Transformation of 3,5-dimethoxy-4-hydroxy cinnamic acid and its derivatives using enzyme from white-rot fungus Trametes versicolor: Enzyme characteristics and its application

Transformation of 3,5-dimethoxy-4-hydroxy cinnamic acid (sinapic acid), sinapaldehyde, sinapine and sinapoyl in the model system containing an enzyme secreted by the fungus Trametes versicolor was investigated. The affinity of this enzyme was highest for sinapic acid followed by sinapaldehyde and sinapine. The optimum temperature and pH for these transformations were 50°C and pH 3·3, 50°C and pH 4·5, 60°C and pH 4·0 for sinapaldehyde, sinapine, and sinapic acid, respectively. The apparent heat of the enzyme-sinapic acid complex formation is −2557·6 J mol⁻¹. Higher concentrations of sinapine and sinapic acid caused enzyme inhibition. When canola meal was treated with this enzyme the phenolics content in this commodity was decreased by 90%.     

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     

脂肪酶降解壳聚糖的反应动力学研究

用还原糖法研究了脂肪酶降解壳聚糖过程中一系列反应条件包括温度、p H值、时间、酶浓度、底物浓度对降解速度的影响 ,比较合适的降解条件是 :最适宜温度 5 5°C,最适宜 p H值 5 .0 ,适当增大酶浓度和底物浓度能够加速壳聚糖的降解 ,而且脂肪酶降解壳聚糖的反应不遵循简单的一级反应动力学     

Zwitterionic poly(carboxybetaine) microgels for enzyme (chymotrypsin) covalent immobilization with extended stability and activity

There is emerging evidence that biocompatible zwitterionic materials can prevent nonspecific interactions within protein systems and increase protein stability. Here, a zwitterionic microgel was synthesized from poly (carboxybetaine methyl methacrylate) (pCB) using an inverse emulsion, free radical polymerization reaction technique. The microgel was loaded with a model enzyme, α-chymotrypsin (ChT), using a post-fabrication loading technique. A reaction scheme was developed and studied for covalent immobilization of ChT within the microgel. Confocal laser microscopy studies showed that immobilized ChT (i-ChT) was distributed within the hydrogel. The enzyme-immobilized microgels showed excellent reusability (72% of its initial activity after 10 uses) and could undergo several freezing/drying/rehydration cycles while retaining enzymatic activity. The i-ChT activity, half-life, and conformational stability were studied at varying pH and temperatures with results compared to free ChT in buffer. ChT immobilized within pCB hydrogel showed increased enzymatic stability as observed by a 13°C increase in the temperature at which i-ChT loses activity compared to free ChT. Furthermore, enzyme half-life increased up to seven-fold for the pCB immobilized ChT, and the increased stability resulted in higher activity at elevated pH. The i-ChT was most active at pH of 8.5 and was partially active up to the pH of 10.2.     

固定化L-天门冬酰胺酶的性质及其填充床反应器的研究

利用海藻酸钙包埋、戊二醛交联的方法对L-天门冬酰胺酶进行固定化。研究了固定化L-天门冬酰胺酶的最适pH、最适温度、米氏常数、半衰期等酶学性质,并考察了影响固定化酶柱式填充床反应器转化率的因素。结果表明:固定化酶最适pH值为8.5,最适温度为67°C,固定化酶的米氏常数Km增大,固定化酶半衰期随着温度的增加而逐渐减小;温度、反应柱内径、底物溶液浓度、流速等因素对填充床反应器转化率均有显著影响。     

Optimization of the enzymatic hydrolysis conditions of steam‐exploded wheat straw for maximum glucose and xylose recovery

