Immobilization of Paenibacillus macerans NRRL B-3186 cyclodextrin glucosyltransferase and properties of the immobilized enzyme

Cyclodextrin glucosyltransferase from Paenibacillus macerans NRRL B-3186 was immobilized on aminated polyvinylchloride (PVC) by covalent binding with a bifunctional agent (glutaraldehyde). The immobilized activity was affected by the length of the hydrocarbon chain attached to the PVC matrix, the amount of the protein loaded on the PVC carrier, and glutaraldehyde concentration. The activity of the immobilized enzyme was 121 units/gram carrier, the specific activity calculated on bound protein basis was 48% of the soluble enzyme. Compared to the free enzyme, the immobilized form exhibited: a higher optimal reaction temperature and energy of activation, a higher K_m (Michaelis constant) and lower V_max (maximal reaction rate), improved thermal stability and resistance to chemical denaturation. The operational stability was evaluated in repeated batch process and the immobilized enzyme retained about 85% of the initial catalytic activity after being used for 14 cycles.     

Immobilization of pancreatic lipase on chitin and chitosan

In this study, porcine pancreatic lipase (EC 3.1.1.3) was immobilized on chitin and chitosan by adsorption and subsequent crosslinking with glutaraldehyde, which was added before (conjugation) or after (crosslinking) washing unbound proteins. Conjugation proved to be the better method for both supports. The properties of free and immobilized enzymes were also investigated and compared. The results showed that the pH optimum was shifted from 8.5 to 9.0 for both the immobilized enzymes. Also, the optimum temperature was shifted from 30 to 40 degrees C for chitin-enzyme and to 45 degrees C for chitosan-enzyme conjugates. The immobilization efficiency is low, but the immobilized enzymes have good reusability and stability (storage and operational). Besides these properties, the immobilized lipases were also suitable for catalyzing esterification reactions of fatty acids and fatty alcohols, both with a medium chain length. According to our results, esterification activities of immobilized lipases were two- and four-fold higher for chitosan- and chitin-enzyme, than for the free enzyme, respectively. The immobilization procedure shows a great potential for commercial applications of the immobilized lipase, a relatively low cost commercial enzyme.     

固定化脂肪酶合成二元酸酯

The syntheses of dicarboxylic esters by immobilized lipase from Candida sp. -1619 were investigated. The reaction system was composed of 1 mmol dicarboxylic acid, 2 mmol alcohol, 3 mL hexane and 15 mg celite-adsorbed im mobilized lipase(300 u), in a closed 100 mL Erlenmeyer flask, shaken at 40°C for 5h. Sebacic acid was the best substrate among nine dicarboxylic acids selected. Among the 18 saturated fatty n-alcohols, the alcohols with carbon chain length rangin from C4～C18 had good reactivity. The primary alcohols had much better reactivities than corresponding secondary alcohols and multihydroxy-alcohols. Tertiary alcohols showed no reactivity. Hydrocarbons, benzene, toluene, xylene and te trachloride were favorite reactants among 15 organic solvents selected, in none-solvent stationary system, (5 mmol sebacic acid, 10 mmol dndecanol, 150 mg immobilized lipase(3000 u))reacted without plug for 3.5h, the optimum temperature was 60°C. The conversion degree was over 92% when reaction carried out at 50～90°C for 17h. The suitable reaction pH ranged from 6～8. The reactant was developed on GF254 plate(hexane ethyl ether acetic acid = 30201 ( V V V).There were three spots with different Rf value at 0.96, 0.55 and 0 corresponding to product, oleyl alcohol and sebacic acids, respectively.     

Immobilization of S-methyltransferase

S-methyltransferase was solubilized from pig liver microsomes by treatment with N-dodecyl-N,N-dimethyl-3-ammonio-1-sulfonate (Zwittergent). The soluble enzyme was immobilized by covalent binding to agarose and by copolymerization with acrylamide. The specific activity for the agarose-bound enzyme towards the substrate ethane thiol was 0.87 nmol/min/mg and for the acrylamide-bound enzyme 0.55 nmol/min/mg. The specific activity of the soluble enzyme was found to vary with increasing chain length of the substrate molecules from 0.5 nmol/min/mg for methane thiol (C1) to 6.3 nmol/min/mg for n-heptane thiol (C7). After binding of the enzyme to agarose beads, the increase in specific activity towards substrates with increasing chain length was no longer detectable. Instead, a relatively constant specific activity of 1.1 nmol/min/mg was observed for the whole range of substrates tested from C1 to C7. The stability of the agarose immobilized enzyme at -20 degrees C is twice as good as the soluble enzyme. The acrylamide immobilized enzyme is less stable than the soluble enzyme.     

