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Novel Geobacillus sp. DS3, isolated from the Sikidang Crater in Dieng, exhibits promising characteristics for industrial applications, particularly in thermostable α-amylase production. Recombinant technology was used to express thermostable α-amylase in E. coli BL21(DE3) to overcome high-temperature production challenges. The study aimed to express, purify, characterize, and explore potential applications of this novel enzyme. The enzyme was successfully expressed in E. coli BL21(DE3) at 18 °C for 20 h with 0.5 mM IPTG induction. Purification with Ni-NTA column yielded 69.23 % from the initial crude enzyme, with a 3.6-fold increase in specific activity. The enzyme has a molecular weight of ±70 kDa (±58 kDa enzyme+11 kDa SUMO protein). It exhibited activity over a wide temperature range (30–90 °C) and pH range (6–8), with optimal activity at 70 °C and pH 6 with great stability at 60 °C. Kinetic analysis revealed Km and Vmax values of 324.03 mg/ml and 36.5 U/mg, respectively, with dextrin as the preferred substrate without cofactor addition. As a metalloenzyme, it showed the best activity in the presence of Ca2+. The enzyme was used for porous starch production and successfully immobilized with chitosan, exhibiting improved thermal stability. After the fourth reuse, the immobilized enzyme maintained 62 % activity compared to the initial immobilization.

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Porous starches are essential modified starches that have potential application as natural absorbents and contain abundant micro sized pores. Increasing interest is directed to utilizing the properties of porous starches in various starchy foods, such as Canna edulis Kerr. To date, no report about edible canna porous starch is available. Therefore, this study aimed to characterize the porous starch from edible canna produced by enzymatic hydrolysis using thermostable α-amylase. A starch suspension (25%, w/v) in phosphate buffer (0.1 M, pH 6.5) was added the thermostable α-amylase (100 U/g, 200 U/g and 300 U/g) and then incubated at 60 °C and 80 rpm for 8 h. The pellets were dried at 40 °C for 48 h. The SEM results displayed that the increase in enzyme concentration caused the formation of larger pores on the starch granule surface due to amylose degradation. FTIR and XRD analysis showed that enzymatic hydrolysis did not affect the crystalline structure of the edible canna porous starch. Furthermore, the edible canna porous starch exhibited high solubility and oil and water absorption capacity. Thus, the porous starch from C. edulis Kerr. could be used as a probiotic, flavoring, or drug delivery agent by microencapsulation.

Copyright of Modern Food Science & Technology is the property of Editorial Office of Modern Food Science & Technology and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

A carbohydrate binding module 68 (CBM68) of pullulanase from Anoxybacillus sp. LM18-11 was used to enhance the secretory expression of a thermostable α-amylase (BLA702) in Bacillus subtilis, through an atypical secretion pathway. The extracellular activity of BLA702 guided by CBM68 was 1248 U/mL, which was 12.6 and 7.2 times higher than that of BLA702 guided by its original signal peptide and the endogenous signal peptide LipA, respectively. A single gene knockout strain library containing 51 genes encoding macromolecular transporters was constructed to detect the effect of each transporter on the secretory expression of CBM68-BLA702. The gene knockout strain 0127 increased the extracellular amylase activity by 2.5 times. On this basis, an engineered strain B. subtilis 0127 (AmyE::BLA702-NprB::CBM68-BLA702-PrsA) was constructed by integrating BLA702 and CBM68-BLA702 at the AmyE and NprB sites in the genome of B. subtilis 0127, respectively. The molecular chaperone PrsA was overexpressed, to reduce the inclusion body formation of the recombinant enzymes. The highest extracellular amylase activity produced by B. subtilis 0127 (AmyE::BLA702-NprB::CBM68-BLA702-PrsA) was 3745.7 U/mL, which was a little lower than that (3825.4 U/mL) of B. subtilis 0127 (pMAC68-BLA702), but showing a better stability of passage. This newly constructed strain has potential for the industrial production of BLA702. [ABSTRACT FROM AUTHOR]

Corn starch was dually modified using thermostable α-amylase and pullulanase to prepare resistant starch (RS). The concentration of starch liquid, the amount of added thermostable α-amylase, the duration of enzymatic hydrolysis and the amount of added pullulanase were optimized using RSM to increase RS content of the treated sample. The optimum pretreatment conditions were 15% starch liquid, 3 U/g thermostable α-amylase, 35 min of enzymatic hydrolysis and 8 U/g pullulanase. The maximum RS content of 10.75% was obtained, and this value was significantly higher than that of native corn starch. The degree of polymerization (DP) of the enzyme-modified starch decreased compared with that of native starch. The scanning electron microscopy (SEM) and differential scanning calorimetry (DSC) were performed to assess structural changes in native and pretreated starch. The effect of dual enzyme pretreatment on the structure and properties of corn starch was significant. Unlike the untreated one, the pretreated corn starch showed clear pores and cracks. Significant differences in RS contents and structural characterization between starch pretreated and untreated with dual enzymes demonstrated that the dual enzyme modification of corn was effective in enhancing RS contents. [ABSTRACT FROM AUTHOR]

