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.     

由淀粉生物合成海藻糖途径的初步验证和环境条件影响的研究

由温泉分离得一株茵WX-10．初步验证含有直接催比淀粉生成海藻糖的酶系。酶的作用受多种条件影响。包括反应时间、温度、pH等。最适反应条件为pH65～7.0，温度35C。反应液中有较高浓度缓冲液存在时，反应速度增快。DE为20浓度为10％的淀粉液化液是酶合适的底物。     

Effect of Pullulanase Debranching of Sago (Metroxylon sagu) Starch at Subgelatinization Temperature on the Yield of Resistant Starch

The effects of pullulanase debranching of sago (Metroxylon sagu) starch in the granular state and subsequent physical treatments on the formation and yield of type III resistant starch (RS 3) have been investigated. Sago starch was enzymatically debranched with pullulanase at 60°C and at pH 5.0 using different enzyme concentrations (24, 30, 40, 50 PUN/g dry starch) which was added to 20% (w/v) starch slurry and incubated for 0 to 48 h. Optimum enzyme concentration of 40 PUN/g dry starch and three debranching times (8, 16 and 24 h) have been selected for subsequent preparation of RS. Granule morphology and molecular weight distribution (MWD) of the debranched and resistant starch were examined. Debranched starch samples showed blurred birefringence patterns, a decrease in amylopectin fraction, an increase in low molecular weight fraction and a broadening of MWD. Debranched starch samples with a maximum RS yield of 7% were obtained at 8 h debranching time. Temperature cycling and incubation at certain temperature and storage time enhanced the formation of RS. Under the conditions used in this study, the optimum conditions to obtain the highest RS yield (11.6%) were 8 h of debranching time, followed by incubation at 80°C for seven days. The MWD analysis showed that RS consisted of material with relatively low degree of polymerization. This study showed that pullulanase treatment of starch in the granular state resulted in limited debranching of amylopectin but the subsequent physical treatments (incubation time/temperature) can be manipulated to promote crystallization and enhance formation of RS 3.     

Synthesis and in vitro digestion of resistant starch type III from enzymatically hydrolysed cassava starch

Resistant starch type III (RS III) was synthesised from cassava starch by autoclaving followed by debranching with pullulanase, at varied concentrations (0.4–12 U g^?1) and times (2–8 h), and recrystallisation (?18 to 90 °C for 1–16 h). The highest RS III yield (22 g/100 g) was obtained at an enzyme concentration of 4 U g^?1 after 8 h incubation, followed by recrystallisation at 25 °C for 16 h. Varying the recrystallisation conditions indicated that higher RS III yields (30–35 g/100 g) could be obtained at 90 °C within 2 h. Thinning cassava starch using α‐amylase prior to debranching using pullulanase did not further increase the RS III content. In vitro digestion data showed that whereas 44% RS III was digested after 6 h, the corresponding value for cassava starch was 89%.     

Inhibition of Raw Starch Digestion by One Glucoamylase Preparation from Black Aspergillus at High Enzyme Concentration

Raw starch digestion by glucoamylase I (Ab. G-I) preparation from black Aspergillus was inhibited significantly at relatively high concentration of the enzyme. The properties of this enzyme were studied together with those of another glucoamylase I (Nor. G-I), also from black Aspergillus. The two glucoamylases do not differ so much in their physico-chemical properties such as molecular weight, pH and thermal stability, pH and temperature optimum, substrate specificity, debranching activity, isoelectric point etc. The adsorption rate of both enzymes on raw starch increased by the increase of enzyme concentration. The raw starch digestion rate by adsorbed Ab. G-I, however, was decreased with the increase of concentration of enzyme whereas the same was increased in case of Nor. G-I. The inhibitory effect was weaker at 60°C or above.     