漆酶在磁性壳聚糖微球上的固定及其酶学性质研究

以磁性壳聚糖微球为载体,戊二醛为交联剂,共价结合制备固定化漆酶。探讨了漆酶固定化的影响因素,并对固定化漆酶的性质进行了研究。确定漆酶固定化适宜条件为：50 mg磁性壳聚糖微球,加入10mL 0.8mg/mL 漆酶磷酸盐缓冲液（0.1mol/L,pH 7.0）,在4℃固定2h。固定化酶最适pH为3.0, 最适温度分别为10℃和55℃,均比游离酶降低5℃。在pH 3.0,温度37℃时,固定化酶对ABTS的表观米氏常数为171.1μmol/L。与游离酶相比,该固定化漆酶热稳定性明显提高,并具有良好的操作和存储稳定性。     

Reactive properties of the organic solvent-soluble lipase

In a previous report, the organic solvent-soluble lipase was prepared using a synthetic detergent, didodecyl glucosyl glutamate, and it was estimated that 150 +/- 30 molecules of the detergent were attached to one lipase molecule based on gel permeation chromatography and chemical analysis. In this paper, the reactivity of the organic solvent-soluble lipase was compared with that of the native lipase to study the effect of the surrounding detergent on the thermostability and enzymatic reactivity. The activity of the organic solvent-soluble lipase was preserved in the organic solvents up to a temperature of 50 degrees C as in the case of the native lipase in buffer (pH 7.0). The influence of the chain length of fatty acids of the substrate triacylglycerols on the hydrolysis activities was studied. The organic solvent-soluble lipase hydrolyzed triacylglycerols with longer chains more rapidly than the native lipase. The presence of Ca2+ at 0.1 mM stimulated the activity of the native lipase, whereas Ca2+ at a high concentration inhibited it. On the other hand, even at a low concentration, Ca2+ inhibited the activity of the organic solvent-soluble lipase. These results suggest that the detergent attached to the lipase molecule affected the reactive properties.     

Production of lipase in a fermentor using a mutant strain of Corynebacterium species: its partial purification and immobilization

Extracellular Corynebacterium lipase was produced using a 2.5 L Chemap fermentor using 1300 ml fermentation medium at temperature 33 degrees C, agitator speed 50 rpm, aeration rate 1 VVM having KLa 16.21 hr(-1). Crude lipase was purified by salting out method followed by dialysis and immobilized using calcium alginate gel matrix followed by glutaraldehyde cross linking Purification process increased specific activity of enzyme from 2.76 to 114.7 IU/mg. Activity of immobilized enzyme was 107.31 IU/mg. Optimum temperature for purified and immobilized enzyme activity were 65 degrees and 50 degrees C respectively. Optimum pH was 8.0 in both the cases, Km and Vmax value for purified lipase were 111.1 micromol/min and 14.7% respectively. Ca2+ (5 mM) was found to be stimulator for enzyme activity. Immobilized lipase retained 68.18% of the original activity when stored for 40 days.     

Immobilization of trypsin on chitosan gels: use of different activation protocols and comparison with other supports

Trypsin was immobilized on chitosan gels coagulated with 0.1 or 1 M NaOH and activated with glutaraldehyde or glycidol. The derivatives were characterized by their recovered activity, thermal (40, 55 and 70 degrees C) and alkaline (pH 11) stabilities, amount of enzyme immobilized on gels for several enzyme loads (8-14 mg(protein)/g(Gel)) and compared to agarose derivatives. Enzyme loads higher than 14 mg(protein)/g(Gel) can be immobilized on glutaraldehyde derivatives, which showed 100% immobilization yield and, for loads up to 8 mg(protein)/g(Gel), 100% recovered activity. Activation with glycidol led to lower immobilization yields than the ones obtained with glutaraldehyde, 61% for agarose-glyoxyl (AgGly) with low grade of activation and 16% for the chitosan-glyoxyl (ChGly), but allowed obtaining the most stable derivative (ChGly), that was 660-fold more stable than the soluble enzyme at 55 and 70 degrees C-approximately threefold more stable than AgGly. The ChGly derivative presented also the highest stability during incubation at pH 11. Analyses of lysine residue contents in soluble and immobilized trypsin indicated formation of multipoint bonds between enzyme and support, for glyoxyl derivatives.     

