Continuous production of maltotetraose using a dual immobilized enzyme system of maltotetraose-forming amylase and pullulanase

In order to produce a product with a high content of maltotetraose, dual-enzyme systems composed of immobilized maltotetraose-forming amylase (G(4)-forming amylase) and pullulanase were studied. The thermostability of individually immobilized enzymes was examined in continuous operation; studies revealed that the enzyme immobilized on "Chitopearl" was much more stable than that immobilized on Diaion HP-50. The effects of operating conditions on the stability of G(4) forming amylase immobilized on "Chitopearl" were examined to confirm that the apparent half-life data could be arranged using the immobilized enzyme stability factor, f(s). As for the dual immobilized enzyme system, six methods of usage were considered, with five yielding a 7-10% (w/w) higher content of maltotetraose product than the single-enzyme system. The effects of operating conditions on the maltotetraose production reaction were examined to confirm that the maltotetraose content of the products could be analyzed using the specific space velocity,SSV. In dual immobilized enzyme systems, pullulanase immobilized on the same carrier as the G(4)-forming amylase was found to be more stable than pullulanase immobilized on separate carriers. The effectiveness of using immobilized pullulanase along with the G(4)-forming amylase was confirmed from constant-conversion operations in which the maltotetraose content in the product was kept at 50% (w/w) in laboratory-scale experimentation.     

Stability of immobilized maltotetraose-forming amylase from Pseudomonas stutzeri

The stability of immobilized maltotetraose (G(4))-forming amylase (1,4-alpha-D-glucan maltoteraohydrolase, EC 3.2.1.60) from Pseudomonas stutzeri was investigated in both batch and continous processes. The inactivation process of the immobilized enzyme seemed to obey first-order kinetics, and the immobilized enzyme became more stable when coexisting with 20-30 wt % substrate and calcium ions. From intensive studies on the operational stability in the continuous process, the apparent half-life of G(4) productivity in a constant-flow system was mainly affected by the reaction temperature, substrate concentration, and initial immobilized enzyme activity. A new factor, immobilized enzyme stability factor f(s), was proposed to evaluate the half-life of the immobilized enzyme system.     

Starch metabolism in Pseudomonas stutzeri. I. Studies on maltotetraose-forming amylase.

The extracellular maltotetraose-forming amylase of Pseudomonas stutzeri was purified to homogeneity by a combination of affinity and hydroxyapatite chromatography. Sodium dodecyl sulfate-gel electrophoresis indicated that the oligomeric enzyme contains two different subunits with molecular weights of 48 000 and 58 000. Cross-linking studies using dimethyl suberimidate have demonstrated that the native enzyme consists of dimers. Seven isozymes of the amylase have been identified after polyacrylamide gel electrophoresis and amylose-digestion zymograms. The amylase of Ps. stutzeri is known to produce maltotetraose from linear and branched alpha-glucans by an exomechanism. The relatively high conversion rate of starch (75% hydrolysis), and the hydrolysis of cross-linked blue starch by this amylase indicate that the enzyme can cleave its substrates also by an endomechanism. Further strong evidence for an endomechanism was obtained from the action of the amylase on maltotetraose units which are located within the pullulan molecule. Dextran, pullulan, and maltotetraose are compeititve inhibitors. EDTA caused reversible inactivation. Amylase activity could be restored by addition of Ca2+. Heavy metals are inhibitory.     

Immobilization of Exo-maltotetraohydrolase and Pullulanase

A dual enzyme system of exo-maltotetraohydrolase [EC 3.2.1.60] and pullulanase [EC 3.2.1.41] was studied for the continuous production of maltotetraose. Porous chitosan beads were selected from among many carriers as the best carrier to immobilize both enzymes.

