Effect of pore diffusion limitation on dextrin hydrolysis by immobilized glucoamylase

Data reported here and previously indicate that when dextrin is hydrolyzed in the presence of immobilized glucoamylase, use of a larger average molecular weight substrate leads to lower overall rates of hydrolysis, while the maltose concentration during the bulk of the reaction and the maximum glucose concentration are lower than when the soluble form of the enzyme is employed under the same conditions. Computer simulation of the system demonstrated that all three observations were caused by pore diffusion limitation: the first by slow diffusion of substrate, the second by slow diffusion of intermediates, and the third by slow diffusion of glucose. Follow-up experiments with glucoamylase immobilized to particles of different sizes confirmed this finding, as results with the smallest beads were identical to those with soluble glucoamylase.     

Immobilization of glucoamylase by adsorption on carbon supports and its application for heterogeneous hydrolysis of dextrin

Glucoamylase (GA) was immobilized by adsorption on carbon support: on Sibunit, on bulk catalytic filamentous carbon (bulk CFC) and on activated carbon (AC). This was used to prepare heterogeneous biocatalysts for the hydrolysis of starch dextrin. The effect of the texture characteristics and chemical properties of the support surface on the enhancement of the thermal stability of the immobilized enzyme was studied, and the rates of the biocatalyst's thermal inactivation at 65-80 degrees C were determined. The thermal stability of glucoamylase immobilized on different carbon supports was found to increase by 2-3 orders of magnitude in comparison with the soluble enzyme, and decrease in the following order: GA on Sibunit>GA on bulk CFC>GA on AC. The presence of the substrate (dextrin) was found to have a significant stabilizing effect. The thermal stability of the immobilized enzyme was found to increase linearly when the concentration of dextrin was increased from 10 wt/vol % to 50 wt/vol %. The total stabilization effect for glucoamylase immobilized on Sibunit in concentrated dextrin solutions was about 10(5) in comparison with the enzyme in a buffer solution. The developed biocatalyst, 'Glucoamylase on Sibunit' was found to have high operational stability during the continuous hydrolysis of 30-35 wt/vol % dextrin at 60 degrees C, its inactivation half-time (t1/2) exceeding 350 h. To improve the starch saccharification productivity, an immersed vortex reactor (IVR) was designed and tested in the heterogeneous process with the biocatalyst 'Glucoamylase on Sibunit'. The dextrin hydrolysis rate, as well as the process productivity in the vortex reactor, was found to increase by a factor of 1.2-1.5 in comparison with the packed-bed reactor.     

Catalytic properties of glucoamylase immobilized on synthetic carbon material Sibunit

Glucoamylase (commercial preparation Glucavamorin) was immobilized by sorption on a carbon support Sibunit. Starch saccharification by the resulting biocatalyst (dextrin hydrolysis) was studied. Investigation of the effect of adsorptional immobilization on kinetic parameters of glucoamylase, including the rate constant of thermal inactivation, showed that immobilization of Glucavamorin on Sibunit resulted in a thousand-fold increase in glucoamylase stability in comparison with the dissolved enzyme. Presence of the substrate (dextrins) in the reaction mixture had a considerable stabilizing effect. Increase in dextrin concentration increases the thermostability of the immobilized enzyme. The overall factor of glucoamylase stabilization adsorbed on Sibunit with the presence of 53% dextrin solutions in comparison with the dissolved enzyme approximated 10⁵. The biocatalyst for starch saccharification made on the base of Subunit-adsorbed Glucavamorin had a high operational stability. Its half-inactivation time at 60°C exceeded 30 days.     

