Academic literature on the topic 'Amylolytic enzymes purification'

When grown in static culture it appears as if Thermomyces lanuginosus has a biphasic secretion of the extracellular starch-degrading activity. This could be due to the presence of at least two different amylases. By ion-exchange chromatography on DEAE-Trisacryl an α-amylase (EC 3.2.1.1) and a glucoamylase (EC 3.2.1.3) were separated and purified from the extracellular protein from 14-day-old static cultures grown on soluble starch. The hydrolysis of soluble starch by the purified glucoamylase resulted in only glucose as the end product, whereas the α-amylase gave maltose as the smallest end product. The molecular weights and isoelectric points of the enzymes were for glucoamylase 70 000 – 76 000 and pH 4.0, and for α-amylase 54 000 – 57 000 and pH 3.4. An α-glucosidase (EC 3.2.1.20) with a molecular weight of 44 000 – 48 000 and an isoelectric point at pH 3.8 was eluted close to the α-amylase fraction on the DEAE-Trisacryl column.

A starch-degrading enzyme produced by the yeast Cryptococcus sp. S-2 was purified in only one step by using an α-cyclodextrin–Sepharose 6B column, and was characterized as an α-amylase (EC 3.2.1.1). The molecular mass and isoelectric point of purified α-amylase (AMY-CS2) were estimated to be 66 kDa and 4.2 respectively. AMY-CS2 has raw-starch-digesting and raw-starch-absorbing activities. Furthermore it was shown to be thermostable. An open reading frame of the cDNA specified 611 amino acids, including a putative signal peptide of 20 amino acids. The N-terminal region of AMY-CS2 (from the N-terminus to position 496) had 49.7% similarity with the whole region of α-amylase from Aspergillus oryzae (Taka-amylase), whereas the C-terminal region had a sequence that was similar to the C-terminal region of glucoamylase G1 from A. niger. In addition, putative raw-starch-binding motifs exist in some amylolytic enzymes. A mutant AMY-CS2 that lacks the C-terminal domain lost not only its ability to bind or digest raw starch, but also its thermostability. Consequently it is possible that the putative raw-starch-binding domain of AMY-CS2 plays a role not only in the molecule's raw-starch-digesting ability but also in its thermostability.

ABSTRACT The gene encoding a thermoactive pullulanase from the hyperthermophilic anaerobic archaeon Desulfurococcus mucosus (apuA) was cloned in Escherichia coli and sequenced. apuA from D. mucosusshowed 45.4% pairwise amino acid identity with the pullulanase fromThermococcus aggregans and contained the four regions conserved among all amylolytic enzymes. apuA encodes a protein of 686 amino acids with a 28-residue signal peptide and has a predicted mass of 74 kDa after signal cleavage. The apuAgene was then expressed in Bacillus subtilis and secreted into the culture fluid. This is one of the first reports on the successful expression and purification of an archaeal amylopullulanase in a Bacillus strain. The purified recombinant enzyme (rapuDm) is composed of two subunits, each having an estimated molecular mass of 66 kDa. Optimal activity was measured at 85°C within a broad pH range from 3.5 to 8.5, with an optimum at pH 5.0. Divalent cations have no influence on the stability or activity of the enzyme. RapuDm was stable at 80°C for 4 h and exhibited a half-life of 50 min at 85°C. By high-pressure liquid chromatography analysis it was observed that rapuDm hydrolyzed α-1,6 glycosidic linkages of pullulan, producing maltotriose, and also α-1,4 glycosidic linkages in starch, amylose, amylopectin, and cyclodextrins, with maltotriose and maltose as the main products. Since the thermoactive pullulanases known so far from Archaeaare not active on cyclodextrins and are in fact inhibited by these cyclic oligosaccharides, the enzyme from D. mucosus should be considered an archaeal pullulanase type II with a wider substrate specificity.

Background Bacterial biofilms have become a major threat to human health. The objective of this study was to isolate amylase-producing bacteria from soil to determine the overall inhibition of certain pathogenic bacterial biofilms. Methods We used serial dilution and the streaking method to obtain a total of 75 positive amylase isolates. The starch-agar plate method was used to screen the amylolytic activities of these isolates, and we used morphological and biochemical methods to characterize the isolates. Optimal conditions for amylase production and purification using Sephadex G-200 and SDS-PAGE were monitored. We screened these isolates’ antagonistic activities and the purified amylase against pathogenic and multi-drug-resistant human bacteria using the agar disk diffusion method. Some standard antibiotics were controlled according to their degree of sensitivity. Finally, we used spectrophotometric methods to screen the antibiofilm 24 and 48 h after application of filtering and purifying enzymes in order to determine its efficacy at human pathogenic bacteria. Results The isolated Bacillus species were Bacillus megaterium (26.7%), Bacillus subtilis (16%), Bacillus cereus (13.3%), Bacillus thuringiesis (10.7%), Bacillus lentus (10.7%), Bacillus mycoides (5.3%), Bacillus alvei (5.3%), Bacillus polymyxa (4%), Bacillus circulans (4%), and Micrococcus roseus (4%). Interestingly, all isolates showed a high antagonism to target pathogens. B. alevi had the highest recorded activity (48 mm) and B. polymyxa had the lowest recorded activity (12 mm) against Staphylococcus aureus (MRSA) and Escherichia coli, respectively. On the other hand, we detected no antibacterial activity for purified amylase. The supernatant of the isolated amylase-producing bacteria and its purified amylase showed significant inhibition for biofilm: 93.7% and 78.8%, respectively. This suggests that supernatant and purified amylase may be effective for clinical and environmental biofilm control. Discussion Our results showed that soil bacterial isolates such as Bacillus sp. supernatant and its purified amylase are good antibiofilm tools that can inhibit multidrug-resistant former strains. They could be beneficial for pharmaceutical use. While purified amylase was effective as an antibiofilm, the isolated supernatant showed better results.

