添加CTAB促进吸水链霉菌产谷氨酰胺转胺酶

研究了添加十六烷基三甲基溴化铵（CTAB）对吸水链霉菌（Streptomyces hygroscopicus）合成谷氨酰胺转胺酶的影响。结果表明,添加CTAB可以提高发酵过程中谷氨酰胺转胺酶的酶活,摇瓶培养中,CTAB的最佳添加时间和添加量分别为32h和1%,发酵终了时,谷氨酰胺转胺酶酶活最高达5.04u/mL,比对照提高了21.8%。初步研究表明,CTAB的主要作用是促使谷氨酰胺转胺酶的酶原转化为成熟酶,因此,在发酵过程中添加适当浓度的CTAB,可使酶原快速、完全地转化为成熟的MTG,解除酶原的产物抑制作用,促进了细胞产酶。     

Antimicrobial cationic surfactant, cetyltrimethylammonium bromide, induces superoxide stress in Escherichia coli cells

Aims: To clarify whether an antibacterial surfactant, cetyltrimethylammonium bromide (CTAB), induces superoxide stress in bacteria, we investigated the generation of superoxide and hydrogen peroxide and expression of soxR, soxS and soxRS regulon genes in Escherichia coli cells with the treatment of CTAB. Methods and Results: In situ oxidative stress analyses with BES fluorescent probes revealed that generation of both superoxide and hydrogen peroxide were significantly increased with the CTAB treatment at a sublethal concentration in wild‐type strain OW6, compared with the CTAB‐resistant strain OW66. The activity of manganese–superoxide dismutase (Mn–SOD), a member of the soxRS regulon proteins, was decreased by the CTAB treatment only in strain OW6. Furthermore, quantitative real‐time PCR analyses revealed that expression of the soxRS regulon genes was not upregulated, although soxS was upregulated by the CTAB treatment in strain OW6. Conclusions: Cetyltrimethylammonium bromide treatment led E. coli cells to a generation state of superoxide and hydrogen peroxide. It was also suggested that superoxide generation was caused by inhibiting SoxS function and decreasing Mn–SOD activity. Significance and Impact of the Study: It was revealed that excess superoxide generation in bacterial cells play a key action of antibacterial surfactants.     

Immobilization of permeabilized whole cell penicillin G acylase from Alcaligenes faecalis using pore matrix crosslinked with glutaraldehyde

The activity of penicillin G acylase from Alcaligenes faecalis increased 7.5-fold when cells were permeabilized with 0.3% (w/v) CTAB. The treated cells were entrapped by polyvinyl alcohol crosslinked with boric acid, and crosslinked with 2% (v/v) glutaraldehyde to increase the stability. The conversion yield of penicillin G to 6-aminopenicillanic acid was 75% by immobilized system in batch reaction. No activity was lost after 15 cycles and about 65% enzyme activity was retained at the end of the 31th cycle.     

Improvement of oxidoreductase stability of cethyltrimethylammoniumbromide permeabilized cells ofZymomonas mobilis through glutaraldehyde crosslinking

Summary Permeabilization ofZymomonas mobilis with CTAB(Cetyltrimethylammoniumbromide) was investigated in order to obtain a stable process for sorbitol production in the immobilized system. The optimum conditions for sorbitol formation were treating cells with 0.2% CTAB at 4°C for 10 min. For the immobilized system permeabilized cells were treated with glutaraldehyde to improve the system with cross-linking of enzymes. In this way, no significant loss of enzyme activity was apparent during 30 day operation in a continuous process. The productivity of the continuous process at a dilution rate 0.2 h^–1 was 6.51g/L-h for sorbitol. The CTAB-permeabilized cells could be used to produce sorbitol and gluconic acid simultaneously in the long term continuous process.     

The removal of lipid from the surface of wool to promote the subsequent enzymatic process with modified protease for wool shrink resistance

Abstract

When treated with modified protease, wool shows shrink resistance without significant damage to the fiber. It was considered that if wool fiber was pre-treated to make it more hydrophilic, the subsequent treatment with modified protease would be more efficient. After wool was pre-treated with cetyltrimethylammonium bromide (CTAB) under alkaline conditions, the fiber became very hydrophilic due to the removal of surface lipid. After CTAB treatment, it was found that residual CTAB on the fiber significantly decreased enzyme activity. Therefore prior to enzyme treatment, CTAB was washed off the fiber with anionic surfactant under acidic conditions. It was found that the activity of modified protease towards wool improved if wool had been pre-treated with CTAB then washed with anionic surfactant. It was concluded that pre-treatment of wool with CTAB under alkaline conditions followed by washing with anionic surfactant improves the wettability of wool and therefore promotes more efficient treatment with modified protease, achieving improved levels of shrink resistance with no effect on strength of the fiber or coloration properties.     

