Preparation of amidoxime-modified polyacrylonitrile nanofibers immobilized with laccase for dye degradation

A novel approach to preparing multifunctional composite nanofibrous membrane was developed. Polyacrylonitrile (PAN) nanofibrous membrane was fabricated by electrospinning and then the nitrile groups in PAN copolymer was chemically modified to obtain amidoxime modified PAN (AOPAN) nanofiber membrane which was further used as a functional support for laccase immobilization. During the process of reactive dye degradation catalyzed by the AOPAN nanofiber membrane immobilized with laccase, metal ion adsorption occurred at the same time. The chemical modification was confirmed by Fourier transform spectroscopy (FTIR). Scanning electron microscope (SEM) was employed to investigate the surface morphologies of the electrospun nanofibers before and after laccase immobilization. The effects of environmental factors on laccase activity were studied in detail. It was found that the optimum pH and temperature for the activity of immobilized laccase was 3.5 and 50 °C. The relative activity retention of the immobilized laccase decreased dramatically during the initial four repeated uses. After 20 days’ storage, the activity retention of immobilized laccase was still high above 60 %. It has also proved that laccase immobilized on AOPAN nanofiber membrane performed well in dye degradation and metal ion adsorption.     

Decolorization of dyes using immobilized laccase enzyme on zinc ferrite nanoparticle from single and binary systems

In this paper, laccase enzyme was immobilized onto zinc ferrite nanoparticle and the characteristics of enzyme immobilized nanoparticle (EIN) were evaluated by Fourier transform infrared (FTIR) and scanning electronic microscope (SEM). Enzymatic decolorization of dyes using EIN from single and binary systems was studied. Direct red 31 (DR31) and Acid blue 92 (AB92) were used as model dyes. The effects of several parameters such as EIN dosage, pH, and dye concentration on decolorization of dyes from single and binary systems were evaluated. The optimized amount of EIN, reaction time, and pH for decolorization of dyes from single and binary systems were 0.2 g (for DR31) and 0.3 g (for AB92), 40 min, and 3 in single systems and 0.2 g (for DR31 and AB92), 40 min, and 3 in binary systems, respectively. Dye decolorization kinetics followed Michaelis-Menten model. The results showed that enzymatic process using EIN was effective method to decolorize dyes from single and binary systems.     

Enhancement biocompatibility of bacterial cellulose membrane via laccase/TEMPO mediated grafting of silk fibroins

Silk fibroin (SF) has the characteristic of moisture penetrability and biocompatibility. To enhance the biocompatibility of bacterial cellulose (BC), silk fibroin is grafted onto BC membrane using laccase and 2,2′,6,6′-tetramethylpiperidine-N-oxyl (TEMPO). As the model compound of BC, cellobiose is incubated with laccase/TEMPO for disclosing the mechanism of enzymatic oxidation. The structure and property of the composite membranes of SF/BC are investigated by means of FTIR, XPS, DSC, and biocompatibility analysis. The results indicate that cellobiose might react with hexamethylenediamine and form Schiff bases. The concentration of amino group in SF solution noticeably decreased after laccase/TEMPO oxidation, indicating the occurrence of self-crosslinking of SF. After enzymatic grafting of SF, the content of atomic nitrogen on BC surface was increased compared to that of the control. Meanwhile, the composite membrane of SF/BC exhibits more satisfactory biocompatibility compared to BC, and it has potential applications in biomedical fields.     

