超声波和超临界流体对酶活性的影响

酶是生物催化剂,具有一般催化剂所没有的独特优点,但由于容易失活而限制了其在工业上的应用。超声波是一种物理能量形式,在一定的条件下可与酶协同作用促进反应。超临界流体作为良好的非水反应溶剂,在生物工程中有着广泛的应用前景。简要介绍了超声波、超临界流体对酶活性的各种影响因素、作用机理及研究进展。     

海参i-型溶菌酶在毕赤酵母基因工程菌中优化表达及纯化

将实验室已构建的毕赤酵母基因工程茵(pPIC9K-SjLys/GS115)作为海参i-型溶茵酶生产菌株,本研究分别从甲醇浓度、培养基pH、温度和诱导时间对其产酶发酵条件进行优化.实验得出甲醇诱导浓度为1.0％,发酵培养基初始pH 6.0,温度30℃,培养96 h为最佳目的蛋白表达条件,其发酵液中海参i-型溶菌酶含量达10.63 mg/L.将发酵液经离心和超滤浓缩后得到上清液,再经离子交换和凝胶过滤层析纯化获得海参i-型溶菌酶产品,其酶活力达826.44 U/mg.经测定该酶对革兰氏阳性菌溶壁微球菌和革兰氏阴性菌副溶血弧菌均具有明显的抑菌作用.     

Effects of supercritical CO2 exposure and depressurization on immobilized lipase activity

Lipozyme IM20 from Novo Nordisk (Denmark) was examined after various treatments. Conditions were chosen to reflect those that would be considered in the design of an industrial process. A two-level factorial design was employed to assess the effects of pressurization/depressurization cycles, rate of depressurization and exposure length. A significant three-factor interaction was observed. Lowest residual activity was observed for runs in which the depressurization rate was 86–89 bar min^–1. Incubation for 12 h also yielded low residual activity but only when exposing the immobilized enzyme to one cycle. The highest residual activity was obtained for immobilized enzymes repeatedly exposed for periods of 12 h (5 times) with a depressurization rate of 4.3 to 4.45 bar min^–1. This effect may be due to the extraction of an inhibiting compound. Tuning process parameters can lead to a seven-fold change in residual activity.     

Irreversible lysozyme inactivation and aggregation induced by stirring: kinetic study and aggregates characterisation

Stirring strongly enhanced irreversible inactivation and aggregation of lysozyme being studied as a model enzyme. From 0 to 740 rpm (equivalent to impeller tip speeds from 0 to 0.77 m s^–1), the inactivation kinetic constant was proportional to the power imparted by the impeller. Collisions between inactive and native molecules induced inactivation of the latter and led to lysozyme aggregation. These fractal aggregates of lysozyme were made of monomers, dimers and trimers.     

羟胺氧化还原酶在生物脱氮中的研究进展

羟胺氧化还原酶(hydroxylamine oxidoreductase,HAO)属于多血红素蛋白酶家族,每个单体由7个电子转移血红素和1个催化血红素组成.HAO既可分别催化羟胺和肼的氧化反应,也可催化羟胺、一氧化氮及亚硝酸盐的还原反应.不同硝化细菌中,HAO的最适温度、pH、底物、产物特异性及酶抑制剂等存在差异.作为...     

Effect of polyols on the conformational stability and biological activity of a model protein lysozyme

The purpose of this study was to investigate the stabilizing action of polyols against various protein degradation mechanisms (eg, aggregation, deamidation, oxidation), using a model protein lysozyme. Differential scanning calorimeter (DSC) was used to measure the thermodynamic parameters, mid point transition temperature and calorimetric enthalpy, in order to evaluate conformational stability. Enzyme activity assay was used to corroborate the DSC results. Mannitol, sucrose, lactose, glycerol, and propylene glycol were used as polyols to stabilize lysozyme against aggregation, deamidation, and oxidation. Mannitol was found to stabilize lysozyme against aggregation, sucrose against deamidation both at neutral pH and at acidic pH, and lactose against oxidation. Stabilizers that provided greater conformational stability of lysozyme against various degradation mechanisms also protected specific enzyme activity to a greater extent. It was concluded that DSC and bioassay could be valuable tools for screening stabilizers in protein formulations.     

Preparation of budesonide and budesonide-PLA microparticles using supercritical fluid precipitation technology

The objective of this study was to prepare and characterize microparticles of budesonide alone and budesonide and polylactic acid (PLA) using supercritical fluid (SCF) technology. A precipitation with a compressed antisolvent (PCA) technique employing supercritical CO₂ and a nozzle with 100-μm internal diameter was used to prepare microparticles of budesonide and budesonide-PLA. The effect of various operating variables (temperature and pressure of CO₂ and flow rates of drug-polymer solution and/or CO₂) and formulation variables (0.25%, 0.5%, and 1% budesonide in methylene chloride) on the morphology and size distribution of the microparticles was determined using scanning electron microscopy. In addition, budesonide-PLA particles were characterized for their surface charge and drug-polymer interactions using a zeta meter and differential scanning calorimetry (DSC), respectively. Furthermore, in vitro budesonide release from budesonide-PLA microparticles was determined at 37°C. Using the PCA process, budesonide and budesonide-PLA microparticles with mean diameters of 1 to 2 μm were prepared. An increase in budesonide concentration (0.25%–1% wt/vol) resulted in budesonide microparticles that were fairly spherical and less aggiomerated. In addition, the size of the microparticles increased with an increase in the drug-polymer solution flow rate (1.4–4.7 mL/min) or with a decrease in the CO₂ flow rate (50–10 mL/min). Budesonide-PLA microparticles had a drug loading of 7.94%, equivalent to ∼80% encapsulation efficiency. Budesonide-PLA microparticles had a zeta potential of— 37±4 mV, and DSC studies indicated that SCF processing of budesonide-PLA microparticles resulted in the loss of budesonide crystallinity. Finally, in vitro drug release studies at 37°C indicated 50% budesonide release from the budesonide-PLA microparticles at the end of 28 days. Thus, the PCA process was successful in producing budesonide and budesonide-PLA microparticles. In addition, budesonide-PLA microparticles sustained budesonide release for 4 weeks.     