Catalytic properties of glucoamylase immobilized on the synthetic carbon material Sibunit

Glucoamylase (commercial preparation Glucavamorin) was immobilized by sorption on a carbon support Sibunit. Starch saccharification by the resulting biocatalyst (dextrin hydrolysis) was studied. Investigation of the effect of adsorptional immobilization on kinetic parameters of glucoamylase, including the rate constant of thermal inactivation, showed that immobilization of Glucavamorin on Sibunit resulted in a thousandfold increase in glucoamylase stability in comparison with the dissolved enzyme. Presence of the substrate (dextrins) in the reaction mixture had a considerable stabilizing effect. Increase in dextrin concentration increases the thermostability of the immobilized enzyme. The overall factor of glucoamylase stabilization adsorbed on Sibunit with the presence of 53% dextrin solutions in comparison with the dissolved enzyme approximated 10(5). The biocatalyst for starch saccharification made on the base of Subunit-adsorbed Glucavamorin had a high operational stability. Its half-inactivation time at 60 degrees C exceeded 30 days.     

A comparative study of immobilized amyloglucosidase in a packed bed reactor and a continuous feed stirred tank reactor

The catalytic activity of amyloglucosidase covalently attached to DEAE-cellulose was studied in a packed bed reactor and a continuous feed stirred tank reactor (CSTR) for the reaction maltose → glucose. At low flow rates mass-transfer limitations in the bed reactor lead to lower conversions for this reactor compared to the CSTR. Simple theoretical expressions for these reactors were compared with the experimental results. There are significant differences between the kinetic parameters and pH profile of the immobilized and free enzyme. The immobilized enzyme also showed greater stability at 50°C than did free amyloglucosidase. The temperature dependence of the reaction rate was the same for immobilized and free enzyme.     

Properties of yeast debranching enzyme and its specificity toward branched cyclodextrins.

Debranching enzyme was purified from Saccharomyces cerevisiae by DEAE-cellulose, omega-aminobutyl agarose and hydroxyapatite column chromatography. The activity of the eluent was monitored by the iodine-staining method which detects both the direct and indirect debranching enzymes. The elution profiles at every step showed a single peak with no shoulder. The crude and the purified enzyme preparations gave a single activity band with the same mobility on PAGE. The crude product produced 80% glucose compared to reducing sugar from glycogen-phosphorylase-limited dextrin while the partially purified and purified preparations produced 100% glucose. The activity of the purified enzyme was characterized and compared with that of the rabbit muscle enzyme by using various branched cyclodextrins as substrates. Both enzymes hydrolyzed 6-O-alpha-D-glucosyl cyclodextrins to glucose and cyclodextrins, but did not act on 6-O-alpha-maltosyl cyclomaltoheptaose. The yeast enzyme gave rise to glucose as a sole reducing sugar from 6-O-alpha-maltotriosyl cyclomaltoheptaose and 6-O-alpha-maltotetraosyl cyclomaltoheptaose, indicating that maltosyl and maltotriosyl transfers, respectively, had occurred, prior to the action of amylo-1,6-glucosidase. 6-O-alpha-D-Glucosyl cyclomaltoheptaose and 6-O-alpha-D-glucosyl cyclomalto-octaose, respectively, were better substrates than glycogen-phosphorylase-limited dextrin for the yeast and muscle enzymes. The yeast enzyme released glucose at a similar rate from 6-O-alpha-maltotriosyl cyclomaltoheptaose as from 6-O-alpha-maltotetraosyl cyclomaltoheptaose, but considerably lower rates than that from limit dextrin. The yeast debranching enzyme appears to be exclusively oligo-1,4----1,4-glucantransferase-amylo-1,6-glucosidase and does not have isoamylase.     

