The effect of additives and mechanical agitation in surface modification of acrylic fibres by cutinase and esterase

The surface of an acrylic fibre containing about 7% of vinyl acetate was modified using Fusarium solani pisi cutinase and a commercial esterase, Texazym PES. The effect of acrylic solvents and stabilising polyols on cutinase operational stability was studied. The half-life time of cutinase increased by 3.5-fold with the addition of 15% N,N-dimethylacetamide (DMA) and by 3-fold with 1M glycerol. The impact of additives and mechanical agitation in the protein adsorption and in the hydrolysis of vinyl acetate from acrylic fabric was investigated. The hydroxyl groups produced on the surface of the fibre were able to react specifically with Remazol Brilliant Blue R (cotton reactive dye) and to increase the colour of the acrylic-treated fabric. The best staining level was obtained with a high level of mechanical agitation and with the addition of 1% DMA. Under these conditions, the raise in the acrylic fabric colour depth was 30% for cutinase and 25% for Texazym. The crystallinity degree, determined by X-ray diffraction, was not significantly changed between control samples and samples treated with cutinase. The results showed that the outcome of the application of these enzymes depends closely on the reaction media conditions.     

Effect of thermal cycling and disinfection on colour stability of denture base acrylic resin

doi: 10.1111/j.1741‐2358.2012.00676.x Effect of thermal cycling and disinfection on colour stability of denture base acrylic resin Objectives: The purpose of this study was to investigate the effect of thermal cycling and disinfection on the colour change of denture base acrylic resin. Materials and Methods: Four different brands of acrylic resins were evaluated (Onda Cryl, QC 20, Classico and Lucitone). All brands were divided into four groups (n = 7) determined according to the disinfection procedure (microwave, Efferdent, 4% chlorhexidine or 1% hypochlorite). The treatments were conducted three times a week for 60 days. All specimens were thermal cycled between 5 and 55°C with 30‐s dwell times for 1000 cycles before and after disinfection. The specimens’ colour was measured with a spectrophotometer using the CIE L*a*b* system. The evaluations were conducted at baseline (B), after first thermal cycling (T₁), after disinfection (D) and after second thermal cycling (T₂). Colour differences (ΔE) were calculated between T₁ and B (T₁B), D and B (DB), and T₂ and B (T₂B) time‐points. Results: The samples submitted to disinfection by microwave and Efferdent exhibited the highest values of colour change. There were significant differences on colour change between the time‐points, except for the Lucitone acrylic resin. Conclusions: The thermal cycling and disinfection procedures significantly affected the colour stability of the samples. However, all values obtained for the acrylic resins are within acceptable clinical parameters.     

Bioconversion of acrylonitrile to acrylic acid by rhodococcus ruber strain AKSH-84

A new versatile acrylonitrile-bioconverting strain isolated from a petroleum-contaminated sludge sample and identified as Rhodococcus ruber AKSH-84 was used for optimization of medium and biotransformation conditions for nitrilase activity to produce acrylic acid. A simple and rapid HPLC protocol was optimized for quantification of acrylic acid, acrylamide, and acrylonitrile. The optimal medium conditions for nitrilase activity were pH of 7.0, temperature of 30degreesC, agitation of 150 rpm, and inoculum level of 2%. Glycerol as a carbon source and sodium nitrate as the nitrogen source provided good nutritional sources for achieving good biotransformation. Nitrilase activity was constitutive in nature and was in the exponential growth phase after 24 h of incubation under optimal conditions without addition of any inducer. The substrate preference was acrylonitrile and acetonitrile. The present work demonstrates the biotransformation of acrylonitrile to acrylic acid with the new strain, R. ruber AKSH-84, which can be used in green biosynthesis of acrylic acid for biotechnological processes. The nitrilase produced by the isolate was purified and characterized.     

Single nucleotide polymorphisms in the Melanocortin 1 Receptor gene are linked with lightness of fibre colour in Peruvian Alpaca (Vicugna pacos)

Melanocortin 1 receptor (alpha melanocyte stimulating hormone receptor) (MC1R) is a gene‐controlling melanogenesis in mammals. However, it is not well characterized in alpacas and its association with colour is not known. The aim of this study was to look for polymorphisms in the MC1R gene in Peruvian Huacaya alpacas and to analyse the relationship between MC1R single nucleotide polymorphisms (SNPs) and the variations in the instrumental measurement of colour of alpaca fibre. Sixty alpaca fibre samples from black, brown, cream and white animals (15 for each colour) were used to extract DNA from hair bulbs. Colour was measured with a spectrophotometer to obtain quantitative values (CieL*a*b*). Sixteen samples, four of each colour group, were sequenced. Eighteen SNP mutations, 10 not previously described, were found in these 16 sequences. Three of them were chosen (c.82A>G, c.865C>T, c.901C>T) to analyse genotypes by PCR‐RFLP in the other 44 fibre samples and to determine the association of mutations with instrumental colour. These three polymorphisms showed association with fibre lightness (P < 0.05), although there was no correlation with colour groups.     

