Effects of reaction conditions on laccase‐catalyzed α‐naphthol polymerization

Enzymatic oxidative polymerization of α‐naphthol was carried out batch‐wise with the laccase enzyme, produced by Trametes versicolor (ATCC 200801). The polymerization reaction was conducted in a closed, temperature controlled system containing acetone (solvent) and sodium acetate buffer for pH control. The effects of the organic solvent (acetone) composition, monomer (α‐naphthol) and enzyme concentrations, buffer pH and temperature on the polymerization rate were investigated with respect to initial reaction conditions and depletion rate of dissolved oxygen. The optimum acetone composition, pH, monomer, dissolved oxygen and enzyme concentrations were determined as 50% (v/v), 5, 3409 gm⁻³, 20.3 gm⁻³ and 0.173 U cm⁻³, respectively; these values provided the most desirable conditions for initial reaction rate. Temperature rise supported the rate increase up to 37 °C, after which the rate tended to be stable due to a drop in dissolved oxygen concentration. The product polymer, poly(α‐naphthol), with an average molecular weight of 4920 Da was soluble in common organic solvents. © 2000 Society of Chemical Industry     

Preparation of glucose oxidase immobilized in different carriers using radiation polymerization

Glucose oxidase (EC 1.1.3.4) was immobilized on different polymeric materials using different immobilization techniques (entrapping by γ‐irradiation, and covalent binding using epichlorohydrin). Studies were carried out to increase the thermal stability of glucose oxidase (GOD) for different applications. The activity and stability of the resulting biopolymers have been compared with those of free GOD. The effect of different polyvinyl alcohol/polyacrylamide (PVA/PAAm) compositions of the copolymer carrier on the enzymatic activity of the immobilized GOD was studied. The maximum enzymatic activity was obtained with the composition ratio of PVA/PAAm of 60:40. The behaviour of the free and immobilized enzyme was analysed as a function of pH. A broadening in the pH profile (5.5–8) was observed for immobilized preparations. The activity and stability of the resulting biopolymers produced by immobilization of GOD onto different carriers have been compared, in both aqueous and organic media, with those of the free GOD. The enzyme's tolerance toward both heat and organic solvent was enhanced by immobilization onto polymers. The addition of different concentrations of organic solvents (10–50%, v/v) to the enzyme at higher temperature (60 °C) was found to stabilize the enzyme molecule. The strongest stabilizing effect on the enzymatic activity was achieved at a concentration of 10%. Copyright © 2005 Society of Chemical Industry     

Pigment removal from canola oil using chlorophyllase

Frost-damaged or prematurely harvested canola seed (rapeseed) may yield oil with a high chlorophyll content (50–60 μg/ml). Enzymatic hydrolysis of chlorophyll, added to buffer/surfactant, buffer/acetone or buffer/acetone/canola oil, to produce water-soluble chlorophyllide (green pigment) was studied using a crude chlorophyllase preparation (acetone-dried chloroplasts) from 15 to 20-day-old sugar beet seedlings. In buffer/surfactant, the optimum pH for enzyme activity was temperature dependent. At 30 C and 0.24% Triton X-100 (or 30% acetone), chlorophyllase showed maximum activity toward a crude chlorophyll preparation over the range of pH 8–10. At 60 C, the activity was more than twofold higher, with a sharp maximum at ∼pH 8. Mg²⁺ enhanced the activity with an optimal concentration of 50 mM. At pH 7.5, 50 C and in the presence of only 6% acetone, the enzyme showed high affinity for chlorophyll (Km=15μM or 13.5 μg/ml), suggesting that the natural chlorophyll concentrations found in green canola oils might facilitate high enzymatic efficiencies. The crude enzyme was stable in buffer/acetone at pH 7.5 and 50 C for at least two hr. With acetone concentrations as low as 6%, maximum enzyme activities in buffer and buffer/canola oil required intensive mixing (homogenization) of the various substrate, enzyme and liquid phases. In general, the rate and extent of chlorophyll hydrolysis were greater in buffer than in buffer/oil. In both reaction systems, chlorophyll hydrolysis slowed down with time due to accumulation of phytol, which proved to be a competitive inhibitor (K_i=11 μM or 3.3 μg/ml). The other hydrolysis product, chlorophyllide, did not affect enzymatic activity. Crude canola oil used in the reconstitution of green oil did not support enzymatic chlorophyll hydrolysis without prior degumming and desoaping. The optimum buffer/oil ratio of the reaction mixtures was above 2/1 (v/v).     

