Kinetics of tributyrin hydrolysis by lipase

The kinetics for the tributyrin hydrolysis using lipase (Pseudomonas fluorscenes CCRC-17015) were investigated in the liquid–liquid and liquid–solid–liquid reaction systems in a batch reactor. The lipase was covalently immobilized onto the surface of porous polymethylacrylamide (PMAA) crosslinking with N,N-methylene biacrylamide with a spacer of ethylenediamine actived by glutaraldehyde. The conditions such as tributyrin concentration, temperature, agitation, and pH value, were evaluated to achieve the optimum reaction conditions for both free lipase and immobilized lipase. The kinetic parameters in the reaction system were also obtained for two reaction systems. The turnover numbers calculated for free lipase and immobilized lipase were 29 and 5.7 s⁻¹, respectively. The parameters of k and k_m obtained using Lineweaver-Burk plot method were 26.2 mol/(mg min) and 1.35 mol/dm³ for free lipase, 5.2 mol/(mg min) and 0.2 mol/dm³ for immobilized lipase, respectively. The experimental results revealed good thermal stability, with greater stability at higher pH value for immobilized lipase in the liquid–solid–liquid reaction.     

Tuning of Lecitase features via solid-phase chemical modification: Effect of the immobilization protocol

Lecitase Ultra (a quimeric fosfolipase commercialized by Novozymes) has been immobilized via two different strategies: mild covalent attachment on cyanogen bromide agarose beads and interfacial activation on octyl-agarose beads. Both immobilized preparations have been submitted to different individual or cascade chemical modifications (amination, glutaraldehyde or 2,4,6-trinitrobenzensulfonic acid (TNBS) modification) in order to check the effect of these modifications on the catalytic features of the immobilized enzymes (including stability and substrate specificity under different conditions). The first point to be remarked is that the immobilization strongly affects the enzyme catalytic features: octyl-Lecitase was more active versus p-nitrophenylbutyrate but less active versus methyl phenylacetate than the covalent preparations. Moreover, the effects of the chemical modifications strongly depend on the immobilization strategy used. For example, using one immobilization protocol a modification improves activity, while for the other immobiled enzyme is even negative. Most of the modifications presented a positive effect on some enzyme properties under certain conditions, although in certain cases that modification presented a negative effect under other conditions. For example, glutaraldehyde modification of immobilized or modified and aminated enzyme permitted to improve enzyme stability of both immobilized enzymes at pH 7 and 9 (around a 10-fold), but only the aminated enzyme improved the enzyme stability at pH 5 by glutaraldehyde treatment. This occurred even though some intermolecular crosslinking could be detected via SDS-PAGE. Amination improved the stability of octyl-Lecitase, while it reduced the stability of the covalent preparation. Modification with TNBS only improved enzyme stability of the covalent preparation at pH 9 (by a 10-fold factor).     

环氧交联剂和氨基载体固定化海洋假丝酵母脂肪酶*

游离酶经过固定化后,稳定性和环境耐受性得到提高,在食品、医药、化工、环境和皮革等领域可以很好的提高酶的利用率并降低生产成本,具有极大的应用潜力。新型交联剂在固定化酶工艺的应用极大推进了固定化酶研究的深入。借助新型交联剂聚乙二醇二缩水甘油醚(PEGDGE),利用氨基载体LX-1000HA固定化海洋假丝酵母脂肪酶,结合单因素和正交试验优化得到交联及固定化条件为:交联温度30℃,交联2h,交联剂浓度0.75%,pH7.0,加酶量800U,载体量0.5g,固定化2h,固定化温度45℃。根据上述最佳固定化工艺,制备得到固定化酶LX-1000HA-PEGDGE-CRL在最适条件下测得酶活达到160.81U/g,约为此前制备的固定化酶LX-1000HA-GA-CRL(由LX-1000HA和戊二醛交联脂肪酶得到)和LX-1000EA-PEGDGE-CRL(由短链氨基载体LX-1000EA和PEGDGE交联脂肪酶得到)酶活的2倍,发现固定化酶LX-1000HA-PEGDGE-CRL的最适反应温度相比于游离酶提高15℃;在70℃的环境中3h后酶活仍存留70%;循环使用6次后残留65%左右的酶活;酸碱耐受性和储存稳定性也表现良好,4℃保存30天后剩余约70%的初始酶活。同时,将制备的固定化酶LX-1000HA-PEGDGE-CRL与游离酶、固定化酶LX-1000HA-GA-CRL、固定化酶LX-1000EA-PEGDGE-CRL进行了比较,发现固定化酶LX-1000HA-PEGDGE-CRL在温度耐受性和重复使用性等方面具有更好的使用效果。     