Isolation and characterization of a novel Arthrobacter nitroguajacolicus ZJUTB06-99, capable of converting acrylonitrile to acrylic acid

A nitrilase-producing strain ZJUTB06-99, capable of biotransforming acrylonitrile into acrylic acid, was newly isolated from soil samples. Based on the morphology, physiological tests, ATB system and its 16S rDNA sequence, strain ZJUTB06-99 was identified as Arthrobacter nitroguajacolicus. Optimal reaction conditions were investigated by the manipulation and measurement of various parameters including pH, temperature and certain cationic metals. The highest nitrilase activity was obtained when reaction was carried out at a pH of 6.5 phosphate buffer and in temperature of 40 °C water bath. The nitrilase of A. nitroguajacolicus ZJUTB06-99 exhibited excellent thermostability. Nitrilase activity was strongly inhibited by Hg²⁺, Ag⁺ and Cu²⁺, but Ni²⁺ and Ca²⁺ increased enzyme activity to 163% and 158%, respectively. The investigation of substrates spectrum showed that A. nitroguajacolicus ZJUTB06-99 exhibited the highest nitrilase activity towards aromatic nitriles such as phenylacetonitrile. However, no detectable activity was recorded when any of the tested amides were used as substrates.     

Induction of auxin biosynthetic enzymes by jasmonic acid and in clubroot diseased Chinese cabbage plants

Nitrilase (NIT) and myrosinase are important enzymes for auxin biosynthesis in Brassicaceae, which is increased during clubroot disease. Therefore, NIT and myrosinase levels during club development and possible regulation mechanisms were investigated. In addition, the occurrence of different nitrilase isoforms in Chinese cabbage has been shown. Nitrilase activity was enhanced in infected roots during later stages of club development (35–42 days after inoculation). However, no differences in nitrilase mRNA levels between infected and healthy roots were found during symptom development. Myrosinase expression was increased in clubbed roots at slightly earlier time points (28 days after inoculation) and also at later time points during infection. The activities of tryptophan oxidizing enzyme (TrpOxE), which catalyzes the first step in tryptophan-dependent auxin biosynthesis in Brassicaceae, and nitrilase were enhanced after treatment with jasmonic acid (JA) and methyl jasmonate. Similarly, the amount of myrosinase mRNA was increased by JA. During clubroot disease the endogenous concentration of JA increased in infected roots 3–5 weeks after inoculation. From our results it can be concluded that: (1) de novo indole-3-acetic acid (IAA) biosynthesis plays a role for symptom development of clubroot disease in Brassicaceae during later developmental stages; and (2) JA which increased during club development, may be involved in the up-regulation of three enzymes important for IAA synthesis.     

Production of acrylic acid and methacrylic acid using Rhodococcus rhodochrous J1 nitrilase

Summary -Caprolactam-induced Rhodococcus rhodochrous J1 cells containing abundant nitrilase were used in the production of acrylic acid and methacrylic acid from acrylonitrile and methacrylonitrile, respectively. Under a periodic substrate feeding system, the highest accumulations, 390 g acrylic acid/l and 260 g methacrylic acid/l, were attained. Offprint requests to: T. Nagasawa     

Bioconversion of Iminodiacetonitrile to Iminodiacetic acid with whole cells of <Emphasis Type="Italic">Lysinibacillus boronitolerans</Emphasis> MTCC 107614 (IICT-akl252)

Biotechnological potential of nitrilases are prompting significant interest in finding the novel microbes capable of hydrolyzing nitriles. In this view, we have screened about 450 bacterial strains for nitrilase production using bioconversion of iminodiacetonitrile (IDAN) to iminodiacetic acid (IDA) through hydrolysis and obtained six nitrilase-producing isolates. Among these six isolates, IICT-akl252 was promising which was identified as Lysinibacillus boronitolerans. This is the first report on L. boronitolerans for nitrilase activity. Optimization of various medium and reaction parameters for maximizing the nitrilase production using whole cells in shake flask was carried out for L. boronitolerans IICT-akl252. Sucrose (2 %) as a carbon source attained better nitrilase yield while IDAN appeared to be the preferable inducer (0.2 %). The maximum IDA formation was achieved with 100 mM IDAN and 150 mg/ml cells at 30 °C and pH 6.5. After optimization of the culture and reaction conditions, the activity of nitrilase was increased by 2.3-fold from 27.2 to 64.5 U. The enzyme was stable up to 1 h at 50 °C. The enzyme was able to hydrolyze aliphatic, aromatic and heterocyclic nitrile substrates.     

