Production of Maltosyl β-Cyclodextrin by a Bioreactor System with Pullulanase Immobilized on Partially Deacetylated Chitin

Thermostable pullulanase (EC 3.2.1.41) from Bacillus acidopullulyticus was well immobilized on partially deacetylated chitin (PDAC-35) with glutaraldehyde. The immobilized enzyme showed the ability of producing maltosyl β-cyclodextrin (6-α-maltosylcyclomaltoheptaose) through a reverse action in a reaction mixture containing high concentration of maltose and β-cyclodextrin, and a bioreactor system with the immobilized enzyme could produce the branched cyclodextrin more efficiently than the native enzyme. The reaction mixture was passed through a charcoal column to remove large amounts of maltose, and cyclodextrins adsorbed in the column were eluted with ethanol solutions. β-Cyclodextrin, maltosyl β-Cyclodextrin, and dimaltosyl β-cyclodextrin were separated by using a ODS or a GPC column.     

Die Wirkung von α-Amylasen auf β-Cyclodextrin und der Nachweis von α-Amylasefremdaktivitäten in ausgewählten Enzympräparaten

The action of α-amylases on β-cyclodextrin and the evidence of foreign activity of α-amylase in selected preparations of enzymes The interaction between cyclodextrins and α-amylases taken from different sources is discribed contradictious in the literature. Some α-amylases e.g. isolated from Aspergillus oryzae, porcine pancreas and saliva hydrolized cyclodextrins to glucose. The hydrolysis of cyclodextrins catalysed by α-amylase from Bacillus species have been described conflicting. In this paper the action of hydrolysis of different preparations of α-amylases on β-cyclodextrin have been investigated. It has been shown that Rohalase M3 (α-amylase from Aspergillus niger) cleaves the ring of β-cyclodextrin. 2 α-amylases from Bacillus subtilis are not able to hydrolyse β-cyclodextrin. The reasons for the different actions of hydrolysis have been discussed with size and structure of the active centre of the enzymes. Moreover, different preparation of hydrolysis have been tested on secondary activity of α-amylase. 2 glucoamylases from Aspergillus oryzae have been shown secondary activity of α-amylase. With the hydrolases α-glucosidase from fungies, β-amylase from malt, saccharase from yeast, invertase from S. cerevisiae and pullulanase from Aerobacter aerogenes no cleavage of the ring of β-cyclodextrin could be detected.     

Production of Maltose from Starch by Simultaneous Action of beta-Amylase and Flavobacterium Isoamylase

Debranching isoamylase was prepared by fermentation of Flavobacterium sp., and purified. Soybean ß-amylase was extracted from defatted soybean meal and purified. 1.6, 4.0, 6.6. and 9.1% DE liquefied corn starch was prepared by bacterial α-amylase. The mixture of liquefied starch. Flavobacterium isoamylase, and soybean ß-amylase was incubated at 40 C, pH 6.0, for 46 h. The maximum yield of hydrolysates from 1.6 and 4.0% DE substrates was maltose, and a considerable amount of maltotriose, but no detectable glucose. 6.6 and 9.1% DE substrates also produced largest amounts of maltose with moderate maltotriose, and small amounts of glucose also formed during hydrolysis. With the increase of DE of the substrate the yield of maltose decreased and the yield of glucose and maltotriose increased.     

普鲁兰酶逆向合成麦芽糖基-α-环糊精

以麦芽糖和α-环糊精作为底物,利用普鲁兰酶逆向合成作用合成了麦芽糖基-α-环糊精。用薄层色谱、液-质联用对产物进行了鉴定,并用薄层色谱对其分离。对影响麦芽糖基-α-环糊精合成的反应物摩尔比(麦芽糖/α-环糊精)、底物浓度、反应温度、反应时间、加酶量、pH等因素分别做了单因素实验,最终确定了反应条件:反应物摩尔比(麦芽糖/α-环糊精)为16:1,反应时间24h,反应温度为58℃,加酶量400U/gα-环糊精,pH4.5。     