A carbohydrate binding module 68 (CBM68) of pullulanase from Anoxybacillus sp. LM18-11 was used to enhance the secretory expression of a thermostable α-amylase (BLA702) in Bacillus subtilis, through an atypical secretion pathway. The extracellular activity of BLA702 guided by CBM68 was 1248 U/mL, which was 12.6 and 7.2 times higher than that of BLA702 guided by its original signal peptide and the endogenous signal peptide LipA, respectively. A single gene knockout strain library containing 51 genes encoding macromolecular transporters was constructed to detect the effect of each transporter on the secretory expression of CBM68-BLA702. The gene knockout strain 0127 increased the extracellular amylase activity by 2.5 times. On this basis, an engineered strain B. subtilis 0127 (AmyE::BLA702-NprB::CBM68-BLA702-PrsA) was constructed by integrating BLA702 and CBM68-BLA702 at the AmyE and NprB sites in the genome of B. subtilis 0127, respectively. The molecular chaperone PrsA was overexpressed, to reduce the inclusion body formation of the recombinant enzymes. The highest extracellular amylase activity produced by B. subtilis 0127 (AmyE::BLA702-NprB::CBM68-BLA702-PrsA) was 3745.7 U/mL, which was a little lower than that (3825.4 U/mL) of B. subtilis 0127 (pMAC68-BLA702), but showing a better stability of passage. This newly constructed strain has potential for the industrial production of BLA702.

Novel Geobacillus sp. DS3, isolated from the Sikidang Crater in Dieng, exhibits promising characteristics for industrial applications, particularly in thermostable α-amylase production. Recombinant technology was used to express thermostable α-amylase in E. coli BL21(DE3) to overcome high-temperature production challenges. The study aimed to express, purify, characterize, and explore potential applications of this novel enzyme. The enzyme was successfully expressed in E. coli BL21(DE3) at 18 °C for 20 h with 0.5 mM IPTG induction. Purification with Ni-NTA column yielded 69.23 % from the initial crude enzyme, with a 3.6-fold increase in specific activity. The enzyme has a molecular weight of ±70 kDa (±58 kDa enzyme+11 kDa SUMO protein). It exhibited activity over a wide temperature range (30-90 °C) and pH range (6-8), with optimal activity at 70 °C and pH 6 with great stability at 60 °C. Kinetic analysis revealed Km and Vmax values of 324.03 mg/ml and 36.5 U/mg, respectively, with dextrin as the preferred substrate without cofactor addition. As a metalloenzyme, it showed the best activity in the presence of Ca 2+ . The enzyme was used for porous starch production and successfully immobilized with chitosan, exhibiting improved thermal stability. After the fourth reuse, the immobilized enzyme maintained 62 % activity compared to the initial immobilization., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2024 The Authors. Published by Elsevier B.V.)

Thermostable α-amylase from germinating Sword bean ( Canavalia gladiata (Jacq.) DC.) seeds has been successfully immobilized on DEAE-cellulose (ICgAmy1) and chitosan bead (ICgAmy2) support materials. Optimum conditions of immobilization for DEAE-cellulose and chitosan bead revealed 97% and 96% immobilization yield, respectively. The optimum pH and temperature of both DEAE-cellulose and chitosan bead immobilized α-amylases were pH 7 and 70°C. Both ICgAmy1 and ICgAmy2 were high stability over a wide pH range of pH 5-9 and a temperature range of 70-90°C. In addition, ICgAmy1 and ICgAmy2 led to an operationally stable biocatalyst with above 74% and 76% residual activity after 10 reuses, respectively. Immobilized α-amylases showed high storage stability with 81% (ICgAmy1) and 85% (ICgAmy2) residual activity after 120 days of storage. The easy immobilization process on low-cost, biodegradable, and renewable support materials exhibited an increase in the enzyme operation range and storage stability which reduces production costs. This makes immobilized amylases an effective biocatalyst in various industrial applications especially a potential candidate for bioethanol production, a key renewable energy source., Competing Interests: Conflict of interestThe authors declare no conflicts of interest., (© 2024 Japanese Society for Plant Biotechnology.)

Corn starch was dually modified using thermostable α-amylase and pullulanase to prepare resistant starch (RS). The concentration of starch liquid, the amount of added thermostable α-amylase, the duration of enzymatic hydrolysis and the amount of added pullulanase were optimized using RSM to increase RS content of the treated sample. The optimum pretreatment conditions were 15% starch liquid, 3 U/g thermostable α-amylase, 35 min of enzymatic hydrolysis and 8 U/g pullulanase. The maximum RS content of 10.75% was obtained, and this value was significantly higher than that of native corn starch. The degree of polymerization (DP) of the enzyme-modified starch decreased compared with that of native starch. The scanning electron microscopy (SEM) and differential scanning calorimetry (DSC) were performed to assess structural changes in native and pretreated starch. The effect of dual enzyme pretreatment on the structure and properties of corn starch was significant. Unlike the untreated one, the pretreated corn starch showed clear pores and cracks. Significant differences in RS contents and structural characterization between starch pretreated and untreated with dual enzymes demonstrated that the dual enzyme modification of corn was effective in enhancing RS contents.