Effect of the physical properties of acrylic resin of overnight immersion in sodium hypochlorite solution

doi:10.1111/j.1741‐2358.2009.00336.x
Effect of the physical properties of acrylic resin of overnight immersion in sodium hypochlorite solution Objectives: This study evaluated colour stability, surface roughness and flexural strength of microwave‐polymerised acrylic resin after overnight immersion in sodium hypochlorite, simulating 180 days use. Materials and methods: Forty disc‐shaped (15 mm × 4 mm) and 40 rectangular specimens (65 mm × 10 mm × 3 mm) were prepared from microwave‐polymerised acrylic resin. The specimens were immersed in 0.5, 1% sodium hypochlorite, Clorox/Calgon and distilled water. Colour measurements (ΔE) were determined by a portable colorimeter. A surface analyser was used to measure roughness (μm). The flexural strength (MPa) was measured using a three‐point bending test in a universal testing machine. Data were evaluated by one‐way anova , followed by Student–Newman–Keuls test (α = 0.05). Results: Statistical analysis found significantly higher colour changes (SNK, p < 0.001) for the 1% sodium hypochlorite, but mean ΔE value quantified by National Bureau of Standards was classified as slight. When comparing the surface roughness, no statistical significance was found among the solutions (anova , p = 0.637). The 1% sodium hypochlorite presented significantly lower flexural strength compared with the control group (SNK, p = 0.034). Conclusion: It was concluded that immersion in 1% sodium hypochlorite solutions for 8 h does influence the colour stability and flexural strength of microwave‐polymerised acrylic resin, during the simulated period of 180 days.     

Resistance of Cyanobacterial Fouling on Architectural Paint Films to Cleaning by Water Jet

Mortar panels painted with three different white acrylic coatings were exposed to the environment in urban (São Paulo) and rural (Pirassununga) sites in Brazil for 7 years. After this time, all panels were almost equally discoloured, and paint detachment was observed to only a small degree. The biofilms were composed mainly of cyanobacteria and filamentous fungi, principal genera being Gloeocapsa and Chroococcidiopsis of the cyanobacteria, and Cladosporium and Alternaria of the fungi. Two of the three paints in Pirassununga became covered by a pink film that contained red-encapsulated Gloeocapsa and clay particles. The third, an 800% elastomeric matt formulation, became discoloured with a grey, only slightly pink, film, although the same cyanobacteria were present. The levels of paint detachments from all films in both locations were low, with rating range of 0–1 of a maximum 5 (100% detachment). After high-pressure water jetting, paint detachments increased at both locations, up to 2 in Pirassununga and 3 in São Paulo. Discoloration decreased; L*A*B* analysis of surface discoloration showed that ΔE (alteration in colour from the original paint film) changed from 28–39 before cleaning to 13–16 afterwards. The pink coloration was not entirely removed from Pirassununga samples, suggesting that cyanobacterial cells are difficult to detach, and microscopic analysis of the biofilms confirmed that Gloeocapsa was still present as the principal contaminant on all surfaces, with Chroococcidiopsis being present as the second most common. Almost no fungi were detected after water jet application.     

Altered fibre types in gastrocnemius muscle of high wheel-running selected mice with mini-muscle phenotypes

Selective breeding of mice for high voluntary wheel running has favoured characteristics that facilitate sustained, aerobically supported activity, including a "mini-muscle" phenotype with markedly reduced hind limb muscle mass, increased mass-specific activities of oxidative enzymes, decreased % myosin heavy chain IIb, and, in the medial gastrocnemius, reduced twitch speed, reduced mass-specific isotonic power, and increased fatigue resistance. To evaluate whether selection has altered fibre type expression in mice with either "mini" or normal muscle phenotypes, we examined fibre types of red and white gastrocnemius. In both the medial and lateral gastrocnemius, the mini-phenotype increased activities of oxidative enzymes and decreased activities of glycolytic enzymes. In red muscle samples, the mini-phenotype markedly changed fibre types, with the % type I and type IIA fibres and the surface area of type IIA fibres increasing; in addition, mice from selected lines in general had an increased % type IIA fibres and larger type I fibres as compared with mice from control lines. White muscle samples from mini-mice showed dramatic structural alterations, with an atypical distribution of extremely small, unidentifiable fibres surrounded by larger, more oxidative fibres than normally present in white muscle. The increased proportion of oxidative fibres and these atypical small fibres together may explain the reduced mass and increased mitochondrial enzyme activities in mini-muscles. These and previous results demonstrate that extension of selective breeding beyond the time when the response of the selected trait (i.e. distance run) has levelled off can still modify the mechanistic underpinnings of this behaviour.     