Effect of laccase on dyeing properties of polyphenol-based natural dye for wool fabric

Chemical mordants are generally used during the dyeing process, to increase the uptake of natural dyes. Traditional mordants include metal salts, such as copper, iron, aluminum, chromium, and other metal ions. Continuous developments in bio-engineering technology focus on methods that lower the impact on the environment. In this regard, enzymatic processes show great promise in textile field, due to their efficacy, mild conditions, and environment friendly nature. Laccase is a multicopper oxidoreductase that catalyzes in-situ polymerization of small phenolic monomers to form a colorful polymer. In this study, effects of laccase treatment on the dyeing properties of wool fabrics, dyed with natural dyes (turmeric, grape seed extract, and Chinese gallnut), were investigated. The dyeing properties of the dyed wool fabrics were evaluated under different conditions, including laccase stoichiometry, temperature, pH, and reaction time. The structural changes of natural dyes, due to laccase catalyst were also examined by FT-IR. The results showed that laccase greatly influenced the dyeing performance of Chinese gallnut. Moreover, dyeing effects of the samples using post-mordanting method was better than the other two methods, under the same conditions. After laccase treatment, the dyeing properties of Chinese gallnut improved. Finally, factors affecting the dyeing process with Chinese gallnut were studied and the optimized conditions were determined through single-factor experiments.     

Thrombin immobilization to enzymatic modified PET and PAN fabrics and their applications

Enzymatic modification of synthetic materials has immense potential both of the functionalization of polymeric materials, such as poly(acrylonitrile) or polyesters, and the production of polymers for special applications, such as medical devices and enzyme immobilization. In this study, poly(ethyleneterephtalate) and poly(acrylonitrile) fabrics were modified with commercial laccase and nitrilase, respectively. Contact angles of enzymatic modified and unmodified fabrics were measured and it was found contact angles of enzymatic modified fabrics were less than those of unmodified fabrics. Attenuated-Total-Reflection-Fourier-Transform infrared spectroscopy showed that carboxylic acid groups occurred on fabrics after enzymatic modifications. Surfaces of modified and unmodified fabrics were investigated using scanning electron microscopy. Surfaces of unmodified fabrics were smooth but surfaces of modified fabrics were rugged and cracked. Thrombin was immobilized in modified fabrics by using 1-Ethyl-3-(3-dimetylaminopropyl)-carbodiimide. Optimization studies were also performed for the immobilization of thrombin. After prepared material was tested to stop bleeding in vitro conditions and it was found that thrombin immobilized poly(ethyleneterephtalate) and poly(acrylonitrile) fabrics had a reduced recalcification time to 51 % and 89 %, respectively. Thrombin immobilized poly(ethyleneterephtalate) fabric was also tested in in vivo conditions by using Cavia porcellus and it was observed that this material caused bleeding to stop at a ratio of 24.6 %. The results were statistically significant.     

A promising approach for bio-finishing of cotton using immobilized acid-cellulase

In this research, cellulases were immobilized on Eudragit S-100 to minimize the tensile strength loss of cotton fabric caused by the enzymatic hydrolysis. About 76 % of the enzyme activity and 81 % of the amount of protein were recovered after the immobilization process, and the immobilized cellulase exhibited good reuse ability. The immobilized cellulase had the better adsorptive performance on cotton than the free cellulase. In addition, the results revealed that the catalytic efficiency of the immobilized cellulase on cotton was degradation, perhaps because the diffusion of the enlarged cellulase molecules is significantly inhibited in the interior of the cotton fiber. Moreover, the cotton fabric treated with the immobilized cellulase showed less weight and strength losses. SEM pictures further indicated that the cotton fabric treated with the immobilized cellulase suffered less damage.     

Chitosan Activated with Genipin: A Nontoxic Natural Carrier for Tannase Immobilization and Its Application in Enhancing Biological Activities of Tea Extract