The Caribbean king crab Maguimithrax spinosissimus is an omnivorous crustacean with high feeding plasticity. We characterized the main digestive enzymes of this crab species. Alpha-amylase and esterase activities were higher in the hepatopancreas, while those of trypsin and chymotrypsin were higher in the gastric fluid. Only one α-amylase isoform of 40 kDa generates a high α-amylase activity in this crab with a maximum at pH 5.5, 40–60°C, and 2.5 mM CaCl2. A high proteolytic activity was observed in the gastric chamber, while the activity of these enzymes was low in hepatopancreas extracts due to the presence of an inhibitor that was further demonstrated by enzyme assays and reverse zymography. Several proteases from 10 to 45 kDa are synthesized in the hepatopancreas and selectively secreted into the gut. Trypsin-like and chymotrypsin-like activities were highest at pH 7, 60°C, 0.02–1.5 M NaCl, 25 mM CaCl2. These activities were poorly influenced by Ca2+. Both enzymes were stable at a neutral to alkaline pH and a temperature up to 40°C. Esterase activity peaked at pH 9.0, 37°C, 2 M NaCl, being also poorly affected by Ca2+ but temperature sensitive. In summary, high α-amylase and protease activities, a high protease abundance, as well as a tight control over their activity by an unknown inhibitor-based mechanism appear suited for M. spinsissimus to feed opportunistically on a diet of benthic algae and epifauna with eventual carnivorous preferences. [ABSTRACT FROM AUTHOR]

Abstract: Problems with rehydration and palatability are considered as unacceptable quality characteristics for the noodles produced using high‐strength extrusion technique. Thus, the aim of this study was to solve these problems by designing a novel method to create a porous structure for the high‐strength extruded noodles (HENs). The quality indices of HENs were significantly improved after adding to them thermostable α‐amylase (TαA) at 0.05% to 0.10%. The microstructure graphs showed that a well‐developed porous structures was successfully created throughout noodle strands. This indicated that the TαA has effectively worked on starch granules in spite of the high‐strength performance of the extrusion process. MALLS‐GFC, X‐ray diffraction, and differential scanning calorimeter investigations showed that the appearance of a porous structure was mainly attributed to the internal collapse of α‐1,4‐glycosidic bonds and the dissolution of water‐soluble degradation products, such as dextrin and oligosaccharides. Moreover, the slight inhibited effect of excess TαA on the starch gelatinization was because of the fact that the high enzyme concentration might cause TαA to adhere or overlay on it. Practical Application: The rehydration and palatability properties of HENs were greatly improved by creating a well‐developed or honeycomb‐like porous structure using TαA at low concentration. The findings of this study could be applied to enhance the quality characteristics of HENs and to encourage the research and development in the noodle industry. [ABSTRACT FROM AUTHOR]

A new Rhizopus oryzae strain FSIS4 isolated from wheat seed cultivated in Algerian Sahara was evaluated for the production of a thermostable α-amylase enzyme. Medium components were optimized by a statistical approach for the optimal production of α-amylase using an agro-industrial residue (decommissioned dates). The Plackett-Burman design was performed to screen the fermentation variables and response surface methodology for optimizing the significant factors affecting the production. The optimum levels for maximum production of α-amylase (approx 10,151 IU) were determined. A 1.8-fold increase of production was obtained in comparison to the non optimized medium. Partial characterization of the enzyme showed optimal temperature and pH at 60 °C and 5.0, respectively. The enzyme was found to be stable at 80 and 90 °C, with a half-life of 165 and 105 min, respectively. According to the large literature, this is the first report on the production of α-amylase by the thermophilic R. oryzae strain FSIS4. The thermo and pH stability of the α-amylase suggest its wide application in the food and pharmaceutical industries. [ABSTRACT FROM AUTHOR]

Gracilaria lemaneiformis ( G. lemaneiformis) is rich in polysaccharides that have many functional activities. In this study, the thermostable α-amylase-assisted extraction and antioxidant activity of water-soluble polysaccharides from G. lemaneiformis were investigated. The extraction conditions were optimized as follows: time, 40 min; temperature, 95 °C; pH, 5 and enzyme amount, 6000 U/g, under which the yield of G. lemaneiformis polysaccharides (GPs) reached 49.15% dry base. The GPs were characterized by monosaccharide composition, FTIR spectrum, UV spectrum and antioxidant activities. The results indicate that the GPs had strong hydroxyl radical activity at a concentration of 100 μg/mL and could be used as potential antioxidants. [ABSTRACT FROM AUTHOR]

Amylases and proteases are utilized in industrial processes such as starch liquefaction or as supplements for washing agents. For these applications it is desirable to have enzymes active at high temperatures (>70 °C). In this work, thermostable α-amylase and neutral proteases were produced using the thermophilic strain Bacillus caldolyticus DSM 405. The goal of this work is to reduce the cost of production media by substituting expensive medium components such as prehydrolyzed starch and peptone, used in control fermentations, by inexpensive food industry wastes such as potato fruit water, potato pulp, cheese whey, draff, pea pulp, pea fruit water, bread residues, and pork blood. Comparative studies were conducted in shake flasks. With the use of such wastes, significant improvements in the activities of the enzyme α-amylase were obtained along with concomitant reductions in medium costs. With the use of pea pulp, 160 % increase in the activity of α-amylase was observed with 97 % reduction in medium costs compared to control medium. The cost of medium for the production of proteases also decreased by more than 50 %.

