Advances in enzyme immobilization based on hierarchical porous metal-organic frameworks / 生物工程学报

As an excellent hosting matrices for enzyme immobilization, metal-organic framework (MOFs) provides superior physical and chemical protection for biocatalytic reactions. In recent years, the hierarchical porous metal-organic frameworks (HP-MOFs) have shown great potential in enzyme immobilization due to their flexible structural advantages. To date, a variety of HP-MOFs with intrinsic or defective porous have been developed for the immobilization of enzymes. The catalytic activity, stability and reusability of enzyme@HP-MOFs composites are significantly enhanced. This review systematically summarized the strategies for developing enzyme@HP-MOFs composites. In addition, the latest applications of enzyme@HP-MOFs composites in catalytic synthesis, biosensing and biomedicine were described. Moreover, the challenges and opportunities in this field were discussed and envisioned.

Recent progress in magnetic nanoparticles and mesoporous materials for enzyme immobilization: an update / Progresso recente em nanopartículas magnéticas e materiais mesoporosos para imobilização de enzimas: uma atualização

Enzymes immobilized onto substrates with excellent selectivity and activity show a high stability and can withstand extreme experimental conditions, and their performance has been shown to be retained after repeated uses. Applications of immobilized enzymes in various fields benefit from their unique characteristics. Common methods, including adsorption, encapsulation, covalent attachment and crosslinking, and other emerging approaches (e.g., MOFs) of enzyme immobilization have been developed mostly in recent years. In accordance with these immobilization methods, the present review elaborates the application of magnetic separable nanoparticles and functionalized SBA-15 and MCM-41 mesoporous materials used in the immobilization of enzymes.

Enzimas imobilizadas em substratos com excelente seletividade e atividade apresentam alta estabilidade e podem suportar condições experimentais extremas, e seu desempenho foi mantido após repetidos usos. As aplicações de enzimas imobilizadas em vários campos se beneficiam de suas características únicas. Métodos comuns, incluindo adsorção, encapsulamento, ligação covalente e reticulação, e outras abordagens emergentes (por exemplo, MOFs) de imobilização de enzima, foram desenvolvidos principalmente nos últimos anos. De acordo com esses métodos de imobilização, a presente revisão elabora a aplicação de nanopartículas magnéticas separáveis e materiais mesoporosos funcionalizados SBA-15 e MCM-41 usados na imobilização de enzimas.

Preparation and catalytic properties of catalase-inorganic hybrid nanoflowers / 生物工程学报

Catalase is widely used in the food, medical, and textile industries. It possesses exceptional properties including high catalytic efficiency, high specificity, and environmental friendliness. Free catalase cannot be recycled and reused in industry, resulting in a costly industrial biotransformation process if catalase is used as a core ingredient. Developing a simple, mild, cost-effective, and environmentally friendly approach to immobilize catalase is anticipated to improve its utilization efficiency and enzymatic performance. In this study, the catalase KatA derived from Bacillus subtilis 168 was expressed in Escherichia coli. Following separation and purification, the purified enzyme was prepared as an immobilized enzyme in the form of enzyme-inorganic hybrid nanoflowers, and the enzymatic properties were investigated. The results indicated that the purified KatA was obtained through a three-step procedure that included ethanol precipitation, DEAE anion exchange chromatography, and hydrophobic chromatography. Then, by optimizing the process parameters, a novel KatA/Ca3(PO4)2 hybrid nanoflower was developed. The optimum reaction temperature of the free KatA was determined to be 35 ℃, the optimum reaction temperature of KatA/Ca3(PO4)2 hybrid nanoflowers was 30-35 ℃, and the optimum reaction pH of both was 11.0. The free KatA and KatA/Ca3(PO4)2 hybrid nanoflowers exhibited excellent stability at pH 4.0-11.0 and 25-50 ℃. The KatA/Ca3(PO4)2 hybrid nanoflowers demonstrated increased storage stability than that of the free KatA, maintaining 82% of the original enzymatic activity after 14 d of storage at 4 ℃, whereas the free KatA has only 50% of the original enzymatic activity. In addition, after 5 catalytic reactions, the nanoflower still maintained 55% of its initial enzymatic activity, indicating that it has good operational stability. The Km of the free KatA to the substrate hydrogen peroxide was (8.80±0.42) mmol/L, and the kcat/Km was (13 151.53± 299.19) L/(mmol·s). The Km of the KatA/Ca3(PO4)2 hybrid nanoflowers was (32.75±2.96) mmol/L, and the kcat/Km was (4 550.67±107.51) L/(mmol·s). Compared to the free KatA, the affinity of KatA/Ca3(PO4)2 hybrid nanoflowers to the substrate hydrogen peroxide was decreased, and the catalytic efficiency was also decreased. In summary, this study developed KatA/Ca3(PO4)2 hybrid nanoflowers using Ca2+ as a self-assembly inducer, which enhanced the enzymatic properties and will facilitate the environmentally friendly preparation and widespread application of immobilized catalase.