Enzyme stabilization by glutaraldehyde crosslinking of adsorbed proteins on aminated supports

The stabilization achieved by different immobilization protocols have been compared using three different enzymes (glutaryl acylase (GAC), D-aminoacid oxidase (DAAO), and glucose oxidase (GOX)): adsorption on aminated supports, treatment of this adsorbed enzymes with glutaraldehyde, and immobilization on glutaraldehyde pre-activated supports. In all cases, the treatment of adsorbed enzymes on amino-supports with glutaraldehyde yielded the higher stabilizations: in the case of GOX, a stabilization over 400-fold was achieved. After this treatment, the enzymes could no longer be desorbed from the supports using high ionic strength (suggesting the support-protein reaction). Modification of the enzymes immobilized on supports that did not offer the possibility of react with glutaraldehyde showed the same stability that the non modified preparations demonstrating that the mere chemical modification did not have effect on the enzyme stability. This simple strategy seems to permit very good results in terms of immobilization rate and stability, offering some advantages when compared to the immobilization on glutaraldehyde pre-activated supports.     

氨基化硅胶载体固定化纤维素酶的研究

用硅胶作载体,戊二醛作交联剂,制备了固定化的纤维素酶。对制备固定化纤维素酶的偶联剂浓度、pH、给酶量3个影响因素进行了研究,通过正交试验优化得出最佳的固定化条件:交联剂戊二醛浓度为1%,固定化pH值为5,固载量为每克载体100mg纤维素酶。     

Mechanical properties and water vapor transmission in some blends of cassava starch edible films

The continuous increase of consumer interest in quality, convenience and food quality has encouraged further research into edible films and coatings from natural polymers, such as polysaccharides. Ecoefficient products are the new generation of biobased products prepared with sustainable materials, that agree with ecological and economic requirements including environmentally acceptable disposal of post-user waste. The numerous potential applications of natural polymers such as polysaccharides stimulated the study with edible films based on cassava starch. Blends of glycerol (GLY) and polyethylene glycol (PEG) as plasticizers, and glutaraldehyde (GLU) as crosslinking agent were prepared in order to determine the mechanical properties and water vapor transmission of those films. A response surface methodology was applied on the results to identify the blend with the best mechanical properties and lowest water vapor transmission. The crosslinking effect of glutaraldehyde in the films can be observed. The plasticizing action of polyethylene glycol was restrained by more than 0.5 g of glutataraldehyde. The use of glycerol was less evident for this property even after 284 h of contact time with water vapor.     

L-DOPA production from tyrosinase immobilized on nylon 6,6

The production of L-DOPA immobilized on chemically modified nylon 6,6 membranes was studied in a batch reactor. Tyrosinase was immobilized on nylon using glutaraldehyde as a crosslinking agent. The effects of membrane pore size and glutaraldehyde concentration upon enzyme uptake and L-DOPA production were investigated. Enzyme uptake was unaffected by glutaraldehyde concentration; approximately 70% uptake was observed when 25% w/v (group 1), 5% (group 2), and 3% (group 3) glutaraldehyde were used, indicating that glutaraldehyde was in excess. Similarly, uptake was the same for membranes with 0.20 and 10 mum pore sizes.Membranes produced using different levels of glutaraldehyde exhibited dramatically different capacities for L-DOPA production, despite the fact that enzyme uptake was equivalent. Membranes from groups 2 and 3 (5% and 3% glutaraldehyde) produced L-DOPA at a rate of 1.70 mg L(-1) h(-1) over 170 h in a 500-mL batch reactor. However, no free L-DOPA was detected when group 1 membranes were used. Experimental evidence suggests that L-DOPA was produced, but remained bound to these membranes via excess glutaraldehyde left over from the immobilization process. Membrane pore size also effected L-DOPA production; less production was observed when 10-mum membranes were used, despite equivalent enzyme uptake. The observed difference in production may be due to differences in the pore density on the two types of membranes which could affect the access of the substrate to the immobilized enzyme.The results of these studies indicate that tyrosinase can be effectively immobilized on nylon 6,6. L-DOPA production was optimal when 0.20-mum-pore-size membranes were activated with 3-5% glutaraldehyde. Stability studies indicated a 20% reduction in activity over 14 days when the immobilized enzyme was used under turnover conditions. (c) 1996 John Wiley & Sons, Inc.     

