Production of lipolytic enzymes in batch cultures of Ophiostoma piceae

After an extensive fungal screening, an Ophiostoma piceae strain was selected for its ability to produce high concentrations of lipase and esterase to remove pitch components of wood pulp. Optimal conditions for growth and enzyme production were established first in shaken flasks. A medium containing rapeseed oil and mycological peptone resulted in a lipase activity of 736 U dm^?3 and an esterase activity of 1569 U dm^?3. When fermentation runs were implemented using the same medium under controlled conditions in 2‐dm³ fermenters, the lipase and esterase activities were increased to 1005 and 4006 U dm^?3 respectively. Further scale‐up was carried out in two stages to 20 dm³ and 72 dm³ (pilot‐scale) stirred tank reactors. The results proved that the cultures could be scaled‐up successfully from shaken flasks to pilot‐scale with increases of 47% and 146% in lipase and esterase activities respectively. © 2001 Society of Chemical Industry     

Influence of lactic acid on the solvent extraction separation of Sm(III) and Co(II) from chloride medium using DEHPA

ABSTRACT

A novel process for the separation of Sm (III) and Co (II) from chloride medium using lactic acid in the aqueous feed employing Di-2-ethylhexyl phosphoric acid (DEHPA) in petrofin as organic phase solvent has been developed. Complete separation has been accomplished with 1 M HLac and 0.1 M DEHPA in the pH range 1–3 and using 2.2 M HLac at pH 3 with 0.2 M DEHPA. Sm (III) could be entirely stripped off the loaded organic solvent by means of 1.2 M HCl and HNO₃. The extraction process is heat releasing for Sm (III) and heat absorbing for Co (II).     

Electrodeposition of poly(n‐methylpyrrole) coatings on steel from aqueous medium

Poly(N‐methylpyrrole) coatings were formed on low carbon steel by an electrochemical method from aqueous oxalate solutions. The electrochemical reactions were performed in a wide range of pH of the reaction medium and applied current density. The formation of poly(N‐methylpyrrole) on steel occurred in three stages: (i) dissolution of the steel, followed by (ii) passivation of the steel, and, finally, (iii) electropolymerization of N‐methylpyrrole on the passivated steel. The time taken to form the passive interphase (induction time) is decreased by an increased applied current. Passivation occurred instantaneously at pH 8.4. Below pH 7, the shortest passivation time occurred at pH 2.6. The quantity of the charge consumed during passivation (passivation charge) remained independent of the applied current at pH ≈ 2.6 and decreased with the applied current at pH 4.1 and 5.7. The polymerization potential increased with the pH and the applied current. Polymerization potentials greater than 2.0 V resulted in film degradation. By controlling the electrochemical process parameters, good quality poly(N‐methylpyrrole) was formed at a controlled induction time. © 1999 John Wiley & Sons, Inc. J Appl Polym Sci 71: 1293–1302, 1999     

聚合物接枝脂肪酶的合成及其对酶活的影响

利用原子转移自由基聚合方法将含C₂～C₄烷基链的单体化合物分别接枝到皱褶假丝酵母脂肪酶（CRL）表面合成了HEMA-g-CRL、GMA-g-CRL、nPMA-g-CRL和BMA-g-CRL四种聚合物接枝CRL。CD和荧光光谱学分析显示,聚合物接枝导致HEMA-g-CRL、nPMA-g-CRL和BMA-g-CRL分子的α-螺旋和β-折叠含量增加。与此同时,聚合物接枝CRL分子荧光发射光谱也发生了蓝移,意味着其具有更加紧密的分子构象。酶活测定结果进一步显示,聚合物接枝增强CRL酶活227%～278%。随着单体化合物烷基链长增加,聚合物接枝CRL的K _m值由0.17 mmol·L^-1降至0.09 mmol·L^-1。与此同时,聚合物接枝CRL的k _cat值则提高至106～182 s^-1。BMA-g-CRL的催化效率达到了野生型CRL的3.28倍。这反映出聚合物接枝助力了CRL“盖子”结构开启和CRL活性中心暴露,促进底物的转化。稳定性测试表明,聚合物接枝提升了CRL的热稳定性并拓宽了其pH操作范围。     

