Pronase-susceptible Floc Forming Bacteria: Relationship between Flocculation and Calcium Ion

Six strains of floc-forming bacteria belonging to Flavobacterium were isolated from activated sludge which were deflocculated by Pronase treatment. The flocculated cells of the strain B, one of the isolates, was deflocculated not only by Pronase, but also by ethylenediaminetetraacetate. Growth was stimulated when Pronase was added in the medium. An adequate amount of calcium ion in the medium was required for flocculation. No flocculation was observed, however, when calcium was added to the cells grown with a low level of calcium. Deflocculation was observed at the late stationary phase and the onset of deflocculation depended on the concentrations of calcium in the medium. The higher concentrations delayed the deflocculation. The floes formed in the presence of calcium over 0.5 nm in the medium became resistant to the Pronase treatment.     

Liver-specific Induction of Ribosomal Protein Gene Expression by Amino Acid Starvation in Rats

An aminoacylase, inducibly formed in Bacillus thermoglucosidius grown with a synthetic compound, acetamidocinnamate, was used for enzymatic synthesis of l-phenylalanine from chloroacetamido-cinnamate. The reaction system consisted of the hydrolysis of chloroacetamidocinnamate to phenylpyruvate by aminoacylase and the reductive amination of phenylpyruvate to l-phenylalanine by phenylalanine dehydrogenase. The coenzyme NADH consumed was regenerated by a coupled reaction with formate dehydrogenase. Under optimum conditions for l-phenylalanine production, more than 98% of the initially added chloroacetamidocinnamate was converted effectively to l-phenylalanine without appreciable decomposition or racemization.     

厌氧真菌菌系乙酰酯酶的特性研究

利用厌氧培养技术,采用产酶培养基,培养课题组自行构建的一组厌氧真菌菌系,使之产乙酰酯酶。采用硫酸铵分级沉淀、透析袋透析、DEAE-纤维素离子交换柱层析、Sephadex G-75凝胶过滤柱层析,分离纯化所得到乙酰酯酶,研究其酶学性质。酶活力动态分析表明,乙酰酯酶在产酶培养基上,培养至第3天酶活力达到最高。乙酰酯酶最适温度为41℃,最适pH为9．0,Mg^2＋、K^＋、Ca^2＋对酶有一定的激活作用,Fe^3＋对酶有很强的抑制作用。该厌氧真菌菌系所产的乙酰酯酶,对于发酵木质纤维素类物质具有潜在应用价值。     

Metabolic and molecular responses in Nile tilapia,Oreochromis niloticus during short and prolonged hypoxia

The strictly aquatic breathing Nile tilapia, Oreochromis niloticus is an extremely hypoxia-tolerant fish. To augment our understanding of the effects of hypoxia on anaerobic glycolysis in the Nile tilapia, we studied the effect of short-term for 1 day (trial 1) and long-term for 30 days (trial 2) hypoxia on a selected glycolytic enzymes activity and mRNA expression in liver and white muscle. The hypoxic oxygen concentrations used in the two trials were 2, 1, and 0.5 mg O₂ L^?1 for comparison with a control normoxic group 8 mg O₂ L^?1. The activity of phosphofructokinase (PFK), pyruvate kinase (PK), and lactate dehydrogenase (LDH) in liver and white muscle except liver LDH decreased in trial 1 and increased in trial 2. Assessments of mRNA levels in trial 1 revealed that PFK was downregulated and LDH was upregulated in liver and white muscle, while PK fluctuated between upregulation in liver and downregulation in white muscle. Meanwhile, PK and LDH were upregulated while PFK was similar to control values in both tissues in trial 2. Comet assay results demonstrated an increase in DNA damage that was directly proportional to increasing hypoxic concentrations. This damage was more pronounced in trial 1. This suggests that the Nile tilapia cope better with long-term hypoxic conditions, possibly as an adaptive response.     