Immobilization strategy of accessible transmission for trypsin to catalyze synthesis of dipeptide in mesoporous support

Immobilized trypsin in mesoporous silica foams was used to catalyze dipeptide synthesis in hydrophilic organic solvent instead of soluble form. The area surface of nano support was measured. The catalytic activity, coupled yield and kinetic characterization of immobilized trypsin were examined. Bz-Arg-OEt was chosen as the acyl donor with Lys-OH as the nucleophile. The trypsin-catalyzed synthesis condition was optimized, such as catalytic temperature, pH, reaction time, physical properties and content of organic solvents, together with the added enzyme amount. The immobilized trypsin showed 112.8% of residual activity with 91.9% of coupled yield, and the kinetic parameters exhibited accessibility for transmission. The product yield of 5.8% was reached at the optimum conditions for enzymatic synthesis of dipeptide: 800 mg of wet immobilized trypsin (200 mg/g support) was used in Tris-HCl buffer (0.1 mol/L, pH 8.0) containing 80% (v/v) ethanol solvents for 6 h of reaction time at 35 °C. This attempt of immobilized strategy for trypsin in nanopores renders the possibility of wide application of inorganic nano-sized support in catalytic synthesis process, which can avoid usage of large amounts of organic solvents in washing steps by chemical methods and reduce the tedious purification process of its soluble form.     

水复合溶剂体系茴脑臭氧化反应合成茴香醛新工艺

以茴脑为原料,有机溶剂与水复合溶剂为溶剂,室温下通过臭氧化分解反应制取茴香醛,并通过GC-MS、FT-IR和1H-NMR对产品进行了表征.对该溶剂体系与传统溶剂体系下茴香醛的产率进行了对比,并确定了丙酮/水体系为最佳溶剂体系.实验详细考察了溶剂用量、臭氧气流量、混合溶剂中水含量和反应时间等工艺参数.优化的工艺条件为:丙酮和水为溶剂,m(茴脑)∶m(混合溶剂)=1∶3,臭氧气流量0.06 m3·h-1,混合溶剂中水的质量含量为15％,反应时间100 min,茴香醛产率82.70％.该反应在水的存在下实现了室温下一步法合成茴香醛,避免了茴脑臭氧化物的分离及还原步骤,工艺简单,洁净环保.     

银杏叶黄酮类化合物提取方法的研究

对纤维素酶预处理和甲基化β-环糊精溶液相结合提取银杏叶总黄酮的工艺进行了探讨,考察了料液比、酶浓度、温度、时间及pH值对酶解效果的影响,以及M-β-环糊精浓度、温度和时间对浸提效果的影响。得到最佳酶解预处理条件为：经料液比（银杏叶质量与纤维素酶溶液体积之比）1／60、酶质量浓度0．2mg／mL、酶解温度40℃、酶解介质pH=6．5、酶解时间150min处理后,在M—β-环糊精质量分数2．0%、温度60℃条件下浸提180min,总黄酮得率可达2．68％。该工艺为银杏叶黄酮类化合物提取提供了新途径,同时避免了有机溶剂的使用,便于纯化,值得推广。     

Optimization of the enzymatic production of isoamyl acetate with novozym 435 from candida antarctica