The properties of the immobilized enzymes were examined and compared with those of the native enzymes. For exo-maltotetraohydrolase, the optimum pH of the immobilized enzyme shifted slightly to the acidic side and the pH stability was improved on the alkaline side. The optimum temperature of the immobilized enzyme increased by about 15°C and thermostability was improved by about 10°C. As for pullulanase, very little difference in thermostability was observed.

The effects of operating conditions on the continuous production of maltotetraose using exo- maltotetraohydrolase immobilized on the porous chitosan beads were examined. Porous chitosan beads were recognized to be superior to Diaion HP-50.

The continuous production of maltotetraose was accomplished using the dual immobilized enzyme system. The dual enzyme system proved to be effective to increase the maltotetraose content in the product. A stable operation was successfully continued for more than 60 days.     

Production, purification, and characterization of alpha-amylase from Thermomonospora curvata.

Thermomonospora curvata produces an extracellular alpha-amylase. Maximal amylase production by cultures in a starch-mineral salts medium occurred at pH 7.5 and 53 degrees C. The crude enzyme was unstable to heating (65 degrees C) at pH 4 to 6, and was activated when heated at pH 8. The enzyme was purified 66-fold with a 9% yield and appeared homogeneous on discontinuous gel electrophoresis. The pH and temperature optima for activity of the purified enzyme were 5.5 to 6.0 and 65 degrees C. The molecular weight was calculated to be 62,000. The Km for starch was 0.39 mg/ml. The amylolytic pattern consisted of a mixture of maltotetraose and maltopentaose.     

Culture Conditions for Production of Thermostable Amylase by Bacillus stearothermophilus

Bacillus stearothermophilus grew better on complex and semisynthetic medium than on synthetic medium supplemented with amino acids. Amylase production on the complex medium containing beef extract or corn steep liquor was higher than on semisynthetic medium containing peptone (0.4%). The synthetic medium, however, did not provide a good yield of extracellular amylase. Among the carbohydrates which favored the production of amylase are, in order starch > dextrin > glycogen > cellobiose > maltohexaose-maltopeptaose > maltotetraose and maltotriose. The monosaccharides repressed the enzyme production, whereas inositol and d-sorbitol favored amylase production. Organic and inorganic salts increased amylase production in the order of KCI > sodium malate > potassium succinate, while the yield was comparatively lower with other organic salts of Na and K. Amino acids, in particular isoleucine, cysteine, phenylalanine, and aspartic acids, were found to be vital for amylase synthesis. Medium containing CaCl(2) 2H(2)O enhanced amylase production over that on Ca -deficient medium. The detergents Tween-80 and Triton X-100 increased biomass but significantly suppressed amylase synthesis. The amylase powder obtained from the culture filtrate by prechilled acetone treatment was stable over a wide pH range and liquefied thick starch slurries at 80 degrees C. The crude amylase, after (NH(4))(2)SO(4) fractionation, had an activity of 210.6 U mg. The optimum temperature and pH of the enzyme were found to be 82 degrees C and 6.9, respectively. Ca was required for the thermostability of the enzyme preparation.     

Haloalkaliphilic maltotriose-forming alpha-amylase from the archaebacterium Natronococcus sp. strain Ah-36.

A haloalkaliphilic archaebacterium, Natronococcus sp. strain Ah-36, produced extracellularly a maltotriose-forming amylase. The amylase was purified to homogeneity by ethanol precipitation, hydroxylapatite chromatography, hydrophobic chromatography, and gel filtration. The molecular weight of the enzyme was estimated to be 74,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The amylase exhibited maximal activity at pH 8.7 and 55 degrees C in the presence of 2.5 M NaCl. The activity was irreversibly lost at low ionic strength. KCl, RbCl, and CsCl could partially substitute for NaCl at higher concentrations. The amylase was stable in the range of pH 6.0 to 8.6 and up to 50 degrees C in the presence of 2.5 M NaCl. Stabilization of the enzyme by soluble starch was observed in all cases. The enzyme activity was inhibited by the addition of 1 mM ZnCl2 or 1 mM N-bromosuccinimide. The amylase hydrolyzed soluble starch, amylose, amylopectin, and, more slowly, glycogen to produce maltotriose with small amounts of maltose and glucose of an alpha-configuration. Malto-oligosaccharides ranging from maltotetraose to maltoheptaose were also hydrolyzed; however, maltotriose and maltose were not hydrolyzed even with a prolonged reaction time. Transferase activity was detected by using maltotetraose or maltopentaose as a substrate. The amylase hydrolyzed gamma-cyclodextrin. alpha-Cyclodextrin and beta-cyclodextrin, however, were not hydrolyzed, although these compounds acted as competitive inhibitors to the amylase activity. Amino acid analysis showed that the amylase was characteristically enriched in glutamic acid or glutamine and in glycine.     

