Purification and biochemical characterization of novel α-amylase and cellulase from Bacillus sp. PM06.

Bacillus sp. PM06, previously isolated from sugarcane waste pressmud, could produce dual enzymes α-amylase and cellulase. The isolate's crude enzymes were purified homogeneously using ammonium sulfate precipitation followed by High Quaternary amine anion exchange chromatography. Purified enzymes revealed the molecular weights of α-amylase and cellulase as 55 and 52 kDa, with a purification fold of 15.4 and 11.5, respectively. The specific activity of purified α-amylase and cellulase were 740.7 and 555.6 U/mg, respectively. It demonstrated a wide range of activity from pH 5.0 to 8.5, with an optimum pH of 5.5 and 6.4 for α-amylase and cellulase. The optimum temperature was 50 °C for α-amylase and 60 °C for cellulase. The kinetic parameters of purified α-amylase were 741.5 ± 3.75 µmol/min/mg, 1.154 ± 0.1 mM, and 589 ± 3.5/(s mM), using starch as a substrate. Whereas cellulase showed 556.3 ± 1.3 µmol/min/mg, 1.78 ± 0.1 mM, and 270.9 ± 3.8/(s mM) of Vmax, Km, Kcat/Km, respectively, using carboxymethyl cellulose (CMC) as substrate. Among the various substrates tested, α-amylase had a higher specificity for amylose and CMC for cellulase. Different inhibitors and activators were also examined. Ca2+ Mg2+, Co2+, and Mn2+ boosted α-amylase and cellulase activities. Cu2+ and Ni2+ both inhibited the enzyme activities. Enzymatic saccharification of wheat bran yielded 253.61 ± 1.7 and 147.5 ± 1.0 mg/g of reducing sugar within 12 and 24 h of incubation when treated with purified α-amylase and cellulase. A more significant amount of 397.7 ± 1.9 mg/g reducing sugars was released from wheat bran due to the synergetic effect of two enzymes. According to scanning electron micrograph analysis, wheat bran was effectively broken down by both enzymes.

Purification and Characterization of Thermostable α-Amylase from Soil Bacterium Bacillus sp.

BACKGROUND: Amylases are used in several industrial and biotechnological sectors, including those producing textiles, detergents, paper and bakery products. OBJECTIVE: This study aimed to purify an industrially important α-amylase from Bacillus sp. For this purpose, a single and rapid α-amylase purification was performed using the starch affinity method. METHODS: Characterization of the purified enzyme was determined by investigating temperature, pH stability, detergents, and metal ions. RESULTS: The purification coefficient of 29.8-fold with a yield of 9.2% was found. The molecular weight of the purified α-amylase was determined to be 53 kDa by SDS-PAGE, and thermostability was confirmed with 100% activity at 30ºC and 40ºC after 1 h. The purified enzyme was stable over a wide range of pH values, with optimum activity at pH 6.0, 7.0 and 8.0 after 2 h. The study also investigated the effects of the metal ions and detergents on the purified amylase and found that Mg2+ and Ca2+ ions were the activators of the enzyme, while Zn2+, Co2+ and Na+ ions decreased the activity. Furthermore, Hg2+; indicated complete inhibition of amylase activity. The detergents Triton X-100 and Tween 20 increased the α-amylase activity, while sodium dodecyl sulfate inhibited the activity. CONCLUSION: The purified α-amylase obtained from Bacillus sp. is considered to be environmentally friendly, can be processed in a short time, and has a low cost.

A simple 2-step purification process of α-amylase from Bacillus subtilis: Optimization by response surface methodology.