Biochemical characterisation of a glycogen branching enzyme from Streptococcus mutans: Enzymatic modification of starch

A gene encoding a putative glycogen branching enzyme (SmGBE) in Streptococcus mutans was expressed in Escherichia coli and purified. The biochemical properties of the purified enzyme were examined relative to its branching specificity for amylose and starch. The activity of the approximately 75 kDa enzyme was optimal at pH 5.0, and stable up to 40 °C. The enzyme predominantly transferred short maltooligosyl chains with a degree of polymerization (dp) of 6 and 7 throughout the branching process for amylose. When incubated with rice starch, the enzyme modified its optimal branch chain-length from dp 12 to 6 with large reductions in the longer chains, and simultaneously increased its branching points. The results indicate that SmGBE can make a modified starch with much shorter branches and a more branched structure than to native starch. In addition, starch retrogradation due to low temperature storage was significantly retarded along with the enzyme reaction.     

Gene cloning and characterization of a trehalose synthase from Corynebacterium glutamicum ATCC13032

Trehalose synthase (TreS) is an enzyme which produces trehalose from maltose through intramolecular transglycosylation. In this study, a gene (cg2529) encoding for TreS from Corynebacterium glutamicum (CgTS) was cloned and expressed in Escherichia coli. The hexahistidinetagged CgTS showed an optimum temperature and pH of 35°C and pH 7.0, respectively. This enzyme was not thermostable, but stable in a broad pH range from pH 5.0 to 8.5. Its activity slightly increased by 5 mM Mg²⁺ and Fe²⁺, while it was strongly inhibited by 5 mM sodium dodecyl sulfate (SDS). CgTS catalyzed the conversion from maltose into trehalose, and vice versa. Lowering reaction temperature by 5°C from the optimum temperature significantly reduced hydrolysis activity to produce glucose as a by-product compared to transglycosylation activity to produce trehalose, leading to increase in the conversion yields from maltose into trehalose. Consequently, the maximum conversion yield by CgTS reached 69% at 25°C after 9 hr of reaction.     

Effect of modified resistant starch of culinary banana on physicochemical,functional, morphological,diffraction, and thermal properties

Resistant starch has received much attention for both its potential health benefits and functional properties. The present study revealed that the culinary banana can be a potential source for production of resistant starch type III. The results showed that resistant starch yield can be increased using debranching enzyme (Pullulanase EC 3.2.1.41) and hydrothermal process. The three repeated autoclaving and cooling (hydrothermal) cycles followed by storage at –20ºC, increased the resistant starch yield from 12.3 to 26.4%. The use of debranching enzyme and subsequent retrogradation at –20ºC increased the resistant starch yield up to 31.2%. The modified resistant starch when compared to culinary banana starch evinced better stability. The modified resistant starch was analyzed using scanning electron microscopy, Fourier transform infrared, x-ray diffraction, and thermogravimetric diffraction analysis to determine the structural changes which proved that the modification occurred substantially.     

Inhibition of Raw Starch Digestion by One Glucoamylase Preparation from Black Aspergillus at High Enzyme Concentration

Raw starch digestion by one glucoamylase preparation from Black Aspergillus was inhibited at relatively high concentration of enzyme. The hydrolysis rate of raw starch, pre-treated with this abnormal glucoamylase preparation, by normal glucoamylase decreased significantly. The glucoamylase preparation was then separated into glucoamylase I and II by DEAE-cellulose chromatography. The phenomenon of inhibition was present in glucoamylase I only. Glucoamylase I had a high debranching activity and was very active in raw starch digestion at low enzyme concentration.     

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.     

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.     

Optimisation of porous starch preparation by ultrasonic pretreatment followed by enzymatic hydrolysis

Porous starch granules were formed by the partial hydrolysis of starch using amylase. Fungal amylase and corn starch were chosen as the original amylase and substance from different sources, respectively. Ultrasonic technique as an assistant of enzymatic hydrolysis was used to pretreat raw starch. Scanning electron microscopy was used to examine the structure of native and treated starch, revealing the size of the pores in each occasion. The extent of enzymatic hydrolysis (y%) was markedly enhanced by statistical optimisation of enzymatic conditions. A significant influence of amount, temperature and pH of enzyme has been noted with Plackett–Burman design. It was then revealed with response surface methodology (RSM) that 503.26 U (g substance)^?1 amount of enzyme, temperature of 55 °C and pH of 5.1 were optimum. This optimisation strategy led to the enhancement of y% from 53.4% to 61.38%.     