Bacterial Strain <Emphasis Type="Italic">Alcaligenes denitrificans</Emphasis> C-32 Containing Two Nitrilases with Different Substrate Specificities

Two genes encoding nitrilases with different properties have been found in an Alcaligenes denitrificans C-32 strain with high nitrilase activity that is currently used as a biocatalyst for commercial ammonium acrylate production. Both genes were expressed in E. coli, and the properties of the recombinant nitrilases were studied. One of these genes, which is designated as nitC1, controlled the formation of nitrilase that preferred aliphatic nitriles (acrylonitrile and butyronitrile) as best substrates. The nucleotide sequence of the gene nitC1 was almost (99%) identical to the gene sequence of an aliphatic nitrilase from Acidovorax facilis 72W (DQ4444267). In turn, nitC2 had a high level of homology (85%) with the arylacetonitrilase gene from Alcaligenes faecalis JM3 (D13419). Benzyl cyanide was shown to be the best substrate for nitC2-encoded nitrilase. In light of the results of DNA homology and differences in substrate specificity, the NitC2 and NitC1 nitrilases from Alcaligenes denitrificans C-32 were allocated to the groups of aliphatic nitrilases and arylacetonitrilases, respectively.     

Random mutagenesis of the arylacetonitrilase from Pseudomonas fluorescens EBC191 and identification of variants,which form increased amounts of mandeloamide from mandelonitrile

The nitrilase from Pseudomonas fluorescens EBC191 was modified by introducing random mutations via error-prone PCR techniques in order to obtain nitrilase variants, which form increased amounts of mandeloamide from racemic mandelonitrile. A screening system was established and experimentally optimized, which allowed the screening of nitrilase variants with the intended phenotype. This system was based on the simultaneous expression of nitrilase variants and the mandeloamide converting amidase from Rhodococcus rhodochrous MP50 in recombinant Escherichia coli cells. The formation of increased amounts of mandeloamide from mandelonitrile by the nitrilase variants was detected after the addition of hydroxylamine and ferric iron ions by taking advantage of the acyltransferase activity of the amidase, which resulted in the formation of coloured iron(III)–hydroxamate complexes from mandeloamide. The system was applied for the screening of libraries of nitrilase variants and 30 enzyme variants identified, which formed increased amounts of mandeloamide from racemic mandelonitrile. The increase in amide formation was quantified by high-performance liquid chromatography and the genes encoding the relevant nitrilase variants sequenced. Thus, different types of mutations were identified. One group of mutants carried different deletions at the carboxy-terminus. The other types of variants carried amino acid exchanges in positions that had not been related previously to an increased amide formation. Finally, a nitrilase variant was created by combining two independently obtained point mutations. This enzyme variant demonstrated a true nitrile hydratase activity as it formed mandeloamide and mandelic acid in a ratio of about 19:1 from racemic mandelonitrile.     

Performance linked to residence time distribution by a novel wool-based bioreactor for tertiary sewage treatment

Laboratory-scale experiments were carried out using up-flow 7 L Submerged Aerated Filter reactors packed with wool fibre or commercial plastic pall rings, Kaldnes, (70% by volume) support media for the tertiary treatment of sewage. The performance of the wool bioreactor was more consistent than that with Kaldnes medium, for both TOC removal (93%) and SS removal (90%). Both plastic and wool-packed bioreactors achieved complete nitrification at the load of about 0.4 kgCOD/m³/day. The sludge yield of the wool bioreactor was almost half that of the bioreactor with Kaldnes suggesting that wool could retain residual organics and particulates. The wool however was degraded and it was concluded that wool would have to be considered as additional sacrificial adsorption capacity rather than an alternative medium. The performance was linked to the residence time distribution studies and these changes in the wool structure. Biomass growth increased the retention of the tracer in the wool reactor by, it was suggested, exposing a greater surface area. Results from the plastic media on the other hand showed increased mixing possibly by increasing the mobility of the plastic. Aeration increased the mixing in both reactors, and patterns were in all cases predominantly well-mixed.     