Purification of extracellular α-amylase from Bacillus subtilis was carried out via fractional precipitation by acetone and ion exchange chromatography. These steps provide fast precipitation as well as purification of α-amylase to improve enzyme purity, activity and stability. Compared with two-phase methods in which the yield was less than 1, this method resulted in a yield of more than 3. Moreover, 95% of acetone was recovered that enhanced the economy of the downstream process. Using the data provided by 2D electrophoresis, purification was done by a single step ion exchange chromatography. The enzyme exhibited a molecular mass (SDS-PAGE) of 50KD and the pI of 5. Maximum "yield" and "purification fold" were achieved through optimization of operation parameters such as volume and flowrate of loaded protein using response surface methodology (RSM). 0.5ml of loaded protein at a flow rate of 0.5 ml/min was purified as 48 folds and achieved a specific activity of 524 U/mg.

Design and synthesis of a novel nanocomposite based on magnetic dopamine nanoparticles for purification of α-amylase from the bovine milk.

In this paper, a novel nanocomposite based on magnetic nanoparticles decorated by dopamine were reported. Three modified magnetic nanocomposites by dopamine were offered with different type of linkers. The mentioned magnetic nanocomposites were applied to separate α-amylase protein from fresh bovine milk. All of the magnetic nanocomposites were characterized and investigated by using Fourier-transform infrared spectroscopy, energy-dispersive X-ray spectroscopy, field-emission scanning microscope, X-ray diffraction pattern, and vibrating-sample magnetometer analyses. To investigate the purifying application, sodium dodecyl sulfate polyacrylamide gel electrophoresis, one-dimensional isoelectric focusing gel electrophoresis, and alpha-amylase activity assay were employed. With paying attention to factors such as yield of purification and concentration of separated protein by each of magnetic nanocomposite, it could be concluded that the length of linkers played an important role in α-amylase protein separation. According to the results, the best separation and purification of α-amylase protein with 49.83% recovery and 40.11-fold purification efficiency was related to longest length linker, 1,4-butanediol diglycidyl ether, because of considerable conjugation with nanocomposite. Also, docking calculation has shown that the binding energy is - 1.697 kcal/mol and ΔG = - 6.844 kcal/mol which result that the interaction process between dopamine and α-amylase protein is spontaneous.

A novel pH and thermo-tolerant halophilic alpha-amylase from moderate halophile Nesterenkonia sp. strain F: gene analysis, molecular cloning, heterologous expression and biochemical characterization.

A novel pH and thermo-tolerate halophilic alpha-amylase from moderately halophilic bacterium, Nesterenkonia sp.strain F was cloned and expressed in Escherichia coli. 16S rRNA sequence of the strain shared 99.46% similarities with closely related type species. Also, the genome sequence shared ANI values below 92% and dDDH values below 52% with the closely related type species. Consequently, it is proposed that strain F represents a novel species. The AmyF gene was 1390 bp long and encodes an alpha-amylase of 463 amino acid residues with pI of 4.62. The deduced AmyF shared very low sequence similarity (< 24%) with functionally characterized recombinant halophilic alpha-amylases. The recombinant alpha-amylase was successfully purified from Ni-NTA columns with a molecular mass of about 52 KDa on sodium dodecyl sulfate polyacrylamide gel electrophoresis. The enzyme was active over a wide range of temperature (25-75 °C) and pH (4-9) with optimum activity at 45 °C and 7.5, respectively. Also, although it was active over a various concentrations of NaCl and KCl (0-4 M), increasing activity of the enzyme was observed with increasing concentration of these salts. Low concentrations of Ca2+ ion had no activating effect, but high concentrations of the ion (40-200 mM) enhanced activity of AmyF. The enzyme activity was increased by increasing concentrations of Mg2+, Zn2+, Hg2+ and Fe3+. However, it was inhibited only at very high concentrations of these metal ions. Cu2+ did not decrease the amylase activity and the highest activity was observed at 100 mM of the ion. These properties indicate wide potential applications of this recombinant enzyme in starch processing industries. This is the first isolation, cloning and characterization of a gene encoding alpha-amylase from Nesternkonia genus.

Biochemical and synergistic properties of a novel alpha-amylase from Chinese nong-flavor Daqu.

