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.

Heterologous expression and enzymatic properties of active inclusion bodies of sucrose isomerase fused with coiled-coil domain / 中国生物制品学杂志

@#Objective To express the sucrose isomerase(SI) fused with the tetrameric coiled-coil domain of the cell surface protein tetrabrachion(TdoT),and study the enzymatic properties of the recombinant enzymes.Methods The gene of SI fused with TdoT at the N/C terminus was cloned into the expression vectors respectively to construct the recombinant expression vectors pET-24a-TdoT-SI and pET-24b-SI-TdoT,which were transformed into E.coli BL21(DE3) and induced to express recombinant enzymes.The enzymatic properties and product specificity of the purified recombinant enzymes were studied.Results TdoT-SI and SI-TdoT were expressed as inclusion bodies with catalytic activity,while SI inclusion bodies without TdoT showed no catalytic activity.The results of enzymatic property analysis showed that the optimum reaction temperature for TdoT-SI and SI-TdoT active inclusion bodies was 40 ℃,and the optimum reaction pH was 5.5 and 5.0,respectively.The K_m of TdoT-SI active inclusion bodies was(103.9±9.5) mmol/L and the k_(cat)/K_m was(0.06±0.002) L/(mmol·s),while the K_m of SI-TdoT active inclusion bodies was(54.4±6.6) mmol/L and the k_(cat)/K_m was(0.03±0.002) L/(mmol·s).The results of product specificity analysis exhibited that the proportion of isomaltulose in the product did not change significantly,while the proportion of trehalose decreased,and the proportion of monosaccharides increased with increasing reaction temperature.Conclusion The active inclusion bodies of SI fused with coiled-coil domain were successfully prepared by fusion expression technology.As a novel self-immobilized enzyme,it has the advantage of simultaneous expression and immobilization,which provides a new strategy for large-scale preparation and efficient utilization of recombinant SI.

Immobilization of lactase on magnetic chitosan microspheres and its effect on enzymatic properties / 中国组织工程研究

BACKGROUND: The free enzyme has the problems of poor stability and inability to be reused during application. Using magnetic polymer microspheres as the carrier of the binding enzyme to prepare immobilized enzyme can maintain the natural activity of the enzyme, and can be reused, but also provide convenient conditions for automatic production management. OBJECTIVE: Magnetic chitosan microspheres as carrier of binding enzyme were used to prepare the immobilized lactase, which is easy to recycle, can be reused, and has high enzyme activity and stability. METHODS: By inputting a certain amount of magnetic chitosan microspheres into the phosphoric acid buffer for swelling for 2 hours, the swelling magnetic chitosan microspheres were collected with a magnet and added to a certain concentration of lactase phosphate buffer. They were shocked in a constant temperature shaker for 1 hour and preserved in refrigerator at 4 °C. The microspheres were precipitated with a magnet to pour out the supernatant. After full washing with buffer solution, the immobilized lactase was obtained. The properties of the magnetic microspheres (the amount of glutaraldehyde used in the preparation of microspheres was 2, 4, 6, 8, 10, 12, 14 mL), the amount of enzymes (0.5, 1, 1.5, 2 g/L) added, the pH (6.4, 6.8, 7.0, 7.2) of the buffer, and the immobilization time (1, 2, 5, 10, 15, 20 hours) were tested to determine the optimal immobilization conditions. RESULTS AND CONCLUSION: (1) The optimum conditions for immobilized lactose with magnetic chitosan microspheres were as follows: magnetic chitosan microspheres prepared with 10 mL of glutaraldehyde were selected as the immobilized carrier of lactase. The amount of enzyme added was 0.3 g/L, pH 7.0 and the immobilization time was 5 hours. (2) Compared with the free enzyme, immobilized lactase showed a wider range of reaction temperature and pH value. (3) The ability of immobilized enzyme binding substrate was enhanced. (4) After repeated use of the immobilized enzyme five times, the enzyme activity remained 65%. (5) The storage stability of lactase was also improved after immobilization.