Immobilized Pseudomonas cepacia lipase for biodiesel fuel production from soybean oil

Enzymatic transesterification of soybean oil with methanol and ethanol was studied. Of the nine lipases that were tested in the initial screening, lipase PS from Pseudomonas cepacia resulted in the highest yield of alkyl esters. Lipase from Pseudomonas cepacia was further investigated in immobilized form within a chemically inert, hydrophobic sol-gel support. The gel-entrapped lipase was prepared by polycondensation of hydrolyzed tetramethoxysilane and iso-butyltrimethoxysilane. Using the immobilized lipase PS, the effects of water and alcohol concentration, enzyme loading, enzyme thermal stability, and temperature in the transesterification reaction were investigated. The optimal conditions for processing 10 g of soybean oil were: 35 degrees C, 1:7.5 oil/methanol molar ratio, 0.5 g water and 475 mg lipase for the reactions with methanol, and 35 degrees C, 1:15.2 oil/ethanol molar ratio, 0.3 g water, 475 mg lipase for the reactions with ethanol. Subject to the optimal conditions, methyl and ethyl esters formation of 67 and 65 mol% in 1h of reaction were obtained for the immobilized enzyme reactions. Upon the reaction with the immobilized lipase, the triglycerides reached negligible levels after the first 30 min of the reaction and the immobilized lipase was consistently more active than the free enzyme. The immobilized lipase also proved to be stable and lost little activity when was subjected to repeated uses.     

酶法制备壳寡糖及其生物学功能

用正交试验方法考察温度、酶浓度、pH对蜗牛酶降解壳聚糖的影响,筛选蜗牛酶降解壳聚糖的最佳反应条件,采用SDS-PAGE方法分析降解产物,制备具有生物学功能的壳寡糖。用不同浓度的壳寡糖处理人肝癌HepG2细胞,观察细胞形态学变化,MTT法检测壳寡糖对其增殖的影响,琼脂糖凝胶电泳检测DNA变化,流式细胞术检测凋亡率(AR)。结果表明:蜗牛酶降解壳聚糖的产物主要是聚合度为4以上的寡糖,更多的接近壳六糖。最佳反应条件为pH 4.0、温度40℃、酶和底物质量比为4∶50;壳寡糖质量浓度在2~4 mg/mL时,对HepG2细胞增殖有抑制效应,细胞经壳寡糖处理48 h后,开始空泡化,DNA出现明显的凋亡条带,AR明显高于对照组。在最佳反应条件下蜗牛酶能较好地降解壳聚糖,制备的壳寡糖在一定浓度范围内能通过诱导HepG2细胞发生凋亡而抑制其增殖,其作用呈浓度依赖性。     

Optimization of lipase pretreatment prior to lipase immobilization to prevent loss of activity

In our previous work, a method of pretreating lipase was developed to prevent loss of its activity during covalent immobilization. In this study, Rhizopus oryzae lipase was pretreated before immobilization and then immobilized on a silica gel surface. The effects of the various materials and conditions used in the pretreatment stage on the activity of immobilized lipase were investigated. Immobilized lipase pretreated with 0.1% of soybean oil had better activity than those pretreated with other materials. The optimal temperature, agitation speed, and pretreating time for lipase pretreatment were determined to be 40 degrees C, 200 rpm, and 45 min, respectively. The activity of immobilized soybean oil pretreated lipase was 630 U/g matrix, which is 20 times higher than that of immobilized non-pretreated lipase. In addition, immobilized lipase activity was maintained at levels exceeding 90% of its original activity after 10 reuses.     

Design of a specific peptide tag that affords covalent and site-specific enzyme immobilization catalyzed by microbial transglutaminase

Transglutaminase-mediated site-specific and covalent immobilization of an enzyme to chemically modified agarose was explored. Using Escherichia coli alkaline phosphatase (AP) as a model, two designed specific peptide tags containing a reactive lysine (Lys) residue with different length Gly-Ser linkers for microbial transglutaminase (MTG) were genetically attached to N- or C-termini. For solid support, agarose gel beads were chemically modified with beta-casein to display reactive glutamine (Gln) residues on the support surface. Recombinant APs were enzymatically and covalently immobilized to casein-grafted agarose beads. Immobilization by MTG markedly depended on either the position or the length of the peptide tags incorporated to AP, suggesting steric constraint upon enzymatic immobilization. Enzymatically immobilized AP showed comparable catalytic turnover (k(cat)) to the soluble counterpart and comparable operational stability with chemically immobilized AP. These results indicate that attachment of a suitable specific peptide tag to the right position of a target protein is crucial for MTG-mediated formulation of highly active immobilized proteins.     