BACKGROUND: Daqu is the most important fermentation starter for Chinese liquor, with large number of microbes and enzymes being openly enriched in the Daqu system over thousands of years. However, only a few enzymes have been analyzed with crude protein for total liquefying power and saccharifying power of Daqu. Therefore, the complex enzymatic system present in Daqu has not been completely characterized. Moreover, their pivotal and complicated functions in Daqu are completely unknown. RESULTS: In this study, a novel α-amylase NFAmy13B, from GH13_5 subfamily (according to the Carbohydrate-Active enZYmes Database, CAZy) was successfully heterologous expressed by Escherichia coli from Chinese Nong-flavor (NF) Daqu. It exhibited high stability ranging from pH 5.5 to 12.5, and higher specific activity, compared to other GH13_5 fungal α-amylases. Moreover, NFAmy13B did not show activity loss and retained 96% residual activity after pre-incubation at pH 11 for 21 h and pH 12 for 10 h, respectively. Additionally, 1.25 mM Ca2+ significantly improved its thermostability. NFAmy13B showed a synergistic effect on degrading wheat starch with NFAmy13A (GH13_1), another α-amylase from Daqu. Both enzymes could cleave maltotetraose and maltopentaose in same degradation pattern, and only NFAmy13A could efficiently degrade maltotriose. Moreover, NFAmy13B showed higher catalytic efficiency on long-chain starch, while NFAmy13A had higher catalytic efficiency on short-chain maltooligosaccharides. Their different catalytic efficiencies on starch and maltooligosaccharides may be caused by their discrepant substrate-binding region. CONCLUSIONS: This study mined a novel GH13_5 fungal α-amylase (NFAmy13B) with outstanding alkali resistance from Nong-flavor (NF) Daqu. Furthermore, its synergistic effect with NFAmy13A (GH13_1) on hydrolyzing wheat starch was confirmed, and their possible contribution in NF Daqu was also speculated. Thus, we not only provide a candidate α-amylase for industry, but also a useful strategy for further studying the interactions in the complex enzyme system of Daqu.

Immobilization of α-amylase on GO-magnetite nanoparticles for the production of high maltose containing syrup.

Robust amylases with stability and catalysis at multitude of extremities are the need of an hour. Enzyme immobilization may prove beneficial at commercial scale to achieve such attributes. In the present study, a commercially available amylase was immobilized on graphene oxide (GO) - magnetite (Fe3O4) nanoparticles through covalent bonding. The structural and morphological characterizations were conducted by XRD, SEM and TEM. Further, FTIR and TGA confirmed the interaction between amylase, GO and nanoparticles. The variables, such as concentrations of GO (1.3 mg), Fe3O4 (58 µg), and amylase (4.5 mg) were optimized by the response surface methodology using central composite design. High loading capacity of 77.58 µg amylase over 1 µg GO-magnetite nanoparticles was achieved under optimum conditions. Biochemically, the pH optimum remained unaltered, i.e., pH 7, whereas, the alkalitolerance was increased by ~20% in relative activities upon immobilization. The half-life of soluble amylase was 13 h, which enhanced to 20 h upon immobilization in 20 mM phosphate buffer, pH 7 at 50 °C. Besides, the thermodynamic parameters supported the stability trends. The immobilized amylase could be used for 11 subsequent cycles. The mentioned attributes and the dextrose equivalent values during the production of high maltose containing syrup highlighted its commercialization.

Natural edible materials made of protein-functionalized aerogel particles for postprandial hyperglycemia management.

