A Novel Debranching Enzyme for Application in the Glucose Syrup Industry

Some of the properties of a novel, microbial, amylopectin debranching enzyme are described. The enzyme belongs to the class of debranching enzymes known as pullulanases (EC 3.2.1.41) and is characterized by being stable at higher temperatures and lower pH's than previously described enzymes of this type. The novel debranching enzyme can be used together with Aspergillus niger glucoamylase to improve the efficiency of starch conversion to glucose or together with cereal ß-amylases to increase the maltose level in high maltose syrups. Examples of these applications are given. It is expected that the enzyme will gain widespread acceptance in the glucose syrup industry.     

各类麦芽糖浆的生产工艺

<正> 麦芽糖是由2个葡萄糖残基通过α-1,4-葡糖基连接而成的二糖,是麦芽糖浆的主要成分。液化淀粉经过酶作用制得不同麦芽糖含量的糖浆,从而形成不同的糖浆类别。关于各类麦芽糖的典型组成成分,可参照表一。     

真菌α－淀粉酶饴糖的制造

真菌α－淀粉酶为内切酶，用它生产麦芽糖时，产品成分受很多因素影响。本文研究了淀粉乳浓度、液化淀粉葡萄糖值（ＤＥ值）、加酶量、反应时间以及脱支酶对真菌α－淀粉酶糖化的影响。实验发现，淀粉浓度、液化淀粉葡萄糖值对反应的影响较小，加酶量影响较大，酶量太少，即使延长反应时间，麦芽糖含量也很低。酶量过高，则会生成较多的葡萄糖。脱支酶有助于糖化，添加后麦芽糖含量增加６％左右。     

Note on the Hydrolysis of Amylose by beta-Amylase

Amylose is thought to be a slightly branched polymer with a ß-amylolysis limit of about 78%. Treating amylose with a mixture of the debranching enzyme pullulanase and ß-amylase will give 100% conversion to maltose. Interesting one does not obtain 100% conversion if the enzymes are used sequentially. We have found that amylose retrogrades and becomes insoluble while being incubated with pullulanase. Minimum debranching time will give 100% ß-amylolysis, which we think explains the previously reported need to use the enzymes simultaneously.     

利用酶制剂提高啤酒糖浆质量

酶制剂是影响啤酒用糖浆的关键因素之一,选用合理的酶制剂组合可以生产优质辅料、营养、特种啤酒专用糖浆。正确选用酶制剂和应用技术将有助于提高啤酒糖浆的质量,降低成本,增加产量。新型酶制剂的应用将与淀粉糖和啤酒互为融合、互为促进、共同进步。     

Heat Denaturation of Durum Wheat Semolina β-Amylase Effects of Chemical Factors and Pasta Processing Conditions

The susceptibility to heat denaturation of durum wheat β-amylase was studied in aqueous solution at 50 to 65°C. The activation energy of the reaction of denatnration was 439 kJ/mole between 56 and 65°C. Additives were tested to determine their protective effects on enzyme activity (at 65°C) in aqueous solution. Maltose, resulting from degradation of starch by β-amylase, appeared to be the most protective. The β-amylase became more resistant to heat with higher concentrations of maltose, indicating an enzyme-maltose complex which was more stable to heat than the enzyme alone. The same mechanism occurred when durum wheat pasta was processed: maltose produced either during mixing and sheeting or during extrusion protected the enzyme. The degree of protection was proportional to the intensity of mechanical work imparted to the dough.     

Immobilised β-Amylase in the Production of Maltose Syrups

In order to have a full continuous starch hydrolysates production process, the use of continuous conversions catalysed by immobilised enzymes is necessary. With respect to this, immobilisation of β-amylase on anion exchange resins and continuous conversion of maltodextrins to maltose syrups is investigated in packed bed column reactors at lab-scale. It is observed that the type of carrier and the source of the β-amylase are important parameters for the required capacity of the continuous process. Results show that, from the investigated enzymes, the commercial Spezyme BBA 1500L® β-amylase preparation immobilised on Duolite A568® as preferred carrier gives the best results in terms of initial performance and stability under realistic conversion conditions (50°C, pH 4.5, 50% d.s. substrate). On basis of the present results, the process is promising in terms of applicability at plant-scale after optimisation.     