不同糖源及糖水平对大菱鲆糖代谢酶活性的影响

采用34双因素实验设计, 以初始质量为(8.060.08) g的大菱鲆幼鱼(Scophthalmus maximus L.)为对象, 研究在饲料中添加3种糖源(葡萄糖、蔗糖和糊精)及4个水平(0、5%、15%、28%)对大菱鲆肝脏糖酵解关键酶己糖激酶(HK)、葡萄糖激酶(GK)、磷酸果糖激酶(PFK)、丙酮酸激酶(PK)和糖异生关键酶磷酸烯醇式丙酮酸羧激酶(PEPCK)、1, 6-二磷酸果糖酶(FBPase)活性的影响。结果表明: 饲料糖添加量从0升高到15%时, 大菱鲆的糖酵解酶GK和PK活性随饲料葡萄糖或糊精含量的增加而增加; 当饲料中葡萄糖或糊精含量为28%时, GK和PK活性有下降的趋势。3种糖源的4个添加水平对HK和PFK活性均无显著影响(P 0.05)。添加不同水平的葡萄糖对大菱鲆糖异生途径的PEPCK活性无显著影响(P 0.05), 但在饲料中葡萄糖添加量为5%时显著促进了FBPase活性(P 0.05), 当葡萄糖添加量升高为15%或28%时, FBPase活性与对照组无显著差异(P 0.05)。糊精作为饲料糖源时抑制了大菱鲆肝脏FBPase和PEPCK的活性, 而添加不同水平的蔗糖对FBPase和PEPCK活性的影响均不显著(P 0.05)。总的来说, 从大菱鲆幼鱼肝脏糖代谢角度而言, 在饲料中添加15%的葡萄糖或糊精时, 可以有效促进大菱鲆肝脏糖酵解能力; 较添加葡萄糖, 糊精在促进大菱鲆肝脏糖酵解的同时对糖异生存在一定程度的抑制。蔗糖作为饲料糖源时, 仅在添加量为28%时显著促进糖酵解酶GK活性, 糖酵解其他酶活性以及糖异生酶活性均不受蔗糖水平的显著影响。       

Detection of glycogen-debranching system in trophozoites of Entamoeba histolytica

Homogenates of trophozoites of Entamoeba histolytica were shown to bring about the total degradation of glycogen while purified phosphorylase of the same source alone yielded a limit dextrin as end product. An enzyme system capable of debranching the limit dextrin was obtained from the 40,000 g pellet by extraction in aqueous medium, purified by gel filtration on Fractogel TSK HW-55(F), and separated from phosphorylase by chromatography on Blue Sepharose CL-6B and aminobutyl Agarose. The glycogen-debranching system was purified 540-fold to a state of homogeneity by criterion of disc-gel electrophoresis. The purified enzyme was able to degrade glycogen-limit dextrin in the presence of phosphorylase and exhibited activities of both amylo-1,6-glucosidase (EC 3.2.1.33) and 4-alpha-glucanotransferase (EC 2.4.1.25). Although amylo-1,6-glucosidase released glucose from a glycogen-phosphorylase limit dextrin, transferase activity moved single glucose residues from the limit dextrin to 4-nitrophenyl-alpha-glucoside yielding successively 4-nitrophenyl-alpha-maltoside and 4-nitrophenyl-alpha-maltotrioside that could be detected by HPLC. Native glycogen-debranching system exhibited a relative molecular mass of Mr = 180,000 +/- 10% by gel filtration and gel electrophoresis in both denaturing and nondenaturating conditions.     

Detection of Glycogen-Debranching System in Trophozoites of Entamoeba histolytica

ABSTRACT. Homogenates of trophozoites of Entamoeba histolytica were shown to bring about the total degradation of glycogen while purified phosphorylase of the same source alone yielded a limit dextrin as end product. An enzyme system capable of debranching the limit dextrin was obtained from the 40,000 g pellet by extraction in aqueous medium, purified by gel filtration on Fractogel TSK HW-55(F), and separated from phosphorylase by chromatography on Blue Sepharose CL-6B and aminobutyl Agarose. The glycogen-debranching system was purified 540-fold to a state of homogeneity by criterion of disc-gel electrophoresis. The purified enzyme was able to degrade glycogen-limit dextrin in the presence of phosphorylase and exhibited activities of both amylo-1,6-glucosidase (EC 3.2.1.33) and 4- α -glucanotransferase (EC 2.4.1.25). Although amylo-1,6-glucosidase released glucose from a glycogen-phosphorylase limit dextrin, transferase activity moved single glucose residues from the limit dextrin to 4-nitrophenyl- α -glucoside yielding successively 4-nitrophenyl- α -maltoside and 4-nitrophenyl- α -maltotrioside that could be detected by HPLC. Native glycogen-debranching system exhibited a relative molecular mass of M_r= 180,000 ± 10% by gel filtration and gel electrophoresis in both denaturing and nondenaturating conditions.     

