Identification of the essential catalytic residues and selectivity-related residues of maltooligosyltrehalose trehalohydrolase from the thermophilic archaeon Sulfolobus solfataricus ATCC 35092

Maltooligosyltrehalose trehalohydrolase (MTHase) catalyzes the release of trehalose by cleaving the alpha-1,4-glucosidic linkage next to the alpha-1,1-linked terminal disaccharide of maltooligosyltrehalose. Mutations at residues D255, E286, and D380 were constructed to identify the essential catalytic residues of MTHase, while mutations at residues W218, A259, Y328, F355, and R356 were constructed to identify selectivity-related residues of the enzyme. The specific activities of the purified D255A, E286A, and D380A MTHases were only 0.15, 0.09 and 0.01%, respectively, of that of wild-type MTHase, suggesting that these three residues are essential catalytic residues. Compared with wild-type MTHase, A259S, Y328F, F355Y, and R356K MTHases had increased selectivity ratios, which were defined as the ratios of the catalytic efficiencies for glucose formation to those for trehalose formation in the hydrolysis of maltooligosaccharides and maltooligosyltrehaloses, respectively, while W218A and W218F MTHases had decreased selectivity ratios. When starch digestion was carried out at 75 degrees C and wild-type and mutant MTHases were, respectively, used with isoamylase and maltooligosyltrehalose synthase (MTSase), the ratios of initial rates of glucose formation to those of trehalose formation were inversely correlated to the peak trehalose yields.     

Amyloglucosidase hydrolysis of high-pressure and thermally gelatinized corn and wheat starches

The study of glucose production using amyloglucosidase as a biocatalyst was carried out using high-pressure and thermally gelatinized corn and wheat starches. For corn starch, the measured initial rate of glucose production obtained from thermal gelatinization is faster than that obtained from the two high-pressure treatments, but the equilibrium yield of glucose was found to be similar for the three treatments. High-pressure treatments of wheat starch significantly improve the equilibrium yield of glucose compared with those obtained from the thermally gelatinized wheat starch. This difference has been related to the formation of amylose-lipid complexes during heating and could also explain previous physicochemical differences observed between high-pressure and thermally gelatinized starch.     

Glucose release of water-soluble starch-related alpha-glucans by pancreatin and amyloglucosidase is affected by the abundance of alpha-1,6-glucosidic linkages

This study tested the hypothesis that an increased branch density (i.e., the percentage of alpha-1,6-glucosidic linkage) in water-soluble, starch-related alpha-glucans leads to reduced glucose release by pancreatin and amyloglucosidase. Malto-oligosaccharides and phytoglycogens were structurally analyzed and compared for their susceptibility to the enzymes. Malto-oligosaccharides were prepared by subjecting starch to alpha-amylase and beta-amylase followed by ultrafiltration to enrich alpha-1,6-glucosidic linkages. The branch density of the oligosaccharide products reached up to 17%, determined by (1)H NMR. Phytoglycogens were extracted from six sweet corn lines, and analysis showed similar chain length distributions and a branch density range from 8.8 to 9.5%, as compared with 4.6% for normal corn starch and 5.7% for waxy corn starch. The digestion behavior of these alpha-glucans was correlated to branch density: Highly branched malto-oligosaccharides had much reduced glucose release as compared with starch, whereas the reduction of glucose release from phytoglycogen was relatively low. Particularly, the reduction of glucose release associated with enhanced branch density was caused by reduced hydrolysis by amyloglucosidase.     