The uses of thermostable starch hydrolytic biocatalysts are steadily increasing for the industrial application because of their obvious need for biocatalytic performance at elevated temperatures. The starch liquefaction and saccharification can be carried out simultaneously by the use of thermostable starch hydrolytic biocatalysts, thus minimizing the unit operations, time, and efforts. The cost factor hampers the industrialization of expensive soluble (free) enzymes for biocatalytic applications and the immobilization of enzymes offers promising alternative to the hurdle. The present investigation was aimed for immobilization of thermostable α-amylase using calcium alginate, and statistical optimization studies were carried out for enhanced biocatalytic performance. Initially, one-parameter at a time optimization studies were carried out for identification of significant factors influencing the immobilization. Furthermore, a statistical approach, response surface methodology, was applied for immobilization of α-amylase. The immobilized α-amylase in alginate microbeads showed enhanced stability to temperature and reusable property for up to seven cycles (with the retention of 50% initial activity). Finally, the kinetic behavior of free and immobilized enzyme showed the Km value of 1.2% and 2.6% (w/v) and Vmax of 1,020 and 1,030 U, respectively. Fifty percent reduction in affinity of the immobilized enzyme toward substrate was compensated by its longer stability. [ABSTRACT FROM AUTHOR]

In order to investigate the effect of thermostable α-amylase on the rice physicochemical properties, free/bound phenolics, and antioxidant capacities during extrusion, seven different gradients of enzyme concentration from 0 to 6 ‰ (db of starch) were used for both hulled and whole rice. For rice treated with the same extrusion conditions, special mechanical energy (SME) input changed slightly in the presence of fiber fraction (bran/husk), while decreased greatly with increasing α-amylase levels due to the reduced viscosity and friction between mechanical elements and materials. Huge physicochemical modifications were achieved in expansion ratio (ER), bulk density (BD), water absorption/solubility indices (WAI/WSI), and viscosity via enzymatic liquefaction. The total phenolic content (TPC) retention increased dramatically (93.06 and 86.33 % for hulled and whole rice, respectively) with increasing enzyme concentrations to 6 ‰, indicating that a milder reaction environment was created with less thermal-mechanical impact on phenolics. By linear analysis, the TPC of extruded rice hulled or not was strongly and positively related with BD ( R = 0.9185 or 0.9719) and WSI ( R = 0.9719 or 0.8857), whereas negatively related with SME ( R = −0.9460 or −0.8906). Besides, the free/bound phenolics combined with antioxidant activities (1,1-diphenyl-2-picryl-hydrazyl (DPPH), 2,2-azino-bis-(3-ethylbenzothiazoline-6-sulphonic acid (ABTS), and ferric reducing antioxidant power (FRAP)) were all improved significantly by the separate/synergetic addition of enzyme and bran/husk used as, respectively, the 'special protective agent' and rich source of bioactive compounds. In summary, thermostable α-amylase could achieve many beneficial effects on both physicochemical properties and antioxidant activities of rice extruded with/without fiber for promoting the development of cake making, wine brewing, infant food industries, or animal husbandry. [ABSTRACT FROM AUTHOR]

Cross-linked enzyme aggregates (CLEAs) of thermostable α-amylase from Bacillus licheniformis have been prepared for starch liquefaction. Among the six different precipitants, tert -butanol performed the best with an aggregation efficiency of 99.54% for starch hydrolysis. The optimal conditions for the immobilization process required 5 mM glutaraldehyde, 0.96 mg/mL enzyme, 1:2 ratio (0.5 ratio) of enzyme/bovine serum albumin (BSA), and 12 h crosslinking at 2–3 °C. Starch was used as the main substrate in enzyme assay. Immobilization did not affect pH (5.5) and temperature (95 °C) optima of free α-amylase thus, these values remained constant for produced CLEAs. Different concentrations of calcium and sodium ions were added to the enzyme during the aggregation process. It was observed that simultaneous addition of both calcium and sodium (1200 ppm of calcium and 400 ppm of sodium ions, in a ratio of 3:1) significantly improved the catalytic efficiency ( k cat / K m ), of CLEAs–BSA–CN from 3.91 × 10 5 to 4.57 × 10 5 (1.2-folds) compared with free enzyme. Although immobilization did not significantly affect V max (5.35–5.25), substrate affinity of the enzyme increased (1.34–1.12) after addition of mixed ions to the prepared CLEAs. Moreover, compared with free α-amylase, enzyme half-life ( t 1/2 ) of CLEAs–BSA–CN increased from 43.31 to 115 min (about 3-folds) at 110 °C. The CLEAs–BSA–CN retained 76% residual activity even after 10 cycles of reuse. [ABSTRACT FROM AUTHOR]

Current work is aimed to evaluate the lactose induced expression of Thermotoga petrophila α-amylase gene under T7 promoter in E. coli. Recombinant protein was expressed in nine different media (LB, ZB, 4xZB, ZBM, ZYB, 3xZYB, M9, ZYBM9 and 3xZYBM9) by using Isopropyl β-D-1-thiogalactopyranoside (IPTG) and lactose as inducer. Lactose was found to be the better inducer in all the media with maximum expression in tryptone rich ZBM medium. The enzyme expression increased when host cells were given heat shock (42°C) for 1 h immediately after induction with lactose. The parameters such as lactose concentration (200 mM), cell density at the time of induction (OD600 0.8), and incubation temperature (37°C) were also optimized, that resulted in 1.5 fold increase in intracellular α-amylase activity. Thus, lactose as inducer is recommended to be used for the enhanced expression of recombinant α-amylase in tryptone rich medium. © 2014 Friends Science Publishers [ABSTRACT FROM AUTHOR]