In this work, a non-toxic chitosan-based carrier was constructed via genipin activation and applied for the immobilization of tannase. The immobilization carriers and immobilized tannase were characterized using Fourier transform infrared spectroscopy and thermogravimetric analysis. Activation conditions (genipin concentration, activation temperature, activation pH and activation time) and immobilizations conditions (enzyme amount, immobilization time, immobilization temperature, immobilization pH, and shaking speed) were optimized. The activity and activity recovery rate of the immobilized tannase prepared using optimal activation and immobilization conditions reached 29.2 U/g and 53.6%, respectively. The immobilized tannase exhibited better environmental adaptability and stability. The immobilized tannase retained 20.1% of the initial activity after 12 cycles and retained 81.12% of residual activity after 30 days storage. The catechins composition analysis of tea extract indicated that the concentration of non-ester-type catechins, EGC and EC, were increased by 1758% and 807% after enzymatic treatment. Biological activity studies of tea extract revealed that tea extract treated with the immobilized tannase possessed higher antioxidant activity, higher inhibitory effect on α-amylase, and lower inhibitory effect on α-glucosidase. Our results demonstrate that chitosan activated with genipin could be an effective non-toxic carrier for tannase immobilization and enhancing biological activities of tea extract.     

Effects of flax lignin addition on enzymatic oxidation of poly(ethylene adipate) urethanes

Lignin use in polymer industry has recently become very attractive from both economical and environmental reasons. In the specific case of blending, the addition of low lignin amounts was found to improve the properties and extends the application field of other natural or synthetic polymers, but the effects of oxidative enzymes on resulting blends are widely unknown. Thereby, the current study was carried out to determine some potential effects of small flax lignin concentrations (4.2 and 9.3 wt%) on the enzymatic degradation of a poly(ethylene adipate) urethane (PU). Thin cast films of PU and its lignin blends were incubated for 3 days at 30 °C with buffered solutions of fungal peroxidase and laccase extracted from Aspergillus sp., and compared with the untreated ones. Changes in surface structure and morphology were investigated by attenuated total reflectance-Fourier transform infrared spectroscopy (ATR-FTIR) and scanning electron microscopy (SEM), while the impact on bulk was assessed from tensile tests and thermogravimetry analysis (TGA). Although the addition of flax lignin reduces the surface structural modifications after enzyme treatment, the morphology, tensile and thermo-oxidative characteristics are still affected, with laccase showing the higher degradative efficiency. The lignin concentration, its high impact on the resulting blends morphology and relative low resistance to laccase and peroxidase degradation was the most important factors proved to driven the enzymatic oxidation.     

Effects of laccase,xylanase and their combination on the rheological properties of wheat doughs

The effects of Trametes hirsuta laccase alone and in combination with Aspergillus oryzae and Bacillus subtilis xylanases on dough extensibility were studied using the Kieffer test to determine the dough extensibility (E_x) and the resistance to stretching (R_max). Laccase treatment resulted in dough hardening: the R_max of dough increased and the E_x at R_max decreased as a function of dosage (5–50 nkat/g flour). Xylanases softened flour and gluten doughs. Hardening by laccases and softening by xylanases was weaker in gluten doughs. Dough hardening, observed in the laccase treatments, decreased as a function of dough resting time. The softening effect occurred especially at higher laccase dosages (≈50 nkat/g flour). The softening phenomenon was related to the laccase-mediated depolymerization of the cross-linked AX network. In combined laccase and xylanase treatments, the effect of laccase was predominant, especially at low xylanase dosage, but when xylanase was added to flour dough at high concentrations, the hardening effect of laccase on dough was decreased. In combined laccase and xylanase treatments in gluten doughs, similar decreases in laccase-mediated hardening were not seen.     

不同状态培养基下pH对香菇、滑菇及金针菇漆酶活性的影响

考察不同物理状态培养基条件下pH对香菇、滑菇、金针菇漆酶活性的影响,为香菇、滑菇、金针菇的栽培提供重要参考。固化培养基以及液体培养基以NaOH作为碱化剂,固体培养基以壳灰作为碱化剂,考察不同pH对3种食用菌漆酶活性的影响。结果表明：微碱化培养可提高3种不同食用菌的漆酶活力,并与培养基的物理状态有关。香菇在固化、液体、固体培养基中,pH分别为6.5、6.5以及6.0时漆酶活性最高,比对照提高5.3%~14.0%;金针菇适宜的pH分别为7.0、6.0和6.5,与对照相比提高35.9%~343.0%;滑菇适宜的pH均为6.0,与对照相比提高3.1%~94.9%。     