In vivo self-aggregation and efficient preparation of recombinant lichenase based on ferritin / 生物工程学报

Enzyme separation, purification, immobilization, and catalytic performance improvement have been the research hotspots and frontiers as well as the challenges in the field of biocatalysis. Thus, the development of novel methods for enzyme purification, immobilization, and improvement of their catalytic performance and storage are of great significance. Herein, ferritin was fused with the lichenase gene to achieve the purpose. The results showed that the fused gene was highly expressed in the cells of host strains, and that the resulted fusion proteins could self-aggregate into carrier-free active immobilized enzymes in vivo. Through low-speed centrifugation, the purity of the enzymes was up to > 90%, and the activity recovery was 61.1%. The activity of the enzymes after storage for 608 h was higher than the initial activity. After being used for 10 cycles, it still maintained 50.0% of the original activity. The insoluble active lichenase aggregates could spontaneously dissolve back into the buffer and formed the soluble polymeric lichenases with the diameter of about 12 nm. The specific activity of them was 12.09 times that of the free lichenase, while the catalytic efficiency was 7.11 times and the half-life at 50 ℃ was improved 11.09 folds. The results prove that the ferritin can be a versatile tag to trigger target enzyme self-aggregation and oligomerization in vivo, which can simplify the preparation of the target enzymes, improve their catalysis performance, and facilitate their storage.

Amplification of urea detection based on pH-sensitive liposomes

BACKGROUND: This study aimed to develop an amplification method of urea detection based on pHsensitive liposomes. RESULTS: The urease covalently immobilized on the magnetic particles and the pH-sensitive liposomes encapsulating ferricyanide were added to the cyclic-voltammeter cell solution where urea was distributed. The conversion of urea into carbonic acid seemed to induce a pH decrease that caused a reduction in the electrostatic repulsion between the headgroups of weakly acidic 1,2-dipalmitoyl-sn-glycero3-succinate. The reduction induced the liposomes to release potassium ferricyanide that was encapsulated inside. The effects of urea concentration and pH value were investigated. A specific concentration (0.5 mg/mL) of the urea solution was set to observe the response. The activity of urease was reversible with respect to the pH change between 7 and 5. The sensitivity of this detection was almost identical to the comparable techniques such as an enzyme-linked immunosorbent assay and a field-effect transistor. CONCLUSIONS: In summary, the methodology developed in this study was feasible as a portable, rapid, and sensitive method.

LXYL-P1-2 immobilized on magnetic nanoparticles and its potential application in paclitaxel production

BACKGROUND: LXYL-P1-2 is the first reported glycoside hydrolase that can catalyze the transformation of 7-b-xylosyl-10-deacetyltaxol (XDT) to 10-deacetyltaxol (DT) by removing the D-xylosyl group at the C7 position. Successful synthesis of paclitaxel by one-pot method combining the LXYL-P1-2 and 10- deacetylbaccatin III-10-b-O-acetyltransferase (DBAT) using XDT as a precursor, making LXYL-P1-2 a highly promising enzyme for the industrial production of paclitaxel. The aim of this study was to investigate the catalytic potential of LXYL-P1-2 stabilized on magnetic nanoparticles, the surface of which was modified by Ni2+-immobilized cross-linked Fe3O4@Histidine. RESULTS: The diameter of matrix was 20­40 nm. The Km value of the immobilized LXYL-P1-2 catalyzing XDT (0.145 mM) was lower than that of the free enzyme (0.452 mM), and the kcat/Km value of immobilized enzyme (12.952 mM s 1 ) was higher than the free form (8.622 mM s 1 ). The immobilized form maintained 50% of its original activity after 15 cycles of reuse. In addition, the stability of immobilized LXYL-P1-2, maintained 84.67% of its initial activity, improved in comparison with free form after 30 d storage at 4 C. CONCLUSIONS: This investigation not only provides an effective procedure for biocatalytic production of DT, but also gives an insight into the application of magnetic material immobilization technology.