In this study the production of isoamyl acetate by esterification of isoamyl alcohol and acetic acid was carried out using immobilized C . antarctica lipase (Novozym 435) as a catalyst without any organic solvent. The esterification yield was optimized with response surface methodology. This method was used with four parameters to evaluate the effects of important variables on the esterification yield. The parameters are acid/alcohol mole ratio (0.2-0.8), enzyme amount (4-12%, w/w), temperature (30-50 °C), and reaction time (4-8 hr). It was found that the most effective parameter was acid/alcohol mole ratio. As acid/alcohol mole ratio increased at any given reaction time and amount of enzyme, ester concentration, C p (mmol ester/g mixture), increased up to an acid/alcohol mole ratio of 0.7 and thereafter decreased. The model indicated the optimum conditions for maximum esterification (3.45 mmol ester/g mixture) in the acid/alcohol mole ratio of 0.52 for 8.15% enzyme at 43.2 °C and after 5.27 hr, which were in good agreement with the experimental value (3.5 mmol ester/g mixture).     

Enzymatic synthesis of geranyl acetate by transesterification with acetic anhydride as acyl donor

Pseudomonas sp. lipase (PS) was immobilized by adsorption technique onto glass beads and tested for its ability to synthesize geranyl acetate by transesterification with acetic anhydride as the acyl donor. Reactions were carried out inn-hexane containing 0.1 M geraniol, 0.1 M acetic anhydride, and 200 units of lipase PS. Enzyme load, effect of substrate concentration, added water, temperature, time course, organic solvent, pH memory, and enzyme reuse were studied. Yields of up to 96% were obtained with 200 units (approximately 11% w/w of reactants) of enzyme. Increasing amounts of geraniol inhibited lipase activity, while excess acyl donor concentration enhanced ester production. Yields as high as 97% were obtained at 50°C, 24 h incubation, with no added water. Solvents with logP values ≥3.0 showed the highest conversion yields. Solvent-free samples also performed well. The pH range of 4–9 gave good yields (92–98.4%). Enzyme reuse studies showed the lipase remained active after 15 runs.     

Mycelium-bound lipase from a locally isolated strain of Aspergillus flavus link: Pattern and factors involved in its production

Aspergillus flavus produces a lipase (EC 3.1.1.3) which is partly bound to the mycelium during growth. The production of the mycelium-bound lipase is concomitant with growth, and declines when growth ceases. Maximum productivity of the enzyme is obtained when the culture is incubated at 30°C, an initial culture pH of 6·5 and with 2% (w/v) each of corn oil and yeast extract as carbon and organic nitrogen source. Yeast extract affects not only the production of lipase but also the secretion of proteases into the culture medium. Production of the latter enzymes, which inactivate the free lipase, is enhanced by adding yeast extract (1–2%, w/v) to the culture medium. However, at 5% (w/v) yeast extract concentration, proteolytic activity is not detected and consequently, the activity of free lipase may easily be measured. Free lipase activity can easily be detected when 0·001 mol dm⁻³ EDTA is added to the culture medium. The presence of the chelating agent enhances the production and maintains the stability of the extracted mycelium-bound lipase.     

香蕉茎秆酶法脱胶工艺及其脱胶纤维性能

为了将生物质废弃物香蕉茎秆进行高值化利用,采用酶法脱胶工艺制备香蕉茎秆纤维。研究超声预处理工艺中频率、处理温度对香蕉茎秆脱胶率的影响,确定较佳的超声预处理工艺。将预处理后的香蕉茎秆粗纤维进行半纤维素酶、果胶酶脱胶,分别考察缓冲液pH、酶浓度、反应温度对脱胶率的影响。结果表明:预处理工艺中超声处理的较佳频率为40 kHz,温度为70℃;酶法脱胶工艺中较佳的半纤维素酶缓冲液pH为5.5,半纤维素酶浓度为0.004 g·ml^-1,反应温度为50℃;较佳的果胶酶缓冲液pH为6.0,果胶酶浓度为0.003 g·ml^-1,反应温度为55℃。采用傅里叶红外光谱(FTIR)、X射线衍射(XRD)和扫描电镜(SEM)分析酶法较佳工艺制备的香蕉茎秆纤维的理化特性;并对其机械性能进行测试与分析。FTIR及XRD结果表明:酶法较佳工艺制备的香蕉茎秆纤维去除了半纤维素和大部分果胶,相对结晶度为66.4%;机械性能测试结果表明酶法较佳工艺制备的香蕉茎秆纤维的拉伸强度、杨氏模量和断裂伸长率分别是118.48 MPa,15.15 GPa,0.67%。     