The raw starch-degrading alkaline amylase ofBacillus sp IMD 370

The amylase ofBacillus sp IMD 370 is the first report of an alkaline amylase with the ability to digest raw starch. The amylase could degrade raw corn and rice starches more effectively than raw potato starch. It showed no adsorb-ability to any type of raw starch at any pH value tested. The enzyme digested raw corn starch to glucose, maltose, maltotriose and maltotetraose. The maximum pH for raw starch hydrolysis was pH 8.0 compared to pH 10.0 for soluble starch hydrolysis. The metal chelator, ethylenediaminetetraacetic acid, strongly inhibited raw starch-digestion and its effect was reversed by the addition of divalent cations. Degradation of raw starch was stimulated six-fold in the presence of -cyclodextrin (17.5 mM).     

Extracellular alkaline amylase from a Bacillus species

A selective medium was used to isolate a bacterium (Bacillus species NRRL B-3881) that produced extracellular alkaline amylase in an alkaline medium (pH 9.5). Maximal enzyme yield was obtained in an aerated medium after 21 hr at 36 C. The enzyme was purified 18-fold by ultrafiltration and ammonium sulfate precipitation. Three active isoenzymes (one major and two minor) of alkaline amylase were detected by disc electrophoresis in polyacrylamide gel. The enzyme was only 12% inactivated by 20 mm ethylenediaminetetraacetic acid after 1 hr at pH 9.2 and 32 C. The optimal temperature was 50 C at pH 9.2, and the optimal pH was 9.2 at 50 C. The enzyme was stable between pH 7.5 and 10. It had an endomechanism of substrate encounter. The products produced from amylose and amylopectin had the beta-configuration. Cyclomaltoheptaose was hydrolyzed to maltotriose, maltose, and glucose. The main final product produced from amylose and amylopectin was beta-maltose; the other final products were maltotriose and small quantities of glucose and maltotetraose. The predominant product at early stages of hydrolysis was maltotetraose; other products were maltose through maltonanaose.     

Production, purification, and characterization of alpha-amylase from Thermomonospora curvata.

Thermomonospora curvata produces an extracellular alpha-amylase. Maximal amylase production by cultures in a starch-mineral salts medium occurred at pH 7.5 and 53 degrees C. The crude enzyme was unstable to heating (65 degrees C) at pH 4 to 6, and was activated when heated at pH 8. The enzyme was purified 66-fold with a 9% yield and appeared homogeneous on discontinuous gel electrophoresis. The pH and temperature optima for activity of the purified enzyme were 5.5 to 6.0 and 65 degrees C. The molecular weight was calculated to be 62,000. The Km for starch was 0.39 mg/ml. The amylolytic pattern consisted of a mixture of maltotetraose and maltopentaose.     