α-Amylase inhibitors (α-AIs) delay digestion of dietary starch by inhibiting α-amylase in the gut, thereby reducing the postprandial glycemia, which is beneficial to the patients with obesity and diabetes. The proteinaceous α-AIs from wheat can effectively control starch digestion and regulate postprandial hyperglycemia. However, their gastric intolerance remains a challenge, which limits its commercial production and industrial application. In this study, sodium alginate/chitosan aerogels loaded with wheat protein α-AIs were prepared and evaluated as potential transportation and protection matrices for important components in food or pharmaceutical applications. Specifically, the biodegradable aerogel cross-linked with sodium alginate-chitosan-calcium chloride, has a large surface area and open porous structure, which can adsorb staple wheat proteins as an integrated edible material to block around 88,660 U/g of α-amylase activity. The aerogel particles were able to protect the activity of wheat α-AIs in the stomach, leading to the slow passage of the wheat α-AIs through the small intestine to inhibit starch digestion more effectively. Animal experiments further showed that the postprandial blood glucose levels in rats were effectively controlled through delayed increase, after administration of wheat protein-functionalized aerogel particles loaded with wheat α-AIs, which are natural biological macromolecules. This is a novel, safe, and economical method for the prevention and pretreatment of diabetes.

Colorado potato beetle alpha-amylase: Purification, action pattern and subsite mapping for exploration of active centre.

Colorado potato beetle is an invasive insect herbivore and one of the most challenging agricultural pests globally. This study is the first characterization of the active centre of Colorado potato beetle (Leptinotarsa decemlineata) α-amylase (LdAmy). Bond cleavage frequency values for LdAmy were determined by HPLC product analysis on a chromophore labelled maltooligomer substrate series. Binding energies between amino acid moieties of subsites and glucose residues of substrate were calculated. Active site contains six subsites in the binding region of LdAmy; four glycone- (-4, -3, -2, -1) and two aglycone-binding sites (+1, +2). Subsite map calculation resulted in apparent binding energies -11.8 and - 11.0 kJ/mol for subsites (+2) and (-3), respectively, which revealed very favorable interactions at these positions. Structures of binding sites of LdAmy and mammalian α-amylases show similarity, but there are variations in the binding energies at subsite (-2) and (-4). Differences were interpreted by comparison of amino acid sequences of human salivary α-amylase (HSA) and porcine pancreatic α-amylase (PPA) and two insect (Leptinotarsa decemlineata and Tenebrio molitor) enzymes. The observed substitution of positively charged His305 in HSA at subsite (-2) with an acidic Asp in LdAmy in the same position may explain the obtained energy reduction.

Characterization and potential application of an α-amylase (BmAmy1) selected during silkworm domestication.

Efficient resource utilization plays a central role in the high productivity of domesticated plants and animals. Whether artificial selection acts on digestive enzymes in the domesticated silkworm (Bombyx mori), which is larger than its wild ancestor, Bombyx mandarina (B. mandarina), remains unknown. In this study, we present the characteristics of a novel alpha-amylase, BmAmy1, in B. mori. The activity of recombinant BmAmy1 was maximal at 35 °C and pH 9.0, and could be suppressed by amylase inhibitors from mulberry, the exclusive food source of silkworms. Three different transposable element fragments, which were independently inserted in the 5'-upstream regulatory region, might be responsible for the enhanced expression of BmAmy1 in different domesticated silkworm strains as revealed by dual-luciferase reporter assay. The BmAmy1 overexpression increased the weight of female and male B. mori by 11.9% and 6.8%, respectively, compared with non-transgenic controls. Our results emphasize that, by exploring the genetic mechanisms of human-selected traits, the domestication process could be further accelerated through genetic engineering and targeted breeding.

Molecular analysis of Bacillus velezensis KB 2216, purification and biochemical characterization of alpha-amylase.