Parameters affecting productivity in the lipase-catalysed synthesis of sucrose palmitate

The industrial application of lipases for the synthesis of sucrose esters is usually limited by its low productivity, as we need a medium where a polar reagent (the sugar) and a non-polar fatty acid donor are soluble and able to react in the presence of the biocatalyst. In this work, we have studied the problems encountered when trying to increase the volumetric productivity of sucrose esters. The synthesis of sucrose palmitate was performed in 2-methyl-2-butanol:dimethylsulfoxide mixtures by transesterification of different palmitic acid donors with sucrose, catalysed by the immobilized lipase from Candida antarctica B (Novozym 435). A protocol for substrate preparation different from that previously reported was found to improve the reaction rate. Several parameters, such as sucrose and acyl donor loadings, the percentage of DMSO in the mixture and the nature of acyl donor, were investigated. Under the best experimental conditions (15% DMSO, 0.1 mol l^?1 sucrose, 0.3 mol l^?1 vinyl palmitate), a maximum of 45 g l^?1 sucrose palmitate was obtained in 120 h. Using methyl or ethyl palmitate, the highest productivity was 7.3 g l^?1 in 120 h using 20% DMSO with 0.2 mol l^?1 sucrose and 0.6 mol l^?1 acyl donor. The formation of free fatty acid, and the effect of the percentage of DMSO on the monoester/diester selectivity were also studied. To our knowledge, this is the first report on enzymatic synthesis of sucrose esters of long fatty acids using alkyl esters as acyl donors.     

Economic comparison of enzymatic reactors and advanced oxidation processes applied to the degradation of phenol as a model compound

Abstract

Some micropollutants present in wastewaters are barely removed in sewage treatment plants. In many cases a post-treatment process based on separation and/or oxidation has to be applied. The aim of this study was the technical and economic comparison of enzymatic technologies with other advanced oxidation processes (AOPs) for the degradation of phenol. Batch and continuous enzymatic reactors, using free and immobilized manganese peroxidase (MnP, EC 1.11.1.13), were considered. Continuous degradation of phenol in an enzymatic membrane reactor was shown to be the fastest process and degradation in a continuous reactor with immobilized enzyme involved the lowest consumption of enzyme. However, the immobilization process increased the enzyme cost 100-fold. A continuous enzymatic membrane reactor gave high degradation efficiency and may be a viable technology for phenol removal when compared with other AOPs from both technical and economic points of view.     

Hydrolysis of crambe oil by enzymatic catalysis: An evaluation of the operational conditions

Abstract

In this work, the enzymatic hydrolysis of the crambe oil by using a commercial immobilized lipase Lipozyme RM IM was evaluated. The effect of the operational conditions, such as temperature, water/oil molar ratio, enzyme/substrate mass ratio and stirring speed were assessed based on the experimental designs. The experiments were performed in a closed and batch system with controlled temperature and stirring speed. In addition, the kinetics of the process was studied in the best operational conditions, wherein the experimental data were obtained and described by a mathematical model. The influence of the operational conditions was assessed based on the measured values of the free fatty acids (FFA) produced by the enzymatic hydrolysis. In 4?h of reaction, a yield of 42.6% was observed and the most significant operational conditions were the enzyme/substrate mass ratio and stirring speed. By the kinetic investigation, an initial reaction rate of 3.5?×?10⁴?mol?mL^?1?h^?1 and a maximum yield of 74% were observed after 40?h of reaction (in the equilibrium condition). The mathematical model was not only able to adequately describe the experimental data of FFA concentrations profiles but also showed predictive capacity to independents assays in different operational conditions. Therefore, based on the simulation analysis of the enzymatic hydrolysis of the crambe oil, the model can be useful for process optimization and phenomenological studies.     