The gene (1,542 bp) encoding thermostable Ca-independent and raw starch hydrolyzing α-amylase of the extremely thermophilic bacterium Geobacillus thermoleovorans encodes for a protein of 50 kDa (Gt-amyII) with 488 amino acids. The enzyme is optimally active at pH 7.0 and 60 °C with a t of 19.4 h at 60 and 4 h at 70 °C. Gt-amyII hydrolyses corn and tapioca raw starches efficiently and therefore finds application in starch saccharification at industrial sub-gelatinisation temperatures. The starch hydrolysis is facilitated following adsorption of the enzyme to starch at the C-terminal domain, as confirmed by the truncation analysis. The adsorption rate constant of Gt-amyII to raw corn starch is 37.6-fold greater than that for the C-terminus truncated enzyme (Gt-amyII-T). Langmuir-Hinshelwood kinetic analysis in terms of equilibrium parameter ( K) suggested that the adsorption of Gt-amyII to corn starch is more favourable than that of Gt-amyII-T. Thermodynamics of temperature inactivation indicated a decrease in thermostabilisation of Gt-amyII upon truncation of its C-terminus. The addition of raw corn starch increased t of Gt-amyII, but it has no such effect on Gt-amyII-T. It can, therefore, be stated that Gt-amyII binds to raw corn starch via C-terminal region that contributes to its thermostability. Phylogenetic analysis confirmed that starch binding region of Gt-amyII is, in fact, the non-catalytic domain C, and not the typical SBD of CBM families. The role of domain C in raw starch binding throws light on the evolutionary path of the known SBDs. [ABSTRACT FROM AUTHOR]

An α-amylase gene was cloned from the thermophilic bacterium Bacillus subtilis isolated from Indonesian oil palm shell waste. The gene expressed an extracellular enzyme. Optimal hydrolysis conditions for the enzyme were 70°C and pH 6.0. The specific activity of the enzyme was 16.0 kU per mg of protein, which was higher than for other thermostable amylases. Hydrolytic products of the enzyme using starch and glycogen were mainly maltohexaose and maltopentaose. The enzyme had a K value of 0.099 mg/mL for amylopectin, more than 10 times lower than for amylose. The catalytic efficiency of the enzyme using amylopectin was 39,200 mL/mg·s and was 3,270 mL/mg·s using amylose. The enzyme liquefied corn starch at pH 5.0, which was successfully converted to glucose using commercial glucoamylase and pullulanase without pH adjustment. The enzyme has advantages for industrial applications. [ABSTRACT FROM AUTHOR]

BACKGROUND In industry, a jet cooker is used to gelatinize starch by mixing the starch slurry with steam under pressure at 100-175 °C. A higher degree of starch hydrolysis in an extruder is possible with glucoamylase. Unfortunately, it is difficult to carry out liquefaction and saccharification in parallel, because the temperature of gelatinization will be too high and will inactivate glucoamylase. Since the temperature for liquefaction and saccharification is different, it is hard to change the temperature from high (required for liquefaction) to low (required for saccharification). The industrial gelatinization process is usually carried out with 30-35% (w/w) dry solids starch slurry. Conventional jet cookers cannot be used any more at high substrate concentrations owing to higher viscosity. In this study, therefore, corn starch was extruded at different melt temperatures to overcome these limitations and to produce the highest enzyme-accessible starch extrudates. RESULTS Significant effects on physical properties (water solubility index, water absorption index and color) and chemical properties (reducing sugar and % increase in reducing sugar after saccharification) were achieved by addition of thermostable α-amylase at melt temperatures of 115 and 135 °C. However, there was no significant effect on % increase in reducing sugar of extruded corn starch at 95 °C. CONCLUSION The results show the great potential of extrusion with thermostable α-amylase injection at 115 and 135 °C as an effective pretreatment for breaking down starch granules, because of the significant increase ( P < 0.05) in % reducing sugar and enzyme-accessible extrudates for saccharification yield. © 2013 Society of Chemical Industry [ABSTRACT FROM AUTHOR]

Expression of foreign enzymes in yeast is a traditional genetic engineering approach; however, useful secretory enzymes are not produced in every case. The hyperthermostable α-amylase encoded by the AmyL gene of Bacillus licheniformis was expressed in Saccharomyces cerevisiae; however, it was only weakly produced and was degraded by the proteasome. To determine the cause of low α-amylase production, AmyL was expressed in a panel of yeast mutants harboring knockouts in non-essential genes. Elevated AmyL production was observed in 44 mutants. The knockout genes were classified into six functional categories. Remarkably, all non-essential genes required for N-linked oligosaccharide synthesis and a gene encoding an oligosaccharyl transferase subunit were identified. Immunoblotting demonstrated that differently underglycosylated forms of AmyL were secreted from oligosaccharide synthesis-deficient mutants, while a fully glycosylated form was produced by wild-type yeast, suggesting that N-linked glycosylation of AmyL inhibited its secretion in yeast. Mutational analysis of six potential N-glycosylation sites in AmyL revealed that the N33Q and N309Q mutations remarkably affected AmyL production. To achieve higher AmyL production in yeast, all six N-glycosylation sites of AmyL were mutated. In wild-type yeast, production of the resulting non-glycosylated form of AmyL was threefold higher than that of the glycosylated form. [ABSTRACT FROM AUTHOR]

