Immobilization of thermostable β-glucosidase from Sulfolobus shibatae by cross-linking with transglutaminase

Thermostable β-glucosidase from Sulfolobus shibatae was immobilized on silica gel modified or not modified with 3-aminopropyl-triethoxysilane using transglutaminase as a cross-linking factor. Obtained preparations had specific activity of 3883 U/g of the support, when measured at 70 °C using o-nitrophenyl β-d-galactopyranoside (GalβoNp) as substrate. The highest immobilization yield of the enzyme was achieved at pH 5.0 in reaction media. The most active preparations of immobilized β-glucosidase were obtained at a transglutaminase concentration of 40 mg/ml at 50 °C. The immobilization was almost completely terminated after 100 min of the reaction and prolonged time of this process did not cause considerable changes of the activity of the preparations. The immobilization did not influence considerably on optimum pH and temperature of GalβoNp hydrolysis catalyzed by the investigated enzyme (98 °C, pH 5.5). The broad substrate specifity and properties of the thermostable β-glucosidase from S. shibatae immobilized on silica-gel indicate its suitability for hydrolysis of lactose during whey processing.     

Specificity of mutations induced by carbon ions in budding yeast Saccharomyces cerevisiae

To investigate the nature of mutations induced by accelerated ions in eukaryotic cells, the effects of carbon-ion irradiation were compared with those of γ-ray irradiation in the budding yeast Saccharomyces cerevisiae.

The mutational effect and specificity of carbon-ion beams were studied in the URA3 gene of the yeast. Our experiments showed that the carbon ions generated more than 10 times the number of mutations induced by γ-rays, and that the types of base changes induced by carbon ions include transversions (68.7%), transitions (13.7%) and deletions/insertions (17.6%). The transversions were mainly G:C → T:A, and all the transitions were G:C → A:T. In comparison with the surrounding sequence context of mutational base sites, the C residues in the 5′-AC(A/T)-3′ sequence were found to be easily changed. Large deletions and duplications were not observed, whereas ion-induced mutations in Arabidopsis thaliana were mainly short deletions and rearrangements. The remarkable feature of yeast mutations induced by carbon ions was that the mutation sites were localized near the linker regions of nucleosomes, whereas mutations induced by γ-ray irradiation were located uniformly throughout the gene.     

Preparation of chitosan particles suitable for enzyme immobilization

Macro-, micro- and nanosized chitosan particles suitable as immobilization carriers were prepared by precipitation, emulsion cross-linking and ionic gelation methods, respectively. Effects of particle preparation parameters on particle size were investigated. Activities of β-galactosidase covalently attached to differently sized particles have been evaluated and compared. The highest activity was shown by the biocatalyst immobilized on nanoparticles obtained by means of the ionotropic gelation method with sodium sulphate as gelation agent. β-Galactosidase fixed on macro- and microspheres exhibited excellent storage stability in aqueous solution, with no more than 5% loss of activity after 3 weeks storage at 4 °C and pH 7.0.     

Influence of the microenvironment on the activity of enzymes immobilized on Teflon membranes grafted by γ-radiation

The effect of the microenvironment and immobilization method on the activity of immobilized β-galactosidase was investigated. Immobilization was done on Teflon membranes grafted with different acrylic monomers by γ-radiation and activated by two different coupling agents through the functional groups of the grafted monomers. 2-Hydroxyethyl methacrylate (HEMA) and methacrylic acid (MAA) were grafted on the membrane, and 1,6-hexamethylenediamine (HMDA) was used as a spacer. Glutaraldehyde or cyanuric chloride were used as coupling agents to bind the enzyme to the membrane. Four different catalytic membranes were obtained using the same solid support. Direct comparison between the isothermal behaviour of the biocatalyst in its free and immobilized form was carried out. In particular the dependence of the isothermal activity on the temperature and pH was studied and the kinetic parameters determined. The influence of the microenvironment on the observed activity of the four membranes was evidenced and discussed. The way of improving the yield of these catalytic membranes is discussed also.     

