometer when grafted with Eu³⁺/Tb³⁺ ions. For the dppz‐vSilica@Eu,Tb material absolute sensitivity S_a of 0.011 K^?1 (210 K) and relative sensitivity S_r of 1.32 %K^?1 (260 K) were calculated showing good sensing capability of the material. Upon temperature change from 10 K to 360 K the emission color of the material changed gradually from yellow to red.     

Biomimetic particles for isolation and reconstitution of receptor function

Biomimetic particles supporting lipid bilayers are becoming increasingly important to isolate and reconstitute protein function. Cholera toxin (CT) from Vibrio cholerae, an 87-kDa AB₅ hexameric protein, and its receptor, the monosialoganglioside GM₁, a cell membrane glycolipid, self-assembled on phosphatidylcholine (PC) bilayer-covered silica particles at 1 CT/5 GM₁ molar ratio in perfect agreement with literature. This receptor-lig-and recognition represented a proof-of-concept that receptors in general can be isolated and their function reconstituted using biomimetic particles, i.e., bilayer-covered silica. After incubation of colloidal silica with small unilamellar PC vesicles in saline solution, pH 7.4, PC adsorption isotherms on silica from inorganic phosphorus analysis showed a high PC affinity for silica with maximal PC adsorption at bilayer deposition. At 0.3 mM PC, fluorescence of pyrene-labeled GM₁ showed that GM₁ incorporation in biomimetic particles increased as a function of particles concentration. At 1 mg/mL silica, receptor incorporation increased to a maximum of 40% at 0.2–0.3 mM PC and then decreased as a function of PC concentration. At 5 μM GM₁, 0.3 mM PC, and 1 mg/mL silica, CT binding increased as a function of CT concentration with a plateau at 2 mg bound CT/m² silica, which corresponded to the 5 GM₁/1 CT molar proportion and showed successful reconstitution of receptor-ligand interaction.     

Molecular‐Level Design of Pyrrhotite Electrocatalyst Decorated Hierarchical Porous Carbon Spheres as Nanoreactors for Lithium–Sulfur Batteries

Lithium–sulfur batteries (LSBs) are a class of new‐generation rechargeable high‐energy‐density batteries. However, the persisting issue of lithium polysulfides (LiPs) dissolution and the shuttling effect that impedes the efficiency of LSBs are challenging to resolve. Herein a general synthesis of highly dispersed pyrrhotite Fe_1?xS nanoparticles embedded in hierarchically porous nitrogen‐doped carbon spheres (Fe_1?xS‐NC) is proposed. Fe_1?xS‐NC has a high specific surface area (627 m² g^?1), large pore volume (0.41 cm³ g^?1), and enhanced adsorption and electrocatalytic transition toward LiPs. Furthermore, in situ generated large mesoporous pores within carbon spheres can accommodate high sulfur loading of up to 75%, and sustain volume variations during charge/discharge cycles as well as improve ionic/mass transfer. The exceptional adsorption properties of Fe_1?xS‐NC for LiPs are predicted theoretically and confirmed experimentally. Subsequently, the electrocatalytic activity of Fe_1?xS‐NC is thoroughly verified. The results confirm Fe_1?xS‐NC is a highly efficient nanoreactor for sulfur loading. Consequently, the Fe_1?xS‐NC nanoreactor performs extremely well as a cathodic material for LSBs, exhibiting a high initial capacity of 1070 mAh g^?1 with nearly no capacity loss after 200 cycles at 0.5 C. Furthermore, the resulting LSBs display remarkably enhanced rate capability and cyclability even at a high sulfur loading of 8.14 mg cm^?2.