I. CONDITIONS OF MASHING AND CARBOHYDRATE COMPOSITION OF THE WORT

Experimental mashings of ungerminated barley and 5–10% of malt with addition of the debranching enzyme pullulanase have been carried out. Worts with high attenuation are obtained in good yield. Of the fermentable sugars, there is less glucose, and more maltose and maltotriose than normally observed. The dextrin pattern is different from, but not necessarily inferior to, that traditionally seen. The worts resulting from the action of pullulanase are deprived of the dextrins with 8–14 glucose units, whereas the amounts of dextrins with 4–6 glucose units are close to those normally observed. The pullulanase preparations used are accompanied by proteolytic activity. It is suggested that debranching enzymes such as pullulanase offer an alternative choice of carbohydrases to be used in brewing from unmalted cereals.     

Production and Application of a Maltogenic Amylase by a Strain of Thermomonospora viridis TF-35

An extracellular and thermostable maltogenic amylase-producing moderate thermophile (Thermomonospora viridis TF-35), which grew well at 28–60°C, with optima at 45°C and pH 7, was isolated from soil. Maximal enzyme production was attained after aerobical cultivation for 32 h at 42°C with a medium (pH 7.3) composed of 2% (w/v) soluble starch, 2% gelatin hydrolyzate, 0.1% K₂HPO₄ and 0.02% MgSO₄ · 7H₂O. The partially purified enzyme, which was most active at 60°C and pH 6.0 and stabilized with Ca²⁺, converted about 65, 80, 75, 75, 65 and 60% of maltotriose, maltotetraose, maltopentaose, amylose, amylopectin and glycogen into maltose as a major product under the conditions used, respectively. Glucose and small amounts of maltooligosaccharides were also formed concomitantly as by-products. The molar ratio of maltose to glucose from maltotriose were larger than 1 during all stages of the hydrolysis. About 70 and 76% of 25% (w/v) potato starch liquefites having a 3.5 DE value were converted into maltose by the enzyme in the absence and presence of pullulanase during the saccharification, respectively. About 90 and 94% of the starch liquefites were also converted into maltose with relatively low contents of maltooligosaccharides by the cooperative 2 step reaction with the enzyme after obtaining starch hydrolyzates containing about 85 and 90% maltose by the simultaneous actions of soybean ß-amylase and debranching enzymes.     

Solubility of Maltose in Water in the Presence of Glucose and Maltotriose

Maltose is used in various forms in the food and pharmaceutical industries. Maltose syrups produced by enzymatic starch conversion usually contain impurity sugars, e.g. glucose, maltotriose, and higher saccharides, which affect the solubility of maltose. Using a simple refractometric technique, solubility data for the pure and impure maltose systems were determined. Glucose and maltotriose were found to decrease the equilibrium concentration of maltose but increase the total dissolved solids concentration.     

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.     

降低冰核生产菌X．ampelina TS206黄原胶产率的研究

培养基中不同的碳源影响冰核细菌的冰核活性、生物量和产胶率，麦芽糖、山梨醇、蔗糖等均可做为促进冰核细菌生长碳源，麦芽糖作为碳源加入发酵培养基中生物活性和生物量均较高，同时产胶率低于葡萄糖和蔗糖十倍左右。麦芽汁代替麦芽糖可以降低产胶率，但效果不如麦芽糖。在培养基中加入0．1ml，100ml乳酸可以有效地抑制黄原胶的产生，而对冰核活性和生物量影响不大。     

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.     