Production of Trehalose from Starch by Thermostable Enzymes from Sulfolobus acidocaldarius

The optimum conditions for the production of trehalose from starch were investigated using two thermostable enzymes, maltooligosyl trehalose synthase (MTSase) and maltooligosyl trehalose trehalohydrolase (MTHase), from Sulfolobus acidocaldarius ATCC 33909. The optimum pH was 5.5 and the optimum temperature was 55—57°C using isoamylase from Pseudomonas amyloderamosa as a debranching enzyme. The addition of CGTase to the reaction mixture during the saccharification process caused an increase in trehalose and a decrease in maltose and maltotriose. Isoamylase was better than pullulanase as a debranching enzyme. The yield of trehalose was independent of the type of starch used. Under optimum conditions, the yield of trehalose from corn starch at 30% concentration was more than 82%.     

Production of Trehalose from Starch by Maltose Phosphorylase and Trehalose Phosphorylase from a Strain of Plesiomonas

The cooperative concomitant action of maltose phosphorylase (MP), trehalose phosphorylase (TP), β-amylase and a starch debranching enzyme (pullulanase, isoamylase) was investigated to develop a more efficient method for preparing trehalose from starch. About 40 and 51—56% as solid basis of 25% (w/v) liquefied potato starch were converted to trehalose by the combination of soybean β-amylase and the crude enzyme preparation (MTA) containing MP, TP and a saccharogenic amylase from a strain (SH-35) of Plesiomonas in the absence and presence of a starch debranching enzyme, respectively. A stable maltose syrup (70%, w/w) containing about 30% trehalose in the dry solid was prepared from starch directly, and about 36% as dry basis of the mother liquor (70%, w/w) containing about 56% trehalose was obtained as crystals of this non-reducing disaccharide by conventional crystallization. Trehalose in the by-product obtained after removing crystals increased up to almost that of the mother liquor by reacting with MTA again. By the method reported here, trehalose was produced from starch on an industrial scale without any remaining by-products.     

超高麦芽糖浆的生产

本文研究了超高麦芽糖浆的生产，结果发现，普通β－淀粉酶和脱支酶共同作用适于制造普通非结晶性麦芽糖浆，组成大致为８０％左右的麦芽糖和１０％左右的麦芽三糖，而外切型α－麦芽糖－α－小淀粉酶（Ｍａｌｔｏ－ｇｅｎａｓｅ）由于价格及水解方式两方面的原因，不适合用于此类麦芽糖浆的生产。但生产用于制造结晶麦芽糖的超高麦芽糖浆时，则宜用β－淀粉酶、Ｍａｌｔｏｇｅｎａｓｅ和脱支酶共同作用，通常的组成为８０％以上的麦芽糖，４％以下的麦芽三糖。     

Interference of Cyclodextrins with Amylolytic Activity Assays of Cyclodextrin Glycosyltransferases

Cyclodextrin glycosyltransferases (CGTases) catalyse the cyclisation of starch dextrins to cyclodextrins and concomitantly show amylolytic activity which is commonly determined by the starch-iodine assay. In the present study it was found that cyclodextrins interfere with this assay. Adding cyclodextrins in concentrations ranging from 0.01mM to 0.6mM to starch-iodine complex solution resulted in a significant decrease in absorbance at 600nm due to α- and β-cyclodextrin competing with starch in the iodine complex formation. The largest reduction was caused by α-cyclodextrin. Therefore, the use of the starch-iodine assay will result in an overestimation of the amylolytic activity of CGTases – particularly when the enzyme has a high α-cyclodextrin production activity. In contrast, determination of the amylolytic activity by measurement of released reducing sugars with CuSO₄/bicinchonate was not disturbed by cyclodextrins.     