Bioactive and Physicochemical Properties of Persimmon as Affected by Drying Methods

In this study, the effects of three different drying methods (freeze drying, oven drying, and vacuum oven drying) on bioactive (total phenolics, total flavonoids, condensed tannin and total hydrolyzable tannin contents, antiradical activity, and antidiabetic activity) and some physicochemical (dry matter, ash, water activity (a _w ), color, protein, hydroxymethylfurfural, glucose and fructose content) properties of persimmon fruit were investigated. Simplex lattice mixture design methodology was applied to determine the best solvent mixture for the extraction of phenolics from the samples. It was found that the mixture of acetone:water at the ratio of 50:50 % (v/v) was the best solvent mixture for the extraction. The persimmon powder sample obtained from freeze drying showed significantly (p <0.05) higher bioactivity values than oven- and vacuum-oven-dried samples. Antiradical activity changed significantly depending on the drying method employed and it was superior in freeze-dried samples than that of the other drying methods.     

Systemic Concocting of Cross-Linked Enzyme Aggregates of Candida antarctica Lipase B (Novozyme 435) for the Biomanufacturing of Rhamnolipids

In the present study, Candida antarctica lipase B was immobilized on amine-functionalized silica microspheres as cross-linked enzyme aggregates (CLEA) and utilized for the biomanufacturing of rhamnolipids (RL). Lipase CLEA synthesized under optimized conditions of 2.0:1.0 by volume of silica microsphere/enzyme concentration, a 1.0:2.5 (v/v) ratio of enzyme/2-propanol, 7 mM glutaraldehyde concentration, when incubated at pH 9.0 and 40 °C, for a cross-linking time of 30 min were observed to exhibit superior biocatalytic properties and a maximum enzyme load of 770 U g⁻¹. Lipase CLEA exhibited enhanced pH stability in acidic and alkaline media and increased temperature resistance as compared to free lipase. Both free and CLEA lipases were used to synthesize RL in different solvent systems. After 12 h, from initiation of the esterification, the degree of esterification (molar conversion yield) reached 46% and 71% in the batch mode. ¹H and ¹³C nuclear magnetic resonance (NMR) and high-performance liquid chromatographic (HPLC) analysis confirm RL production by CLEA lipase. The CLEA showed greater confrontation to enzyme-mediated bioprocess approach as compared to its soluble counterpart and exhibited excellent RL production and catalytic activity even after its tenth successive reuse.     

Comparison of soybean peroxidase with laccase in the removal of phenol from synthetic and refinery wastewater samples

BACKGROUND: Several studies have demonstrated the feasibility of treating aqueous phenols and aromatic amines with oxidoreductases in synthetic wastewater samples. However, little work has been done on the effectiveness of enzymatic treatment on real wastewater. Here a comparison was made of the oxidative coupling of phenol catalyzed by laccase or soybean peroxidase (SBP) using synthetic and refinery wastewaters. RESULTS: Optimization of pH, enzyme concentration, effect of polyethylene glycol (PEG) addition, and reducing anions were examined for a 3 h reaction time. Laccase had an optimum pH of 5.6–6.0, while SBP had a broad optimum from 6.0 to 8.0. In synthetic samples in tap water to achieve ≥ 95% removal of 1.0 mmol L^?1 phenol in 3 h required 0.12 and 1.5 U mL^?1 of catalytic activity of laccase and SBP, respectively. In refinery samples comparable removals required 1.2‐ to 1.8‐fold more enzyme than in synthetic tap water samples. Added PEG allowed for a small reduction in the SBP concentration for synthetic wastewater but was ineffective with either enzyme in treating refinery samples. Reducing ions increased the demand for oxidant but, with the exception of cyanide, phenol removal still occurred. CONCLUSION: Both laccase and SBP were effective in removing phenol from aqueous refinery samples, albeit at slightly higher concentrations than required for the corresponding synthetic samples. Copyright © 2008 Society of Chemical Industry     