Immobilization on Eupergit C of cyclodextrin glucosyltransferase (CGTase) and properties of the immobilized biocatalyst

The extreme thermophilic cyclodextrin glucanotransferase (CGTase) from Thermoanaerobacter sp. was covalently attached to Eupergit C. Different immobilization parameters (incubation time, ionic strength, pH, ratio enzyme/support, etc.) were optimized. The maximum yield of bound protein was around 80% (8.1 mg/g support), although the recovery of β-cyclodextrin cyclization activity was not higher than 11%. The catalytic efficiency was lower than 15%. Results were compared with previous studies on covalent immobilization of CGTase.

The enzymatic properties of immobilized CGTase were investigated and compared with those of the soluble enzyme. Soluble and immobilized CGTases showed similar optimum temperature (80–85 °C) and pH (5.5) values, but the pH profile of the immobilized CGTase was broader at higher pH values. The thermoinactivation of the CGTase coupled to Eupergit C was slower than the observed with the native enzyme. The half-life of the immobilized enzyme at 95 °C was five times higher than that of the soluble enzyme. The immobilized CGTase maintained 40% of its initial activity after 10 cycles of 24 h each. After immobilization, the selectivity of CGTase (determined by the ratio CDs/oligosaccharides) was notably shifted towards oligosaccharide production.     

Comparative study of spent grains and delignified spent grains as yeast supports for alcohol production from molasses

Fresh, defrosted and delignified brewer's spent grains (BSG) were used as yeast supports for alcoholic fermentation of molasses. Glucose solution (12%) with and without nutrients was used for cell immobilization on fresh BSG, without nutrients for cell immobilization on defrosted and with nutrients for cell immobilization on delignified BSG. Repeated fermentation batches were performed by the immobilized biocatalysts in molasses of 7, 10 and 12 initial Baume density without additional nutrients at 30 and 20 degrees C. Defrosted BSG immobilized biocatalyst was used only for repeated fermentation batches of 7 initial Baume density of molasses without nutrients at 30 and 20 degrees C. After immobilization, the immobilized microorganism population was at 10(9) cells/g support for all immobilized biocatalysts. Fresh BSG immobilized biocatalyst without additional nutrients for yeast immobilization resulted in higher fermentation rates, lower final Baume densities and higher ethanol productivities in molasses fermentation at 7, 10 and 12 initial degrees Be densities than the other above biocatalysts. Adaptation of defrosted BSG immobilized biocatalyst in the molasses fermentation system was observed from batch to batch approaching kinetic parameters reported in fresh BSG immobilized biocatalyst. The results of this study concerning the use of fresh or defrosted BSG as yeast supports could be promising for scale-up operation.     

Room and low temperature brewing with yeast immobilized on gluten pellets

A biocatalyst prepared by the immobilization of a cryotolerant strain of Saccharomyces cerevisiae on gluten pellets was used for batch and continuous fermentation at low temperatures. The immobilized yeast showed important operational stability in repeated batch fermentations without a decrease of activity even at 0 and 5°C. Repeated batch fermentations using the biocatalyst resulted in improvement of ethanol productivity in comparison with bottom brewing fermentation and free cells using the same yeast strain. At 0 and 10°C, the fermentation rate was four and seven times higher than that of free cells, respectively. For immobilized yeast, diacetyl and polyphenol contents were lower and the alcohol concentration higher at low temperatures (0–7°C) when compared to free cells. Fine clarity was also observed in the beers. Continuous brewing using gluten-supported biocatalyst had an operational stability of 3 months with relatively high productivity and without contamination. Polyphenol and bitterness contents were lower in the continuous process than those of batch fermentations, but at low temperature (5°C) they were higher. The diacetyl content was higher than in batch fermentations and beers had a fine aroma and taste.     