Properties and Application of a Thermostable Maltogenic Amylase Produced by a Strain of Bacillus Modified by Recombinant-DNA Techniques

During a screening programme for amylase producing microorganisms a strain of Bacillus was isolated which produced a maltogenic amylase which had possible industrial potential. Unlike the microbial β-amylases from B. cereus, B. megaterium, B. polymyxa and B. circulans this enzyme was characterized by being stable at 60–70°C at pH 4.5.-5.5. Unfortunately the microorganism proved to be difficult to cultivate. Using recombinant-DNA techniques it was possible to transfer the gene, which coded for the enzyme, to a host organism which was easier to cultivate, thus making industrial production a viable proposition. The enzyme is suitable for producing high maltose syrups from liquefied starch. When used together with an amylopectin debranching enzyme such as pullulanase, more than 75% maltose can be obtained at 30% D. S.     

大豆β-淀粉酶、产气杆菌异淀粉酶在虾壳几丁质上的固定化及在麦芽糖生产中的应用

本文研究了大豆β—淀粉酶和产气杆菌异淀粉酶在同一载体上的固定化作用。以虾壳几丁质和壳聚糖为载体,证明几丁质更适于固定化异淀粉酶。用5％甲酸和50％乙酸预处理载体可增加固定化异淀粉酶的活性和稳定性,这可能是由于载体中壳聚糖的溶解造成的。经甲酸处理的几丁质先与4～6％pH7～9的戊二醛反应1小时,再同酶液(10mg/ml,pH6)反应2小时,并添加0.2mol/1Ca~#,将有利于固定化作用。用本方法得到的固定化酶具有高度活性和稳定性。固定化异淀粉酶最适pH由6.5变为5.0,而固定化β—淀粉酶最适pH变化不大。固定化酶在60℃稳定,在70℃5分钟失活。固定化酶在水中贮藏稳定,并具有良好的操作稳定性,使其在麦芽糖生产中具有潜在的应用价值。     

Cyclodextrins as resveratrol carrier system

The formation of resveratrol–cyclodextrin inclusion complexes in aqueous solutions has been characterized using the hydroperoxidase activity of lipoxygenase as the enzymatic system. The addition of cyclodextrins to the reaction medium had an inhibitory effect on resveratrol oxidation by lipoxygenase due to the complexation of phytoalexin into the cyclodextrin cavity, which is in equilibrium with free cyclodextrins and free resveratrol, the only effective substrate for lipoxygenase. This inhibitory effect depends on the complexation constant K_c between resveratrol and the type of cyclodextrins used. In the present work β- and G₂-β-cyclodextrins were used and their K_c were calculated by nonlinear regression of the inhibition curves obtained in the presence of cyclodextrins. The values obtained were 4317 and 5130 M⁻¹ for β- and G₂-β-cyclodextrin, respectively, values which were checked and confirmed by the “cyclodextrin assay”.     

酶法测定葛根支链淀粉分支化度

通过异淀粉酶和普鲁兰酶分别对葛根支链淀粉β 限制糊精进行彻底的脱支水解 ,测得葛根支链淀粉的分支化度为 1 2∶1。 2种酶对脱支反应作用有不同的影响 ,异淀粉酶的酶活对脱支反应的影响较大 ,而普鲁兰酶的酶活影响相对较小。为保证脱支反应进行完全 ,对保温时间进行了研究。结果表明 ,在酶促反应条件下 ,使反应液中的还原力达到一定值 ,对于1mg/mL葛根支链淀粉 β 限制糊精 ,异淀粉酶和普鲁兰酶的用量分别不得少于 1 2U/mL和0 4U/mL ,保温时间分别不得少于 15h和 2 0h。     

Factors controlling flavors binding constants to cyclodextrins and their applications in foods

The binding constants of 14 different flavors (Maltol, Furaneol, Vanillin, Methyl Cinnamate, Cineole, Citral, Menthol, Geraniol, Camphor, Nootkatone, Eugenol, p-vinil Guayacol and Limonene) to cyclodextrins (α-cyclodextrin and β-cyclodextrin) have been determined by an UV–Vis spectrophotometric technique. In all cases the binding constant of flavors to β-cyclodextrin are bigger than the corresponding one to α-cyclodextrin. This fact is due to the different size of cyclodextrin cavity. As well as, a relationship between log P of each flavor and the binding constants was found, proving that the driving force for host–guest complex formation is hydrophobic/hydrophilic interactions.     