Use of solvent mixtures in supercritical antisolvent process to modify precipitates morphology: Cellulose acetate microparticles

In the supercritical antisolvent precipitation (SAS), the jet fluid dynamics is characterized by two-phase mixing at subcritical conditions, and by one-phase mixing at completely developed supercritical conditions. The amplitude of the pressure range, in which binary systems organic solvent/scCO₂ exhibit the transition between two-phase to one-phase mixing, depends on the organic solvent that is in contact with supercritical carbon dioxide (scCO₂) and conditions the morphology of the microparticles produced by SAS. When this pressure range is wide, as in the case of dimethylsulfoxide (DMSO), solutes solubilized in the organic solvent can be precipitated as microparticles by atomization, droplets formation and drying; when this pressure range is narrow, as for acetone, gas mixing prevails and only nanoparticles are generally observed. Therefore, generally speaking, solutes that are soluble only in solvents exhibiting gas mixing in scCO₂, do not exhibit microparticles morphology and this fact is a limitation for several industrial applications.In this work, a model compound, cellulose acetate (CA), that is slightly soluble in DMSO and freely soluble in acetone, was processed by SAS using mixtures of the two solvents that exhibit intermediate behaviors between the two pure solvents, to extend two phase mixing and produce CA microparticles. Using different DMSO/acetone mixture percentages, the effects of the polymer concentration in the liquid solution and of the pressure were studied. A mixture of DMSO/Acetone 50/50 (v/v), at a pressure of 85 bar and a concentration of the liquid solution equal to 40 mg/mL, efficiently produced non-coalescing CA microparticles with a mean diameter of 0.42 μm and a standard deviation of about 0.15 μm, demonstrating that this SAS strategy can be successful.     

Lipase PS-catalyzed transesterification of citronellyl butyrate and geranyl caproate: Effect of reaction parameters

Pseudomonas sp. lipase PS was immobilized by adsorption and tested for its ability to catalyze the synthesis of citronellyl butyrate and geranyl caproate by transesterification in n-hexane. The reaction parameters investigated were: enzyme load, effect of substrate concentration, added water, temperature, time course, organic solvent, pH memory, and enzyme reuse. Yields as high as 96 and 99% were obtained for citronellyl butyrate and geranyl caproate, respectively, with 300 units (approx. 15% w/w of reactants) of lipase PS. Increasing amounts of terpene alcohol inhibited lipase activity, while excess acyl donor (triacylglycerol) concentration enhanced ester production. Optimal yields were obtained at temperatures from 30–50°C after 24-h incubation time. Yields of 90 and 99% were obtained for citronellyl and geranyl esters, respectively, with 2% added water. Solvents with log P values ≥ 2.5 showed the highest conversion yields. pH 7 and 6–8 seemed to be ideal for citronellyl butyrate and geraniol caproate, respectively. The lipase remained active after reusing 12 times.     

混合溶剂提取催吐萝芙木水溶性生物碱的工艺研究

用混合溶剂提取法研究催吐萝芙木水溶性生物碱的浸提工艺。考察了不同混合溶剂对水溶性生物碱提取率的影响,确定了丙酮-水（v：v=10：1）混合溶剂为适宜溶媒,并通过正交实验,确立了该溶媒提取催吐萝芙木水溶性生物碱的优化条件：8倍量的混合溶剂,提取时间10h,温度40℃,pH=10。在优化后的条件下,采用该混合溶剂对水溶性生物碱提取率为3．22％,是采用乙醇的1．35倍,蒸馏水的1．69倍。     

Development of wood-products adhesives from mangrove bark

Mangrove bark was extracted using either water, ethanol, acetone, or various binary mixtures of these, as solvent. With plywood panels as substrates, joints prepared with adhesives derived from these extracts were tested for strength properties. In terms of the quantity of extracts from mangrove bark, water was the most effective solvent, followed sequentially by ethanol and acetone. However, in terms of the tannin content of these extracts, the order of extraction effectiveness of these solvents was reversed. The use of solvent mixtures in tannin extraction did not produce an observable synergistic effect. The quantity of extract and its tannin content were dictated primarily by the predominant solvent in the binary mixture. The reactivity of mangrove bark extracts is influenced significantly by formaldehyde concentration, cure temperature, type, and pH of the adhesive medium. Adhesive joints of significant bond strength were obtained from mangrove tannin adhesives. Prolonged cure periods, particularly at elevated temperatures, have deleterious effects on the strength properties of these adhesive joints.     