Reusable Enzymatic Strip for Detection of Arsenic

HIGHLIGHTS Arsenic is considered as one of the highly hazardous elements in the environment and a serious carcinogen for the human health. An enzymatic method has been described by using arsenite oxidase for arsenic detection. Residual activity of the immobilized enzyme was 43% of the initial activity after being recycled 10 times.

Abstract Arsenic is considered as one of the highly hazardous elements in the environment and a serious carcinogen for the human health. More attention has taken towards the arsenic due to its presence in ground water in India, China, Bangladesh, Inner Mongolia and several other regions of the world. It's been a challenge to remove arsenic due to the lack of its efficient detection approach in the complicated environmental matrix. The proposed method describes an enzymatic method for arsenic determination using arsenite oxidase, which catalyzes the oxidation of arsenite to arsenate. Hence, a colorimetric PVC strip with immobilized arsenite oxidase has been developed to detect the arsenic concentration and also having potential for the field-testing. The influence of the optimal conditions i.e. pH, temperature, storage stability, and reusability of free and immobilized enzyme were evaluated and compared. The results have shown that the stabilities were significantly enhanced compared with free counterpart. Residual activity of the immobilized enzyme was 43% of the initial activity after being recycled 10 times. We approve that this novel low cost immobilized carrier presents a new approach in large scale applications and expected to act as a model for establishment of indigenous arsenic sensor in miniature form.

Synthesis of (S)-4-fluorophenylglycine by using immobilized amidase based on metal-organic framework / 生物工程学报

A stable Zr-based metal-organic framework (MOF, UiO-66-NH2) synthesized via micro-water solvothermal method was used to immobilize amidase by using the glutaraldehyde crosslinking method. The effect of immoblization conditions on enzyme immoblization efficiency was studied. An activity recovery rate of 86.4% and an enzyme loading of 115.3 mg/g were achieved under the optimal conditions: glutaraldehyde concentration of 1.0%, cross-linking time of 180 min, and the weight ratio of MOF to enzyme of 8:1. The optimal temperature and optimal pH of the immobilized amidase were determined to be 40 °C and 9.0, respectively, and the Km, Vmax and kcat of the immoblized amidase were 58.32 mmol/L, 16.23 μmol/(min·mg), and 1 670 s⁻¹, respectively. The immobilized enzyme was used for (S)-4-fluorophenylglycine synthesis and the optimal reaction conditions were 300 mmol/L of N-phenylacetyl-4-fluorophenylglycine, 10 g/L of immobilized enzyme loading, and reacting for 180 min at pH 9.0 and 40 °C. A conversion rate of 49.9% was achieved under the optimal conditions, and the conversion rate can be increased to 99.9% under the conditions of enantiomeric excess. The immobilized enzyme can be repeatedly used, 95.8% of its original activity can be retained after 20 cycles.

Construction and immobilization of recombinant Bacillus subtilis with D-allulose 3-epimerase / 生物工程学报