Alpha-amylase producing strain KB 2216 was identified as Bacillus velezensis. The growth pattern showed that 72 h is the optimum incubation period of amylase production, which is a stationary period for the strain. By the purification process, maximum alpha-amylase activity was achieved up to 418.25 U/mL at 72 h of incubation, which was purified with 4.74 folds, 4230.32 U/mg specific activity, with 31.35% yield. The strain was found to produce an oligomeric alpha-amylase, namely Amy3. Amy3 was a trimeric macromolecule of 195 kDa with 62, 64, and 66 kDa subunits, as revealed by zymogram and SDS PAGE analyses. Amy3 was highly active at 55 °C and pH 5.5. It had shown the highest stability at pH 5.0-5.5 and between 0 ̊C and 4 ̊C. It did not require any metal cofactors, but it was inhibited by Ag2+, Hg2+ and Cd2+ divalent cations. Glucose and maltose were shown to be the end products of soluble starch digestion by Amy3. These interesting properties of Amy3 may be useful for many biotechnological applications in the future.

A highly divergent α-amylase from Streptomyces spp.: An evolutionary perspective.

The present study deals with the genetic changes observed in the protein sequence of an α-amylase from Streptomyces spp. and its structural homologs from Pseudoalteromonas haloplanktis, invertebrates and mammals. The structural homologs are renowned for their important features such as chloride binding triad and a serine-protease like catalytic triad (a triad which is reported to be strictly conserved in all chloride-dependent α-amylases). These conserved regions are essential for allosteric activation of enzyme and conformational stability, respectively. An evaluation of these distinctive features in Streptomyces α-amylases revealed the role of mutations in conserved regions and evolution of chloride-independent α-amylases in Streptomyces spp. Besides, the study also discovers a highly divergent α-amylase from Streptomyces spp. which varies greatly even within the homologs of the same genus. Another very important feature is the number of disulfide bridges in which the structural homologs own eight Cys residues to form four disulfide bridges whereas Streptomyces α-amylases possess only seven Cys to form three disulfide bridges. The study also highlights the unique evolution of carbohydrate binding module 20 domain (CBM20 also known as raw starch binding domain or E domain) in Streptomyces α-amylases which is completely absent in α-amylases of other structural homologs.

Purification and biochemical characterization of Arabian balsam α-amylase and enhancing the retention and reusability via encapsulation onto calcium alginate/Fe2O3 nanocomposite beads.

In this study, Arabian balsam α-amylase was purified using the three-step purification method with 9.8-fold purification and 7% recovery. The purified α-amylase's molecular weight was 85 kDa. Calcium alginate incorporated with iron (III) oxide nanoparticles was applied as an immobilizing support for α-amylase. The immobilized α-amylase was characterized by Fourier transform infrared spectroscopy, thermogravimetric analysis, and scanning electron microscopy. In addition, the optimum conditions for immobilization efficiency, immobilization time, reusability, kinetic parameters, and the effect of pH for the immobilization process were examined. After storage, about 87% of the initial activity was maintained at 4 °C for 60 days. The immobilized enzyme exhibited enhanced stability compared to the soluble enzyme in relation to pH and temperature. The immobilized enzyme provided the following kinetic variables: 0.455 mg/mL, 4050 s-1, 28.57 µmol maltose/mL, and 8900 s-1 mg-1 mL for Km, kcat, Vmax, and kcat/Km, respectively, compared with 1.798 mg/mL, 5980 s-1, 42.19 µmol maltose/mL, and 3326 s-1 mg-1 mL for the soluble enzyme. The total phenolic contents of the soluble and immobilized α-amylase-treated wheat kernels were increased by 1.26 and 1.31 fold, respectively. Purified α-amylase from Arabian balsam can thus be successfully used to enhance the antioxidant capacity of cereals.

A Mesohaline Thraustochytrid Produces Extremely Halophilic Alpha-Amylases.