Comparative study of digestive enzymes of squid (Todarodes pacificus) viscera after supercritical carbon dioxide and organic solvent extraction

Three major classes of digestive enzymes of squid viscera were characterized following extraction of oil by supercritical carbon dioxide (SCO₂) and organic solvent, n-hexane. Squid viscera were extracted at temperature, 35∼45°C and pressure, 15∼25 MPa for 2.5 h by SCO₂ with a constant flow rate of 22 g/min. Oil extraction yield increased with the increasing of extraction pressure and temperature. The highest oil extracted residues of squid viscera were used for characterization of digestive enzymes. The activities of protease, lipase, and amylase were highest in n-hexane treated squid viscera samples and lowest in SCO₂ treated samples. The crude extracts of SCO₂ and n-hexane treated squid viscera samples showed almost same optimum pH and pH stability for each of the digestive enzymes. The optimum temperature of protease, lipase, and amylase were found to almost similar in SCO₂ and n-hexane treated samples. But the thermal stability for each digestive enzyme in SCO₂ treated squid viscera were slightly higher than that of n-hexane treated squid viscera. Studies using SDS-PAGE showed no significant differences in protein patterns of the crude extracts of untreated and SCO₂ and n-hexane treated squid viscera indicating no denaturation of proteins.     

Expression of functional recombinant human lysozyme in transgenic rice cell culture

Using particle bombardment-mediated transformation, a codon-optimized synthetic gene for human lysozyme was introduced into the calli of rice (Oryza sativa) cultivar Taipei 309. The expression levels of recombinant human lysozyme in the transformed rice suspension cell culture approached approximately 4% of total soluble protein. Recombinant human lysozyme was purified to greater than 95% homogeneity using a two-step chromatography process. Amino acid sequencing verified that the N-terminus of the mature recombinant human lysozyme was identical to native human lysozyme. This indicates that the rice RAmy3D signal peptide was correctly cleaved off from the human lysozyme preprotein by endogenous rice signal peptidase. Recombinant human lysozyme was found to have the same molecular mass, isoelectric point and specific activity as native human lysozyme. The bactericidal activity of recombinant human lysozyme was determined by turbidimetric assay using Micrococcus lysodeikticus in 96-well microtiter plates. The bactericidal activity of lysozyme on Gram-negative bacteria was examined by adding purified lysozyme to mid-log phase cultures of E. coli strain JM109. In this study, significant bactericidal activity was observed after E.coli cells were exposed to recombinant human lysozyme for 60min. Both native and recombinant human lysozyme displayed the same thermostability and resistance to degradation by low pH. The potential for using rice-derived lysozyme as an antimicrobial food supplement, particularly for infant formula and baby foods, is discussed.     

Changes of Hexosamine and Acetyl Contents during Cultivation of Extracellular Galactosamine-containing Polymer from Aspergillus parasiticus

The reactions of N-acetylchitooligosaccharides with chitinolytic enzyme were analyzed by HPLC using a Tosoh TSK-Gel amide-80 column with 70% acetonitrile as an eluent. We separated α and β anomeric forms of N-acetylchitooligosaccharides, and obtain the following advantages of this HPLC method.

1. We can easily identify the reaction mechanism of chitinolytic enzymes by this method, distinguishing the inverting mechanism showing α anomer formation from the retaining mechanism showing β anomer formation.

2. We can also estimate the cleavage patterns of N-acetylchitooligosaccharides by chitinolytic enzymes by using natural substrates.     

Solvent effects on the structure,dynamics and activity of lysozyme

Kuntz and Kauzmann have argued that dehydrating a protein results in conformational changes. In contrast, Rupleyet al. have developed a hydration model which involves no significant change in conformation; the onset of enzyme activity in hen egg-white lysozyme at hydration values of about 0.2 g water/g protein they ascribe rather to a solvation effect. Using a direct difference infra-red technique we can follow specific hydration events as water is added to a dry protein. Conformational studies of lysozyme using laser Raman spectroscopy indicate changes in conformation with hydration that are complete just before measurable activity is found. Parallel nuclear magnetic resonance measurements of exchangeability of the main chain amide hydrogens, as a function of hydration from near dryness, suggest a hydration-related increase in conformational flexibility which occurs before-and is probably necessary for-the Raman-detected conformational changes. Very recent inelastic neutron scattering measurements provides direct evidence of a flexibility change induced by hydration, which is apparently necessary before the enzyme can achieve adequate flexibility for it to begin to function.     

链霉菌MIUG4.46胞内葡萄糖异构酶的提取条件研究

比较了采用不同方法处理链霉菌MIUG4．46的生物量时,细胞内葡萄糖异构酶的释放能力。当联合采用溶菌酶和高压力来破碎细胞时,提取物中葡萄糖异构酶的总酶活力为91．3％,酶的回收率为87％。用溶菌酶溶菌和高压破碎细胞后,在Mg~(2＋)和Co~(2＋)存在的情况下,对细胞悬浮液进行热处理（70℃下加热10min）时,有利于提高提取物中葡萄糖异构酶的纯度,酶的比活力达3．275U／mg蛋白质。