磁性纳米颗粒固定化乙醇脱氢酶的研究

本研究采用3-丙氨基三乙氧基硅烷(APTES)和戊二醛修饰包裹有SiO2磁性Fe3O4纳米颗粒表面,将其作为固定化载体固定化乙醇脱氢酶,研究固定化条件对固定化效率的影响,并对固定化酶性质进行分析。研究发现,当Fe3O4@SiO2纳米颗粒修饰上氨基和醛基后依然具有良好的水分散性和胶体稳定性,适合作为固定化载体。通过单因素优化,发现当最适给酶量为11. 3U/100 mg,搅拌转速为150 r/min,固定化p H和固定化温度分别控制在6. 5和5℃～15℃,固定化时长为45 min时,具有较好的固定化效果,固定化率可达到60. 2%。在此条件下制备得到的固定化酶与游离酶相比,固定化酶具有良好的耐高温和耐碱性。所得固定化乙醇脱氢酶在连续使用8次后,固定化率仍保留在57%左右,表明该固定化酶具有较好的操作稳定性,可为连续生产NADH提供技术依据。     

A constitutive maltotetraose-producing amylase fromPseudomonas sp. IMD 353

Pseudomonas sp. IMD 353, secretes an extracellular maltotetraose-producing amylase. One of the most outstanding features of this enzyme is that it is produced constitutively (29 units/ml), using glucose (3%, w/v) as the carbon source. The amylase was purified to homogeneity and its enzymic properties examined. It had maxima for activity at pH 7.0 and 50°C, a relative molecular mass of 63,000 and an isoelectric point at pH 5.0. Specific amylase inhibitors, tendamistat and -amylase wheat inhibitor, activated the enzyme. Starch was hydrolysed from the non-reducing chain ends, by an endo-acting mechanism, producing maltotetraose in the -anomeric form. Yields of 65% (w/v), and 62% (w/v) were obtained on hydrolysis of starch (1%, w/v) and dextrin (15%, w/v), respectively. This enzyme failed to hydrolyse mmaltotetraose, even on prolonged incubation.     

Purification, partial characterization, and covalent immobilization–stabilization of an extracellular α-amylase from Aspergillus niveus

An extracellular amylase secreted by Aspergillus niveus was purified using DEAE fractogel ion exchange chromatography and Sephacryl S-200 gel filtration. The purified protein migrated as a single band in 5 % polyacrylamide gel electrophoresis (PAGE) and 10 % sodium dodecyl sulfate (SDS-PAGE). The enzyme exhibited 4.5 % carbohydrate content, 6.6 isoelectric point, and 60 and 52 kDa molar mass estimated by SDS-PAGE and Bio-Sil-Sec-400 gel filtration column, respectively. The amylase efficiently hydrolyzed glycogen, amylose, and amylopectin. The end-products formed after 24 h of starch hydrolysis, analyzed by thin layer chromatography, were maltose, maltotriose, maltotetraose, and maltopentaose, which classified the studied amylase as an α-amylase. Thermal stability of the α-amylase was improved by covalent immobilization on glyoxyl agarose (half-life of 169 min, at 70 °C). On the other hand, the free α-amylase showed a half-life of 20 min at the same temperature. The optima of pH and temperature were 6.0 and 65 °C for both free and immobilized forms.     

Alteration of bond-cleavage pattern in the hydrolysis catalyzed by Saccharomycopsis alpha-amylase altered by site-directed mutagenesis.

The 210th lysine (K) residue in the Saccharomycopsis alpha-amylase (Sfamy) molecule was replaced by arginine (R) and asparagine (N) residues by site-directed mutagenesis. The influences of the replacements on the bond-cleavage pattern for several substrates were analyzed. Both mutant enzymes, K210R and K210N, cleave mainly the first glycosidic bond from the reducing end of maltotetraose (G4), while the native enzyme hydrolyzes mainly the second bond from the reducing end. We changed successfully the major cleavage point in the hydrolysis reaction of G4. The 8th subsite affinities of the K210R and K210N enzymes are calculated to be +2.52 and -0.01 kcal/mol, respectively, whereas that of the native enzyme is +3.32 kcal/mol as reported in the previous paper. These affinity values suggest that the K210 residue composes the 8th subsite, one of major subsites, and that a positively charged amino residue is necessary for the 8th subsite affinity. The K210N enzyme is found to be less active for short substrates like maltotetraose (G4) than for long substrates like amylose A (approximately G18). The reduced catalytic activity specifically for the short substrates is also attributable to the remarkable decrease in the affinity of the 8th subsite.     