D-allulose-3-epimerase (DPEase) is the key enzyme for isomerization of D-fructose to D-allulose. In order to improve its thermal stability, short amphiphilic peptides (SAP) were fused to the N-terminal of DPEase. SDS-PAGE analysis showed that the heterologously expressed DPEase folded correctly in Bacillus subtilis, and the protein size was 33 kDa. After incubation at 40 °C for 48 h, the residual enzyme activity of SAP1-DSDPEase was 58%. To make the recombinant B. subtilis strain reusable, cells were immobilized with a composite carrier of sodium alginate (SA) and titanium dioxide (TiO2). The results showed that 2% SA, 2% CaCl2, 0.03% glutaraldehyde solution and a ratio of TiO2 to SA of 1:4 were optimal for immobilization. Under these conditions, up to 82% of the activity of immobilized cells could be retained. Compared with free cells, the optimal reaction temperature of immobilized cells remained unchanged at 80 °C but the thermal stability improved. After 10 consecutive cycles, the mechanical strength remained unchanged, while 58% of the enzyme activity could be retained, with a conversion rate of 28.8% achieved. This study demonstrated a simple approach for using SAPs to improve the thermal stability of recombinant enzymes. Moreover, addition of TiO2 into SA during immobilization was demonstrated to increase the mechanical strength and reduce cell leakage.

Application of immobilized glycosidase in the synthesis of glycoside compounds / 生物工程学报

Glycoside compounds are widely used in medicine, food, surfactant, and cosmetics. The glycosidase-catalyzed synthesis of glycoside can be operated at mild reaction conditions with low material cost. The glycosidase-catalyzed processes include reverse hydrolysis and transglycosylation, appropriately reducing the water activity in both processes may effectively improve the catalytic efficiency of glucosidase. However, glucosidase is prone to be deactivated at low water activity. Thus, glucosidase was immobilized to maintain its activity in the low water activity environment, and even in neat organic solvent system. This article summarizes the advances in glycosidase immobilization in the past 30 years, including single or comprehensive immobilization techniques, and immobilization techniques combined with genetic engineering, with the aim to provide a reference for the synthesis of glycosides using immobilized glycosidases.

Enzymatic synthesis of coconut oil based wax esters by immobilized lipase EQ3 and commercial lipozyme RMIM

BACKGROUND: Liquid wax esters are widely used in cosmetic as well as pharmaceutical and other industries. The demand of organic and natural products is increasing nowadays. Coconut oil contains benefit fatty acids and has been mainly used for oil-based and moisturizer products. Liquid wax esters from coconut oil and unsaturated fatty alcohol can be synthesized by enzymatic reaction; and it is interesting for using as an alternative natural ingredient in these industries. RESULTS: Optimal condition for coconut oil based wax ester synthesis by immobilized lipase EQ3 was 10 U of enzyme, temperature at 30°C and molar ratio of coconut oil to oleyl alcohol at 1:3 (mol/mol) (0.33X) dissolved in isooctane for 12 h, while for Lipozyme RM IM optimal condition was 10 U of enzyme, temperature at 45°C and oil/alcohol molar ratio at 1:3 (0.33X) dissolved in isooctane for 3 h. Percentage of wax esters synthesized by both lipases reached more than 88%. Both immobilized lipases catalyzed high yield of wax esters within the 2nd batch; after that, the immobilized lipases showed reduced activity and synthesized b60% of wax esters from the 3rd to 5th batch. The main composition of wax esters was ~48% oleyl laurate with 10% degradation at ~250°C. CONCLUSIONS: The liquid wax ester synthesis by commercial Lipozyme RM IM had higher effect than immobilized lipase EQ3, but both catalysts were stable within 2 batches in the optimum condition. The characteristic properties of wax esters showed potential for use as components in cosmetics and skin care products.

Modeling of lactose enzymatic hydrolysis using Monte Carlo method

Background: Mathematical modeling is useful in the analysis, prediction, and optimization of an enzymatic process. Unlike the conventional modeling methods, Monte Carlo method has special advantages in providing representations of the molecule's spatial distribution. However, thus far, Monte Carlo modeling of enzymatic system is namely based on unimolecular basis, not suitable for practical applications. In this research, Monte Carlo modeling is performed for enzymatic hydrolysis of lactose for the purpose of real-time applications. Results: The enzyme hydrolysis of lactose, which is conformed to Michaelis­Menten kinetics, is modeled using the Monte Carlo modeling method, and the simulation results prove that the model predicts the reaction kinetics very well. Conclusions: Monte Carlo modeling method can be used to model enzymatic reactions in a simple way for real-time applications.