Hydrolysis of milk lactose by immobilized beta-galactosidase-hen egg white powder

Immobilized beta-galactosidase was obtained by crosslinking the enzyme with hen egg white using 2% glutaraldehyde. The gel obtained could be lyophilized to give a dry enzyme powder. The pH optimum of both the soluble and immobilized enzyme was found to be 6.8. The immobilized enzyme showed a higher K(m) for the substrates. The extent of enzyme inhibition by galactose was reduced upon immobilization. The stability towards inactivation by heat, urea, gamma irradiation, and protease treatment were enhanced. The bound enzyme as tested in a batch reactor could be used repeatedly for the hydrolysis of milk lactose. The possible application of this system for small-scale domestic use has been suggested.     

Production of L-lysine by immobilized trypsin. Study of DL-lysine methyl ester resolution

The possibility of producing L-lysine from chemically synthesized DL-lysine has been investigated. Optical resolution of racemic DK-lysine may be achieved by using the stereospecific esterasic activity of trypsin on DL-lysine methyl ester, which gives L-lysine and unchanged D-lysine methyl ester. SL-lysine methyl ester spontaneous hydrolysis may be neglected when operating at pH 5.5 and 30 degrees C. Effect of pH and substrate concentration on hydrolysis rate has been investigated when using as a catalyst either soluble or immobilized trypsin. For this purpose, trypsin was coupled onto an amine porous silica, Spherosil, activated with glutaraldehyde. The optimal pH is 5.8 for soluble trypsin and 6.0 for immobilized trypsin. It was yet possible to lower the parent optimal pH of immobilized trypsin, and thus increase its activity at 5.5, by co-grafting onto Spherosil an aminosilane, for enzyme coupling via glutaraldehyde activation and a positively charged diethyl amino ethyl (DEAE) silane, for decreasing the pH of trypsin microenvironment.     

金属框架结构材料MOF-199对漆酶的固定化及其性质

以金属框架结构材料MOF-199为载体对漆酶进行固定化,并对固定化酶的性质进行初步研究。首先,以3-氨基丙基三乙氧基硅烷对载体MOF-199进行表面氨基化修饰,再用戊二醛对载体进行活化,最后对漆酶进行固定化。固定化条件优化结果表明:在漆酶质量浓度0.3 g/L,戊二醛用量1%(体积分数),pH 4.8下固定7 h,制得固定化酶活性最高。对固定化酶的研究发现:最适反应温度为40℃,最适pH为5.2,在连续操作7次后,固定化酶的活力仍能保持在51%。固定化漆酶热稳定性,pH耐受性,贮存稳定性均明显高于游离漆酶。     

Cell-Bound Lipase and Esterase of Brevibacterium linens

The activities of glycerol ester hydrolase, lipase (EC 3.1.1.3) and carboxylesterase, and esterase (EC 3.1.1.1) were determined for whole cell preparations of Brevibacterium linens by using the pH-stat assay. The culture growth liquors were inactive against the three substrates, tributyrin emulsion, triacetin, and methyl butyrate. Cells washed in water had less activity than cells washed in 5% NaCl; the ratio of activities was close to 1:2 for all strains using tributyrin emulsion as the substrate. For the esterase substrates, this relationship varied widely and was strain dependent. The ability to hydrolyze the two esterase substrates varied independently of the level of lipase activity.     