Crosslinked poly(vinyl alcohol) supports for the immobilization of a lipolytic enzyme

Different esters of crosslinked poly(vinyl alcohol) (PVA) were synthetized. They were developed for protein fractionation and immobilization. PVA was crosslinked with epichlorohydrin (CL‐PVA) and esterified with linear fatty acids of different length (Cn‐CL‐PVA). A characterization of the obtained products was made. The swelling behavior, the solubility, and the percentage of esterification were examined. Values of equilibrium water content of about 81% were reached for CL‐PVA samples. The polymers' stability and morpholgy were also investigated. Thermal analysis showed an increase in matrices stability, while SEM data showed the superficial development due to crosslinking and esterification reactions. Moreover, evident morphological inhomogeneities, mainly in the commercial and crosslinked products rather than in the final polymer, were present. Finally, immobilization experiments with a commercial crude of Candida rugosa were performed. Experiments showed a greater affinity of the protein for carbon chain length ranging from 8 to 12. Data indicated that compared to Celite 545, C8‐CL‐PVA was a better support for enzyme immobilization by physical adsorption, confirming the fact that microbial lipases prefer hydrophobic supports. © 1999 John Wiley & Sons, Inc. J Appl Polym Sci 74: 1881–1889, 1999     

重介选煤中固液比的影响研究

介绍了固液比对重介选煤的影响。结合淮北选煤厂的实际生产情况,对理论固液比和实际固液比进行对比分析。结果表明,在实际固液比情况下,选煤厂每年节约电量1.5708×106 kWh,实际介耗量减少,固定投资和单位能耗明显降低,经济效益显著。     

Influence of substrate dideuteration on the reaction of the bifunctional heme enzyme psi factor producing oxygenase A (PpoA)

PpoA is a bifunctional enzyme that catalyzes the dioxygenation of unsaturated C18 fatty acids. The products of this reaction are termed psi factors and have been shown to play a crucial role in conferring a balance between sexual and asexual spore development as well as production of secondary metabolites in the fungus Aspergillus nidulans. Studies on the reaction mechanism revealed that PpoA uses two different heme domains to catalyze two subsequent reactions. Initially, the fatty acid substrate is dioxygenated at C8, yielding an 8‐hydroperoxy fatty acid at the N‐terminal domain. This reaction is catalyzed by a peroxidase/dioxygenase‐type domain that exhibits many similarities to prostaglandin H2 synthases and involves a stereospecific homolytic hydrogen abstraction from C8 of the substrate. The C terminus harbors a heme thiolate P450 domain in which rearrangement of the 8‐hydroperoxide to the final product, a 5,8‐dihydroxy fatty acid, takes place. To obtain further information about the intrinsic kinetics and reaction mechanism of PpoA, we synthesized C5‐dideutero‐ and C8‐dideutero‐oleic acid by a novel protocol that offers a straightforward synthesis without employing the toxic additive hexamethylphosphoramide (HMPA) during C? C coupling reactions or mercury salts upon thioketal deprotection. These deuterated fatty acids were then employed for kinetic analysis under multiple‐turnover conditions. The results indicate that the hydrogen abstraction at C8 is the rate‐determining step of the overall reaction because we observed a KIE (V_H/V_D) of ～33 at substrate saturation that suggests extensive nuclear tunneling contributions for hydrogen transfer. Deuteration of the substrate at C5, however, had little effect on V_H/V_D but resulted in a different product pattern presumably due to an altered lifetime and partitioning of a reaction intermediate.     