Continuous sucrose hydrolysis by an immobilized whole yeast cell biocatalyst

An immobilized biocatalyst with invertase activity prepared by immobilization of whole yeast cells without use of any insoluble carrier was tested in tubular fixed-bed reactors from the point of view of possible application for continuous full-scale sucrose hydrolysis. At inlet sucrose concentration above 60% (w/w) and reaction temperature 60–70°C, total sucrose hydrolysis was achieved at a flow rate of 0.6–1.5 bed volumes per hour. At a flow rate about 10 bed volumes per hour, the conversion was still 0.5. The specific productivity of the biocatalyst was 3–25 h⁻¹; the productivity of the reactor was 1–9 kg l⁻¹ h⁻¹. The half-life of the biocatalyst invertase activity was 815 h at 70°C. The specific pressure drop over the biocatalyst bed was less than 23 kPa m⁻¹. The biocatalyst was proved to be fully capable of continuous sucrose hydrolysis in fixed-bed reactors.     

Effect of γ-irradiation on some physiochemical properties of konjac glucomannan

Konjac glucomannan (KGM) was irradiated at 5, 20, 50 and 100 kGy and the effects of γ-irradiation on some physiochemical properties of KGM were studied by using viscosimeter, colorimeter, gel permeation chromatography (GPC), Fourier transform infrared (FT-IR) spectroscopy, ultraviolet (UV) spectroscopy, thermal gravimetric (TG) analysis and scanning electron microscopy (SEM). γ-irradiation led to significant degradation of KGM according to the significant reduction of the weight-average molecular weight (M_w). The apparent viscosity of KGM decreased with increasing dose, while the viscosity stability was improved after irradiation. The colour of KGM became more intense brown with increasing dose up to 20 kGy. FT-IR spectra indicated that γ-irradiation introduced no significant changes into the structure but UV spectra showed a distinct absorption peak at about 265 nm, increasing with irradiation dose, which was attributed to the formation of carbonyl groups or double bond. High irradiation dose (100 kGy) caused a small decrease of thermal stability but presented no visible fissures or splitting of KGM granules according to the TG analysis and SEM microphotographs.     

Immobilization of invertase onto poly(3-methylthienyl methacrylate)/poly(3-thiopheneacetic acid) matrix

A novel immobilization matrix, poly(3-methylthienyl methacrylate)–poly(3-thiopheneacetic acid) (PMTM–PTAA), was synthesized and used for the covalent immobilization of Saccharomyces cerevisiae invertase to produce invert sugar. The immobilization resulted in 87% immobilization efficiency. Optimum conditions for activity were not affected by immobilization and the optimum pH and temperature for both free and immobilized enzyme were found to be 4.5 and 55 °C, respectively. However, immobilized invertase was more stable at high pH and temperatures. The kinetic parameters for free and immobilized invertase were also determined using the Lineweaver–Burk plot. The K_m values were 35 and 38 mM for free and immobilized enzyme, respectively. The V_max values were 29 and 24 mg glucose/mg enzyme min for free and immobilized enzyme, respectively. Immobilized enzyme could be used for the production of glucose and fructose from sucrose since it retained almost all the initial activity for a month in storage and retained the whole activity in repeated 50 batch reactions.     

Biocatalytic properties of native and immobilized subtilisin 72 in aqueous-organic and low water media

Subtilisin 72 was immobilized on cryogel of poly(vinyl alcohol), the macroporous carrier prepared by the freeze-thaw-treatment of concentrated aqueous solution of the polymer. The obtained biocatalyst was active and stable in aqueous, aqueous-organic, as well as in low water media. The stability of immobilized biocatalyst was substantially higher than that of native enzyme in all mixtures especially in aqueous buffer containing 5–8 M Urea and in acetonitrile/60–90%DMF mixtures. The ability of native and immobilized subtilisin to catalyze peptide bond formation between Z-Ala-Ala-Leu-OMe and Phe-pNA was studied in non-aqueous media. Considerable enzyme stabilization in acetonitrile/90%DMF mixture, induced by the immobilization, resulted in higher product yield (57%) than in case of native subtilisin suspension (32%). Detailed study of synthesis reaction revealed that notable increase in product yield could be reached using increase in both substrate concentrations up to 200 mM.     