饲料糖对彭泽鲫生长和生理机能的影响

用分别含20%(低)、40%(高)葡萄糖和糊精的等氮(粗蛋白为35%风干物质)等能(16.4kJ/g)饲料饲养平均尾重为(2.40±0.16)g澎泽鲫Carassius auratus var. Pengze 16周,研究饲料糖种类和水平对鲫鱼生长、抗氧化力、非特异性免疫力和镉耐受性的影响,探讨鲫鱼对糖的利用能力及糖的生理功效。另用相同的饲料在相似的条件下饲养初重为(7.14±0.85)g的鲫鱼8周后胸腔注射12.5mg/kg体重的氯化镉溶液,观察其24h的存活率。实验结果表明饲料糖种类和水平都没有对鲫鱼的生长、脾指数和肝指数造成显著影响。高糖组糖化血红蛋白GHb值较高(P<0.05)。肝胰脏超氧化物歧化酶SOD活性、血浆SOD活性和总抗氧化力T-AOC各组差异不显著,肝胰脏的T-AOC为葡萄糖组显著高于糊精组(P<0.05)。补体溶血活性、血浆溶菌酶活性各组差异不显著。红细胞脆性高糖组显著低于低糖组(P<0.05)。镉染毒存活率各组差异不显著。上述结果提示40%饲料糖对鲫鱼生理功能没有不良影响,一定量的饲料糖有利于提高鲫鱼的抗氧化力。       

Structures of singly branched heptaoses produced by bacterial liquefying alpha-amylase.

1. A singly branched heptaose produced as a limit dextrin in the digest of beta-limit dextrin with liquefying alpha-amylase [EC 3.2.1.1] of Bacillus amyloliquefaciens was isolated in a paper chromatographically pure state. 2. Analysis using several enzymes revealed that the isolated branched dextrin was a mixture of six singly branched heptaoses with different ramifying points. 3. All the branched heptaoses contained a 62-alpha-maltosylmaltotriose moiety in their molecules, differing only in the mode of attachment of one maltose or two glucose residues by alpha-1,4-glucosidic bonds from this core dextrin. 4. The formation of various singly branched heptaoses (the present paper) and hexaoses (the previous paper) is discussed regarding the attack site specificity of the enzyme on beta-limit dextrin.     

Immobilization of glucoamylase onto polyaniline-grafted magnetic hydrogel via adsorption and adsorption/cross-linking

Glucoamylase (GA) was immobilized onto polyaniline (PANI)-grafted magnetic poly(2-hydroxyethylmethacrylate-co-glycidylmethacrylate) hydrogel (m-p(HEMA-GMA)-PANI) with two different methods (i.e., adsorption and adsorption/cross-linking). The immobilized enzyme preparations were used for the hydrolysis of “starch” dextrin. The amount of enzyme loading on the ferrogel was affected by the medium pH and the initial concentration of enzyme. The maximum loading capacity of the enzyme on the ferrogel was found to be 36.7 mg/g from 2.0 mg/mL enzyme solution at pH 4.0. The adsorbed GA demonstrated higher activity (59%) compared to adsorbed/cross-linked GA (43%). Finally, the immobilized GA preparations exhibited greater stability against heat at 55 °C and pH 4.5 compared to free enzyme (50 °C and pH 5.5), suggesting that the ferrogel was suitable support for immobilization of glucoamylase.     

Effects of immobilization on the kinetics of enzyme-catalyzed reactions. I. Glucose oxidase in a recirculation reactor system.