Conformational heterogeneity of the protein-free human spliceosomal U2-U6 snRNA complex

The complex formed between the U2 and U6 small nuclear (sn)RNA molecules of the eukaryotic spliceosome plays a critical role in the catalysis of precursor mRNA splicing. Here, we have used enzymatic structure probing, ¹⁹F NMR, and analytical ultracentrifugation techniques to characterize the fold of a protein-free biophysically tractable paired construct representing the human U2-U6 snRNA complex. Results from enzymatic probing and ¹⁹F NMR for the complex in the absence of Mg²⁺ are consistent with formation of a four-helix junction structure as a predominant conformation. However, ¹⁹F NMR data also identify a lesser fraction (up to 14% at 25°C) of a three-helix conformation. Based upon this distribution, the calculated ΔG for inter-conversion to the four-helix structure from the three-helix structure is approximately −4.6 kJ/mol. In the presence of 5 mM Mg²⁺, the fraction of the three-helix conformation increased to ∼17% and the Stokes radius, measured by analytical ultracentrifugation, decreased by 2%, suggesting a slight shift to an alternative conformation. NMR measurements demonstrated that addition of an intron fragment to the U2-U6 snRNA complex results in displacement of U6 snRNA from the region of Helix III immediately 5′ of the ACAGAGA sequence of U6 snRNA, which may facilitate binding of the segment of the intron adjacent to the 5′ splice site to the ACAGAGA sequence. Taken together, these observations indicate conformational heterogeneity in the protein-free human U2-U6 snRNA complex consistent with a model in which the RNA has sufficient conformational flexibility to facilitate inter-conversion between steps of splicing in situ.     

Optimization of Ethanol Production From Microfluidized Wheat Straw by Response Surface Methodology

In this study, wheat straw was pretreated with a microfluidizer to improve its enzymatic hydrolysis and ethanol yields. The pretreatment was performed at various pressures (500, 1000, and 1500 bar) and solid loadings (1, 2, and 3%). The microfluidized biomass was then subjected to hydrolysis and simultaneous saccharification and co-fermentation (SSCF) experiments at different enzyme loadings (5, 10, and 15 FPU/g dry wheat straw) using a mutant yeast. The results indicated that the microfluidization method alters the structure of biomass and leads to a reduction in lignin content. The samples pretreated at 1% solid loading contained the minimum lignin concentration and provided the maximum sugar and ethanol yields. These results signified that the microfluidization method is more effective on biomass at low solid loadings. The process conditions were optimized for higher ethanol and sugar yields using response surface methodology (RSM). The optimum pressure and solid and enzyme loadings were found as 1500 bar, 1%, and 15 FPU/g dry wheat straw, respectively. The yields obtained at this condition were 82%, 94%, and 65% for glucose, xylose, and ethanol, respectively. High sugar yields implied that microfluidization is an effective pretreatment method for cellulosic ethanol production. On the other hand, low ethanol yield may indicate that the microorganism was sensitive to inhibitory compounds present in the fermentation medium.     

Process design and optimization of bioethanol production from cassava bagasse using statistical design and genetic algorithm

Abstract

Bioethanol production from agro-industrial residues is gaining attention because of the limited production of starch grains and sugarcane, and food–fuel conflict. The aim of the present study is to maximize the bioethanol production using cassava bagasse as a feedstock. Enzymatic liquefaction, by α-amylase, followed by simultaneous saccharification and fermentation (SSF), using glucoamylase and Zymomonas mobilis MTCC 2427, was investigated for bioethanol production from cassava bagasse. The factors influencing ethanol production process were identified and screened for significant factors using Plackett–Burman design. The significant factors (cassava bagasse concentration (10–50?g/L), concentration of α-amylase (5–25% (v/v), and temperature of fermentation (27–37?°C)) were optimized by employing Box–Behnken design and genetic algorithm. The maximum ethanol concentrations of 25.594?g/L and 25.910?g/L were obtained from Box–Behnken design and genetic algorithm, respectively, under optimum conditions. Thus, the study provides valuable insights in utilizing the cost-effective industrial residue, cassava bagasse, for the bioethanol production.