Immobilization of lipase on hydrogels: Structural aspects of polymeric matrices as determinants of enzyme activity in different physical environments

Well‐defined and characterized polymeric matrices showing close chemical similarities but wide differences in water uptake and swellability in aqueous medium were used for lipase immobilization. Biphasic networks of 2‐hydroxypropylcellulose (HPC) were synthesized with acrylamide (AAm), methacrylamide (MAAm), N‐isopropylacrylamide (N‐i‐PAAm), and 2‐acrylamido‐2‐methyl‐1‐propanesulfonic acid (AMPSA) and simultaneously crosslinked with N,N‐methylene bisacrylamide in aqueous medium by using simultaneous γ‐radiation technique. Lipase enzyme was produced from a mesophilic bacterial isolate (HBK‐8) and was immobilized onto all the matrices by adsorption method. The activity of the immobilized enzyme was optimized for pH, temperature, and amount of crude enzyme and effect of dehydration. High relative activity for the immobilized enzymes was observed and loss of activity with time was minimal; reusability was found to be good. The activity of the immobilized enzyme was also observed to be good in both esterification and hydrolysis of esters. In the present study, lipase immobilization, hydrolysis of p‐nitrophenyl palmitate, and optimum pH and temperature for substrate hydrolysis were evaluated for different matrices to study polymer structure and enzyme activity relationship in diverse physical environments. © 2004 Wiley Periodicals, Inc. J Appl Polym Sci 92: 3135–3143, 2004     

Influence of hydrodynamic stress in the growth of Scenedesmus obliquus using a culture medium based on olive-mill wastewater

The wastewater from olive oil elaboration (OMW) constitutes a major environmental problem in producing countries of olive oil. OMW from the three-phase system is harmful due to its high organic load and the presence of phytotoxic phenolic compounds. The culture medium was constituted with 5% OMW. The effects of mechanical agitation and aeration rates on the microalga Scenedesmus obliquus growth, in a batch photobioreactor were studied. A maximum specific growth rate, μ_m = 0.031 h⁻¹, was obtained using a rotational impeller speed around 350 rpm without aeration and was greater than that found in the absence of stirring 0.024 h⁻¹. The chlorophyll contents were independent of the stress and light intensity in the cultures, but did boost the nutrient concentration in the medium. The stress induced by stirring or by aeration of the cultures did not alter the final cell-protein and carbohydrate content, 18% and 48% as mean value, respectively.     

Influence of nanoparticle diameter on conjugated enzyme activity

Lactase conjugated to nanomaterials represents an area of significant potential to the food processing as a means to produce novel value-added products, reduce waste, and enable diagnostics. While it is recognized that, in general, matter exhibits unique properties when manipulated at the nanoscale, little is known about how reducing the size of the carrier to the nanoscale effects attached lactase. The purpose of this work is to investigate the influence of particle size on activity retention of lactase (Aspergillus oryzae) covalently conjugated to magnetic nanoparticles of varying sizes. Lactase was attached to carboxylic acid functionalized magnetic nanoparticles 18 nm, 50 nm, and 200 nm in diameter using carbodiimide chemistry. After attachment, activity retention was 73%, 39%, and 14% compared to the free enzyme for the 18 nm, 50 nm, and 200 nm conjugates, respectively. The apparent K_m was not significantly different as a function of particle size while the apparent k_cat decreased with increasing particle size. Reducing the particle size of magnetic nanoparticles can increase the activity retention of conjugated lactase. This work provides improved understanding of enzyme-nanoparticles systems and allows for enhanced design of lactase-conjugated materials.     

Influence of varying surface hydrophobicity of chitosan membranes on the adsorption and activity of lipase

The hydrophobic surface modification of chitosan membranes was performed using the amidating reaction of amino groups on a membrane surface with stearic acid activated by 1‐ethyl‐3‐(3‐dimethylaminopropyl)‐carbodiimide hydrochloride (EDC) and N‐hydroxysuccinimide (NHS). The varying surface hydrophobicity of chitosan membranes was achieved by changing the degree of amidation and evaluated by the water contact angle analysis and the adsorption experiment of the hydrophobic dye, Rose Bengal. The effects of the surface hydrophobicity of chitosan membranes on the adsorption behaviors, activity and stability of Candida rugosa lipases were investigated. The experimental results suggested that the increased surface hydrophobicity of chitosan membranes improved the adsorption capacity and activity of the immobilized lipase. The modified chitosan membranes with 30.36% amidation exhibited the maximum activity retention of 83.87%. In addition, a desirable thermal stability was also achieved for the adsorbed lipase. © 2009 Wiley Periodicals, Inc. J Appl Polym Sci, 2010     