Expression, purification, and characterization of the maltooligosyltrehalose trehalohydrolase from the thermophilic archaeon Sulfolobus solfataricus ATCC 35092

The maltooligosyltrehalose trehalohydrolase (MTHase) mainly cleaves the alpha-1,4-glucosidic linkage next to the alpha-1,1-linked terminal disaccharide of maltooligosyltrehalose to produce trehalose and the maltooligosaccharide with lower molecular mass. In this study, the treZ gene encoding MTHase was PCR-cloned from Sulfolobus solfataricus ATCC 35092 and then expressed in Escherichia coli. A high yield of the active wild-type MTHase, 13300 units/g of wet cells, was obtained in the absence of IPTG induction. Wild-type MTHase was purified sequentially using heat treatment, nucleic acid precipitation, and ion-exchange chromatography. The purified wild-type MTHase showed an apparent optimal pH of 5 and an optimal temperature at 85 degrees C. The enzyme was stable at pH values ranging from 3.5 to 11, and the activity was fully retained after a 2-h incubation at 45-85 degrees C. The k(cat) values of the enzyme for hydrolysis of maltooligosyltrehaloses with degree of polymerization (DP) 4-7 were 193, 1030, 1190, and 1230 s(-1), respectively, whereas the k(cat) values for glucose formation during hydrolysis of DP 4-7 maltooligosaccharides were 5.49, 17.7, 18.2, and 6.01 s(-1), respectively. The K(M) values of the enzyme for hydrolysis of DP 4-7 maltooligosyltrehaloses and those for maltooligosaccharides are similar at the same corresponding DPs. These results suggest that this MTHase could be used to produce trehalose at high temperatures.     

Kinetics of the surface hydrolysis of raw starch by glucoamylase

The hydrolysis of raw starch catalyzed by glucoamylase has been studied with starch granules of different sizes by use of an amperometric glucose sensor by which the direct and continuous observation of the concentration of glucose can be achieved even in a thick raw starch suspension. The initial rate of the enzymatic hydrolysis in the raw starch suspension increased with increasing concentration of the enzyme to approach a saturation value and was proportional to the amount of substrate. Also, the rate was proportional to the specific surface area of the substrate. The experimental results can be explained well by the rate equations derived from a three-step mechanism, which consists of adsorption of the free enzyme onto the surface of the substrate, reaction of the adsorbed enzyme with the substrate, and liberation of the product.     

Activity‐Guided Fractionation to Identify Blue Wheat (UC66049 Triticum aestivum L.) Constituents Capable of Inhibiting In Vitro Starch Digestion

The inhibition or delay of starch digestion by dietary compounds could help manage postprandial blood glucose levels. The objective of this study was to identify constituents from whole grain blue wheat capable of decreasing α‐amylase‐catalyzed starch digestion. An activity‐guided fractionation approach based on liquid chromatography was used to identify solvent‐ and alkaline‐extractable blue wheat constituents reducing α‐amylase‐mediated starch digestion in vitro. Fatty acids, potentially released from cell wall polymers by alkaline hydrolysis, inhibited the digestion of amylose, probably through the formation of amylose‐lipid complexes. However, the degradation of amylopectin was not affected by fatty acids. In addition, 1‐(3,5‐dihydroxyphenyl)heneicosan‐2‐one, a 5‐(2′‐oxoalkyl)resorcinol, was found to reduce starch digestion. However, because the digestion of both amylopectin and amylose was reduced, the inhibition mechanism was different from that of fatty acids. Further research is needed to evaluate whether this component also reduces starch digestion in vivo. Other phenolic compounds of blue wheat such as anthocyanins or hydroxycinnamates were not identified as major starch digestion inhibitors by using the activity‐guided fractionation approach.     

两步法高效水解水稻秸秆制取木糖和葡萄糖

在序批式高压反应釜反应器中,采用两步法研究了水稻秸秆在稀硫酸水溶液中水解为木糖和葡萄糖,系统考察了反应温度、反应时间、水稻秸秆用量和稀酸浓度对水稻秸秆水解反应的影响。实验结果表明:与传统一步法酸水解相比,采用两步法酸水解水稻秸秆,既可以得到很高的木糖产率,又可以得到较高的葡萄糖产率;反应温度、反应时间和酸浓度对水稻秸秆酸水解产物的分布和产率有着重要影响。第一步水解反应中,当底物用量为1.5 g,酸浓度为0.5%wt时,140℃反应120 min,木糖产率高达162.6 g·kg-1;第二步水解反应中,当底物用量为0.5 g,酸浓度为1.0%wt时,180℃反应120 min,葡萄糖产率高达216.5 g·kg-1。本研究为农业废弃物水稻秸秆的高效和高值资源化利用提供了新的策略。     