固定化黑曲霉β-葡萄糖苷酶制备染料木素活性苷元的研究

比较了以海藻酸钠为载体,用胶囊法、包埋-交联法、交联-包埋法三种不同方法固定化黑曲霉β-葡萄糖苷酶的效果,并研究了最佳固定化方法的固定化条件和固定化酶的部分性质。结果表明,交联-包埋法即β-葡萄糖苷酶与0.20%戊二醛交联后再用2.0%海藻酸钠包埋的固定化方法中酶结合效率和酶活力回收率最高。海藻酸钠浓度和戊二醛浓度对酶结合效率影响较大,戊二醛浓度和包埋颗粒直径大小对酶活力回收率影响显著。与游离酶相比,制备的固定化酶最适温度、最适pH值和Km值分别由50℃、4.5和2.57μg/mL下降到40℃、4.0和2.02μg/mL。固定化酶具有更强的耐酸性和稳定性。该固定化酶用于大豆异黄酮活性苷元染料木素的合成,重复使用6次后,固定化酶的活力仍保持84.94%,染料木苷转化率为56.04%。     

Surface immobilization and entrapping of enzymes on glutaraldehyde crosslinked gelatin particles

A mild and reproducible method has been developed for the surface-immobilization of enzymes on glutaraldehyde crosslinked gelatin beads. In this method glutaraldehyde is used in a dual capacity, as crosslinking agent and as the enzyme coupling agent. Glucoamylase (exo-α-1,4-d-glucosidase, EC 3.2.1.3), β-d-fructofuranosidase (invertase, EC 3.2.1.26) and β-d-glucoside (cellobiase, β-d-glucoside glucohydrolase, EC 3.2.1.21) have been successfully immobilized by this method, on the surface of the crosslinked gelatin particles. The method can be combined with the existing technology for the production of gelatin-entrapped enzymes. Thus, dual immobilized enzyme conjugates of glucoamylase and invertase have been prepared using this method, by entrapment of one enzyme in, and surface-binding of the other to, the gelatin matrix. The coupling of glucoamylase onto cross-linked gelatin particles by precipitation with poly(hexamethylenebiguanide hydrochloride) was also tested.     

Immobilization and kinetics of catalase onto magnesium silicate

Bovine liver catalase was immobilized covalently with glutaraldehyde, or glutaraldehyde+3-aminopropionic acid as a spacer, onto magnesium silicate. The coupling time was determined as 2 h for immobilization. The pH and temperature optima as well as the changes in the kinetics (K_m, V_max, E_a) of the immobilized catalase was observed and discussed. Immobilized catalase preparations showed higher storage stabilities than free catalase. The half-life of free catalase, catalase immobilized via glutaraldehyde and catalase immobilized via glutaraldehyde+spacer were calculated as 2, 55 and 10 days at room temperature and 4, 85 and 107 days at 5 °C, respectively. The operational stability of the catalase immobilized via glutaraldehyde was higher than the catalase immobilized via glutaraldehyde+spacer. The remaining activity of the catalase immobilized via glutaraldehyde was about 90% and that of the catalase immobilized via glutaraldeyde+spacer was about 30% after 20 cycles of batch operation.     

Urease immobilization on macroporous silicas

Urease was immobilized on macroporous silicas using gamma-aminopropyl triethoxysilane and glutaraldehyde. The amount of protein on the surface, the structure of pores of the support and the purity of the initial enzyme were varied, the enzymic activity of the immobilized preparations being controlled. After the immobilization of sufficiently large quantities of the enzyme (about 3 mg protein per m2 support) about 35% of the specific activity was retained. The maximum activity per unit weight of the support was observed for silicagels and silochromes with the mean diameter of pores 70-90 nm and the specific surface area about 70 m2/g. The use of purified urease produced highly active preparations of the immobilized enzyme (17,000 U per g dry support). Freeze-drying of the immobilized enzyme in the presence of sorbitol yielded dry preparations retaining their activity.     

Hydrolysis of particulate tributyrin in a fluidized lipase reactor.