BACKGROUND: The enzymatic hydrolysis of steam‐exploded wheat straw using commercial enzyme complexes has been studied. A cellulase enzyme complex (Accellerase 1500), along with specific xylanase complements (Accellerase‐XC and Accellerase‐XY) provided by Genencor, have been used to enhance glucose and xylose recovery. A systematic study with response surface methodology (RSM) was used to check the effect of the operating conditions: pH (4–5), temperature (50–60 °C) and enzyme/substrate ratio (0.1–0.5 mL g_cellulose^?1) on the enzymatic hydrolysis with Acellerase 1500 to maximize the sugar yield. Xylanases were used as complements to increase the release of xylose. RESULT: Statistical results from ANOVA analysis demonstrated that the enzymatic hydrolysis was clearly improved by temperature and enzyme/substrate ratio. The optimum conditions for higher glucose and xylose releases were obtained with the higher enzyme dosage ratio (0.5 mL g^?1_cellulose), 50 °C and pH 4. CONCLUSION: Model validation at optimum operating conditions showed good agreement between the experimental results and the predicted responses for a confidence level of 95%. The use of the xylanase complements, Accellerase‐XY (accessory xylanase enzyme complex) and Accellerase‐XC (accessory xylanase/cellulase enzyme complex), increases the conversion of hemicellulose. Accellerase‐XC supplementation was more effective, obtaining an increase in yields of glucose and xylose of 11.8% and 23.6%, respectively, using a dosage of 0.125 mL g^?1_cellulose. © 2012 Society of Chemical Industry     

Crystallization of alkaline protease as a means of purification process

A purification protocol of alkaline protease purification using crystallization was developed by investigating the effects of pH, temperature, initial enzyme concentration, salt (as crystal inducer) concentration, and the presence of impurity proteins. A commercial alkaline protease solution was used as a starting material and NaCl was used as a crystal inducing salt. The crude enzyme solution was first diafiltered against deionized water and then concentrated by ultrafiltration. To the enzyme concentrate appropriate amount of NaCl was added to induce the crystallization which was lasted for 24 hours, and the enzyme crystals formed were filtered and washed with deionized water before being resolubilized. Crystal habit was typical needle shape, and the reaction order of its formation was estimated to be 1.53. The crystallization was strongly influenced by initial enzyme concentration. Solubility of alkaline protease at 25°C was 24.8 mg/ml, which was about one half of that of 4°C. Enzyme recovery yield of the purification process including the crystallization step ranged 50 to 60 %. The crystallization step was shown to successfully exclude impurity proteins from their habits as evidenced by gel permeation chromatography. The optimum condition for the crystallization was: pH 9.0, 25°C temperature, ca. 53 mg/ml or higher enzyme concentration, and minimum 5 % (w/w) NaCl concentration. In summary, an enzyme purification protocol based on crystallization was established, which can be applied to obtain a higher-purity alkaline protease solution on a large scale.     

Study of simultaneous saccharification and fermentation for steam exploded wheat straw to ethanol

Although simultaneous saccharification and fermentation (SSF) has been investigated extensively, the optimum condition for SSF of wheat straw has not yet been determined. Dilute sulfuric acid impregnated and steam explosion pretreated wheat straw was used as a substrate for the production of ethanol by SSF through orthogonal experiment design in this study. Cellulase mixture (Celluclast 1.5 l and ?-glucosidase Novozym 188) were adopted in combination with the yeast Saccharomyces cerevisiae AS2.1. The effects of reaction temperature, substrate concentration, initial fermentation liquid pH value and enzyme loading were evaluated and the SSF conditions were optimized. The ranking, from high to low, of influential extent of the SSF affecting factors to ethanol concentration and yield was substrate concentration, enzyme loading, initial fermentation liquid pH value and reaction temperature, respectively. The optimal SSF conditions were: reaction temperature, 35°C; substrate concentration, 100 g·L^-1; initial fermentation liquid pH, 5.0; enzyme loading, 30 FPU·g^-1. Under these conditions, the ethanol concentration increased with reaction time, and after 72 h, ethanol was obtained in 65.8% yield with a concentration of 22.7 g·L^-1.     

Urease immobilized on chitosan membrane: Preparation and properties

Urease was covalently immobilized on glutaraldehyde-pretreated chitosan membranes. The optimum immobilization conditions were determined with respect to glutaraldehyde pretreatment of membranes and to reaction of glutaraldehyde-pretreated membranes with urease. The immobilized enzyme retained 94% of its original activity. The properties of free and immobilized urease were compared. The Michaelis constant was about five times higher for immobilized urease than for the free enzyme, while the maximum reaction rate was lower for the immobilized enzyme. The stability of urease at low pH values was improved by immobilization; temperature stability was also improved. The optimum temperature was determined to be 65°C for the free urease and 75°C for the immobilized form. The immobilized enzyme had good storage and operational stability and good reusability, properties that offer potential for practical application.     