Separation of astaxanthin from cells of Phaffia rhodozyma using colloidal gas aphrons in a flotation column

The aim of this study is to investigate the separation of astaxanthin from the cells of Phaffia rhodozyma using colloidal gas aphrons (CGA), which are surfactant stabilized microbubbles, in a flotation column. It was reported in previous studies that optimum recoveries are achieved at conditions that favor electrostatic interactions. Therefore, in this study, CGA generated from the cationic surfactant hexadecyl trimethyl ammonium bromide (CTAB) were applied to suspensions of cells pretreated with NaOH. The different operation modes (batch or continuous) and the effect of volumetric ratio of CGA to feed, initial concentration of feed, operating height, and flow rate of CGA on the separation of astaxanthin were investigated. The volumetric ratio was found to have a significant effect on the separation of astaxanthin for both batch and continuous experiments. Additionally, the effect of homogenization of the cells on the purity of the recovered fractions was investigated, showing that the homogenization resulted in increased purity. Moreover, different concentrations of surfactant were used for the generation of CGA for the recovery of astaxanthin on batch mode; it was found that recoveries up to 98% could be achieved using CGA generated from a CTAB solution 0.8 mM, which is below the CTAB critical micellar concentration (CMC). These results offer important information for the scale‐up of the separation of astaxanthin from the cells of P. rhodozyma using CGA. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Flow injection methods for the determination of retinol and α‐tocopherol using lucigenin‐enhanced chemiluminescence

Flow injection (FI) methods are reported to determine retinol and α‐tocopherol based on its enhancement affect of lucigenin chemiluminescence (CL) in alkaline medium. Surfactants including Brij‐35, Triton X‐100, cetyltrimethyl ammonium bromide (CTAB) and sodium dodecyl sulfate have been reported for the first time to enhance lucigenin CL intensity in the presence of retinol and α‐tocopherol. With Brij‐35, the CL intensity was enhanced by 67% for retinol and 58% for α‐tocopherol. CTAB was found to enhance the CL intensity by 16% for retinol whereas for α‐tocopherol, the CL intensity was quenched up to 95%. Retinol could be determined specifically in the presence of α‐tocopherol using CTAB. The calibration graphs were found to be linear up to 1.43 mg/L (R² = 0.9985, n = 8) with a detection limit (3s) of 1.43 × 10^?3 mg/L for retinol and 2.15 mg/L (R² = 0.9989; n = 8) with a detection limit (3s) of 4.31 × 10^?4 mg/L for α‐tocopherol. An injection throughput of 120/h, and relative standard deviations of 0.9–2.8% (n = 4) were achieved in the concentration range studied. The influence of common ions, excipients in pharmaceutical formulations and related organic compounds on the determination of retinol and α‐tocopherol individually was studied. The proposed methods were applied to determine retinol and α‐tocopherol in pharmaceutical formulations and human blood serum. The results did not differ significantly from the CL method and HPLC reference method at 95% confidence level. Copyright © 2010 John Wiley & Sons, Ltd.     

Biocatalytic synthesis of (<Emphasis Type="Italic">R</Emphasis>)-(−)-mandelic acid from racemic mandelonitrile by cetyltrimethylammonium bromide-permeabilized cells of <Emphasis Type="Italic">Alcaligenes faecalis</Emphasis> ECU0401

The nitrilase from Alcaligenes faecalis ECU0401 belongs to the category of arylacetonitrilase, which could hydrolyze 2-chloromandelonitrile, 3,4-dimethoxyphenylacetonitrile, mandelonitrile, and phenylacetonitrile into the corresponding arylacetic acids. To overcome the permeability barrier and prepare whole cell biocatalysts with high activities, permeabilization of Alcaligenes faecalis ECU0401 in relation to nitrilase activity was optimized by using cetyltrimethylammonium bromide (CTAB) as permeabilizing agent. The nitrilase activity from Alcaligenes faecalis ECU0401 increased 4.5-fold when the cells were permeabilized with 0.3% (w/v) CTAB for 20 min at 25°C and pH 6.5. Consequently, almost all the mandelonitrile was consumed and converted to (R)-(−)-mandelic acid with greater than 99.9% enantiomeric excess (e.e.) by the CTAB-permeabilized cells. The permeability barrier has been significantly reduced in the hydrolysis of mandelonitrile by using CTAB-permeabilized cells and a dynamic resolution was successfully achieved, giving a 100% theoretical yield of (R)-(−)-mandelic acid. Efficient biocatalyst recycling was achieved as a result of cell immobilization in calcium alginate, with a product-to-biocatalyst ratio of 3.82 g (R)-(−)-mandelic acid g⁻¹ dry cell weight (dcw) cell after 20 cycles of repeated use.     