全酶法制备超高麦芽糖浆工艺

以碎米为原料,采用全酶法制备超高麦芽糖浆。以麦芽糖含量为指标,采用正交试验对耐高温α- 淀粉酶、大麦β- 淀粉酶、普鲁兰酶的添加量和糖化结束DE 值4 个因素进行研究,确定最佳工艺为3 种酶的添加量分别为0.20、0.50、1.05kg/t 原料,糖化结束DE 值控制在48% 左右。选用高效阴离子交换色谱法分析制备的麦芽糖浆中各糖组分含量,以峰面积外标法得出样品中麦芽糖含量为70.7%,符合超高麦芽糖标准。     

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%.     

Interference of Non-enzyme Protein in the Reaction of α-Amylase on Amyloses

In a previous communication [1] the assessment of amyloses as potential chromogenic substrates for the assay of α-amylase was described. An α-amylase suscetibility test for amyloses using a highly purified α-amylase from Bacillus subtilis was used. During the course of these investigations commercially available control sera containing α-amylase were used, However, these preparations proved to be highly unsuccessful due to having both low enzyme activity and high level of extraneous protein. Therefore, the difficulties encountered using these preparations are reported in this communication as illustration of a wider principle of superfluous protein in enzyme preparations. In this context it should be noted that industrial preparations of starch degrading enzymes also contain non-enzyme protein and such preparations are used for the production of e. g. glucose and high-fructose syrups.     

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.     

PRODUCTION OF AMYLOLYTIC ENZYMES BY SEVERAL YEAST SPECIES

Several amylolytic yeast species, endomycopsis spp, schwanniomyces spp, pichia spp and saccharomyces spp have been compared for their ability to synthesise α-amylase (E.C. 3.2.1.1), glucoamylase (E.C. 3.2.1.3) and pullulanase (E.C. 3.2.1.9). Endomycopsis fibuligera strain 240 possessed the highest glucoamylase activity (208 nmoles glucose/min) and second highest α-amylase activity (128 nmoles maltose/min) and produced the largest amount of biomass. Schwanniomyces spp were the only yeast species studied which exhibited significant debranching activity. It was found that in endomycopsis spp, schwanniomyces spp and pichia spp, glucose, at a concentration above 3.0 × 10^?3 m , appeared to repress α-amylase activity. However, glucoamylase activity was not repressed at that glucose concentration.     

酶制剂制备马铃薯高麦芽糖浆的研究

以马铃薯淀粉为原料,利用酶制剂进行了制备高麦芽糖浆的研究,摸清了马铃薯高麦芽糖浆的工艺条件和参数,得出了马铃薯可作为淀粉糖食品开发的理论依据。     

Substrate-accelerated death of Saccharomyces cerevisiae CBS 8066 under maltose stress

When Saccharomyces cerevisiae CBS 8066 was grown under maltose limitation, two enzymes specific for maltose utilization were present: a maltose carrier, and the maltose-hydrolysing alpha-glucosidase. The role of these two enzymes in the physiology of S. cerevisiae was investigated in a comparative study in which Candida utilis CBS 621 was used as a reference organism. Maltose pulses to a maltose-limited chemostat culture of S. cerevisiae resulted in 'substrate-accelerated death'. This was evident from: (1) enhanced protein release from cells; (2) excretion of glucose into the medium; (3) decreased viability. These effects wee specific with respect to both substrate and organism: pulses of glucose to maltose-limited cultures of S. cerevisiae did not result in cell death, neither did maltose pulses to maltose-limited cultures of C. utilis. The maltose-accelerated death of s. cerevisiae is most likely explained in terms of an uncontrolled uptake of maltose into the cell, resulting in an osmotic burst. Our results also provide evidence that the aerobic alcoholic fermentation that occurs after pulsing sugars to sugar-limited cultures of s. cerevisiae (short-term Crabtree effect) cannot solely be explained in terms of the mechanism of sugar transport. Both glucose and maltose pulses to maltose-limited cultures triggered aerobic alcohol formation. However, glucose transport by S. cerevisiae occurs via facilitated diffusion, whereas maltose entry into this yeast is mediated by a maltose/proton symport system.