重组真菌腈水解酶的发酵工艺条件及生物催化特性初探【需加急】

对产真菌腈水解酶的重组大肠杆菌的培养基种类、培养基成分、诱导剂种类和浓度、诱导条件、p H和温度进行了系统考察。摇瓶发酵优化结果显示:以甘油作为主要碳源,蛋白胨和酵母膏作为主要氮源,并添加微量元素的SOC培养基作为发酵培养基,最适接种量为0.5%;较优的诱导剂诱导条件为:采用0.5 mmol/L的IPTG诱导12 h,发酵p H=7.5,诱导温度25℃时产酶效果最佳。经过优化后,重组酶的酶活得到了显著提高,总酶活最高达到了3.84 U/m L,相比初始水平(0.84 U/m L)提高约4倍。5 L发酵罐的放大实验表明,产酶效果良好,总酶活和比酶活均与摇瓶水平基本持平。全细胞催化性质考察研究结果表明,该菌株所产腈水解酶催化反应的最适催化反应温度是45℃,最适反应p H约为7.2。     

The preparation of an immobilised glucose isomerase II. Immobilisation and properties of the immobilised enzyme

Glucose isomerase ex Lactobacillus brevis was successfully immobilised on microcrystalline cellulose, using the transition metal-link method. Immobilisation could be performed over a pH range of 5 to 9, and usually resulted in an apparent specific activity increase. The immobilised glucose isomerase generally displayed properties similar to those of the soluble enzyme, with the exception of the following differences:

• (i) a pH optimum at pH = 6, an acid shift of 0.5 units on immobilisation;
• (ii) an optimum reaction temperature at 50 °C, lower than that for the soluble enzyme;
• (iii) on incubation at 4 °C, a retention of 53% of the initial specific activity, when stored in 0.02 M, pH = 7, Tris buffer, after 8 weeks, compared with an apparent activation of the soluble enzyme after 10 and 19 weeks' storage.

Storage properties of the immobilised enzyme at 4 °C in Tris were apparently improved by the presence of Mn⁺⁺ and Co⁺⁺, although associated with some protein release. Storage at 4 °C in water alone, as opposed to Tris, resulted in a more rapid activity loss.     

聚乙烯醇脱氢酶的分离纯化及其酶学性质

采用(NH4)2SO4分级沉淀及Q-Sepharose HP柱层析两步纯化,首次从可高效降解聚乙烯醇(PVA)的混合菌系发酵产物中分离纯化获得了纯蛋白聚乙烯醇脱氢酶(PVADH),并对其酶学性质进行了研究. 结果表明,PVADH分子量为134.3 kDa,最适作用温度为35℃,最适作用pH值为7.5;PVADH以仲醇为底物时酶活性普遍高于以其他醇类为底物时,PVADH与PVA反应产物中检测到羰基化合物,证实PVADH对PVA有降解作用.     

Immobilization of Phospholipase C for the Production of Ceramide from Sphingomyelin Hydrolysis

The immobilization of Clostridium perfringens phospholipase C was studied for the first time and the catalytic properties of the immobilized enzyme were investigated for the hydrolysis of sphingomyelin to produce ceramide. Ceramide is of great commercial value in the cosmetic and pharmaceutical industries for use in, for example, hair and skin care products, owing to its major role in maintaining the water-retaining properties of the epidermis. The feasibility of enzymatic production of ceramide through hydrolysis of sphingomyelin has previously been proven. In order to improve the reusability of the enzyme, the present study focused on the immobilization of phospholipase C in the production of ceramide from sphingomyelin. By screening nine different carriers, we found that the enzyme immobilized on Lewatit had the highest catalytic activity towards sphingomyelin hydrolysis. Prewetting Lewatit with ethanol led to higher enzyme fixation on the carrier, but the activity of the enzyme was decreased. Increasing the initial enzyme concentration resulted in more enzyme adsorption on the carrier, where the specific activity was increased. Through optimization of the reaction using the immobilized enzyme, the optimal temperature was around 46 °C and the optimal water volume was 3.5%. The reaction had little dependence on pH. After seven recycles, immobilized enzyme retained around 70% of the initial activity. Immobilized enzyme was deactivated irregularly when stored at room temperature, but followed first-order deactivation when stored at 40 °C.     