Study on the stability and reusability of Glutamate Dehydrogenase immobilized on bacterial cellulose nanofiber

In this study, a simple method with improved properties for Glutamate Dehydrogenase immobilization is proposed by using bacterial cellulosic nanofiberas as a novel and inexpensive biomatrix. Acetobacter xylinum ATCC 10245 has been chosen and cultivated for synthesis of this nanofiber with the diameter of 30-70 nm. Thereafter, Glutamate Dehydrogenase was immobilized into the nanofiber pores in two steps: 1) enzyme adsorption and 2) crosslinking by glutaraldehyde. Optimum pH for the activity of immobilized enzyme and optimum temperature was reported in this work. Also the relative activity of immobilized enzyme was about 38 % in comparison to the enzyme activity of the first day after about two months. Moreover after reusing of the enzyme for 9 cycles, the immobilized enzyme retained over 83 % of its initial activity. Glutamate Dehydrogenase is immobilized on bacterial cellulosic nanofiber for the first time which can affect on facilitation of Glutamate production. Also can be a good method for having a stable and robust immobilized Glutamate Dehydrogenase that can withstand harsh operating conditions in processes which are common in these kind of reactions.     

Biochemical Properties and Anti-Biofilm Activity of Chitosan-Immobilized Papain

Chitosan, the product of chitin deacetylation, is an excellent candidate for enzyme immobilization purposes. Here we demonstrate that papain, an endolytic cysteine protease (EC: 3.4.22.2) from Carica papaya latex immobilized on the matrixes of medium molecular (200 kDa) and high molecular (350 kDa) weight chitosans exhibits anti-biofilm activity and increases the antimicrobials efficiency against biofilm-embedded bacteria. Immobilization in glycine buffer (pH 9.0) allowed adsorption up to 30% of the total protein (mg g chitosan⁻¹) and specific activity (U mg protein⁻¹), leading to the preservation of more than 90% of the initial total activity (U mL⁻¹). While optimal pH and temperature of the immobilized papain did not change, the immobilized enzyme exhibited elevated thermal stability and 6–7-fold longer half-life time in comparison with the soluble papain. While one-half of the total enzyme dissociates from both carriers in 24 h, this property could be used for wound-dressing materials design with dosed release of the enzyme to overcome the relatively high cytotoxicity of soluble papain. Our results indicate that both soluble and immobilized papain efficiently destroy biofilms formed by Staphylococcus aureus and Staphylococcus epidermidis. As a consequence, papain, both soluble and immobilized on medium molecular weight chitosan, is capable of potentiating the efficacy of antimicrobials against biofilm-embedded Staphylococci. Thus, papain immobilized on medium molecular weight chitosan appears a presumably beneficial agent for outer wound treatment for biofilms destruction, increasing antimicrobial treatment effectiveness.     

油菜叶绿体同工酶的研究

本实验采用聚丙烯酰胺凝胶盘状电泳的方法和酶电游图谱扫描以及对酶液中酶活和蛋白质含量的测定,研究了杂种油菜F_1(S45×81008—34)及其亲本在初花期的叶绿体过氧化物酶同工酶。结果表明:(1)过氧化物酶同工酶在叶绿体中是真实存在的。(2)F_1叶绿体的过氧化物酶同工酶图谱显示出杂种油菜F_1不仅具有它的亲本的互补酶而且还具有它的亲本没有的杂种酶,它明显地表现出杂种优势。(3)杂种油菜F_1的酶液中的酶活及蛋白质的含量皆高于它的亲本的酶活和蛋白质含量。     

Effect of arabinoxylans and laccase on batter rheology and quality of yeast-leavened gluten-free breads