Optimization of maltooligosyl fructofuranosidesproduction by recombinant β-cyclodextrin glycosyltransferase / 生物工程学报

Coupling sugar is a kind of new sweetener which can substitute sucrose. It has a good application prospect in food, medicine and other fields because of its good coloration, water retention and anti caries. The purpose of this study was to find cheap and easily available donor and acceptor, and to optimize the preparation process of coupling sugar by using β-cyclodextrin glycosyltransferase from Bacilluscirculans 251. Using sucrose as acceptor, the factors of preparing coupling sugar was optimized, including enzyme dosage, starch types, temperature, pH, ratio of starch/sucrose, and cooperation of isoamylase and β-CGTase. When 105 g/L potato starch and 95 g/L sucrose was used as substrates, the yield of coupling sugar reached 88.4%, which was catalyzed by 13.5 U/g immobilized β-CGTase and 45.0 U/g isoamylase under the conditions of pH 5.5 and 40 °C for 21 h. In this study, isoamylase and β-CGTase were used to prepare coupling sugar innovatively. This method had obvious advantages in yield and cost, which laid both theoretical and experimental foundation for the industrial enzymatic preparation of coupling sugars.

Concomitant use of immobilized uridine-cytidine kinase and polyphosphate kinase for 5'-cytidine monophosphate production / 生物工程学报

Uridine-cytidine kinase, an important catalyst in the compensation pathway of nucleotide metabolism, can catalyze the phosphorylation reaction of cytidine to 5'-cytidine monophosphate (CMP), but the reaction needs NTP as the phosphate donor. To increase the production efficiency of CMP, uridine-cytidine kinase gene from Thermus thermophilus HB8 and polyphosphate kinase gene from Rhodobacter sphaeroides were cloned and expressed in Escherichia coli BL21(DE3). Uridine-cytidine kinase was used for the generation of CMP from cytidine and ATP, and polyphosphate kinase was used for the regeneration of ATP. Then, the D403 metal chelate resin was used to adsorb Ni²⁺ to form an immobilized carrier, and the immobilized carrier was specifically combined with the recombinant enzymes to form the immobilized enzymes. Finally, single-factor optimization experiment was carried out to determine the reaction conditions of the immobilized enzyme. At 30 °C and pH 8.0, 60 mmol/L cytidine and 0.5 mmol/L ATP were used as substrates to achieve 5 batches of high-efficiency continuous catalytic reaction, and the average molar yield of CMP reached 91.2%. The above method has the advantages of low reaction cost, high product yield and high enzyme utilization rate, and has good applied value for industrial production.

Advances in chromatography-based methods for screening active compounds from natural products / 药学学报

Natural products have been a major source of leading compounds in drug discovery. How to effectively screen active compounds from complex matrix remains an interesting topic. In this review, we comprehensively summarized advanced liquid chromatography based approaches in natural products screening, including pre-column, on-column and post-column screening methods. Their advantages, disadvantages and prospect are also discussed.

Screening, identification and activity evaluation of pancreatic lipase inhibition in Prunella vulgaris / 中国中药杂志

Pancreatic lipase (PL) inhibitors were firstly screened from Prunella vulgaris with PL immobilized on carboxylic acid-terminated magnetic nanoparticles, then these possible inhibitors were identified by LC-MS/MS and mixed standards. Finally, their inhibitory effects and types on PL were tested by p-nitrophenol method. The results showed that four PL inhibitors were screened out from P. vulgaris and confirmed by LC-MS/MS and mixed standards. The IC₅₈ and inhibition types were as follows: caffeic acid [(252.3±3.6) mg·L⁻¹, anti-competitive inhibition], rutin [(91.2±1.6)mg·L⁻¹, competitive inhibition], hesperidin [(31.5±4.4) mg·L⁻¹, competitive inhibition] and ursolic acid [(41.3±2.2) mg·L⁻¹, competitive inhibition]. Their inhibitive types and abilities on PL were related to their molecular size, hydrophobicity and the number of hydrogen bond with PL triplet.