Halophilic bacteria are well known to produce highly salt-tolerant enzymes that have unusual applications in biotechnology. Production of halophilic proteins is generally not expected in mesohaline microorganisms. Ulkenia sp. AH-2, a mesohaline, marine straminipilan thraustochytrid isolated from waters of a mangrove ecosystem, produces halophilic alpha-amylases. Four enzyme fractions, viz.., A, B, C, and D, were obtained upon ammonium sulfate fractionation and gel filtration. These had a broad salinity tolerance ranging from 0 to 4 M NaCl, with an optimum at 3 M NaCl. Pools A, C, and D each resolved as a single band on PAGE and zymogram analysis, and the purified proteins were designated Amy a, Amy c, and Amy h. The major activity resided in "pool B," consisting of several amylases which could not be further resolved into pure fractions. Together, these had an optimum at 2 M NaCl. All the enzymes were stable to storage for 2 to 24 h at 4 °C in a range of salt concentrations and even showed enhanced activity following such incubations. True to halophilic enzymes, the complex of "pool B" amylases showed improved activity in the presence of a wide range of organic solvents at 20% concentration. These enzymes are of particular interest by virtue of their constitutive nature as well as production under culture conditions that do not require salinity beyond that of seawater.

Purification and Characterization of Natural Solid-Substrate Degrading and Alcohol Producing Hyperthermostable Alkaline Amylase from Bacillus cereus (sm-sr14).

OBJECTIVE: Amylases enzymes hydrolyze starch molecules to produce diverse products including dextrins, and progressively smaller polymers. These include glucose units linked through α-1- 1, α-1-4, α-1-6, glycosidic bonds. METHODS: This enzyme carrying an (α /ß) 8 or TIM barrel structure is also produced containing the catalytic site residues. These groups of enzymes possess four conserved regions in their primary sequence. In the Carbohydrate-Degrading Enzyme (CAZy) database, α-amylases are classified into different Glycoside Hydrolase Families (GHF) based on their amino acid sequence. The present objective was to study one such enzyme based on its molecular characterization after purification in our laboratory. Its main property of solid-natural starch degradation was extensively investigated for its pharmaceutical/ industrial applications. RESULTS: Amylase producing bacteria Bacillus cereus sm-sr14 (Accession no. KM251578.1) was purified to homogeneity on a Seralose 6B-150 gel-matrix and gave a single peak during HPLC. MALDITOF mass-spectrometry with bioinformatics studies revealed its significant similarity to α/ß hydrolase family. The enzyme showed an efficient application; favourable Km, Vmax and Kcat during the catalysis of different natural solid starch materials. Analysis for hydrolytic product showed that this enzyme can be classified as the exo-amylase asit produced a significant amount of glucose. CONCLUSION: Besides the purified enzyme, the present organism Bacillus cereus sm-sr14 could degrade natural solid starch materials like potato and rice up to the application level in the pharmaceutical/ industrial field for alcohol production.

Starch-degrading enzymes from the brown-rot fungus Fomitopsis palustris.

Brown-rot fungi preferentially degrade softwood and cause severe breakdown of wooden structures. At the initial stage of the brown-rot decay, penetrating hyphae of the fungi are observed in ray parenchyma. Since starch grains are known to be present in the ray parenchyma of sapwood, investigation of the functions and roles of the starch-degrading enzymes is important to understand the initial stage of brown-rot decay. We purified and characterized two starch-degrading enzymes, an α-amylase (FpAmy13A) and a glucoamylase (FpGLA15A), from the brown-rot fungus, Fomitopsis palustris, and cloned the corresponding genes. The optimal temperature for both enzymes was 60 °C. FpAmy13A showed higher activity at a broad range of pH from 2.0 to 5.0, whereas FpGLA15A was most active at pH 5.0-6.0. Notable thermal stability was found for FpGLA15A. Approximately 25% of the activity remained even after treatment at 100 °C for 30 min in sodium phosphate buffer at pH 7.0. These different characteristics imply the different roles of these enzymes in the starch degradation of wood.

Production, characterization and application of thermostable, alkaline α-amylase (AA11) from Bacillus cereus strain SP-CH11 isolated from Chilika Lake.