Abstract: A gene encoding acidic, thermostable and raw starch hydrolysing α-amylase was cloned from an extreme thermophile Geobacillus thermoleovorans and expressed. The ORF of 1650bp encodes a 515 amino acid protein (Gt-amy) with a signal peptide of 34 amino acids at the N-terminus. Seven conserved sequences of GH-13 family have been found in its sequence. The specific enzyme activity of recombinant Gt-amy is 1723Umg−1 protein with a molecular mass of 59kDa. It is optimally active at pH 5.0 and 80°C with t 1/2 values of 283, 184 and 56min at 70, 80 and 90°C, respectively. The activation energy required for its temperature deactivation is 84.96kJmol−1. Ca2+ strongly inhibits Gt-amy at 10mM concentration, and inhibition kinetics with Ca2+ reveals that inhibition occurs as a result of binding to a lower affinity secondary Ca2+ binding site in the active centre in a mixed-type inhibition manner. The K m and k cat of the Gt-amy are 0.315mgmL−1 and 2.62×103 s−1, respectively. Gt-amy is Ca2+-independent at the concentration used in industrial starch saccharification, and hydrolyses raw corn and wheat starches efficiently, and thus, is applicable in starch saccharification at the industrial sub-gelatinization temperatures. [Copyright &y& Elsevier]

Two genes that encode α-amylases from two Anoxybacillus species were cloned and expressed in Escherichia coli. The genes are 1,518 bp long and encode 506 amino acids. Both sequences are 98% similar but are distinct from other well-known α-amylases. Both of the recombinant enzymes, ASKA and ADTA, were purified using an α-CD-Sepharose column. They exhibited an optimum activity at 60°C and pH 8. Both amylases were stable at pH 6-10. At 60°C in the absence of Ca, negligible reduction in activity for up to 48 h was observed. The activity half-life at 65°C was 48 and 3 h for ASKA and ADTA, respectively. In the presence of Ca ions, both amylases were highly stable for at least 48 h and had less than a 10% decrease in activity at 70°C. Both enzymes exhibited similar end-product profiles, and the predominant yield was maltose (69%) from starch hydrolysis. To the best of our knowledge, most α-amylases that produce high levels of maltose are active at an acidic to neutral pH. This is the first report of two thermostable, alkalitolerant recombinant α-amylases from Anoxybacillus that produce high levels of maltose and have an atypical protein sequence compared with known α-amylases. [ABSTRACT FROM AUTHOR]

This study is concerned with the co-production of alkaline proteases and thermostable α-amylase by some feather-degrading Bacillus strains: B. mojavensis A21, B. licheniformis NH1, B. subtilis A26, B. amyloliquefaciens An6 and B. pumilus A1. All strains produced both enzymes, except B. pumilus A1, which did not exhibit amylolytic activity. The best enzyme co-production was obtained by the NH1 strain when chicken feathers were used as nitrogen and carbon sources in the fermentation medium. The higher co-production of both enzymes by B. licheniformis NH1 strain was achieved in the presence of 7.5 g/l chicken feathers and 1 g/l yeast extract. Strong catabolic repression on protease and α-amylase production was observed with glucose. Addition of 0.5% glucose to the feather medium suppressed enzyme production by B. licheniformis NH1. The growth of B. licheniformis NH1 using chicken feathers as nitrogen and carbon sources resulted in its complete degradation after 24 h of incubation at 37°C. However, maximum protease and amylase activities were attained after 30 h and 48 h, respectively. Proteolytic activity profiles of NH1 enzymatic preparation grown on chicken feather or casein-based medium are different. As far as we know, this is the first contribution towards the co-production of α-amylase and proteases using keratinous waste. Strain NH1 shows potential use for biotechnological processes involving keratin hydrolysis and industrial α-amylase and proteases co-production. Thus, the utilization of chicken feathers may result in a cost-effective process suitable for large-scale production. [ABSTRACT FROM AUTHOR]

Abstract: This paper reports the effect of α-amylase encapsulation on the degradation rate of a starch-based biomaterial. The encapsulation method consisted in mixing a thermostable α-amylase with a blend of corn starch and polycaprolactone (SPCL), which were processed by compression moulding to produce circular disks. The presence of water was avoided to keep the water activity low and consequently to minimize the enzyme activity during the encapsulation process. No degradation of the starch matrix occurred during processing and storage (the encapsulated enzyme remained inactive due to the absence of water), since no significant amount of reducing sugars was detected in solution. After the encapsulation process, the released enzyme activity from the SPCL disks after 28days was found to be 40% comparatively to the free enzyme (unprocessed). Degradation studies on SPCL disks, with α-amylase encapsulated or free in solution, showed no significant differences on the degradation behaviour between both conditions. This indicates that α-amylase enzyme was successfully encapsulated with almost full retention of its enzymatic activity and the encapsulation of α-amylase clearly accelerates the degradation rate of the SPCL disks, when compared with the enzyme-free disks. The results obtained in this work show that degradation kinetics of the starch polymer can be controlled by the amount of encapsulated α-amylase into the matrix. [Copyright &y& Elsevier]

The recombinant plasmid pC194Amy harbouring 5.25 kbp DNA fragment encoding the thermostable α-amylase gene of Bacillus stearothermophilus was introduced into Bacillus subtilis YB886 and B. subtilis BR151 strains by electroporation. The expressing of the α-amylase gene in recombinant strains were confirmed on starch plate, SDS-PAGE and zymogram analysis. In the condition of 100°C and 15 min, YB886Amy, BR151Amy and ATCC31786 α-amylases were maintained up to 80, 67 and 52% of their maximum activity respectively. The specific enzyme activities of YB886Amy, BR151Amy and ATCC 31786 were estimated as 2.59, 2.98 and 2.9 μM/mg protein/min respectively. [ABSTRACT FROM AUTHOR]