Characterization of Mycobacterium tuberculosis dihydrofolate reductase immobilized on magnetic nanoparticles / 生物工程学报

To explore the immobilization of target proteins for screening libraries of ligand mixtures, magnetic submicron particles (MSP) functionalized with Ni²⁺-NTA and carboxyl were compared for the immobilization of Mycobacterium tuberculosis dihydrofolate reductase (MtDHFR). MtDHFR fused with 6×His was expressed, purified and characterized for kinetics. MtDHFR was immobilized on Ni²⁺-NTA-functionalized MSP directly and carboxyl-functionalized MSP upon activation. The immobilization capacity, residual activity, thermostability and affinities for putative inhibitors were characterized. MtDHFR immobilized on Ni²⁺-NTA-functionalized MSP retained about 32% activity of the free one with the immobilization capacity of (93±12) mg/g of MSP (n=3). Ni²⁺ and EDTA synergistically inhibited MtDHFR activity, while Fe³⁺ had no obvious interference. MtDHFR immobilized on carboxyl-functionalized MSP retained (87±4)% activity of the free one with the immobilization capacity of (8.6±0.6) mg/g MSP (n=3). In 100 mmol/L HEPES (pH 7.0) containing 50 mmol/L KCl, there was no significant loss of the activities of the free and immobilized MtDHFR after storage at 0 °C for 16 h, but nearly 60% and 35% loss of their activities after storage at 25 °C for 16 h, respectively. The inhibition effects of methotrexate on the immobilized and free MtDHFR were consistent (P>0.05). The immobilization of MtDHFR on carboxyl-functionalized MSP was thus favorable for higher retained activity and better thermostability, with promise for rapid screening of its ligand mixtures.

Poly(DL-lactide)-degrading enzyme production by immobilized Actinomadura keratinilytica strain T16-1 in a 5-L fermenter under various fermentation processes

Background: Poly(DL-lactic acid), or PDLLA, is a biodegradable polymer that can be hydrolyzed by various types of enzymes. The protease produced by Actinomadura keratinilytica strain T16-1 was previously reported to have PDLLA depolymerase activity. However, few studies have reported on PDLLA-degrading enzyme production by bacteria. Therefore, the aims of this study were to determine a suitable immobilization material for PDLLA-degrading enzyme production and optimize PDLLA-degrading enzyme production by using immobilized A. keratinilytica strain T16-1 under various fermentation process conditions in a stirrer fermenter. Results: Among the tested immobilization materials, a scrub pad was the best immobilizer, giving an enzyme activity of 30.03 U/mL in a shake-flask scale. The maximum enzyme activity was obtained at aeration 0.25 vvm, agitation 170 rpm, 45°C, and 48 h of cultivation time. Under these conditions, a PDLLA-degrading enzyme production of 766.33 U/mL with 15.97 U/mL·h productivity was observed using batch fermentation in a 5-L stirrer fermenter. Increased enzyme activity and productivity were observed in repeated-batch (942.67 U/mL and 19.64 U/mL·h) and continuous fermentation (796.43 U/mL and 16.58 U/mL·h) at a dilution rate of 0.013/h. Scaled-up production of the enzyme in a 10-L stirrer bioreactor using the optimized conditions showed a maximum enzyme activity of 578.67 U/mL and a productivity of 12.06 U/mL·h. Conclusions: This research successfully scaled-up the enzyme production to 5 and 10 L in a stirrer fermenter and is helpful for many applications of poly(lactic acid).

Molecular cloning, expression, and immobilization of glutamate decarboxylase from Lactobacillus fermentum YS2