Effects of Milling and Cooking Conditions of Rice on In Vitro Starch Digestibility and Blood Glucose Response

The objective of this study was to investigate the effects of milling and cooking conditions of cooked rice prepared from cultivar Koshihikari on in vitro starch digestibility and in vivo glucose response in humans. In addition, compression and adhesiveness tests were conducted for texture analysis of the cooked rice. Brown rice (BR) and surface‐abraded BR (SABR, ≥99.5% of the original weight) were digested more slowly than white rice (91% of the original weight) when cooked rice grain was used for the in vitro test, but they were digested more rapidly in the initial stage of the reaction when cooked rice ground by a meat grinder was used. The increase in water added for cooking significantly increased the extent of starch digestion with BR and SABR. The changes in blood glucose levels after the ingestion of cooked rice were dependent on the sample type. The cooking conditions dramatically influenced the glucose response after the ingestion of BR. A significant correlation was found between blood glucose levels at 45 min and the extent of starch digestion with ground samples, whereas no relationship was found with cooked rice grain samples for in vitro digestibility.     

Biocatalysis for the production of industrial products and functional foods from rice and other agricultural produce

Many industrial products and functional foods can be obtained from cheap and renewable raw agricultural materials. For example, starch can be converted to bioethanol as biofuel to reduce the current demand for petroleum or fossil fuel energy. On the other hand, starch can also be converted to useful functional ingredients, such as high fructose and high maltose syrups, wine, glucose, and trehalose. The conversion process involves fermentation by microorganisms and use of biocatalysts such as hydrolases of the amylase superfamily. Amylases catalyze the process of liquefaction and saccharification of starch. It is possible to perform complete hydrolysis of starch by using the fusion product of both linear and debranching thermostable enzymes. This will result in saving energy otherwise needed for cooling before the next enzyme can act on the substrate, if a sequential process is utilized. Recombinant enzyme technology, protein engineering, and enzyme immobilization are powerful tools available to enhance the activity of enzymes, lower the cost of enzyme through large scale production in a heterologous host, increase their thermostability, improve pH stability, enhance their productivity, and hence making it competitive with the chemical processes involved in starch hydrolysis and conversions. This review emphasizes the potential of using biocatalysis for the production of useful industrial products and functional foods from cheap agricultural produce and transgenic plants. Rice was selected as a typical example to illustrate many applications of biocatalysis in converting low-value agricultural produce to high-value commercial food and industrial products. The greatest advantages of using enzymes for food processing and for industrial production of biobased products are their environmental friendliness and consumer acceptance as being a natural process.     

Native or raw starch digestion: a key step in energy efficient biorefining of grain

Improved molecular disassembly and depolymerization of grain starch to glucose are key to reducing energy use in the bioconversion of glucose to chemicals, ingredients, and fuels. In fuel ethanol production, these biorefining steps use 10-20% of the energy content of the fuel ethanol. The need to minimize energy use and to raise the net yield of energy can be met by replacing high-temperature, liquid-phase, enzymatic digestion with low temperature, solid-phase, enzymatic digestion. Also called cold hydrolysis, the approach is a step toward a "green" method for the production of fuel ethanol. There has been substantial prior and increased recent interest in this approach that is presented in this first review of the subject. We include incentives, developmental research, fundamental factors of raw starch digestion, and novel approaches in enzymology and processing. The discussion draws on resources found in enzymology, engineering, plant physiology, cereal chemistry, and kinetics.     

Time course study of substrate utilization by Aspergillus flavus in medium simulating corn (Zea mays) kernels.