The Biological Reductive Capacity of Tissues is Decreased Following Exposure to Oxidative Stress: A Cyclic Voltammetry Study of Irradiated Rats

The reductive capacity of rat tissue homogenates and body fluids was determined by cyclic voltammetric measurements. The reductive capacity of rat lung, liver and kidney homogenates was significantly reduced four days after total body γ-ray irradiation with 5.5 Gy as compared to controls. In parallel, reduced ability of the irradiated organ homogenates to scavenge hydroxyl radicals and to destroy hydrogen peroxide was recorded. However, no difference in their superoxide dismutase activity was found. The possible use of cyclic voltammetry as a method for qualitative evaluation of the ability of biological tissues to cope with oxidative stress is discussed.     

Electrospun polyacrylonitrile nanofibrous membranes for lipase immobilization

Polyacrylonitrile (PAN) nanofibers could be fabricated by electrospinning with fiber diameter in the range of 150–300 nm, providing huge surface area for enzyme immobilization and catalytic reactions. Lipase from Candida rugosa was covalently immobilized onto PAN nanofibers by amidination reaction. Aggregates of enzyme molecules were found on nanofiber surface from field emission scanning electron microscopy and covalent bond formation between enzyme molecule and the nanofiber was confirmed from FTIR measurements. After 5 min activation and 60 min reaction with enzyme-containing solution, the protein loading efficiency was quantitative and the activity retention of the immobilized lipase was 81% that of free enzyme. The mechanical strength of the NFM improved after lipase immobilization where tensile stress at break and Young's modulus were almost doubled. The immobilized lipase retained >95% of its initial activity when stored in buffer at 30 °C for 20 days, whereas free lipase lost 80% of its initial activity. The immobilized lipase still retained 70% of its specific activity after 10 repeated batches of reaction. This lipase immobilization method shows the best performance among various immobilized lipase systems using the same source of lipase and substrate when considering protein loading, activity retention, and kinetic parameters.     

The use of immobilized Saccharomyces cerevisiae on radiation crosslinked acrylamide–maleic acid hydrogel carriers for production of ethyl alcohol

Radiation crosslinked acrylamide/maleic acid (AAm/MA) copolymers were prepared by γ-irradiation. They were used in experiments on swelling, diffusion, and immobilization of yeast cells (Saccharomyces cerevisiae) for the production of ethyl alcohol. AAm/MA hydrogels containing different amount of MA, irradiated at different doses, were used for swelling and diffusion studies. The parameters of swelling, diffusional exponents, network constants, diffusion coefficients and percent porosity of the hydrogel/penetrant systems were calculated and evaluated. Yeast cells were immobilized on to the hydrogels by adsorption during multiplication, and ethyl alcohol production by the hydrogels was investigated. Swelling of AAm/MA increased with increase in MA content. Ethyl alcohol production also increased with increasing MA in the hydrogels but decreased with an increase of irradiation dose.     

Synthesis and characterization of cyclodextrin-based polymers as a support for immobilization of Candida rugosa lipase

The objective of this study was to prepare cross-linked β-cyclodextrin polymers for immobilization of Candida rugosa lipase. The structures of synthesized macrocyclic compounds were characterized by Fourier transform infrared spectroscopy (FTIR), thermal gravimetric analysis (TGA) and scanning electron microscope (SEM) techniques. Properties of the immobilized systems were assessed and their performance on hydrolytic reaction were evaluated and compared with the free enzyme. The influence of activation agents (glutaraldehyde (GA) and hexamethylene diisocyanate (HMDI)) and thermal and pH stabilities of the biocatalyst was evaluated. After the optimization of immobilization process, the physical and chemical characterization of immobilized lipase was performed. Obtained data showed that the immobilized enzyme seemed better and offered some advantages in comparison with free enzyme. It can be observed that the free lipase loses its initial activity within around 80 min at 60 °C, while the immobilized lipases retain their initial activities of about 56% by HMDI and 82% by GA after 120 min of heat treatment at 60 °C.Results showed that the specific activity of the immobilized lipase with glutaraldehyde was 62.75 U/mg protein, which is 28.13 times higher than that of the immobilized lipase with HMDI.     