Recent advances in screening of enzymes inhibitors based on capillary electrophoresis / 药物分析学报

Capillary electrophoresis with many advantages plays an important role in pharmaceutical analysis and drug screening. This review gives an overview on the recent advances in the developments and applications of capillary electrophoresis in the field of enzyme inhibitor screening. The period covers 2013 to 2017. Both the pre-capillary enzyme assays and in-capillary enzyme assays which include electrophoretically mediated microanalysis (EMMA) and immobilized enzyme microreactor (IMER) are summarized in this article.

Immobilized enzyme reactors in HPLC and its application in inhibitor screening: A review / 药物分析学报

This paper sets out to summarize the literatures based on immobilized enzyme biochromatography and its application in inhibitors screening in the last decade.In order to screen enzyme inhibitors from a mass of compounds in preliminary screening,multi-pore materials with good biocompatibility are used for the supports of immobilizing enzymes,and then the immobilized enzyme reactor applied as the inmobilized enzyme stationary phase in HPLC.Therefore,a technology platform of high throughput screening is gradually established to screen the enzyme inhibitors as new anti-tumor drugs.Here,we briefly summarize the selective methods of supports,immobilization techniques,co-immobilized enzymes system and the screening model.

Diffusional restrictions in glyoxyl-agarose immobilized penicillin G acylase of different particle size and protein loading / Restricciones para la difusión de penicilina G acilasa inmovilizada en glicosil agarosa

Particle size and enzyme protein loading are design parameters of enzyme immobilization affecting biocatalyst performance that can be varied within broad margins. Their effect on mass transfer limitations at different bulk penicillin G concentrations has been studied with glyoxyl agarose immobilized penicillin G acylase biocatalysts of average particle size of 5·10-5m and 10·10-4m at protein loadings from 15 to 130 mg/g gel. Internal diffusional restrictions were evaluated for such biocatalysts: Thiele modulus varied from 1.17 for the small particles at the lower protein load to 5.84 for the large particles at the higher protein load. Effectiveness factors at different bulk substrate concentrations were determined for all biocatalysts, values ranging from 0.78 for small particle size at 25 mM penicillin G to 0.15 for large particle size at 2 mM penicillin G. Enzyme protein loading had a strong impact on the effectiveness factors of immobilized penicillin G acylase, being it more pronounced in the case of large particle size biocatalysts. At conditions in which 6-aminopenicillanic acid is industrially produced, all biocatalysts tested were mass-transfer limited, being this information valuable for reactor design and performance evaluation.

Determination of Cholesterol in Human Serum Based on Flow-injection Chemiluminescence Method by Sol-gel Immobilized Enzyme / 分析化学

The cholesterol esterase and its oxidase were respectively immobilized onto sol-gel(tetraethyl orthosilicate)and then an enzymatic reaction column was prepared. Hydrolysis of cholesterol ester took place in the presence of cholesterol esterase to form cholesterol under catalysis of cholesterol oxidase, and Cholesterol was oxidized to produce hydrogen peroxide which reacted with luminal in the presence of simulated enzyme hemoglobin to result in the emission of light. Thereupon, a chemiluminescence system combined with flow injection technology by sol-gel immobilized enzyme was developed for the determination of cholesterol including free and total amount in human serum. The CL intensity was well linear with the concentration of cholesterol;liner range of total and free cholesterol was 1.01×10~(-6)～2.02×10~(-4) mol/L with a detection limit of 7.5×10~(-7) mol/L) and 5.0×10~(-8)～2.18×10~(-5) mol/L) with a detection limit of 5.0×10~(-9) mol/L respectively. Contrasted the method) with the biochemical analyzer (TBA-120FR), both the methods had no significant difference. This method has been successfully applied for the detection of cholesterol in clinic serum samples.