An alkaliphile bacterial strain designated as CH11 was isolated from the sediments of Chilika Lake, Odisha. The isolate showed stupendous growth and production of α-amylase at pH 10.0. Through 16S rRNA gene based molecular technique this isolate was identified as Bacillus cereus strain SP-CH11 having GenBank Accession No. KT992791. Homogenous ~55 kDa extracellular α-amylase was extracted with 241.304, 26.26 and 3.2-fold acceleration in specific activity, purification fold and yield respectively. The alkaline α-amylase AA11 was further characterized. At pH 9.0 the purified enzyme AA11 was highly stable while retaining 88-100% functional viability at temperature range from 35 to 65 °C, confirming its thermostability nature. It showed stability with powdered and liquid detergents at 7 mg/mL and 100-fold dilutions respectively. AA11 efficiently removed the starch stain from cotton fabrics. The findings of this study indicate that the isolate CH11 is a source of novel alkaline α-amylase that has promising application in food and detergent industries.

Purification and immobilization of α-amylase in one step by gram-positive enhancer matrix (GEM) particles from the soluble protein and the inclusion body.

Immobilization of the enzyme benefits the catalytic industry a lot. The gram-positive enhancer matrix (GEM) particles could purify and immobilize the recombinant α-amylase in one step without changing the enzymatic character. The enzyme immobilized by GEM particles exhibited good reusability and storage stability. The denaturants dissolved some of the GEM particles and a part of the GEM particles could bear the denaturants. The GEM particles had strong binding ability to the recombination protein with the AcmA tag even when the denaturants existed. The inclusion body was dissolved by urea and then bound by the GEM particles. The GEM particles binding the recombination protein were separated by centrifugation and resuspended in the renaturation solution. GEM particles were recycled by repeating the boiling procedure used in preparing them. The recombination α-amylase without any tag was obtained by digestion and separated via centrifugation. Altogether, our findings suggest that GEM particles have the potential to function as both immobilization and purification materials to bind the soluble recombinant protein with the AcmA tag and the inclusion body dissolved in the denaturants.

Impact of maltogenic α-amylase on the structure of potato starch and its retrogradation properties.

Structural modification of starch using efficient α-amylases to improve its properties is an established method in the starch industry. In our previous research, the novel maltogenic α-amylase CoMA that catalyzes multi-molecular reactions has been identified. In this study, the impact of CoMA on the structure and retrogradation properties of potato starch was evaluated. CoMA cleaves internal starch chains to change the proportion of amylose and amylopectin in starch. Following treatment, visible pores and microporous on the surface of starch granules were observed from SEM analysis. CoMA modification led to increased insoluble blue complex formation and hydrolysis to shorten the outer chains, which was found to reduce the development rate of starch according to network interactions from the dynamic rheological analysis. Furthermore, maltose accumulation with water competition was also deduced to be involved in the inhibition of retrogradation. Its activities in the cleavage of internal starch granules, shortening of outer chains of starch, and maltose formation make CoMA a powerful agent for the inhibition of starch retrogradation with a very low effective dose of 0.5 mg/kg, which may find potential applications in the starch processing industry.

Cloning, Expression and Characterization of a Novel α-Amylase from Salinispora arenicola CNP193.

α-Amylases are used in various biotechnological processes including the textile, paper, food, biofuels, detergents and pharmaceutical industries. In this study, a novel gene encoding α-amylase was cloned from marine bacterium Salinispora arenicola CNP193 and the protein was expressed in Escherichia coli. The α-amylase gene from S. arenicola CNP193 had a length of 1839 bp and encoded a α-amylase with an estimated molecular mass of 74 kDa. The optimum temperature and pH for the recombinant α-amylase was 50 °C and 7 respectively. Na+, K+ and Ca2+ increased the activity of the recombinant α-amylase whereas the enzyme was inhibited by Cu2+, Zn2+, Hg2+, Pb2+, Fe3+ and Mn2+. Thin layer chromatography results confirmed that monosaccharide, disaccharide and maltotriose are the hydrolysis products. The results of our study suggest that this enzyme has considerable potential in industrial applications.