Optimization of L-Asparaginase Immobilization onto Calcium Alginate Beads

In this study, anti-leukemic enzyme L-asparaginase (E.C.3.5.1.1) from Escherichia coli ATCC 11303 was modified by the microencapsulation technique onto calcium alginate beads. Using response surface methodology (RSM), a three-level full factorial design, the values of concentration of sodium alginate, concentration of calcium chloride, and enzyme loading were investigated to obtain the highest residual L-asparaginase (L-ASNase) activity % (immobilized enzyme activity/free enzyme activity). The effects of the studied factors on immobilization were evaluated The predicted values by the model were close to the experimental values, indicating suitability of the model. The results presented that an increase in sodium alginate concentration increased the percent of residual activity of L-ASNase at any given calcium chloride concentration and the moderate amount of enzyme loading increased the percent residual activity. The optimal immobilization conditions were as follows: sodium alginate 1.98% (w/v), calcium chloride concentration 3.70% (w/v), and enzyme load 46.91% (v/v). The highest residual L-ASNase activity % obtained was 34.49%.     

Studies on preparation and emulsifying properties of guar galactomannan ester of palmitic acid

Various degrees of palmitoylated guar galactomannan (PGGM) are prepared by a heterogeneous method. Both differential thermal analysis and X‐ray diffraction spectra show substituted palmitoyl chains played a role in the new crystallization and the molecular aggregated structure of GGM was changed. Furthermore, the emulsifying properties of both water‐soluble and oil‐soluble PGGM were investigated in detail. It is demonstrated that PGGM has good emulsifying activity, even in high salt concentration or in acidic pH. In addition, the emulsifying properties of water‐soluble PGGM–protein mixture was also studied systematically. It is proven that water‐soluble PGGM is a kind of good oil in water (o/w) emulsion stabilizer. With water‐soluble PGGM–casein mixture (0.1 wt %) as emulsifier, clove oil–water (v : v, 1 : 9) emulsion had good stability within 4 weeks at room temperature, and the breakage of emulsion was not brought about. © 1999 John Wiley & Sons, Inc. J Appl Polym Sci 72: 639–645, 1999     

Production of chitinolytic enzymes by Serratia marcescens QMB1466 using various chitinous substrates

The chitinolytic activity of submerged cultures of Serratia marcescens QMB1466 in media containing four forms of chitin as the main substrate was investigated via a full factorial design experiment with pH, temperature and substrate concentration as the main parameters. At the optimum conditions (pH 7.0, 32.5 °C and 1.0% (w/v) substrate), bioprocessed chitin (BP), isolated by lactic acid fermentation of prawn shell (Nephrops sp), induced a higher level of enzyme activity than untreated prawn shell and colloidal chitin but not that of a chemically isolated chitin (CP). The optimal conditions of pH and temperature were then applied in a bench‐top bioreactor and the chitinolytic activity monitored temporally under the influence of higher concentrations of BP and CP. Increasing the concentration of substrate in the bioreactor (>1.0% w/v) was found to inhibit the enzyme activity of the bacteria. The enzyme mixtures in selected 120‐h culture supernatants were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and the main proteins characterised by molecular weight. The electrophoretic patterns obtained from cultures from different experiments and by the different chitin substrates showed marked similarity and the main proteins isolated were largely homologous to well‐documented chitinases found in the literature. BP chitin was found to be an efficient elicitor of chitinolytic activity from this bacterium and hence is a suitable substrate to employ in an integrated biotechnological process, whereby several commercially applicable products can be obtained from a waste product of the fishing industry. Copyright © 2004 Society of Chemical Industry