Enzymatic Characterisation of Novamyl®, a Thermostable α-Amylase

The thermostable -amylase Novamyl^® is used in the baking industry as an antistaling agent due to its ability to reduce retrogradation of amylopectin. We have studied its enzymatic properties at pH 5.0. We make two main conclusions: (1) Novamyl^® shows sequence homology to cycloglycosyl transferases (CGTases); like these enzymes, Novamyl^® cleaves cyclodextrins, forms transglycosylation products and is subject to product inhibition by maltose. Novamyl^® has 5 subsites in the active site and is also subject to substrate inhibition. (2) Novamyl^® is clearly different from exoglucanases like β-amylase and glucoamylase. It is able to hydrolyse a pentasaccharide with bulky substituents at both ends (INdp5) and is inhibited by the α-amylase inhibitor Trestatin A. Although Novamyl^® appears unable to hydrolyse α-1,6-linkages, it is able to degrade amylopectin to a greater extent than β-amylase as well as β-limit dextrins. Novamyl^® degrades amylose in such a manner that initially the molecular weight is drastically reduced while β-amylase does not show any detectable effect on the molecular weight of this substrate. Products of the degradation of amylopectin and amylose by Novamyl^® are maltose and oligosaccharides, whereas β-amylase and glucoamylase produce only maltose and glucose, respectively. This was shown in baking experiments as well. The new data presented here clearly show that unlike exoamylases, Novamyl^® does not require a non-reducing end and attacks amylose, Indp5 and cyclodextrins in an endo-like manner. Based on these results Novamyl^® should be reclassified.     

Chain Length Distribution of Amylopectins of Several Single Mutants and the Normal Counterpart,and Sugary-1 Phytoglycogen in Maize (Zea mays L.)

β-Limit dextrins of starches of normal (nonmutant), amylose-extender (ae), dull (du), sugary-2 (su₂) and waxy (wx) maize, and phytoglycogen of sugar-1 (su₁) maize were prepared. The β-limit dextrins were successively debranched by isoamylase and pullulanase, and followed by quantitative gel-filtration. The ratio of A to B chains for the ae starch appeared to be high and that for su₁ phytoglycogen was low. The ratio of short B to long B chains for the du starch was high and that for the ae starch appeared to be low. The phytoglycogen did not contain long B chains. The unit chain-length distribution of amylopectins of the normal, wx, and su₂ starches were similar. Fine structures of maize amylopectins and su₁ phytoglycogen were modeled and described.     

Purification and Characterization of Extracellular Isoamylase from Flavobacterium sp

An extracellular isoamylase from Flavobacterium sp., was purified by fractionation with ammonium sulfate, DEAE-cellulose, DEAE-Sephadex A-50, and CM-cellulose column chromatography. Single band of the debranching activity of the purified enzyme was detected by polyacrylamide gel electrophoresis. The enzyme efficiently hydrolyzed α-1,6-glucosidic linkage of glycogen and amylopectin and formed amylose chains, but did not hydrolyze pullulan. The enzyme released maltotriose from ß-limit dextrin of waxy maize amylopectin and glycogen, but no detectable maltose and glucose. Action of the isoamylase is similar to other microbial isoamylases but its physical properties are different.     

Coupling Reaction of Bacillus macerans Cyclodextrin Glucanotransferase on Glucosyl-α-cyclodextrin and Glucose