The supplementation effects of maize fiber arabinoxylans (MFAX, 0%–6%), laccase (0–2 U/g flour) and water absorption level (90%–100%) on gluten-free (GF) batter rheology and bread quality were analyzed. From viscoamylograph analysis, lower starch amount in GF flour due to MFAX addition decreased peak viscosity and retrogradation. Surface response plots showed that laccase did not have significant effect on GF batter rheology and bread quality, whilst water was the most important variable. Higher levels of water absorption benefited bread texture. Higher water level (>100 mL/100 g flour) was needed in the experimental design to evaluate correctly the effect of 6% MFAX replacement on GF bread quality. Further analyses were carried in order to adjust water absorption of batters according to their consistency index (K ≈ 100 Pa sⁿ), resulting an optimal water absorptions of 95%, 100% and 105% for control flour and flours supplemented with 3% or 6% MFAX, respectively. Thus, MFAX addition enhanced water-binding capacity of flour and yielded GF breads with higher specific volume and softer crumb texture. These quality parameters were best rated with 6% MFAX addition to flours. This research demonstrated the potential of MFAX to develop GF breads with improved quality, when optimal water level is used.     

Reversible changes in cell morphology due to cytoskeletal rearrangements measured in real-time by QCM-D

The mechanical properties and responses of cells to external stimuli (including drugs) are closely connected to important phenomena such as cell spreading, motility, activity, and potentially even differentiation. Here, reversible changes in the viscoelastic properties of surface-attached fibroblasts were induced by the cytoskeleton-perturbing agent cytochalasin D, and studied in real-time by the quartz crystal microbalance with dissipation (QCM-D) technique. QCM-D is a surface sensitive technique that measures changes in (dynamically coupled) mass and viscoelastic properties close to the sensor surface, within a distance into the cell that is usually only a fraction of its size. In this work, QCM-D was combined with light microscopy to study in situ cell attachment and spreading. Overtone-dependent changes of the QCM-D responses (frequency and dissipation shifts) were first recorded, as fibroblast cells attached to protein-coated sensors in a window equipped flow module. Then, as the cell layer had stabilised, morphological changes were induced in the cells by injecting cytochalasin D. This caused changes in the QCM-D signals that were reversible in the sense that they disappeared upon removal of cytochalasin D. These results are compared to other cell QCM-D studies. Our results stress the combination of QCM-D and light microscopy to help interpret QCM-D results obtained in cell assays and thus suggests a direction to develop the QCM-D technique as an even more useful tool for real-time cell studies.     

花生蛋白碱性蛋白酶水解物对血管紧张素转化酶抑制作用的初步研究

分别以碱性蛋白酶Alcalase和中性蛋白酶Neutrase对花生分离蛋白进行水解．制备花生分离蛋白水解物．并测定不同水解时间所得产物对血管紧张素转化酶(ACE)的抑制活性。未水解的花生分离蛋白没有ACE抑制活性．用中性蛋白酶Neutrase水解所得的水解物显示弱ACE抑制活性。然而，碱性蛋白酶Alcalasc水解物具有很强的ACE抑制活性．水解0．5h时水解物活性最高，其半抑制浓度为(IC50)0．56mg／mL。本研究表明，当用碱性蛋白酶Alcalase水解时，花生分离蛋白是生产ACE抑制肽的良好蛋白质来源，花生分离蛋白碱性蛋白酶Alcalase水解物可作为具有降压功能的功能食品添料。     

荔枝果实复合保鲜剂的筛选及其常温保鲜效果的研究

为了筛选出有利于荔枝贮藏的复合保鲜剂,以‘井岗红糯’荔枝果实为试验材料,通过正交试验研究了不同浓度的油菜素内酯（brassinolide, BL）和曲酸（kojic acid, KA）配比对采后荔枝果实的保鲜效果。结果表明,在25 ℃贮藏条件下,对荔枝果实的最佳保鲜配方为：油菜素内酯40 μmol/L、曲酸35 mmol/L,浸泡时间为3 min,该复合保鲜剂配方能较好地抑制荔枝果实褐变和腐烂,降低果皮相对电导率、果皮pH和果皮丙二醛（malonaldehyde, MDA）含量,延缓果肉总可溶性固形物（total soluble solids, TSS）和维生素C（vitamin C, VC）含量的下降,维持较高的果皮色度L ^*值、a ^*值、C ^*值和花色苷含量,抑制了多酚氧化酶（polyphenol oxidase, PPO）、过氧化物酶（peroxidase, POD）及漆酶（laccase, Lac）的活性。     