Immobilization in the presence of Triton X-100: modifications in activity and thermostability of <Emphasis Type="Italic">Geobacillus thermoleovorans</Emphasis> CCR11 lipase

A partially purified lipase produced by the thermophile Geobacillus thermoleovorans CCR11 was immobilized by adsorption on porous polypropylene (Accurel EP-100) in the presence and absence of 0.1% Triton X-100. Lipase production was induced in a 2.5% high oleic safflower oil medium and the enzyme was partially purified by diafiltration (co. 500,000 Da). Immobilization conditions were established at 25 °C, pH 6, and a protein concentration of 0.9 mg/mL in the presence and absence of 0.1% Triton X-100. Immobilization increased enzyme thermostability but there was no change in neither the optimum pH nor in pH resistance irrelevant to the presence of the detergent during immobilization. Immobilization with or without Triton X-100 allowed the reuse of the lipase preparation for 11 and 8 cycles, respectively. There was a significant difference between residual activity of immobilized and soluble enzyme after 36 days of storage at 4 °C (P < 0.05). With respect to chain length specificity, the immobilized lipase showed less activity over short chain esters than the soluble lipase. The immobilized lipase showed good resistance to desorption with phosphate buffer and NaCl; minor loses with detergents were observed (less than 50% with Triton X-100 and Tween-80), but activity was completely lost with SDS. Immobilization of G. thermoleovorans CCR11 lipase in porous polypropylene is a simple and easy method to obtain a biocatalyst with increased stability, improved performance, with the possibility for re-use, and therefore an interesting potential use in commercial conditions.     

Preparation and properties of immobilized papain and lipase.

Papain and lipase were immobilized on derivatized Sepharose 4-B. The activated agarose had a binding capacity of 1.2 micronmol amino groups/ml packed agarose or 17 mg proteins/g dry agarose. The immobilized enzyme preparations were tested for the effects of pH of assay, temperature of assay, and substrate concentrations. The effect of 6M urea on the activity of papain was also determined. Soluble forms of the enzymes were used for comparison. Immobilization of the enzymes resulted in slightly different pH and temperature optima for activities. For immobilized papain Km(app) was similar to the one observed with soluble papain. Immobilization of lipase, however, cause a decrease in Km values. The immobilized enzyme preparations were stable when stored at 4 degrees C and pH 7.5 for periods up to eight months. The soluble enzymes lost their activity within 96 hr under similar storage conditions. Immobilized papain did not lose any activity after treatment with 6M urea for 270 min, whereas soluble papain lost 81% of its activity after the urea treatment, indicating that the immobilization of papain imparted structural and conformational stability to this enzyme.     

Influence of mass transfer limitations on the enzymatic synthesis of β-lactam antibiotics catalyzed by penicillin G acylase immobilized on glioxil-agarose

Mass transfer effects were investigated for the synthesis of ampicillin and amoxicillin, at pH 6.5 and 25 degrees C, catalyzed by penicillin G acylase immobilized on agarose. The influence of external mass transfer was analysed using different stirring rates, ranging form 200 to 800 rpm. Above 400 rpm, the film resistance may be neglected. Intra-particle diffusion limitation was investigated using biocatalysts prepared with different enzyme loads and agarose with different mean pore diameters. When agarose with 6, 8 and 10% of crosslinking were used, for the same enzyme load, substrates and products concentration profiles presented no expressive differences, suggesting pore diameter is not important parameter. An increase on enzyme load showed that when more than 90 IU of enzyme activity were used per mL of support, the system was influenced by intra-particle mass transfer. A reactive-diffusive model was used to estimate effective diffusivities of substrates and products.     

Coupling of glycosaminoglycans to agarose beads (Sepharose 4B)

1. Heparin, heparan sulphate, chondroitin sulphate and dermatan sulphate were covalently attached to beads of agarose activated by cyanogen bromide. The bond is probably mediated by the amino group of a serine or peptide residue at the reducing end of the polysaccharide chain. 2. The uptake of glycosaminoglycan during the coupling procedure is about 0.9mg/ml of wet gel. However, direct analysis of washed and freeze-dried gels reveals that only about one-third of this amount is firmly attached to the gel. 3. The use of the gels for polysaccharidase analyses is exemplified by a hyaluronidase assay. Further applications, e.g. interaction studies and preparative purposes, are discussed.     