Pancreatic lipase has been immobilized onto stainless steel beads by adsorption followed by crosslinking, and onto polyacrylamide by covalent bonding. The activities of the two types of immobilized enzyme toward the particulate substrate, tributyrin emulsion droplets, were determined experimentally, and rate constants based on Michaelis-Menten kinetics were calculated. The activity of the stainless steel-lipase was determined for various flow conditions and for various support sizes by the use of a differential fluidized bed recycle reactor. The rate constants calculated indicate that the experimental reaction rate is free from mass transfer influences, since the observed Michaelis constant does not vary with the fluidization velocity or with the support particle size. In addition, the Michaelis constant of the stainless steel-lipase was found to be equal to that of the free enzyme, suggesting that adsorption and subsequent crosslinking does not alter the enzyme-substrate affinity. The emulsion substrate mass transfer rates, calculated from the filtration theory, indicate that each substrate particle which contact the immobilized enzyme is hydrolyzed to a significant extent. The experimentally determined kinetic rate constants may be used directly to predict the size of integral fluidized bed reactors.     

Polyethylene beads as supports for enzyme immobilization

Summary Acid oxidation of polyethylene beads generated surface carboxylic groups which were reacted with excess ethylenediamine via carbodiimide promoted reactions. Glucose oxidase was covalently immobilized on the amine substituted beads using either glutaraldehyde, triazine trichloride, or dimethylsuberimidate as crosslinkers. The kinetic properties, pH-profile and the stability of the immobilized enzymes were reported.     

Immobilization of lactase from Kluyveromyces lactis greatly reduces the inhibition promoted by glucose. full hydrolysis of lactose in milk

The kinetic constants (Km, Vmax, and inhibition constants for the different products) of soluble and different immobilized preparations of beta-galactosidase from Kluyveromyces lactis were determined. For the soluble enzyme, the Km was 3.6 mM, while the competitive inhibition constant by galactose was 45 mM and the noncompetitive one by glucose was 758 mM. The immobilized preparations conserved similar values of Km and competitive inhibition, but in some instances much higher values for the noncompetitive inhibition constants were obtained. Thus, when glyoxyl or glutaraldehyde supports were used to immobilize the enzyme, the noncompetitive inhibition was greatly reduced (Ki approximately 15,000 and >40,000 mM, respectively), whereas when using sugar chains to immobilize the enzyme the behavior had an effect very similar to the soluble enzyme. These results presented a great practical relevance. While using the soluble enzyme or the enzyme immobilized via the sugar chain as biocatalysts in the hydrolysis of lactose in milk only around 90% of the substrate was hydrolyzed, by using of these the enzyme immobilized via the glyoxyl or the glutaraldehyde groups, more than 99% of the lactose in milk was hydrolyzed.     

Immobilization of catalases from Bacillus SF on alumina for the treatment of textile bleaching effluents

A catalase preparation from a newly isolated Bacillus sp. was covalently immobilized on silanized alumina using glutaraldehyde as crosslinking agent. The effect of the coupling time of the enzyme-support reaction was determined in terms of protein recovery and immobilization yield and a certain balance point was found after which the activity recovery decreased. The activity profile of the immobilized catalase at high pH and temperature was investigated. The immobilized enzyme showed higher stabilities (214 h at pH 11, 30°C) at alkaline pH than the free enzyme (10 h at pH 11, 30°C). The immobilized catalase was inhibited by anionic stabilizers or surfactants added to the hydrogen peroxide substrate solution.     

Immobilization of Escherichia coli cells containing aspartase activity with kappa-carrageenan. Enzymic properties and application for L-aspartic acid production.

Whole cells of Escherichia coli having high aspartase (L-asparate ammonialyase, EC 4.3.1.1) activity were immobilized by entrapping into a kappa-carrageenan gel. The obtained immobilized cells were treated with glutaraldehyde or with glutaraldehyde and hexamethylenediamine. The enzymic properties of three immobilized cell preparations were investigated, and compared with those of the soluble aspartate. The optimum pH of the aspartase reaction was 9.0 for the three immobilized cell preparations and 9.5 for the soluble enzyme. The optimum temperature for three immobilized cell preparations was 5--10 degrees C higher than that for the soluble enzyme. The apparent Km values of immobilized cell preparations were about five times higher than that of the soluble enzyme. The heat stability of intact cells was increased by immobilization. The operational stability of the immobilized cell columns was higher at pH 8.5 than at optimum pH of the aspartase reaction. From the column effluents, L-aspartic acid was obtained in a good yield.