Background: GABA (γ-aminobutyric acid) is a four-carbon nonprotein amino acid that has hypotensive, diuretic, and tranquilizing properties. Glutamate decarboxylase (GAD) is the key enzyme to generate GABA. A simple and economical method of preparing and immobilizing GAD would be helpful for GABA production. In this study, the GAD from Lactobacillus fermentum YS2 was expressed under the control of a stress-inducible promoter and was purified and immobilized in a fusion form, and its reusability was investigated. Results: The fusion protein CBM-GAD was expressed in Escherichia coli DH5α carrying pCROCB-gadB, which contained promoter PrpoS, cbm3 (family 3 carbohydrate-binding module from Clostridium thermocellum) coding sequence, the gadB gene from L. fermentum YS2 coding for GAD, and the T7 terminator. After a one-step purification of CBM-GAD using regenerated amorphous cellulose (RAC) as an adsorbent, SDS-PAGE analysis revealed a clear band of 71 kDa; the specific activity of the purified fusion protein CBM-GAD reached 83.6 ± 0.7 U·mg-1. After adsorption onto RAC, the immobilized GAD with CBM3 tag was repeatedly used for GABA synthesis. The protein-binding capacity of RAC was 174 ± 8 mg·g-1. The immobilized CBM-GAD could repeatedly catalyze GABA synthesis, and 8% of the initial activities was retained after 10 uses. We tested the conversion of monosodium glutamate to GABA by the immobilized enzyme; the yield reached 5.15 g/L and the productivity reached 3.09 g/L·h. Conclusions: RAC could be used as an adsorbent in one-step purification and immobilization of CBM-GAD, and the immobilized enzyme could be repeatedly used to catalyze the conversion of glutamate to GABA.

Immobilization of horseradish peroxidase on Fe3O4 magnetic nanoparticles

Background: Iron magnetic nanoparticles have attracted much attention. They have been used in enzyme immobilization because of their properties such as product is easily separated from the medium by magnetic separation. The present work was designed to immobilize horseradish peroxidase on Fe3O4 magnetic nanopraticles without modification. Results: In the present study, horseradish peroxidase (HRP) was immobilized on non-modified Fe3O4 magnetic nanoparticles. The immobilized HRP was characterized by FT-IR spectroscopy, scanning electron microscopy, and energy dispersive X-ray. In addition, it retained 55% of its initial activity after 10 reuses. The optimal pH shifted from 7.0 for soluble HRP to 7.5 for the immobilized HRP, and the optimal temperature shifted from 40°C to 50°C. The immobilized HRP is more thermostable than soluble HRP. Various substrates were oxidized by the immobilized HRP with higher efficiencies than by soluble HRP. Km values of the soluble and immobilized HRP were 31 and 45 mM for guaiacol and 5.0 and 7.0 mM for H2O2, respectively. The effect of metals on soluble and immobilized HRP was studied. Moreover, the immobilized HRP was more stable against high concentrations of urea, Triton X-100, and isopropanol. Conclusions: Physical immobilization of HRP on iron magnetic nanoparticles improved the stability toward the denaturation induced by pH, heat, metal ions, urea, detergent, and water-miscible organic solvent.

Efficient immobilization of agarase using carboxyl-functionalized magnetic nanoparticles as support

Background: A simple and efficient strategy for agarase immobilization was developed with carboxyl-functionalized magnetic nanoparticles (CMNPs) as support. The CMNPs and immobilized agarase (agarase-CMNPs) were characterized by transmission electron microscopy, dynamic light scattering, vibrating sample magnetometry, scanning electron microscopy, X-ray diffraction, thermogravimetric analysis, and zeta-potential analysis. The hydrolyzed products were separated and detected by ESI-TOF-MS. Results: The agarase-CMNPs exhibited a regular spherical shape with a mean diameter of 12 nm, whereas their average size in the aqueous solution was 43.7 nm as measured by dynamic light scattering. These results indicated that agarase-CMNPs had water swelling properties. Saturation magnetizations were 44 and 29 emu/g for the carriers and agarase-CMNPs, respectively. Thus, the particles had superparamagnetic characteristics, and agarase was successfully immobilized onto the supports. Agaro-oligosaccharides were prepared with agar as substrate using agarase-CMNPs as biocatalyst. The catalytic activity of agarase-CMNPs was unchanged after six reuses. The ESI-TOF mass spectrogram showed that the major products hydrolyzed by agarase-CMNPs after six recycle uses were neoagarotetraose, neoagarohexaose, and neoagarooctaose. Meanwhile, the end-products after 90 min of enzymatic treatment by agarase-CMNPs were neoagarobiose and neoagarotetraose. Conclusions: The enhanced agarase properties upon immobilization suggested that CMNPs can be effective carriers for agarase immobilization. Agarase-CMNPs can be remarkably used in developing systems for repeated batch production of agar-derived oligosaccharides.