Bioconversão de sacarose em ácido glicônico e frutose usando reator com membrana / Sucrose bioconversion into gluconic acid and fructose using a membrane reactor

A conversão enzimática da sacarose pela ação sucessiva da invertase e da glicose oxidase (GOD), permite obter produtos de maior valor agregado, a saber, frutose e o ácido glicônico, dois produtos de amplo uso na indústria farmacêutica, alimentícia e química. Foi estudada a aplicação da invertase imobilizada em resinas aniônicas do tipo Dowex® (um copolímero de poliestireno-divinilbenzeno) sobre a hidrólise da sacarose bem como a oxidação da glicose pela glicose oxidase solúvel ou imobilizada no mesmo suporte em separado (sistema bifásico), utilizando-se um reator de membrana acoplado à membrana de ultrafiltração (100kDa) ou de microfitração (5µm). Posteriormente, avaliou-se o desempenho de ambas as formas de enzimas, solúveis ou imobilizadas num sistema monofásico empregando o mesmo reator...

The enzymatic conversion of sucrose through a successive action of invertase and glucose oxidase (GOO) allows the obtainment of products with higher commercial value, fructose and gluconic acid, which are widely used in pharmaceutical, food and chemical industries. Invertase and GOO immobilized on Dowex® anionic resin (a polystyrene divinylbenzene copolymer) as well as soluble GOD were used in a membrane bioreactor (MS) for sucrose hydrolysis and glucose oxidation. The MB was coupled with a UF-membrane (100kDa) or a MF-membrane (5µm). The bioconversion was conducted in two steps (biphasic system) as well as in one step (monophasic system). The bioconversion operated in a biphasic system permitted obtaining a fructose syrup with a concentration of about 70% through a separation of glucose and fructose using a cationic resin, 50W:8-100. As for the monophasic system, the yield of 96.6% and 67.4% for soluble and immobilized forms were attained respectively. No leakage of the enzymes from the support allowed the use of a microfiltration membrane, adding advantages to the membrane bioreactor operation.

Immobilization of ?-glucosidase with Nano SiO_2 and Its Application in Soybean Isoflavone Hydrolysis in Two-phase-system / 中国生物工程杂志

Nano materials are popular with its interesting properties. Large specific surface area, high mechanical strength, good absorbability make them good potencial carriers of catalyst. With the help of chemical modification, nano SiO2 particals were used as carrier to immobilize ?-glucosidase with covalent binding. It is found that the load of nano particals can reach 1/8 of its weight, and the recovered activity of enzyme is 70%. The resistence of immobilized enzyme to organic solvent wase improved obviously, which was then used in Ethyl Acetate-Water-two-phase-system to hydrolyzed soybean isoflavones.

Applications of chitosan and its derivatives in enzyme immobilization / 中国海洋药物

The recent applications of chitosan and its derivatives in enzyme immobilization are reviewed in this paper. It is introduced that the immobilized enzyme on different kinds of chitosan and its derivitaves have been prepared by various immobilization methods. Their applications are also discussed. The chitosan and its derivatives are admirable carriers for enzyme immobilization with many advantages, such as various and simple immobilized methods? good biocompatibility and abundant resources.

Purification and properties of polyphenoloxidase of mango peel (Mangifera indica).

Polyphenoloxidase from mango (Mangifera indica) peel was purified to homogeneity by ammonium sulphate fractionation, chromatography on DEAE-Sephadex and gel filtration of Sephadex G-200. The enzyme had an apparent molecular weight of 136,000. Its pH and temperature optimum were 5.4 and 50°C, respectively. The enzyme possessed catecholase activity and was specific to o-dihydroxy phenols. The enzyme also exhibited peroxidase activity. Some non-oxidizable phenolic compounds inhibited the enzyme competitively. High inhibitory effects were also shown by some metal chelators and reducing agents, Mango peel polyphenol oxidase when immobilized onto DEAE Sephadex showed slightly higher Km for catechol and lower pH and temperature optima.