产腈水解酶基因工程菌的产酶诱导条件及培养基优化

本文通过对产酶诱导条件及发酵培养基进行优化,成功提高了产腈水解酶基因工程菌E. coli BL21(DE3)-pETNYNit的产酶水平。研究结果显示,最佳发酵培养基为：葡萄糖0.2%、甘油0.7%(v/v)、蛋白胨1.2%、酵母膏0.8%、NaCl 0.3%、(NH4)2SO40.3%、NH4Cl 0.13%、Na2 HPO4·12H2 O 1.04%、KH2 PO40.39%、MgSO4·7H2 O 0.03%,pH 7.2。最佳产酶诱导条件为：发酵4 h时加入0.5 mmol/L IPTG,然后在28℃、240 r/min下诱导腈水解酶基因表达14 h~16 h。采用优化方案,重组菌产酶水平可提升至0.9~1×105 U,与野生菌株的产酶水平相比,提高幅度超过50%。同时重组菌培养仅需24 h,培养周期缩短超过50 h。     

金属螯合载体固定重组腈水解酶

以琼脂粉为基质制备金属螯合载体,并用于固定重组腈水解酶。研究发现:制备金属螯合载体最合适的金属离子为Zn2+。当Zn2+离子浓度0.3 mol/L、给酶量15.6 mg/g、固定化pH 8.0、固定化温度40℃时,制得的固定化酶活性最高。固定化酶最适反应温度为50℃、最适反应pH为7.0。当扁桃腈浓度为10 mmol/L、反应1 h时,固定化酶最大产率为0.041 mmol/(g·h);在反应12 h时,产物e.e.值可达到99%以上。固定化酶重复使用8次以后,酶活力仍保持在45%。     

Biotransformation of β-keto nitriles to chiral (<Emphasis Type="Italic">S</Emphasis>)-β-amino acids using nitrilase and ω-transaminase

Objective

To enzymatically synthesize enantiomerically pure β-amino acids from β-keto nitriles using nitrilase and ω-transaminase.

Results

An enzyme cascade system was designed where in β-keto nitriles are initially hydrolyzed to β-keto acids using nitrilase from Bradyrhizobium japonicum and subsequently β-keto acids were converted to β-amino acids using ω-transaminases. Five different ω-transaminases were tested for this cascade reaction, To enhance the yields of β-amino acids, the concentrations of nitrilase and amino donor were optimized. Using this enzymatic reaction, enantiomerically pure (S)-β-amino acids (ee > 99%) were generated. As nitrilase is the bottleneck in this reaction, molecular docking analysis was carried out to depict the poor affinity of nitrilase towards β-keto acids.

Conclusions

A novel enzymatic route to generate enantiomerically pure aromatic (S)-β-amino acids from β-keto nitriles is demonstrated for the first time.

     

Effects of doxycycline on articular cartilage GAG release and mechanical properties following impact

The effects of doxycycline were examined on articular cartilage glycosaminoglycan (GAG) release and biphasic mechanical properties following two levels of impact loading at 1 and 2 weeks post-injury. Further, treatment for two continuous weeks was compared to treatment for only the 1st week of a 2-week culture period. Following impact at two levels, articular cartilage explants were cultured for 1 or 2 weeks with 0, 50, or 100 microM doxycycline. Histology, GAG release to the media, and creep indentation biomechanical properties were examined. The "High" (2.8 J) impact level had gross surface damage, whereas "Low" (1.1 J) impact was indiscernible from non-impacted controls. GAG staining decreased after High impact, but doxycycline did not visibly affect staining. High impact resulted in decreased aggregate moduli at both 1 and 2 weeks and increased permeability at 2 weeks, but tissue mechanical properties were not affected by doxycycline treatment. At 1 week, High impact resulted in more GAG release compared to non-impacted controls. However, following High impact, 100 microM doxycycline reduced cumulative GAG release at 1 and 2 weeks by 30% and 38%, respectively, compared to no treatment. Interestingly, there was no difference in GAG release comparing 2 weeks continuous treatment with 1 week on, 1 week off. These results support the hypothesis that doxycycline can mitigate GAG release from articular cartilage following impact loads. However, doxycycline was unable to prevent the loss of tissue stiffness observed post-impact, presumably due to initial matrix damage resulting solely from mechanical trauma.