Treatment of glucosyl-α-cyclodextrin with radioactive glucose in the presence of Bacillus macerans cyclodextrin glucanotransferase, yielded radioactive maltose, maltotriose, and four kinds of glucosyl branched oligosaccharides. The branched oligosaccharides formed had identical Rf values as those of branched oligosaccharides of 5-8 glucose unit on paper chromatography using the solvent system of 1-butanol: pyridine: water (6/4/4), (Fractions B₅-B₈). Individual oligosaccharides were isolated and their structures determined using the action of porcine pancreatic α-amylase, β-amylase, pullulanase, isopullulanase and glucoamylase. The structure of the main component of the fraction B₅ was 6⁴-O-α-glucosylmaltotetraose, B₆ was 6⁴-O-α-glucosylmatopentaose, B₇ was 6⁵-O-α-glucosylmaltohexaose, and B₈ was 6⁶-O-α-glucosylmaltoheptaose. Fraction B₈, which formed at the initial stage of the reaction, contained 6⁴, 6⁵ and 6⁶-O-α-glucosylmaltoheptaose. Reducing end ¹⁴C-labelled 6⁴-O-α-glucosylmaltotetraose was degraded to 6³-O-α-glucosylmaltotriose and radioactive glucose by the exhaustive action of Bacillus macerans cycloamylose glucanotransferase, and 6³-O-α-glucosylmaltotriose was completely non-reactive to the enzyme. From these results, we proposed a model of the active site of the enzyme.     

α-Anomer-selective glucosylation of (+)-catechin by the crude enzyme, showing glucosyl transfer activity, of Xanthomonas campestris WU-9701

α-Anomer-selective glucosylation of (+)-catechin was carried out using the crude enzyme, showing α-glucose transferring activity, of Xanthomonas campestris WU-9701 with maltose as a glucosyl donor. When 60 mg of (+)-catechin and 50 mg of the enzyme (5.25 units as maltose hydrolysing activity) were incubated in 10 ml of 10 mM citrate-Na₂HPO₄ buffer (pH 6.5) containing 1.2 M maltose at 45°C, only one (+)-catechin glucoside was selectively obtained as a product. The (+)-catechin glucoside was identified as (+)-catechin 3′-O-α- -glucopyranoside (α-C-G) by ¹³C-NMR, ¹H-NMR and two-dimensional HMBC analysis. The reaction at 45°C for 36 h under the optimum conditions gave 12 mM α-C-G, 5.4 mg/ml in the reaction mixture, and the maximum molar conversion yield based on the amount of (+)-catechin supplied reached 57.1%. At 20°C, the solubility in pure water of α-C-G, of 450 mg/ml, was approximately 100 fold higher than that of (+)-catechin, of 4.6 mg/ml. Since α-C-G has no bitter taste and a slight sweet taste compared with (+)-catechin which has a very bitter taste, α-C-G may be a desirable additive for foods, particularly sweet foods.     

Analysis of the Amylolytic Enzyme System of Clostridium thermosulfurogenes EM1: Purification and Synergistic Action of Pullulanases and Maltohexaose Forming α-Amylase

During growth of C. thermosulfurogenes EM1 on starch, seven forms of pullulanase and one form of α-amylase were detected after gel electrophoretic separation of proteins. By determining the isoelectric points and N-terminal sequences of various pullulanase species it was evident that no structural differences exist between these proteins. These pullulanases hydrolyzed α-1,4- and α-1,6-linkages in various soluble sugar polymers causing their breakdown to maltose and maltotriose. Unlike these enzymes, the α-amylase produced by C. thermosulfurogenes EM1 preferentially attacked non-soluble starch. The main product of hydrolysis was maltohexaose. The combined action of pullulanases and α-amylase on native starch showed synergistic effect. This synergistic effect was not observed if soluble starch was used as substrate.     

叶黄素酯/α-环糊精包合研究

目的:研究叶黄素酯与α-环糊精的包合性能。方法:紫外法测定包合行为并计算表观包合常数;研磨法制备包合物;溶解度为指标确定包合比;DTA、DR-FTIR、UV-vis DRS验证包合物;与β-环糊精和混合环糊精的包合作用比较,测定其溶解度、稳定性。结果:叶黄素酯与α-环糊精表观包合常数Ka为127 L/mol;包合比1∶5时溶解度最佳;α-环糊精使叶黄素酯稳定性增强,溶解度提高为叶黄素酯原料的6倍以上。结论:α-环糊精应用于叶黄素酯性能优于β-环糊精和混合环糊精。