Stimulation of Bacillus subtilis membrane adenosine triphosphatase by cationic bactericidal agents

The environmental Mg2+ used in preparation of Bacillus subtilis membranes was found to influence the responses of the associated ATPase to cetyltrimethylammonium bromide (CTAB). Membranes prepared using fluids containing higher Mg2+ levels exhibited lower control activity than was seen with low Mg2+ membranes. Increased environmental Mg2+ resulted in higher stimulations with lower doses of the agent. ATPase of all three membrane types was stimulated in two concentration ranges, but in the doses tested, CTAB inhibited the ATPase of only those membranes obtained using fluids containing high Mg2+ for every stage of the isolation. Sonication of membranes for 25 s at half maximum output yielded three fractions, consisting of a soluble form which was sensitive to CTAB stimulation at 25 microg/ml of assay mixture; small, 95-110 nm, vesicles, which were resistant to CTAB at 25, 75, and 150 microg/ml, and large vesicles, similar to untreated membranes both in morphology and responses to detergent. Combinations of detergent and protein (beta-lysin or arginine-rich histone) produced activity appearing to be additive when the protein level was present in a high concentration and the detergent was present at levels corresponding to the minimum influence. Mixtures of a maximally stimulating dose (75 or 100 microg/ ml) of detergent and a small amount of protein produced activities that were at least 92% or more of the expected sums of individual stimulations. Interference occurred with the following mixtures: high amounts of detergent and protein; high protein and 10 or 15 microg/ml CTAB; and beta-lysin and arginine-rich histone, both at high levels. These data are consistent with a hypothesis that the two peaks in CTAB stimulation reflect two adjacent ATPase sites, one of which is also susceptible to stimulation by cationic protein.     

Comparative antibacterial potential of selected aldehyde-based biocides and surfactants against planktonic Pseudomonas fluorescens

The antimicrobial efficacy of two aldehyde-based biocides (glutaraldehyde, GTA, and ortho-phthalaldehyde, OPA) and two surfactants (cetyltrimethyl ammonium bromide, CTAB, and sodium dodecyl sulphate, SDS) was tested against planktonic Pseudomonas fluorescens. The antimicrobial effects were evaluated by respiratory activity as a measure of the oxygen uptake rate, adenosine triphosphate (ATP) release, outer membrane proteins (OMP) expression and cellular colour changes. The results were compared with the bacterial characteristics without chemical treatment. Tests in the presence of bovine serum albumin (BSA), in order to mimic a disinfection process in the real situation under dirty conditions, were performed according to the European Standard EN-1276. P. fluorescens was completely inactivated with OPA (minimum bactericidal concentration, MBC = 0.5 mM) and CTAB (MBC = 5 mM) and was resistant to GTA and SDS. Only CTAB promoted cellular disruption and consequent ATP release. The antimicrobial action of the chemicals tested was significantly reduced when BSA was introduced into the bacterial cultures, increasing markedly the MBC values. Additionally, the presence of BSA acted as a disruption protective agent when CTAB was applied and stimulated the bacterial respiratory activity when lower concentrations of SDS were tested. The OMP of the bacterial cells was affected by the application of both surfactants. OMP expression remained unaltered after biocide treatment. Bacterial colour change was noticed after treatment with biocides and surfactants. In summary, P. fluorescens was extremely resistant to GTA and SDS, with antimicrobial action being quenched markedly by the reaction with BSA.     

Hydrolysis of whey lactose using CTAB-permeabilized yeast cells

Disposal of lactose in whey and whey permeates is one of the most significant problems with regard to economics and environmental impact faced by the dairy industries. The enzymatic hydrolysis of whey lactose to glucose and galactose by β-galactosidase constitutes the basis of the most biotechnological processes currently developed to exploit the sugar content of whey. Keeping this in view, lactose hydrolysis in whey was performed using CTAB permeabilized Kluyveromyces marxianus cells. Permeabilization of K. marxianus cells in relation to β-galactosidase activity was carried out using cetyltrimethyl ammonium bromide (CTAB) to avoid the problem of enzyme extraction. Different process parameters (biomass load, pH, temperature, and incubation time) were optimized to enhance the lactose hydrolysis in whey. Maximum hydrolysis (90.5%) of whey lactose was observed with 200 mg DW yeast biomass after 90 min of incubation period at optimum pH of 6.5 and temperature of 40 °C.     