Identification of carboxylesterase activities of commercial triacylglycerol hydrolase (lipase) preparations

The carboxylesterase activities of 34 commercial lipase preparations (CLP), from bacterial, fungal, and mammalian sources, were characterized by isoelectric focusing and nonreducing SDS‐PAGE followed by alpha‐naphthyl acetate zymography. Substantial differences in both the numbers of stained proteins and the enzymatic activity banding patterns were seen, even among preparations from the same organism, indicating a great heterogeneity of enzyme activities in commercial lipase preparations. CLP displayed between 1 and 16 carboxylesterase activity bands of discrete isoelectric points, many of which were not detected by silver stain. Most carboxylesterase bands did not display lipase activity: although 26 CLP contained carboxylesterase bands with isoelectric points identical to previously documented lipase activity bands, there were many more carboxylesterase bands than lipase bands. Conversely, twenty CLP lacked carboxylesterase activity at one or more isoelectric points previously shown to exhibit lipase activity. Only in 11 CLP did all electrofocused lipase bands also display carboxylesterase activity. Few carboxylesterase bands (0—4) were detected after nonreducing SDS‐PAGE and alpha‐naphthyl acetate zymography. About half of these were undetectable by silver stain, suggesting high activities. The range of apparent molecular mass (AMM) of active carboxylesterases was 24—67 kDa, lower than the AMM of protease activities present in these lipase preparations, indicating that most esterase activities were not caused by the proteases in the preparations. Seven preparations demonstrated carboxylesterase activity at the same AMM as proteases.     

Oxygen reduction on bornite (Cu5FeS4) in alkaline medium

The electroreduction of molecular oxygen is investigated between –0.1 and –0.5V vs SHE on bornite, Cu5FeS4, at pH9.2 and 14, by means of cyclic voltammetry (CV) and stationary voltammetry (SV), using a double channel electrode flow cell (DCEFC). Using an E/pH diagram established in this work, the CV results suggest that the bornite surface is stable between –0.1 and –0.5V then oxidized to CuS and Fe(OH)3 above –0.1V whereas, below –0.5V the mineral reduces to metal sulphides: Cu2S and FeS. The SV results show that oxygen is reduced to peroxide ions, HO2–. At pH9.2 the generated sulphide ions hinder the oxidation of HO2– on the collector electrode of the DCEFC, due to the formation of a blocking surface layer of elemental sulphur, S, impeding the determination of the kinetic parameters, k1 (direct way) and k 2 (indirect way) of the oxygen electroreduction reaction. In contrast, at pH14, as soluble polysulphides are formed, it was possible to determine these parameters, showing that the bornite is a poor catalyst for oxygen reduction. At pH14, in the presence of potassium ethylxanthate, generally used as a flotation collector, the ethylxanthate ions, C2H5OCSSO–, are oxidized by HO2– to perxanthates, ROCSSO–, while at pH9.2 the oxygen reduction is inhibited due to ethylxanthate chemisoption on the bornite surface.     

Mixing of a wet granular medium: Influence of the liquid addition method

Mixing is an important operation in the elaboration of concrete. This process generally involves high energy consumption. The method by which the liquid is added influences this consumption. The liquid can be added progressively with a constant flow, instantaneously at the beginning of the mixing process, or in several stages.Our results show that the power usually consumed with a progressive liquid addition can be reduced by adding liquid in several stages, by modifying the number of additions and by varying the amount of liquid added for each addition.For a final-state mixture, oversaturated in liquid, instantaneous addition provides the lowest energy consumption. However, for a final state that is less saturated, the lowest energy intensity is obtained by adding the liquid in two stages. The first amount corresponds to a saturation rate located in the funicular regime, and is higher than the second amount.Finally, for saturation states located before the capillary regime, a progressive increase in the energy signal is observed after an instantaneous addition of the liquid. Moreover, the capillary state is obtained using an amount of liquid for which this state is not usually reached by progressive addition. This phenomenon is more pronounced where the particle size is reduced and the rotation velocity increased.     