Increase in conformational stability of enzymes immobilized on epoxy-activated supports by favoring additional multipoint covalent attachment*

Epoxy supports (Eupergit C) may be very suitable to achieve the multipoint covalent attachment of proteins and enzymes, therefore, to stabilize their three-dimensional structure. To achieve a significant multipoint covalent attachment, the control of the experimental conditions was found to be critical. A three-step immobilization/stabilization procedure is here proposed: 1) the enzyme is firstly covalently immobilized under very mild experimental conditions (e.g. pH 7.0 and 20 degrees C); 2) the already immobilized enzyme is further incubated under more drastic conditions (higher pH values, longer incubation periods, etc.) to "facilitate" the formation of new covalent linkages between the immobilized enzyme molecule and the support; 3) the remaining groups of the support are blocked to stop any additional interaction between the enzyme and the support. Progressive establishment of new enzyme-support attachments was showed by the progressive irreversible covalent immobilization of several subunits of multi-subunits proteins (all non-covalent structures contained in crude extracts of different microorganism, penicillin G acylase and chymotrypsin). This multipoint covalent attachment enabled the significant thermostabilization of two relevant enzymes, (compared with the just immobilized derivatives): chymotrypsin (5-fold factor) and penicillin G acylase (18-fold factor). Bearing in mind that this stabilization was additive to that achieved by conventional immobilization, the final stabilization factor become 100-fold comparing soluble penicillin G acylase and optimal derivative. These stabilizations were observed also when the inactivations were promoted by the enzyme exposure to drastic pH values or the presence of cosolvents.     

Flow-injection-type biosensor system for salivary amylase activity

The authors aim to establish a method that can quantitatively evaluate vital reactions to stress. We have been examining the correlation between stress and salivary amylase activity in order to verify its validity as a stress index. In order to quantify human stress, which changes over time, the relationship between stress and salivary amylase activity must be verified by fast and repeated analysis of salivary amylase activity. Standard biosensors are designed such that the enzyme immobilized on an electrode (enzyme electrode) and the substrate-dependent activity is measured. The reverse approach of measuring the alpha-amylase-dependent activity was adopted. We fabricated an amylase activity analytical system. Maltopentaose was selected as a substrate for alpha-amylase and a flow-injection-type device was used to supply maltopentaose continuously. alpha-Glucosidase, having relatively low enzyme activity, was immobilized on a pre-activated membrane so that it could be enclosed in a pre-column, Glucose oxidase, having higher enzyme activity, was immobilized on a working electrode so that it could function as an amperometric biosensor. A saliva-collecting device was fabricated to make saliva pretreatment unnecessary. As a result, an amylase activity analytical system was fabricated that enabled us to measure salivary amylase activity from 0 to 30 kU/l, with an R(2) value of 0.97. The time-course changes in the salivary amylase activities for 1 week were 5.1%, and the initial sensitivity remained nearly constant. Through this study, we were able to verify the possible development of the amylase activity analytical system.     