Lipolytic activity of suspended and membrane immobilized lipase originating from indigenous Burkholderia sp. C20

In this work, a simple, inexpensive, and efficient method of preparing immobilized lipase is presented. The lipase originating from a newly isolated indigenous strain Burkholderia sp. C20 was immobilized onto cellulose nitrate (CN) membrane via filtration. The CN-immobilized lipase was able to retain 60% of its original activity after repeated uses for nine times. The thermal stability of the lipase was also slightly improved after immobilization. The optimal reaction conditions of CN-lipase were pH 9.0 and 55 degrees C, which are similar to those for the suspended lipase. Both suspended and immobilized lipase could hydrolyze the six oil substrates examined, while immobilized lipase displayed less specificity over the oil substrates. Kinetic analysis shows that the dependence of lipolytic activity of both suspended and immobilized lipase on oil substrate concentration can be described by Michaelis-Menten model with good agreement. The estimated kinetic constants for suspended lipase (v(max)=243.9 U/mg, K(m)=0.024 mM) and immobilized lipase (v(max)=32.8 U/mg, K(m)=5.61 mM) were quite different. Employment of immobilization seemed to result in a decrease in v(max) and an increase in K(m), most likely due to the mass transfer resistance arising from formation of micelles during the lipase immobilization process.     

酶法合成糖及糖醇酯

以脂肪酸为酰基供体,糖和糖醇为酰基受体,利用吸附到涤棉布上的假丝酵母脂肪酶作催化剂,在含叔丁醇的系统中,研究了酯化反应条件。酯化最适温度和pH值分别为40℃～45℃和50～75。在酰基供体中,以亚油酸和油酸最好,C8到C22的饱和脂肪酸的酯化程度相仿。在23种糖和糖醇中,果糖、木糖、海藻糖、山梨糖、木糖醇、甘露醇以及异丙基葡萄糖和甲基葡萄糖比其它酰基受体的酯化率高。糖醇的酯化程度明显高于相应的糖。此外,酰基供体与受体的摩尔比大于2∶1时,有利于酯化。在由30mmol(085g)油酸,02mmol山梨醇(0036g),3mL叔丁醇和30mg固定化酯肪酶(600u)组成的反应系统中,40℃震荡反应48h,以等摩尔的底物计算,酯化程度达到90%以上。反应产物经薄层色谱鉴定为单酯和双酯。     

Factors affecting the composition of oligosaccharides produced in chitosan hydrolysis using immobilized chitosanases

The hydrolysis reaction of chitosan using immobilized chitosanases with regard to the composition of its products and the yield of the intermediate target products, pentamer and hexamer of chitosan oligosaccharides, was investigated. Chitosanase was immobilized onto agar or agarose gel particles by the multipoint attachment method. In batch experiments, surface enzyme density, support particle size, temperature, agitator speed, and initial substrate concentration significantly affected the composition of the oligosaccharides produced. It was believed that these factors all related to the reaction rate and mass transfer rate at the surface of the support materials immobilizing the enzymes. These effects were summarized as a correlation with Damk?hler number (Da), defined as the ratio of the maximum reaction rate to the maximum mass transfer rate. The result showed that the reaction conditions that give a low value of Da provide a high yield of pentamer and hexamer oligosaccharides.     

Purification and properties of a novel extra-cellular thermotolerant metallolipase of Bacillus coagulans MTCC-6375 isolate

A novel extra-cellular lipase from Bacillus coagulans MTCC-6375 was purified 76.4-fold by DEAE anion exchange and Octyl Sepharose chromatography. The purified enzyme was found to be electrophoretically pure by denaturing gel electrophoresis and possessed a molecular mass of approximately 103 kDa. The lipase was optimally active at 45 degrees C and retained approximately 50% of its original activity after 20 min of incubation at 55 degrees C. The enzyme was optimally active at pH 8.5. Mg2+, Cu2+, Ca2+, Hg2+, Al3+, and Fe3+ at 1mM enhanced hydrolytic activity of the lipase. Interestingly, Hg2+ ions resulted in a maximal increase in lipase activity but Zn2+ and Co2+ ions showed an antagonistic effect on this enzyme. EDTA at 150 mM concentration inhibited the activity of lipase but Hg2+ or Al3+ (10mM) restored most of the activity of EDTA-quenched lipase. Phenyl methyl sulfonyl fluoride (PMSF, 15 mM) decreased 98% of original activity of lipase. The lipase was more specific to p-nitrophenyl esters of 8 (pNPC) and 16 (pNPP) carbon chain length esters. The lipase had a Vmax and Km of 0.44 mmol mg(-1)min(-1) and 28 mM for hydrolysis of pNPP, and 0.7 mmol mg(-1)min(-1) and 32 mM for hydrolysis of pNPC, respectively.