Simultaneous saccharification and fermentation of wheat bran flour into ethanol using coculture of amylotic Aspergillus niger and thermotolerant Kluyveromyces marxianus

Studies on simultaneous saccharification and fermentation (SSF) of wheat bran flour, a grain milling residue as the substrate using coculture method were carried out with strains of starch digesting Aspergillus niger and nonstarch digesting and sugar fermenting Kluyveromyces marxianus in batch fermentation. Experiments based on central composite design (CCD) were conducted to maximize the glucose yield and to study the effects of substrate concentration, pH, temperature, and enzyme concentration on percentage conversion of wheat bran flour starch to glucose by treatment with fungal α-amylase and the above parameters were optimized using response surface methodology (RSM). The optimum values of substrate concentration, pH, temperature, and enzyme concentration were found to be 200 g/L, 5.5, 65°C and 7.5 IU, respectively, in the starch saccharification step. The effects of pH, temperature and substrate concentration on ethanol concentration, biomass and reducing sugar concentration were also investigated. The optimum temperature and pH were found to be 30°C and 5.5, respectively. The wheat bran flour solution equivalent to 6% (w/V) initial starch concentration gave the highest ethanol concentration of 23.1 g/L after 48 h of fermentation at optimum conditions of pH and temperature. The growth kinetics was modeled using Monod model and Logistic model and product formation kinetics using Leudeking-Piret model. Simultaneous saccharificiation and fermentation of liquefied wheat bran starch to bioethanol was studied using coculture of amylolytic fungus A. niger and nonamylolytic sugar fermenting K. marxianus.     

响应曲面法优化小麦秸秆纤维素酶水解条件

利用响应曲面试验设计方法(RSM),选择底物质量分数、酶投加量、温度、pH值及水解时间为试验因子,还原糖(RS)产率为响应值,考察小麦秸秆纤维素酶水解过程中各影响因子对还原糖产率的影响,对小麦秸秆纤维素酶水解条件进行优化。结果表明,所考察的5个影响因子对还原糖产率均具有显著影响(p<0.05)。所得回归方程R² 值为 0.946 9,p<0.05,变异系数(C_V)值为4.37%,足够精度值为26.396,说明模型高度显著,可以在设计范围内对响应值进行预测。模型预测最佳水解条件为底物质量分数8.0%,酶投加量为35 FPU/g(以秸杆质量计),温度50 ℃,pH值5.4,水解时间96 h。利用最优水解条件进行验证试验,所得还原糖产率为60.73%,水解液中葡萄糖和木糖质量浓度分别为31.84和 16.74 g/L。     

A kinetic model for enzymatic wheat starch saccharification

A kinetic model describing the enzymatic saccharification of wheat starch by a mixture of α‐amylase and amyloglucosidase has been developed. The model describes the influence of pH, glucose inhibition and starch and enzyme concentration. The results of experimental saccharification under different physical conditions, eg pH and temperature, were used to determine the parameters in the model. The dominant enzyme in the mixture was amyloglucosidase and the maximum rate of saccharification due to this enzyme was found to be optimal at pH 5, and increased Five‐Fold when the temperature was increased from 30 to 55 °C. Saccharification due to the action of amyloglucosidase was inhibited by the glucose produced and simulation showed that the maximum rate of saccharification decreased by 58% at a starch concentration of 140 g dm⁻³ compared with a starch concentration much less than 110 g dm⁻³ where the effect of glucose inhibition was negligible. © 2000 Society of Chemical Industry     

Kinetic study of the enantioselective hydrolysis of a meso‐diester using pig liver esterase