Immobilization of α-amylase on gum acacia stabilized magnetite nanoparticles,an easily recoverable and reusable support

In this work, α-amylase is immobilized, using glutaraldehyde, onto magnetite nanoparticles prepared using gum acacia as the steric stabilizer (GA-MN), for the first time. The immobilization of amylase to GA-MN is very fast and the synthesis of GA-MN is very simple. The use of GA enables higher immobilization of α-amylase (60%), in contrast to the unmodified magnetite nanoparticles (∼20%). The optimum pH and temperature for maximum enzyme activity for the immobilized amylase are identified to be 7.0 and 40 °C, respectively, for the hydrolysis of starch. The kinetic studies confirm the Michaelis–Menten behavior and suggests overall enhancement in the performance of the immobilized enzyme with reference to the free enzyme. Similarly the thermal stability of the enzyme is found to increase after the immobilization. The GA-MN bound amylase has also been demonstrated to be capable of being reused for at least six cycles while retaining ∼70% of the initial activity. By using a magnetically active support, quick separation of amylase from reaction mixture is enabled. The catalytic rate of amylase is actually found to enhance by twofold after the immobilization, which is extremely advantageous in industry. At higher temperature, the immobilized enzyme exhibits higher enzyme activity than that of the free enzyme.     

Studies of the inhibition by malto-oligosaccharides of the cyclisation reaction catalysed by the cyclodextrin glycosyltransferase from Klebsiella pneumoniae M 5 al with glycogen

The substrate qualities of malto-oligosaccharides for the disproportionation reaction catalysed by the cyclodextrin glycosyltransferase [(1----4)-alpha-D-glucan:[(1----4)-alpha-D-glucopyranosyl]transferase (cyclising) EC 2.4.1.19] from Klebsiella pneumoniae M 5 al have been re-investigated. Maltose failed to be homologised with measurable velocity. The initial rates of disproportionation and the affinities of the enzyme increased with the chain lengths of the substrates. Maltopentaose was the smallest saccharide which, by disproportionation, yielded longer chains being cyclised initially. D-Glucose did not affect the initial cyclisation from glycogen, but served as acceptor for the "chain-shortening" reaction. Maltose inhibited the initial cyclisation reaction in a linearly competitive manner. Maltotriose and maltotetraose inhibited the cyclisation reaction competitively, the inhibition kinetics pointing to the binding of two effector-molecules to the enzyme. Competitive inhibition was also found with malto-pentaose, -hexaose, and -heptaose. The degrees of inhibition increased from maltose to maltotetraose, and decreased with the larger saccharides; maltotriose and maltotetraose were the most effective inhibitors of the initial cyclisation. Some possibilities for the subsite-mechanisms are discussed.     

活性炭吸附固定化粪产碱杆菌青霉素G酰化酶

目的：以活性炭为载体固定化粪产碱杆菌来源的青霉素G酰化酶,考察固定化酶的性质。方法：对影响酶固定化的因素优化筛选,确定有显著影响的因素：pH、离子强度、酶量、固定化时间进行L934的正交实验,获得最佳固定化条件,并对固定化酶的最适反应温度、pH及批次稳定性进行研究。结果：最佳固定化条件为：载体0.3g,酶量5mL,总反应体系为12mL,离子强度1mol/L,温度4℃,pH 7.0,固定化40h;最高固定化酶活性为135.9U/g湿载体。固定化酶性最适反应温度为55℃,最适pH为10,重复使用12次后没有活性损失。结论：活性炭吸附固定化青霉素G酰化酶的活性高,批次反应稳定,具有工业应用潜力。     

Enzymic activity of salivary amylase when bound to the surface of oral streptococci

The enzymatic activity of salivary amylase bound to the surface of several species of oral streptococci was determined by the production of acid from starch and by the degradation of maltotetraose to glucose in a coupled, spectrophotometric assay. Most strains able to bind amylase exhibited functional enzyme on their surface and produced acid from the products of amylolytic degradation. These strains were unable to utilise starch in the absence of salivary amylase. Two strains failed to produce acid from starch, despite the presence of functional salivary amylase, because they could not utilise maltose. Strains that could not bind salivary amylase failed to produce acid from starch. In no case was all the bound salivary amylase active, and two strains of Streptococcus mitis which bound amylase did not exhibit any enzyme activity on their cell surface. The ability to bind amylase may confer a survival advantage on oral bacteria which inhabit hosts that consume diets containing starch.