Reverse micellar extraction for downstream processing of lipase: Effect of various parameters on extraction

Reverse micellar extraction of lipase using cationic surfactant cetyltrimethylammonium bromide (CTAB) was investigated. The effect of various process parameters on both forward and backward extraction of lipase from crude extract was studied to optimize its yield and purity. Forward extraction of lipase was found to be maximum using Tris buffer at pH 9.0 containing 0.10 M NaCl in aqueous phase and 0.20 M CTAB in organic phase consisting of isooctane, butanol and hexanol. In case of backward extraction, lipase was extracted from the organic phase to a fresh aqueous phase in 0.05 M potassium phosphate buffer (pH 7.0) containing 1.0 M KCl. The activity recovery, extraction efficiency and purification factor of lipase were found to be 82.72%, 40.27% and 4.09-fold, respectively. The studies also indicated that the organic phase recovered after back extraction could be reused for the extraction of lipase from crude extract.     

Stimulation of Bacillus subtilis membrane adenosine triphosphatase by cationic bactericidal agents

The environmental Mg²⁺ used in preparation of Bacillus subtilis membranes was found to influence the responses of the associated ATPase to cetyltrimethylammonium bromide (CTAB). Membranes prepared using fluids containing higher Mg²⁺ levels exhibited lower control activity than was seen with low Mg²⁺ membranes. Increased environmental Mg²⁺ resulted in higher stimulations with lower doses of the agent. ATPase of all three membrane types was stimulated in two concentration ranges, but in the doses tested, CTAB inhibited the ATPase of only those membranes obtained using fluids containing high Mg²⁺ for every stage of the isolation. Sonication of membranes for 25 s at half maximum output yielded three fractions, consisting of a soluble form which was sensitive to CTAB stimulation at 25 μg/ml of assay mixture; small, 95–110 nm, vesicles, which were resistant to CTAB at 25, 75, and 150 μg/ml, and large vesicles, similar to untreated membranes both in morphology and responses to detergent. Combinations of detergent and protein (β-lysin or arginine-rich histone) produced activity appearing to be additive when the protein level was present in a high concentration and the detergent was present at levels corresponding to the minimum influence. Mixtures of a maximally stimulating dose (75 or 100 μg/ml) of detergent and a small amount of protein produced activities that were at least 92% or more of the expected sums of individual stimulations. Interference occurred with the following mixtures: high amounts of detergent and protein; high protein and 10 or 15 μg/ml CTAB; and β-lysin and arginine-rich histone, both at high levels. These data are consistent with a hypothesis that the two peaks in CTAB stimulation reflect two adjacent ATPase sites, one of which is also susceptible to stimulation by cationic protein.     

添加CTAB促进吸水链霉菌产谷氨酰胺转胺酶

研究了添加十六烷基三甲基溴化铵(CTAB)对吸水链霉菌(Streptomyces hygroscopicus)合成谷氨酰胺转胺酶的影响。结果表明,添加CTAB可以提高发酵过程中谷氨酰胺转胺酶的酶活,摇瓶培养中,CTAB的最佳添加时间和添加量分别为32h和1%,发酵终了时,谷氨酰胺转胺酶酶活最高达5.04u/mL,比对照提高了21.8%。初步研究表明,CTAB的主要作用是促使谷氨酰胺转胺酶的酶原转化为成熟酶,因此,在发酵过程中添加适当浓度的CTAB,可使酶原快速、完全地转化为成熟的MTG,解除酶原的产物抑制作用,促进了细胞产酶。     

The effect of beta-tubulin-specific antisense oligonucleotide encapsulated in different cationic liposomes on the suppression [correction of supression] of intracellular L. donovani parasites in vitro

An antisense oligonucleotide (20 mer) targeted to the parasite beta-tubulin gene and encapsulated in cationic liposomes, was used to test its antileishmanial activity in vitro. Cationic liposomes containing dioleyl trimethyl ammonium propane (DOTAP) were found to have higher antileishmanial activity (88% at 4 microM oligonucleotide) compared to two other liposomes with stearyl amine (SA) and cetyl trimethyl ammonium bromide (CTAB) as cations. Dot-blot experiments were performed to analyse the expression of beta-tubulin mRNA using beta-tubulin-specific radiolabelled DNA as a probe. When compared with their respective controls, beta-tubulin-specific gene expression was found to be diminished by treatment with a specific antisense oligonucleotide encapsulated in cationic liposomes (CTAB:DOPE) in a concentration-dependent manner. These experiments show that antisense oligonucleotides targeted to the beta-tubulin gene of Leishmania donovani inhibit beta-tubulin synthesis leading to the arrest of multiplication of intracellular parasites.     