Utilization of the three major corn reserve materials, starch, triglycerides (refined corn oil), and zein (storage protein), by Aspergillus flavus was monitored in vitro over a 7-day fermentation. Medium composition in which proportions of reserve materials initially approximated proportions in mature corn kernels changed little over the first 18 h. Subsequently, hydrolysis of both starch and triglycerides occurred simultaneously, with peak concentrations of glucose and free fatty acids on day 2 of the fermentation period. Fatty acid concentrations dropped relatively rapidly after day 2 but increased again after day 6. Aflatoxin B(1) production increased after 36 h, with a peak at day 4. Aflatoxin B(1) production paralleled fungal biomass production during the exponential growth phase. A. flavus did not appear to preferentially utilize any of the released fatty acids. A number of fungus-specific metabolites were detected, including arabitol, erythritol, mannitol, trehalose, and kojic acid. Mannitol exceeded the other metabolites in concentration, and the timing of mannitol production closely paralleled that of aflatoxin B(1). Kojic acid concentrations peaked at day 6. In contrast to previously described selective use of simple carbohydrates by A. flavus, less discrimination was displayed when faced with utilization of complex substrates such as starch or triglycerides.     

废弃食用油脂两相厌氧发酵酸化条件优化

油脂的水解和长链脂肪酸的降解是油脂厌氧发酵过程中的限速步骤,提高水解酸化阶段挥发性脂肪酸(volatile fatty acid,VFA)的产率,有助于后续甲烷化反应的进行。利用响应面方法(response surface methodology,RSM)对废弃食用油脂两相厌氧发酵水解产挥发酸条件进行优化,考察了初始p H值、原料负荷、反应时间和接种量对产生挥发酸浓度的影响,提出采用该工艺的数学模型及优化后的工艺参数。结果表明,各影响因子对挥发酸的影响顺序为:接种量反应时间原料负荷初始p H值,方程的F值为15.65,相关系数为0.9359,调整相关系数为0.8761,说明数学模型可以较好地模拟真实的反应曲面。优化得到最佳的工艺参数为初始p H值6.2、负荷300 g/L、反应时间8 d、接种量40%,在该条件下,实际产挥发酸7 221.0 mg/L,与预测值7 224.0 mg/L吻合且重现性较好。厌氧产甲烷试验表明,酸化后废弃油脂较未酸化油脂在甲烷产量、甲烷含量、总化学需氧量(chemical oxygen demand,COD)去除率及挥发性固体(volatile solid,VS)去除率方面分别提高了44%、11%、28%和51%,经酸化处理比未酸化油脂的厌氧发酵时间(完成一个厌氧发酵周期内总产气量的80%的时间)缩短了28%。该研究结果为废弃食用油脂的两相厌氧发酵中试提供了参考。     

Synergistic Hydrolysis of Crude Corn Starch by α‐Amylases and Glucoamylases of Various Origins

Four α‐amylases and two glucoamylases from various sources, in eight combinations, were used to study the synergistic hydrolysis of crude corn starch at various temperatures. At 40 and 50°C, the combinations containing Rhizopus mold glucoamylase enhanced hydrolysis of corn starch compared wth that obtained with the combinations from Aspergillus niger. At 60°C, Rhizopus mold combinations gave low reaction yields as the enzyme was inactivated. The differences observed between α‐amylases are smaller, with the exception of Bacillus licheniformis α‐amylase, which presented more than twice the productivity of the other α‐amylases, at all temperatures. In terms of substrate conversion at 5 hr of hydrolysis, the combination of B. licheniformis α‐amylase with Rhizopus mold glucoamylase at 50°C presents 76% substrate conversion, whereas, with all the other combinations, starch conversion was 13–73%. HPLC analysis of the reaction products obtained at 50°C showed that the main product of corn starch hydrolysis was glucose at 85–100%. Further experiments showed that A. niger glucoamylase and B. licheniformis α‐amylase were the only enzymes that retained their initial activity after incubation at the temperatures studied.     