Scaling-up batch conditions for efficient sucrose hydrolysis catalyzed by an immobilized recombinant Pichia pastoris cells in a stirrer tank reactor

Background: Invert sugar is used greatly in food and pharmaceutical industries. This paper describes scaling-up batch conditions for sucrose inversion catalyzed by the recombinant Pichia pastoris BfrA4X whole cells expressing Thermotoga maritima invertase entrapped in calcium alginate beads. For the first time, we describe the application of a kinetic model to predict the fractional conversion expected during sucrose hydrolysis reaction in both, a model and a prototype bioreactor with 0.5- and 5-L working volume, respectively. Results: Different scaled-up criteria used to operate the 0.5-L bioreactor were analyzed to explore the invert sugar large scale production. After model inversion studies, a 5-L scaled-up reaction system was performed in a 7-L stirred reactor. Both scaled-up criteria, immobilized biocatalyst dosage and stirring speed, were analyzed in each type of bioreactors and the collected data were used to ensure an efficient scale-up of this biocatalyst. Conclusions: To date, there is not enough information to describe the large-scale production of invert sugar using different scaled-up criteria such as dose of immobilized biocatalyst and stirring speed effect on mass transfer. The present study results constitute a valuable tool to successfully carry out this type of high-scale operation for industrial purposes.

Synthesis of structured triacylglycerols enriched in n-3 fatty acids by immobilized microbial lipase

Abstract The search for new biocatalysts has aroused great interest due to the variety of micro-organisms and their role as enzyme producers. Native lipases from Aspergillus niger and Rhizopus javanicus were used to enrich the n-3 long-chain polyunsaturated fatty acids content in the triacylglycerols of soybean oil by acidolysis with free fatty acids from sardine oil in solvent-free media. For the immobilization process, the best lipase/support ratios were 1:3 (w/w) for Aspergillus niger lipase and 1:5 (w/w) for Rhizopus javanicus lipase using Amberlite MB-1. Both lipases maintained constant activity for 6 months at 4 °C. Reaction time, sardine-free fatty acids:soybean oil mole ratio and initial water content of the lipase were investigated to determine their effects on n-3 long-chain polyunsaturated fatty acids incorporation into soybean oil. Structured triacylglycerols with 11.7 and 7.2% of eicosapentaenoic acid + docosahexaenoic acid were obtained using Aspergillus niger lipase and Rhizopus javanicus lipase, decreasing the n-6/n-3 fatty acids ratio of soybean oil (11:1 to 3.5:1 and 4.7:1, respectively). The best reaction conditions were: initial water content of lipase of 0.86% (w/w), sardine-free faty acids:soybean oil mole ratio of 3:1 and reaction time of 36 h, at 40 °C. The significant factors for the acidolysis reaction were the sardine-free fatty acids:soybean oil mole ratio and reaction time. The characterization of structured triacylglycerols was obtained using easy ambient sonic-spray ionization mass spectrometry. The enzymatic reaction led to the formation of many structured triacylglycerols containing eicosapentaenoic acid, docosahexaenoic acid or both polyunsaturated fatty acids.

Structural regulation by calcium ion in preparing cross-linked enzyme aggregates / 生物工程学报

We studied the effect of calcium ion on particle size and pore structure of cross-linked enzyme aggregates (CLEAs) of glucose oxidase, with activity and stability of the enzyme as evaluation criteria. With calcium ion to prepare CLEA significantly decreased particle sizes of CLEAs whilst the pore structures of CLEAs gradually disappeared with the increase of calcium concentration. When glucose oxidase was precipitated at 0.1 mmol/L Ca²⁺, glucose oxidase in CLEA showed the definitive pore structure. Moreover, glucose oxidase activity in CLEA with Ca²⁺ was 1.69 times higher than that without Ca²⁺. Even at Ca²⁺ as high as 1.0 mmol/L, glucose oxidase activity in CLEA was 42% higher than that of CLEA without Ca²⁺. Furthermore, CLEA prepared with 0.1 mmol/L Ca²⁺ not only exhibited higher substrate conversion and operational stability, but also increased the maximum reaction speed. Therefore, calcium ion improved the performance of glucose oxidase in CLEAs.