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.     

酶脱支处理对玉米缓慢消化淀粉形成的影响

以玉米原淀粉为原料,研究普鲁兰酶脱支处理糊化后制备缓慢消化淀粉(slowly digestible starch,SDS)过程中各影响因素(温度、p H值、酶用量、贮藏及干燥条件)对SDS形成的影响。结果表明,在57.5℃、p H 4.9、酶用量60 U/g的条件下脱支8 h,然后煮沸灭酶30 min,再经4℃冷藏、60℃干燥后,可得SDS含量为31.09%的产品。原淀粉、酶脱支处理样品及脱支并去除快速消化淀粉样品的X射线衍射图谱表明,脱支处理后,玉米淀粉结晶结构由A型向B型转变。因此,通过酶脱支处理提高SDS含量的可能原因是形成了新的结晶结构,SDS含量与结晶的数量和质量有关。采用酶法制备SDS具有较好的工业化应用前景。     

Resistant Starch Made from Banana Starch by Autoclaving and Debranching

Resistant starches (RS) were prepared from banana starch by debranching with pullulanase for different times and after autoclaving treatment. The different treatments produced seven RS products, which were tested with respect to available starch (AS), RS and in vitro hydrolysis rate. The control sample (without debranching) had the highest AS (80.5%) and the lowest RS content (9.1%). The samples debranched for 5 h and longer did not show significant differences (α = 0.05) in AS (approximately 72%) and RS (approximately 18%). The RS values obtained in the samples prepared were twice as high as that of the control sample. However, the sample debranched for the longest time had the highest hydrolysis rate, demonstrating that this product has a high digestion rate. Banana starch is a good source for RS preparation by autoclaving due to its high RS content and can be an alternative source in developing countries for obtaining a nutraceutic ingredient for functional food preparation.     

A New Approach to Limit Dextrinase and its Role in Mashing*

Limit dextrinase (EC 3.1.2.41) is a debranching enzyme catalyzing the hydrolysis of α-1,6-glucosidic linkages in starch. The role of this debranching enzyme in beer brewing has been questioned due to its assumed heat lability. In the present work the effectiveness of limit dextrinase was studied under conditions mimicking brewery practice rather than in a buffer solution. It was demonstrated that typical conversion temperatures of 63–65 °C and a mash pH of 5.4–5.5 favour the action of malt limit dextrinase. The temperature optimum for the limit dextrinase of a malt extract was 60–62.5 °C, as opposed to 50 °C for purified limit dextrinase. Lowering the mash pH from 5.8 to 5.4 increased wort fermentability due to increased limit dextrinase activity. Wort fermentability was more strongly correlated to the free limit dextrinase activity of malt than to the α- and β-amylase activities.     

Extraction of Lipids from Cereal Starches with Hot Aqueous Alcohols

Cereal starches were extracted at 100°C with various proportions of lower alcohols and water to establish optimum conditions. Lipid yields generally exceeding 90% were obtained with one two-hour extraction of maize starch at 100°C when using not less than 14 ml of 75–85% methanol, 10 ml of 75% ethanol, 12 ml of 74–78% n-propanol, 14 ml of 74–78% isopropanol, or 17 ml of 84% n-butanol per gram of starch. Similar results were obtained with wheat A-starch ( > 8 μm granule diameter), wheat B-starch ( < 8 μm granule diameter) and rice starch using n-propanol-water, and with rice starch using methanol-water. Yields decreased if the proportions of alcohol and water were not kept within well-defined limits, especially with butanol and ethanol. The preferred conditions for complete recovery of lipids were two 2-hour extractions and one 1-hour extraction at 100°C with not less than 16 ml of 75% n-propanol per gram of starch.     