Abstract: A newly isolated Bacillus subtilis JS-2004 strain was cultured in liquid media containing waste potato starch to produce α-amylase. The effect of calcium, yeast extract and glucose supplementation of the production medium on bacterial growth and enzyme production was studied. Maximum enzyme production 72U/mL was achieved after 48h cultivation at pH 7.0 and 50°C. Addition of calcium and yeast extract enhanced microbial growth and enzyme production, where as glucose at 1.0% level showed a strong repression. Studies on crude α-amylase characterization revealed that optimum activity was at pH 8.0 and 70°C. The enzyme was quite stable for 1h at 60 and 70°C, while at 80 and 90°C, 12% and 48% of the original activities were lost, respectively. After incubation of crude enzyme solution for 24h at pH 8.0 at 70°C, a decrease of about 6% of its original activity was observed. The enzyme was activated by Ca2+ (relative activity 117%). It was strongly inhibited by Co2+, Cu2+, and Hg2+ but less affected by Mg2+, Zn2+, Ni2+, Fe2+, and Mn2+. The B. subtilis JS-2004 strain produced high levels of thermostable α-amylase with characteristics suitable for application in starch processing and food industries. [Copyright &y& Elsevier]

Abstract: A novel thermostable α-amylase, D45 was studied for its reaction pattern on starch hydrolysis. Fine structures of the dextrins and oligosaccharides produced by D45 were determined and compared with those produced by other thermostable α-amylases, Termamyl®LC (LC) and Termamyl®SC (SC). Waxy maize starch dispersion was hydrolyzed with LC, SC and D45 at different concentrations to obtain hydrolysates with the same dextrose equivalent value (DE). At DE ∼13, molecular weight distribution of dextrins produced by D45 displayed a mono-distribution with a peak centered at degree of polymerization (DP) of 7, whereas LC and SC hydrolysates displayed a bimodal-distribution of the molecular weight profiles with one peak centered at DP 5 and the other at DP 34. Thin-layer chromatograms (TLC) showed that DP 2, 3, 5, 6 and 7 were the primary oligosaccharides produced in LC hydrolysate, DP 4–7 in SC hydrolysate, and DP 6–9 in D45 hydrolysate. Comparison of the decrease in the blue color of amylose-iodine complex at 620nm (blue value) with the increase in reducing value for the hydrolysis of amylose by LC, SC and D45 showed that for an equivalent decrease in blue value, LC and SC produced a higher percentage of reducing sugar than did D45. The results suggest that D45 has a greater degree of random attack (multichain) reaction, whereas LC and SC have more multiple-attack reactions. [Copyright &y& Elsevier]

Abstract: A new strain of Bacillus sp. I-3, isolated from natural soil samples, showed a high raw starch digesting activity towards potato starch. Upon optimization of various environmental and cultural conditions, the yield of α-amylase reached 642U/mL. The kinetic characterization of partially purified enzyme exhibited the maximum activity at 70°C, pH 7.0 and revealed a high thermostability in the presence of 10mM CaCl2·2H2O where it could retain more than 90% residual activity at 70°C after 3.5h. At 80, 90 and 100°C, the enzyme retained 80, 59 and 26% of its maximum activity after 2.5, 0.5 and 0.5h, respectively. The enzyme preparation had a strong affinity towards raw potato starch granules and was almost completely adsorbed onto it. It also hydrolyzed raw potato starch at a concentration of 12.5% significantly in a short period of time of 12h. [Copyright &y& Elsevier]

Thermostable α-amylases have application in a variety of industrial processes and enzymes from a substantial number of thermophilic bacteria and fungi have been screened and characterized to varying degrees. The characteristics of these enzymes are summarized in this review. The genetics of α-amylase production in Bacillus subtilis is reviewed and classical and recombinant DNA approaches to increasing α-amylase production are discussed. [ABSTRACT FROM AUTHOR]

α-amylase which catalyzes the hydrolysis of α-1,4-glycosidic bonds in starch have frequently been cloned into various microbial workhorses to yield a higher recombinant titer. A thermostable SR74 α-amylase from Geobacillus stearothermophilus was found to have a huge potential in detergent industries due to its thermostability properties. The gene was cloned into a CTG-clade yeast Meyerozyma guilliermondii strain SO. However, the CUG ambiguity present in the strain SO has possibly altered the amino acid residues in SR74 amylase wild type (WT) encoded by CUG the codon from the leucine to serine. From the multiple sequence alignment, six mutations were found in recombinant SR74 α-amylase (rc). Their effects on SR74 α-amylase structure and function remain unknown. Herein, we predicted the structures of the SR74 amylases (WT and rc) using the template 6ag0.1.A (PDB ID: 6ag0). We sought to decipher the possible effects of CUG ambiguity in strain SO via in silico analysis. They are structurally identical, and the metal triad (CaI–CaIII) might contribute to the thermostability while CaIV was attributed to substrate specificity. Since the pairwise root mean square deviation (RMSD) between the WT and rc SR74 α-amylase was lower than the template, we suggest that the biochemical properties of rc SR74 α-amylase were better deduced from its WT, especially its thermostability. [ABSTRACT FROM AUTHOR]