Starch characteristics and their influences on in vitro and pig prececal starch digestion

The main objective of this research was to study the characteristics of starch granules and their influences on in vitro and pig prececal starch digestion of corn, dehulled barley, wheat, and potato. Scanning electron microscopy was used to study the starch endosperm structure in the parent material as well as in vitro starch digestion. The results showed that corn starch granules were polyhedral, with a diameter ranging from 2 to 10 μm, whereas those of dehulled barley and wheat were spherical, with a diameter ranging from 5 to 20 μm. Potato had the largest starch granules among starch sources reported herein, with oval spheres of 10-50 μm in diameter. In vitro starch hydrolysis showed that starch granules of corn degraded faster than the starch of dehulled barley and wheat, with the potato starch being degraded the slowest. The in vivo digestibility trial using ileal-cannulated pigs confirmed the starch degradation of grains. The in vitro (x, %) and in vivo (y, %) digestibility were highly correlated [y = 6.5304x - 538.48 (R(2) = 0.9924)]. On the basis of the results, in vitro starch hydrolysis might be useful in predicting in vivo prececal starch digestibility. The digestion kinetic characteristics of different starch sources might be employed to evaluate the starch digestive rate at the pig ileum.     

Enzymatic synthesis and identification of two trisaccharides produced from lactulose by transgalactosylation

The enzymatic transgalactosylation during lactulose hydrolysis was studied using the beta-galactosidase from Kluyveromyces lactis and an initial lactulose concentration of 250 g/L. During hydrolysis of lactulose, the formation of two novel trisaccharides was followed by high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD). A maximum trisaccharide yield of 14.05% was observed at 91.9% of lactulose hydrolysis. The two novel trisaccharides obtained by transglycosylation of lactulose were isolated and fully characterized by an extensive nuclear magnetic resonance (NMR) study. Complete structure elucidation and full proton and carbon assignment were carried out using 1D ( 1H, 13C, and 1D TOCSY) and 2D (gCOSY, TOCSY, ROESY, gHSQC, and gHMBC) NMR experiments. The trisaccharides were shown to be lactulose-based structures; the main one has a Gal unit linked to C-6 of the galactose moiety, and the other one has a Gal unit linked to C-1 of the fructose moiety. Transglycosylation of lactulose allows for the obtention of galacto-oligosaccharides with new glycosidic structures and would open new routes to the synthesis of prebiotics.     

Development of an oat-based biorefinery for the production of L(+)-lactic acid by Rhizopus oryzae and various value-added coproducts

A novel oat-based biorefinery producing L(+)-lactic acid and various value-added coproducts (e.g., beta-glucan, anti-irritant solution) is proposed. Pearling is employed for sequential separation of bran-rich fractions for the extraction of value-added coproducts. Lactic acid production is achieved via fungal fermentation of Rhizopus oryzae on pearled oat flour. Maximum lactic acid concentration (51.7 g/L) and starch conversion yield (0.68 g/g) were achieved when an oat flour concentration of 116.5 g/L was used. Oxygen transfer played a significant role with respect to lactic acid production and starch conversion yield. Rhizopus oryzae produced a range of enzymes (glucoamylase, protease, phosphatase) for the hydrolysis of cereal flour macromolecules. Enzyme production during fungal fermentation has been reported. The proposed biorefining strategy could lead to significant operating cost reduction as compared to current industrial practices for lactic acid production from pure glucose achieved by bacterial fermentations.     

Pretreatment and Enzymatic Hydrolysis of Sorghum Bran

Sorghum bran has potential to serve as a low‐cost feedstock for production of fuel ethanol. Sorghum bran from a decortication process (10%) was used for this study. The approximate chemical composition of sorghum bran was 30% starch, 18% hemicellulose, 11% cellulose, 11% protein, 10% crude fat, and 3% ash. The objective of this research was to evaluate the effectiveness of selected pretreatment methods such as hot water, starch degradation, dilute acid hydrolysis, and combination of those methods on enzymatic hydrolysis of sorghum bran. Methods for pretreatment and enzymatic hydrolysis of sorghum bran involved hot water treatment (10% solid, w/v) at 130°C for 20 min, acid hydrolysis (H₂SO₄), starch degradation, and enzymatic hydrolysis (60 hr, 50°C, 0.9%, v/v) with commercial cellulase and hemicellulose enzymes. Total sugar yield by using enzymatic hydrolysis alone was 9%, obtained from 60 hr of enzyme hydrolysis. Hot water treatment facilitated and increased access of the enzymes to hemicellulose and cellulose, improving total sugar yield up to 34%. Using a combination of starch degradation, optimum hot water treatment, and optimum enzymatic hydrolysis resulted in maximum total sugar yield of up to 75%.     