A kinetic study of the hydrolysis of the diester dimethyl cis‐cyclohex‐4‐ene‐1,2‐dicarboxylate, to the (1S,2R)‐monoester, catalysed by the enzyme Pig Liver Esterase (PLE) was performed. The effects of the most relevant parameters that influence the enzymatic conversion were studied, such as pH, temperature and concentration of substrate and reaction products. It was concluded that the pH at which the enzyme exhibits a maximum activity is pH 7. At 25 °C PLE presents a better long‐term stability and enantioselectivity than at higher temperatures, although the reaction rate is slower. The kinetic results obtained are well described by the Michaelis–Menten equation, although a slight deviation to this model was observed for low substrate concentrations. Methanol, a co‐product of the enzymatic hydrolysis, was found to act as a non‐competitive inhibitor of the reaction. The Michaelis–Menten parameters were determined and a comprehensive kinetic model, which already accounts for methanol inhibition, is presented. © 2000 Society of Chemical Industry     

Stabilization of penicillin V acylase from Streptomyces lavendulae by covalent immobilization

Penicillin V acylase from the actinomycete Streptomyces lavendulae ATCC 13664 has been immobilized to epoxy‐activated acrylic beads (Eupergit C®) by covalent binding. Further linkage of bovine serum albumin after enzyme immobilization was carried out in order to remove the remaining oxirane groups of the support. The obtained immobilized biocatalyst displayed double exponential deactivation kinetics at temperatures below 55 °C, while the native enzyme followed single exponential decay at the same temperatures. We concluded that soluble penicillin acylase was deactivated in one step, whereas the immobilized enzyme showed an enzymatic intermediate state which is highly thermostable. As a consequence of the immobilization process, the enzyme displayed a 10‐fold increase in its half‐life at 40 °C. At this temperature, the enzymatic intermediate state was progressively destabilized as the pH of the medium was increased. Thus, the optimum pH range for the immobilized enzyme preparation was established as being from 7.0 to 8.0. Higher pH values led to quicker enzyme deactivation. © 2001 Society of Chemical Industry     

A modelling study on hydrolysis of whey lactose and stability of β-galactosidase

In the present study, the effect of process conditions on whey lactose hydrolysis and enzyme inactivation were investigated. The experiments were carried out in 250 mL of 25 mM phosphate buffer solution by using β-galactosidase produced from Kluyveromyces marxianus lactis in a batch reactor system. The degree of lactose hydrolysis (%) and residual enzyme activity (%) against time were investigated versus lactose concentration, enzyme concentration, temperature and pH. The mathematical models were derived from the experimental data to show the effect of process conditions on lactose hydrolysis and residual enzyme activity (in the presence and absence of lactose). At the optimum process conditions obtained (50 g/L of lactose concentration, 1 mL/L of enzyme concentration, 37 °C of temperature and pH 6.5), 81% of lactose was hydrolyzed and enzyme lost its activity by 32%. The activation energy for hydrolysis reaction (E _A ) and the enzymatic inactivation energy (E _D ) were calculated as 52.7 kJ/mol and 96.7 kJ/mol. Mathematical models at various process conditions have been confirmed with the experimental results.     

Immobilization of Candida rugosa lipase on modified natural wool fibers

A method has been developed to immobilize lipase from Candida rugosa on modified natural wool fibers by means of graft copolymerization of poly ethylacrylate in presence of potassium persulphate and Mohr’s salt redox initiator. The activities of free and immobilized lipase have been studied. FTIR spectroscopy, scanning electron microscopy, and the Bradford method were used to characterize lipase immobilization. The efficiency of the immobilization was evaluated by examining the relative enzymatic activity of free enzyme before and after the immobilization of lipase. The results showed that the optimum temperature of immobilized lipase was 40 °C, which was identical to that of the free enzyme, and the immobilized lipase exhibited a higher relative activity than that of free lipase over 40 °C. The optimal pH for immobilized lipase was 8.0, which was higher than that of the free lipase (pH 7.5), and the immobilization resulted in stabilization of enzyme over a broader pH range. The kinetic constant value (km) of immobilized lipase was higher than that of the free lipase. However, the thermal and operational stabilities of immobilized lipase have been improved greatly.