• Low-water solid mixing was used for starch-(thermostable α-amylase)-Ca system of bioextrusion. • CaSO 4, C 6 H 10 O 6 Ca and CaCl 2 increased enzyme activity during bioextrusion. • The increased activity (low enzyme level): SO 4 2ˉ> C 6 H 10 O 6 2ˉ > Clˉ > control > NO 3 ˉ > OHˉ ≈ PO 4 3ˉ. • The increased activity (high enzyme level): C 6 H 10 O 6 2ˉ≈ Clˉ ≈ SO 4 2ˉ > control > PO 4 3 > OHˉ > NO 3 ˉ. The effect of six anions (Clˉ, OHˉ, NO 3 ˉ, SO 4 2ˉ, C 6 H 10 O 6 2ˉ and PO 4 3ˉ) on a starch (St)-enzyme (thermostable α -amylase, TαA)-calcium (Ca) system was investigated in a low-moisture solid state. Two levels of Ca salts (1 and 10 mmol/100 g St) added to potato starch with and without TαA were analyzed by FT-IR, DSC and SEM. The surface morphologies of the St-Ca complexes were different in the presence of various anions, and the residual Ca salts around the St granules might decrease the enzymatic action. For bioextrusion, TαA (0.5‰ and 1.5‰) were introduced for a relatively low Ca content (1 mmol/100 g). Significant differences in enzyme activity were observed, increasing the activity of TαA by SO 4 2ˉ (146.54 %) > C 6 H 10 O 6 2ˉ > Clˉ > control > NO 3 ˉ > OHˉ ≈ PO 4 3ˉ and C 6 H 10 O 6 2ˉ (123.20 %) ≈ Clˉ ≈ SO 4 2ˉ > control > PO 4 3 > OHˉ > NO 3 ˉ for the low and high enzyme levels, respectively. [ABSTRACT FROM AUTHOR]

Most commonly used methods for the measurement of starch in food, feeds, and ingredients employ the combined action of α‐amylase and amyloglucosidase to hydrolyze the starch to glucose, followed by glucose determination with a glucose oxidase/peroxidase reagent. Recently, a number of questions have been raised concerning possible complications in starch analytical methods. In this paper, each of these concerns, including starch hydrolysis, isomerization of maltose to maltulose, effective hydrolysis of maltodextrins by amyloglucosidase, enzyme purity and hydrolysis of sucrose, and β‐glucans have been studied in detailed. Results obtained for a range of starch containing samples using AOAC Methods 996.11 and 2014.10 are compared and a new simpler format for starch measurement is introduced. With this method that employs a thermostable α‐amylase (as distinct from a heat stable α‐amylase) which is both stable and active at 100 °C and pH 5.0, 10 samples can be analyzed within 2 h, as compared to the 6 h required with AOAC Method 2014.10. Commonly employed methods for starch analysis have been critically evaluated to ensure starch is measured accurately. Factors such as starch dextrinization, α‐amylase stability, isomerization of maltodextrins, hydrolysis of sucrose, purity of amyloglucosidase and linearity of the glucose/GOPOD standard curve were evaluated. AOAC Method 996.11 and 2014.10 show similar repeatability and reproducibility, but the former method is easier to use and more robust. A simpler version of AOAC Method 996.11 is introduced. [ABSTRACT FROM AUTHOR]

In the AOAC Method 2002.02 assay for resistant starch (RS), the enzyme-resistant starch is solubilized by cold 2 M KOH, so that it can be converted enzymatically to glucose. However, cross-linked phosphorylated (CLP) starch is not soluble in cold 2 M KOH and therefore cannot be assayed directly. In this study, the enzyme-resistant starch from CLP starch was treated by 1) the directions in AOAC Method 996.11for total starch assay using hot DMSO or cold KOH; 2) two back-to-back incubations with thermal-stable α-amylase in a boiling water bath; and 3) a KOH treatment followed by two back-to-back incubations with thermal-stable α-amylase in a boiling water bath. In the last approach, the alkaline was neutralized with HCl, and the salt was removed by centrifugation. More than 95% of the RS from the CLP starch was converted to glucose. This improved method is recommended to directly determine RS in CLP starch., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Metal-rich thermal-solid enzymatic processing of rice combined with yeast fermentation was investigated. 8 Metal ions were exogenously supplied at 0.05, 0.5 and 5mmol/100g (MG) rice prior to static high pressure enzymatic cooking (HPEC) and dynamic enzymatic extrusion cooking (EEC). Treated rice and its fermentation efficiency (FE) were characterized by rapid viscosity analyzer (RVA), UV-Vis, FT-IR and atomic absorption spectrophotometer (AAS). The optimum pH range of enzyme in solid system (>4.9) was broader than in a liquid system (>5.5). Cations decreased enzymatic activity in HPEC probably due to metal-induced aggregation of rice matrix with reduced reacting area as well as strengthened structure of starch/polysaccharides modified by metals. While using the EEC with mechanical mixing/shearing, relative activity was activated to 110 and 120% by Mg 2+ (0.05-0.5MG) and Ca 2+ (0.05-5MG), respectively. Furthermore, the effectiveness of residual ions to promote further FE was found to follow the order: Ca 2+ >K + >Zn 2+ >Mg 2+ >Mn 2+ >Na + ≈Control>Fe 2+ >Cu 2+ , individually., (Copyright © 2017 Elsevier Ltd. All rights reserved.)