Intact carbohydrate structures as part of the melanoidin skeleton

Model melanoidins from monomeric, oligomeric, and polymeric carbohydrates, and amino acids formed under aqueous as well as water-free reaction conditions, were submitted to acidic catalyzed hydrolysis. Their degradation products were detected qualitatively and quantitatively by HPTLC and HPLC-DAD. A considerable amount of monomer carbohydrates from hydrolysis of model melanoidins formed under water-free reaction conditions was detected. It can be seen clearly that the amount of carbohydrates released increased with increasing degree of polymerization of the carbohydrates used as starting material. In comparison, the hydrolysis of melanoidins formed in aqueous condition resulted in only a small glucose release. It seems that in the Maillard reaction under water-free conditions, a significant amount of di- and oligomer carbohydrates were incorporated into the melanoidin skeleton as complete oligomer with intact glycosidic bond, forming side chains at the melanoidin skeleton. Additional side chains could be formed by transglycosylation reactions. With increasing water content, hydrothermolytic as well as retro-aldol reactions of the starting carbonyl components became significant, and therefore the possibility of forming side chains decreased. The results are consistent with the postulated melanoidin structure being built up mainly from sugar degradation products, probably branched via amino compounds.     

Effects of β-amylolysis on the resistant starch formation of debranched corn starches

Retrograded amylose is resistant to digestion by amylolytic enzymes, which is known as resistant starch type III (RS3). In this study we investigated the effect of β-amylase hydrolysis on the formation and physicochemical properties of RS3 from debranched corn starches. Three types of corn starch (Hylon VII, Hylon V, and common corn) were first gelatinized and then hydrolyzed using β-amylase to varying degrees. The resultant hydrolyzed starch was debranched with isoamylase and then exposed to temperature cycling to promote RS formation. A broad endotherm from approximately 45 to 120 °C and a small endotherm above 150 °C were noted for all retrograded starches. All three corn starches had increased RS contents after moderate β-amylolysis, with Hylon V having the highest RS content at 70.7% after 4 h of β-amylolysis. The results suggest that RS3 formation is affected by the starch composition as well as the starch structure and can be increased by moderate β-amylolysis.     

Inhibitory action of palatinose and its hydrogenated derivatives on the hydrolysis of alpha-glucosylsaccharides in the small intestine

This study was conducted to investigate the inhibitory effects of palatinose and Palatinit, which are disaccharides (or disaccharide alcohol) connected through an alpha-1,6-glucosyl linkage, on the hydrolysis of other carbohydrates, using an enzyme extract from the rat small intestine and a purified sucrase-isomaltase complex. Palatinose and its hydrogenated product, Palatinit, an equimolar mixture of alpha-O-D-glucopyranosyl-1,6-D-sorbitol (GPS) and alpha-O-D-glucopyranosyl-1,6-D-mannitol (GPM), inhibited the hydrolysis of sucrose and maltose. Palatinose and Palatinit also inhibited the hydrolysis of dextrin and soluble starch. Kinetic analysis of the enzymatic inhibition by GPS and GPM on sucrose hydrolysis revealed that both GPS and GPM competitively inhibit sucrase catalytic activity. These results suggest that disaccharides with an alpha-1,6-glucosyl linkage competitively inhibit intestinal alpha-glucosidases and may reduce the rate of hydrolysis of sucrose and other alpha-glucosylsaccharides.