陶瓷研磨体对水泥熟料粉磨性能影响的研究

在辊压机+球磨机水泥联合粉磨系统中,辊压机系统的主要任务是进行物料的预粉碎,以大大减小入磨物料的粒度,球磨机系统的主要任务是进行物料的研磨和整形。因此,使用低密度、高硬度的陶瓷研磨体可以提高球磨机的粉磨效率。本文分别以相近的陶瓷研磨体和金属研磨体的级配和填充率进行了水泥熟料的粉磨实验。实验结果表明,陶瓷研磨体粉磨的水泥颗粒级配更加合理,3~32μm颗粒含量可以提高3%左右,28d抗压强度可以提高4MPa左右,水泥标准稠度用水量可以降低2%左右,水泥颗粒的圆形度可以提高8.5%左右。     

Influence of polymerization conditions on the viscosity and yield of poly(phenylene sulfide ether)

High molecular weight poly(phenylene sulfide ether) (PPSE) was successfully synthesized by reaction of 4,4′‐dihydroxy diphenyl sulfide with 4,4′‐dichloro diphenyl sulfide in N‐methyl‐2‐pyrrolidone (NMP). The influence of polymerization conditions on the intrinsic viscosity and yield of PPSE was investigated and the optimized reaction condition was concluded. Reactions at about 180°C for 6 h along with sodium benzoate as an additive and monomer concentration of 0.588 mol/L NMP were found to produce the highest intrinsic viscosity (0.55 dL/g). Longer reaction time and/or higher temperature reduced the intrinsic viscosity and yield of the resulting product, probably due to side reactions, such as reductive dehalogenation and chemical degradation. X‐ray diffraction indicated that the polymer possessed of orthorhombic cell and had a high crystallinity of 65.8%. The high molecular weight PPSE is a crystalline polymer with T_m of 252°C and T_mc of 224°C. The polymer shows good chemical resistance, but is soluble in organic amide, halo‐hydrocarbon and oxohydrocarbon solvent at a temperature over 150°C. © 2008 Wiley Periodicals, Inc. J Appl Polym Sci, 2008     

Studies on the crystal growth of rice bran wax in a hexane medium

Rice bran oil (RBO) is well known for its high wax content (2–4%). A good separation of waxes must be ensured through the formation of crystals that can be removed with a minimal retention of oil to maintain high yields of refined oil. In the present study, the form and size distribution of RBO wax crystals were investigated using a laser diffraction technique. An attempt was made to study the effect of cooling on growth and size distribution of RBO wax crystals in hexane medium, and it showed that high cooling rate and low temperature induces the formation of a great number of small nuclei. In addition, experiments were performed to evaluate the effect on the growth of wax crystals of successive additions of gum and pure TG to the medium. The entire experiment was designed to optimize the temperature and incubation time of wax crystallization to facilitate the efficient separation of wax from crude RBO-hexane miscella using membrane technology.     

触角状环氧链结构对固定化脂肪酶(YCJ01)酶活的影响

为了改善酶的刚性固定化对酶蛋白构象的负面影响,以原子转移自由基聚合法（ATRP）合成了亲水性、柔性、触角状环氧化酶固定化载体PS-acyl-P（AM-co-GMA）,通过改变单体总量与引发剂量比例得到不同链长触角状环氧化酶固定化载体,并将其用于耐有机溶剂脂肪酶（YCJ01）的共价柔性固定化,重点考察了链长对固定化酶酶活的影响。结果表明,链长（增重率不超过3200%）越长,酶活越高。