Luminescent yeast cells entrapped in hydrogels for estrogenic endocrine disrupting chemical biodetection

In the construction of luminescent yeast cell based fibre-optic biosensors, we demonstrate a novel approach for estrogenic endocrine disrupting chemical (EDC) biodetection by entrapping genetically modified Saccharomyces cerevisiae cells, containing the estrogen receptor alpha-mediated expression of the luc reporter gene, in hydrogel matrices based on calcium alginate or PVA. In order to insure a significant signal, an optimal immobilization ratio of 1:2 alginate 3% (w/v): 5 × 10⁶ [cells/ml], respectively, was used with the highest 17-β-estradiol (β-E2) induction factor after 2.5 h of incubation with 10 [nM] β-E2. It was shown that biocompatible alginate beads, 4.27–4.55 × 10⁵ [CFU/bead], which were characterized by a detection limit of 0.08 [μg l⁻¹] and an EC50 of 0.64 [μg l⁻¹] for β-E2, retained their viability for luminescence measurements after 1 month of storage at −80 °C slow freeze condition, and thus repeated cell cultivations were not required. The assay reproducibility for each tested EDC, represented by the coefficients of variation (CV), ranged from 4.35 to 18.47%. An alternative immobilization method, based on a room temperature partial drying of polyvinyl alcohol (PVA) solution (LentiKat^® Liquid) and cell suspension mix, was investigated with only a slightly lower detection limit for β-E2 than that reported with alginate beads. Alginate yeast based hydrogels may also be applicable to the analysis of environmental water samples since the trend of detected estrogenic activities with alginate beads roughly correlated with LC–MS–MS analytical results.     

Bystander response in human lymphoblastoid TK6 cells

The mechanisms of the medium-mediated bystander response induced by γ-rays in non-irradiated TK6 cells were investigated. Cell cultures were irradiated and the culture medium discarded immediately after irradiation and replaced with a fresh one. In cells incubated with conditioned medium from irradiated cells (CM), a significant decrease in cell viability and cloning efficiency was observed, together with a significant increase in apoptosis, also in directly irradiated cells. To examine whether bystander apoptosis involved the extrinsic pathway, an inhibitor of caspase-8 was added to CM cultures, which significantly decreased apoptosis to control levels. The addition to CM of ROS scavengers, Cu–Zn superoxide dismutase and N-acetylcysteine did not affect the induction of apoptosis. To assess whether CM treatment activates a DNA damage response, also the formation of γ-H2AX foci, as markers of double-strand breaks and their colocalisation with 53-binding protein 1 (53BP1) and the protein mutated in the Nijmegen breakage syndrome 1 (NBS1) was analysed. In cultures treated for 2 h with CM, 9–11% of cells showed γ-H2AX foci, which partially or totally lacked colocalisation with 53BP1 and NBS1 foci. About 85% of irradiated cells were positive for γ-H2AX foci, which colocalised with 53BP1 and NBS1 proteins. At 24 h from irradiation, very few irradiated cells retained foci, fitting DNA repair kinetics. The number of foci-positive bystander cells also decreased to background values 24 h after CM incubation. Our results suggest that irradiated TK6 cells release into the medium some soluble factors, not ROS, which are responsible for the cytotoxic effects induced in bystander cells. In our experimental system, the role of ROS appeared to be of minor importance in inducing cell mortality, but probably critical in activating the DNA damage response in the responsive fraction of bystander cells.     