茶树花青素还原酶的酶学特性研究

茶树花青素还原酶(CsANR)作为原花青素生物合成途径中的关键酶,催化花青素为相应的2,3-顺式-黄烷-3-醇。为了研究该酶的酶学特性,本文采用原核表达及钴离子亲和柱纯化技术,表达并纯化出目的蛋白;重点对CsANR1酶学特性进行研究分析。结果表明,CsANR1的最适反应温度为40℃,最适pH值为6.5;对底物矢车菊色素的亲和力高于飞燕草色素。Cu2+、Co2+、Fe2+、Mn2+、Zn2+和Hg2+等金属离子对酶有抑制作用,存放15d后酶活下降50%。     

Preparation of bacterial cellulose/carbon nanotube nanocomposite for biological fuel cell

In this study, electrically conducting composite membranes were prepared by incorporating carboxylic multi-walled carbon nanotubes (c-MWCNTs) into Bacterial Cellulose (BC) pellicles. The biocathode and bioanode were prepared by a simple method of adsorption. An enzyme biological fuel cell (EBFC) composed of a biocathode and an enzymatic bioanode were developed and tested. The materials was characterised by field emission scanning electron microscope (FESEM), Fourier Transform Infrared (FTIR) Spectroscopy and Thermogravimetric analysis (TGA). The results showed that the presence of c-MWCNTs on BC was certified, on which c-MWCNTs loading was calculated as 30.02/100 g. The BC/c-MWCNTs/Lac composite membranes was characterized by cyclic voltammetry (CV). An EBFC was characterized by linear sweep voltammetry (LSV). The results showed EBFC exhibited excellent performance with the largest open circuit voltage (0.76 V) and a maximum power density value (55 uW/cm³). Additionally, the cell also exhibited acceptable stability over the recording of 30 days. BC was considered to be suitable for advanced applications such as an enzymatic carrier of biological fuel cells.     

Immobilization of Mucor miehei esterase on core-shell magnetic beads via adsorption and covalent binding: Application in esters synthesis

In this work, poly(hydroxyethyl methacrylate/glycidylmethacrylate), p(HPMA-GMA), magnetic beads were prepared via suspension polymerization, and grafted with poly(methacrylic acid), p(MMAc) via redox polymerization. Esterase from M. miehei was immobilized onto core-shell magnetic beads with two different methods (i.e., adsorption and covalent binding). For adsorption, the effects of pH and initial esterase concentration on the immobilization efficiency via adsorption have been studied. The maximum adsorption capacity of the magnetic beads for esterase was found to be 27.6 mg/g magnetic beads at pH 7.0. The covalent immobilization of esterase via carbodiimide coupling on mp(HEMA-GMA)-g-p(MAAc) beads was found to be the more effective than the direct immobilization of the enzyme on the mp(HEMA-GMA) beads. The covalently immobilized esterase on the mp(HEMA-GMA)-g-p(MAAc) beads exhibited higher activity compared to the adsorbed esterase on the same magnetic beads. Tributyrin hydrolysis properties of the free and both immobilized esterase preparations were determined under different experimental conditions. The operational, thermal and storage stabilities of the immobilized esterase preparations were increased compared with the free esterase. Finally, esterification reactions have been performed to produce citronellyl-esters (namely: acetate, butyrate and propionate) in n-hexane medium using covalently immobilized esterase. The immobilized esterase was effectively reused in successive batch runs in n-hexane medium for citronellyl-butyrate synthesis, and only 23 % activity was lost after 6 cycles.