Glucose oxidase from Aspergillus niger was immobilized on nonporous glass beads by covalent bonding and its kinetics were studied in a packed-column recycle reactor. The optimum pH of the immobilized enzyme was the same as that of soluble enzyme; however, immobilized glucose oxidase showed a sharper pH-activity profile than that of the soluble enzyme. The kinetic behavior of immobilized glucose oxidase at optimum pH and 25 degrees C was similar to that of the soluble enzyme, but the immobilized material showed increased temperature sensitivity. Immobilized glucose oxidase showed no loss in activity on storage at 4 degrees C for nearly ten weeks. On continuous use for 60 hr, the immobilized enzyme showed about a 40% loss in activity but no change in the kinetic constant.     

Immobilization of glucose oxidase on silica-based supports

Glucose oxidase (beta-D-glucose: oxygen 1-oxidoreductase, EC 1.1.3.4) was covalently coupled to silica-based supports containing aldehyde functional groups. The activity of the immobilized enzyme was about 1000 U/g support. The optimum pH of the catalytic activity was 5.5 for the soluble enzyme and 6.0 for the immobilized enzyme. With glucose as a substrate the Km value of the immobilized enzyme was higher than in case of the soluble enzyme. The immobilized enzyme was found to be more thermostable than the soluble one. The immobilization did not affect the stability of glucose oxidase against the denaturing effect of urea.     

Biosynthesis of glycogen in Neurospora crassa. Purification and properties of the UDPglucose:glycogen 4-alpha-glucosyltransferase.

The Neurospora crassa glycogen synthase (UDPglucose:glycogen 4-alpha-glucosyltransferase, EC 2.4.1.11) was purified to electrophoretic homogeneity by a procedure involving ultracentrifugation, DEAE-cellulose column chromatography, (NH4)2SO4 fractionation and 3-aminopropyl-Sepharose column chromatography. The final purified enzyme preparation was almost entirely dependent on glucose-6-P and had a specific activity of 6.9 units per mg of protein. The subunit molecular weight of the glycogen synthase was determined by electrophoresis in sodium dodecyl sulfate-polyacrylamide gel to be 88 000--90 000. The native enzyme was shown to have a molecular weight of 270 000 as determined by sucrose density gradient centrifugation. Thus, the glucose-6-P-dependent form of the N. crassa glycogen synthase can exist as trimer of the subunit. Limited proteolysis with trypsin or chymotrypsin converted the glucose-6-P-dependent form of the enzyme into an apparent glucose-6-P-independent form. The enzyme was shown to catalyze transfer of glucose from UDPglucose to glycogen as well as to its phosphorylase limit dextrin, but not to its beta-amylase limit dextrin. Moreover, glucose, maltose and maltotriose were not active as acceptors.     

Preparation of some immobilized linked enzyme systems and their use in the automated determination of disaccharides

1. Glucose oxidase (EC 1.1.3.4), amyloglucosidase (EC 3.2.1.3), invertase (EC 3.2.1.26) and beta-galactosidase (EC 3.2.1.23) were covalently attached via glutaraldehyde to the inside surface of nylon tube. 2. The linked enzyme system, comprising invertase immobilized within a nylon tube acting in series with glucose oxidase immobilized in a similar way, was used for the automated determination of sucrose. 3. The linked enzyme system, comprising beta-galactosidase immobilized within a nylon tube acting in series with glucose oxidase immobilized in a similar way, was used for the automated determination of lactose. 4. The linked enzyme system, comprising amyloglucosidase immobilized within a nylon tube acting in series with glucose oxidase immobilized in a similar way, was used for the automated determination of maltose. 5. Mixtures of glucose oxidase and amyloglucosidase were immobilized within the same piece of nylon tube and used for the automated determination of maltose. 6. Mixtures of glucose oxidase and invertase were immobilized within the same piece of nylon tube and used for the automated determination of sucrose.     

Microcalorimetric methods for substrate determination in flow systems with immobilized enzymes.