氨基树脂固定化L-苏氨酸醛缩酶及其应用*

L-苏氨酸醛缩酶(L-Threonine aldolase,L-TA)可以催化甘氨酸和醛合成β-羟基-α-氨基酸。β-羟基-α-氨基酸具有两个手性中心,是多种手性药物的中间体。但是,游离的L-TA难以重复利用,分离纯化困难,严重阻碍了工业化应用。固定化技术可以有效解决这些问题。利用氨基树脂NAA固定化来源于Bacillus nealsonii的L-苏氨酸醛缩酶,采用戊二醛作为交联剂,经过条件优化确定最佳固定化条件为:加酶量13 U、载体量0.6 g、0.4%(V/V)戊二醛、活化时间2 h、pH 8.5、35℃、固定化5 h。在此条件下,固定化酶酶活回收率为85.7%。在30℃下半衰期可达59天,为游离酶的6.5倍。将其应用于合成L-syn-对甲砜基苯丝氨酸,使用460 h后,残余酶活为79.4%。进一步开发了载体再利用策略,将失活固定化酶表面的氨基用戊二醛活化后,再与新的游离酶进行固定化,实现载体的再利用。利用该方法载体可重复利用两次,制备的固定化酶仍能使用460 h。该方法大大降低了固定化成本,为固定化L-TA的工业化应用打下坚实的基础。     

Multipoint covalent immobilization of microbial lipase on chitosan and agarose activated by different methods

In this work Candida antarctica lipase type B (CALB) was immobilized on agarose and chitosan. The influence of activation agents (glycidol, glutaraldehyde and epichlorohydrin) and immobilization time (5, 24 and 72 h) on hydrolytic activity, thermal and alkaline stabilities of the biocatalyst was evaluated. Protein concentration and enzymatic activity in the supernatant were determined during the immobilization process. More active derivatives were attained when the enzymatic extract was first purified through dialysis. The highest activities achieved were: for agarose-glyoxyl (with glycidol), 845 U/g of gel, after 72 h of immobilization; for chitosan-glutaraldehyde and agarose-glutaraldehyde, respectively, 1209 U/g of gel and 2716 U/g of gel, after 5 h of immobilization. Thermal stability was significantly increased, when compared to the soluble enzyme: 20-fold for agarose-glyoxyl (with glycidol)-CALB, 18-fold for chitosan-glutaraldehyde-CALB and 21-fold for agarose-glutaraldehyde. The best derivative, 58-fold more stable than the soluble enzyme, was obtained when CALB was immobilized on chitosan activated in two steps, using glycidol and glutaraldehyde, 72 h immobilization time. The stabilization degree of the derivative increased with the immobilization time, an indication that a multipoint covalent attachment between enzyme and the support had really occurred.     

Freezing resistance improvement of Lactobacillus reuteri by using cell immobilization

Lactobacillus reuteri shows certain beneficial effects to human health and is recognized as a probiotic. However, its application in frozen foods is still not popular because of its low survival during freezing and frozen storage. Cell immobilization technique could effectively exert protection effects to microbial cells in order to enhance their endurance to unfavorable environmental conditions as well as to improve their viability and cell concentration. Ca-alginate and κ-carrageenan were used to immobilize L. reuteri in this research, and the immobilized cells were exposed to different freezing temperatures, i.e. − 20 °C, − 40 °C, − 60 °C, − 80 °C, and stored at − 40 °C and − 80 °C for 12 weeks. The objectives were to study the protection effects of cell immobilization against the adverse conditions of freezing and frozen storage, and the effects of freezing temperatures to the immobilized cells. Cell immobilization was used to raise the survival of L. reuteri during freezing and frozen storage in order to develop frozen foods with the probiotic effects of L. reuteri. Results indicated that immobilized L. reuteri possessed better survival in both freezing and frozen storage. The survival of immobilized L. reuteri was higher than that of free cells, and the effects of lower freezing temperature were better than higher freezing temperature. The immobilization effects of Ca-alginate were found to be superior to κ-carrageenan. Cell immobilized L. reuteri exhibits potential to be used in frozen foods.