ACTIVITY AND PRIMARY CHARACTERIZATION OF ENZYME FROM THERMUS RUBER CELLS CATALYZING CONVERSION OF MALTOSE INTO TREHALOSE

Thermophilic bacteria Thermus ruber produces enzyme, which catalyzes the conversion of maltose into trehalose. The specific activity of the cell-free extract from this bacteria growing without inducers was 0.028 U/mg protein and it was increased to up to 0.086 U/mg in the presence of 0.5% of maltose in the culture broth. The maximum degree of maltose conversion of about 90% was attained at 10% substrate concentration. The enzyme from Thermus ruber does not catalyze formation of trehalose from maltotetraose and maltopentaose. The optimum temperature for the enzyme activity was 65C. A maximum activity of the maltose conversion was performed at pH 6.5. The highest enzyme activity was achieved during cell cultivation at 55C on a media composed from 0.5% of peptone, 0.1% yeast extract and 0.5% of maltose or starch.

PRACTICAL APPLICATIONS

Trehalose is a chemically stable nonreducing disaccharide which can be used in food, cosmetics, medical and biotechnological industries. Extraction of this carbohydrate from yeast cells or other natural sources is unsuitable for trehalose production because of low process yield and high cost. Thus, the enzymatic methods of trehalose production are developed. In the current study the thermophilic bacteria Thermus ruber has been examined as a new source of the enzyme catalyzing conversion of maltose into trehalose.     

Production of high-fructose rice syrup and high-protein rice flour from broken rice

The objective of this study was to develop a process for the production of both high-fructose rice syrup and high-protein rice flour from broken rice. The rice flour was obtained from broken rice by using either a dry or wet milling method. The glucose produced from the slurry of various raw materials by treating with α-amylase and glucoamylase was compared. Results indicated that cassava and corn starch were better raw materials than rice flour. However, the filtered residue of liquefied rice slurry could be recovered as high-protein rice flour. The particle size of rice flour had a small effect on the glucose yield. The orthogonal-array table (L₂₇) method of experimental design was employed to determine optimum conditions for liquefaction. The glucose yield based on starch was 90.8±3.6% under the following optimum conditions α-amylase, 0.12%; rice flour, 20%; temperature, 96°C; time, 90 min. The filtrate from liquefied rice slurry was saccharified at 60°C with three different concentrations of glucoamylase. The higher the enzyme concentration, the shorter the time required to reach the maximum yield. After saccharification, the glucose solution was decolourised, desalted and concentrated to 40% d.s. and then isomerised to fructose at 60°C under continuous operation by using immobilised glucose isomerase packed in a column. The isomerised syrup was then purified and concentrated to 71% d.s. The final high-fructose rice syrup contained 50% glucose, 42% fructose and 3% maltose. After liquefaction, the rice slurry was centrifuged and the precipitate was dried by either spray or drum drying. The composition of these two high-protein rice flours was almost the same and the protein content was about three times as high as the raw material. There were significant differences in surface structure of rice flour and high-protein rice flours, as observed by the scanning electron microscope.     

Multiplicity of Glucoamylase of Aspergillus oryzae Part 2. Enzymatic and Physicochemical Properties of Three Forms of Glucoamylase

Three forms of glucoamylase of Aspergillus oryzae had optimum pH range between 4.5 to 5.0 at 40°C and optimum temperature range between 40 and 60°C. They were most stable at pH range between 4.0 and 7.0 and temperatures up to 40°C. The molecular weights were 76.000, 38.000 and 38.000, respectively. Hydrolysis activities of the three glucoamylases on different substrates were studied. They differed in their rate of attack on many substrates. Glucoamylase I had strong debranching activity and was highly active in raw corn starch digestion. Glucoamylase II and III had weak debranching activities and could hardly digest raw starch.