The enthalpy of processes catalyzed by immobilized enzymes in the reaction cell of a LKB-flow calorimeter is used for determination of urea (0.5-5 mumol) and glucose (0.03-0.5 mumol). Accuracy is 2-5% and the time needed for one analysis is 20 min. A sensitive "enzyme thermistor" consisting of a flow through cell with an immobilized enzyme and two thermistors is described, which permits glucose determinations (0.05-1 mumol +/- 0.03 mumol) by means of temperature difference caused by reaction heat. Coupling of enzyme reactions for increasing reaction heat and consequently sensitivity in calorimetric determinations is demonstrated.     

Studies on the Amylolytic System of the Black-koji Molds

Saccharogenic and dextrinogenic amylase fractions were prepared from Black-koji amylase system and their actions investigated with a number of different substrates.

It was found that saccharogenic amylase fraction completely hydrolyzes glutinous rice starch and glycogen to glucose, without leaving any limit dextrin. On the other hand, this enzyme fraction converts potato starch to an extent of about 90% theoretical glucose, the remainder being left as limit dextrin, which is colored purple by iodine. The complete hydrolysis of the branched substrates except potato starch shows that the saccharogenic amylase fraction is capable of hydrolyzing the l,6-α-d-glucosidic linkage besides the 1,4-linkage, while the branched fraction of potato starch may contain some sort of anomaly to the enzyme. Dextrinogenic amylase fraction hydrolyzes starch and glycogen just as malt α-amylase.     

鳜对葡萄糖和糊精利用差异比较研究

研究通过比较鳜(Siniperca chuatsi)对不同碳水化合物的利用差异, 探究肉食性鱼类对碳水化合物利用的分子机制。按照1670 mg/kg剂量对鳜灌喂葡萄糖和糊精后, 分别在0、1h、2h、3h、4h、8h、12h和24h收集水样、血浆、肝脏和肌肉, 检测尿糖、血糖、血甘油三酯、血胰岛素、肝糖原、肌糖原含量及糖代谢相关基因表达水平等指标。结果显示: (1) 灌喂后1—12h内, 两组鳜相比, 葡萄糖组尿糖显著高于糊精组, 血糖及胰岛素含量在两组间无显著差异; (2) 两组鳜甘油三酯含量在2h时达到最大值, 糊精组甘油三酯含量在4h时显著高于葡萄糖组, 糊精组肝糖原含量在1h时显著高于葡萄糖组, 且糊精组肌糖原含量在24h内均显著高于葡萄糖组; (3) 灌喂后1h, 灌喂糊精组葡萄糖激酶(Glucokinase, GK)、脂肪酸合成酶(Fatty Acid Synthetase, FAS)、乙酰辅酶A羧化酶Ⅰ型(Acetyl-CoA Carboxylase Type Ⅰ, ACC1)、柠檬酸合成酶(Citroyl Synthetase, CS)基因表达水平显著高于葡萄糖组, 而在灌喂后8h, 糊精组糖原合酶(Glycogen Synthase, GS)和CS基因表达水平却显著低于葡萄糖组。结果表明, 肉食性鱼类鳜摄入糖后可以促进糖原和脂肪的合成, 转化为糖原和甘油三酯, 从而减少未利用糖的排出, 且鳜对葡萄糖的利用效率低于糊精。     

Estimation of intrinsic kinetic constants for pore diffusion-limited immobilized enzyme reactions

A simple method is presented that establishes intrinsic rate parameters when slow pore diffusion of substrate limits immobilized enzyme reactions that obey Michaelis-Menten kinetics. The Aris-Bischoff modulus is employed. Data at high substrate concentrations, where the enzyme would be saturated in the absence of diffusion limitation, and at low substrate concentrations, where effectiveness factors are inversely proportional to reaction modulus, are used to determine maximum rate and Michaelis constant, respectively. Because Michaelis-Menten and Langmuir-Hinshelwood kinetics are formally identical, this method may be used to estimate intrinsic rate parameters of many heterogeneous catalysts. The technique is demonstrated using experimental data from the hydrolysis of maize dextrin with diffusion-limited immobilized glucoamylase. This system yields a Michaelis constant of 0.14%, compared to 0.11% for soluble glucoamylase and 0.24% for immobilized glucoamylase free of diffusional effects.