Sanitization of immobilized biocatalysts for the production of 6‐aminopenicillanic acid

Five different chemical reagents and γ‐rays were tested for the sanitization of immobilized biocatalysts with high penicillin G acylase (PGA) activity. The most effective chemical reagents were N‐cetyl‐N,N,N‐trimethylammonium bromide (CTAB) and 2‐isopropyl‐5‐methylphenol (thymol). The optimum concentration of CTAB for the treatment of the immobilized enzyme was 0.25% [w/v] and 1 h, for immobilized cells 0. [w/v] and 3 h. The optimum concentration of thymol for the immobilized enzyme was found to be 0.1% [w/v] and 1 h, for immobilized cells 0.27% [w/v] and 2 h. The optimum dose of γ‐rays for the sanitization of the immobilized enzyme was established as 3.2 kGy, for immobilized cells as 4.5 kGy.     

Reverse micellar extraction of papain with cationic detergent based system: An optimization approach

In this study, reverse micellar extraction of papain model system was performed using cetyltrimethylammonium bromide (CTAB)/iso-octane/hexanol/butanol system to optimize the forward and back extraction efficiency (BEE). A maximum forward extraction efficiency of 55.0, 61.0, and 54% was achieved with an aqueous phase pH of 11.0, 150?mM CTAB/iso-octane and 0.1?M NaCl, respectively. Taguchi’s orthogonal array was applied to optimize the pH of stripping phase, concentration of isopropyl alcohol (IPA) and potassium chloride (KCl) for maximizing BEE. The optimal levels of stripping phase pH, concentration of IPA and KCl were found to be 6, 20% (v/v), and 0.8?M, respectively. Under these optimal levels, the BEE was found to be 88% after which enzyme activity was recovered with 2.5-fold purification. Further optimization was performed using artificial neural network-linked genetic algorithm, where the BEE was improved to 90.52% with pH 6, IPA (%)?=?19.938, and KCl (M)?=?0.729.     

Stabilizing hyperactivated lecitase structures through physical treatment with ionic polymers

Lecitase Ultra has been covalently immobilized on cyanogen bromide cross-linked 4% agarose (CNBr) beads, maintaining 70% of the initial activity. The activity of the immobilized enzyme was improved in the presence of Triton X-100, sodium dodecyl sulfate (SDS), and cetyltrimethyl ammonium bromide (CTAB) (e.g., up to 800% when using CTAB). However, CTAB and Triton X-100 presented a negative effect on enzyme stability even at low concentrations, and SDS cannot be used for a long time at 1% concentration. To maintain the hyperactivated conformation of the enzyme in the absence of detergent, ionic polymers were added during incubation of the immobilized enzyme in the presence of detergents. Coating the immobilized enzyme with polyethylenimine in aqueous buffer (PEI) produced a 3-fold increase in enzyme activity. However, in the presence of 0.1% SDS (v/v), this coating produced a 50-fold increase in enzyme activity. Using PEI and 0.01% (v/v) CTAB, the Lecitase activity decreased to 10%. Using irreversible inhibitors, it could be shown that the PEI/SDS-CNBr-Lecitase preparation allowed its catalytic Ser to be more accessible to the reaction medium than the unmodified CNBr-Lecitase.     

Morphological Alterations in Euglena gracilis Induced by Treatment with CTAB (Cetyltrimethylammonium Bromide) and Triton X-100: Correlations with Effects on Photophobic Behavioral Responses*

SYNOPSIS. Treatment of Euglena gracilis with the cationic detergent CTAB at concentrations of 0.05 mM or higher selectively inhibited the ability of the cells to respond with flagellar reorientation to a sudden decrease of light intensity (step-down photophobic response). The ability to respond similarly to an increase in light intensity (step-up photophobic response) was unaffected even at detergent concentrations at which the step-down response was completely inhibited. Electron microscopy of cells treated with 1.0 mM CTAB revealed selective destruction of the membrane of the reservoir and flagellum. No selective effects upon the step-down or step-up photophobic responses were found upon treatment of the cells with Triton X-100.