A purification scheme has been developed for the extracellular carbohydrases; aglucosidase and a-amylase, secreted by the ascosporogenous soil yeast Lipomyces starkeyi NCYC 1436. Growth and optimum enzyme yield conditions were determined with cultures of L. starkeyi grown at 30°C on a medium containing 2% soluble starch, 1% yeast extract and 1% Bactopeptone, appropriate enzyme assay procedures having been devised. Both enzymes were initially precipitated from the cell free supernatant by an 85%(w/v) ammonium sulphate precipitation. Many different chromatographic media were then assessed, but the choice of a hydrophobic (Phenyl Sepharose CL-4B) column had the advantage of utilizing the ammonium sulphate precipitate with minimum sample preparation. The pellet was adjusted to 1M salt and adsorbed to the hydrophobic column. Elution of the two activities was carried out by a series of decreasing salt washes. Although fractionation of the two enzymes was not complete, the column was effective in eliminating a substantial quantity of inactive material, whilst maintaining good recoveries of approximately 59% for cc-glucosidase and 72% for a-amylase. Both enzymes were substantially purified using ion exchange chromatography (QSepharose fast flow medium), with a-glucosidase being apparently close to electrophoretic homogeneity; elution of the two enzyme activities was carried out at 4°C, using a linear gradient with sodium acetate buffer, pH 5.0. Individual HIC fractions purified by ion exchange showed between a 0.3 to 24.6-fold increase in purity for a-glucosidase, and between a 2.8 to 7.6 increase in purity for aamylase. However, by selecting the most active a-glucosidase fraction isolated after running the 0.01M HIC fraction on ion exchange, the purification factor rose to 83.4. Several properties of the purified a-glucosidase enzyme were investigated. The molecular weight of the a-glucosidase was determined by electrophoresis under denaturing and non-denaturing conditions, after ion exchange chromatography. A single major band was detected by SDS-PAGE with an estimated molecular weight of 93,000 ± 5,000 Daltons, and under native conditions, the molecular weight was estimated at 162,000 Daltons. Specific enzyme activity staining of the native gel confirmed that the single band was an a-glucosidase. However, isoelectric focusing of the purified Lipomyces starkeyi a-glucosidase, detected three bands - within the pi range of 4.6-5.0. Further characterisation studies revealed the pH and temperature optimum of the purified L starkeyi a-glucosidase at 4.5 and 55°C respectively. Km and Fmax measurements using a range of substrates suggested that the aglucosidase had a broad substrate specificity but showed a marked preference for substrates with a-1,4 linkages such as soluble starch, PNPG, and a-1,3 linkages, such as nigerose SDS and native gel electrophoresis (combined with specific activity staining) revealed 3 distinct a-amylase activities, and the molecular weights were determined as 82,000, 68,000 and 48,500 Daltons.

A maca (Lepidium meyenii Walpers) é uma planta herbácea bienal da família Brassicae, cultivada principalmente na região dos Andes da América do Sul. A parte subterrânea vem sendo consumida por muito tempo devido a seu valor nutricional e energético, mas é mais conhecida no mercado peruano e internacional por alegadas propriedades terapêuticas. Esta raiz apresenta até 76% de carboidratos, dos quais 30% é amido. Este trabalho teve como objetivos estudar: as propriedades físico-químicas e funcionais do amido isolado; os parâmetros enzimáticos durante o armazenamento e a purificação parcial de enzimas amilolíticas. Em relação às propriedades do amido, este apresentou um teor de amilose de 20% valor semelhante aos encontrados em raízes e tubérculos similares. A turbidez das suspensões de amido apresentou estabilidade durante o armazenamento. A temperatura de gelatinização e a viscosidade da pasta foram a 45,7° e 46°C, respectivamente. Com base nos dados obtidos, o amido de maca seria indicado para alimentos que requeiram temperaturas moderadas no processamento, não sendo apropriado para o emprego em alimentos congelados. Os parâmetros enzimáticos medidos tais como teor de amido total, teor de açúcares solúveis, atividade amilolítica total, atividade de α e β amilases, não mostraram diferenças significativas entre as medidas durante um período de armazenamento de 16 dias. As microscopias eletrônicas de varredura (MEV) dos grânulos de amido mostraram grãos íntegros com superfícies lisas, com algumas depressões ao redor dos grânulos os quais poderiam indicar o inicio de ataque enzimático, ou fraturas na purificação. Em relação à purificação de enzimas amilolíticas, foi possível separar uma fração ativa com a carboximetilcelulose (CMC) seguida de cromatografia liquida de alta resolução (CLAE) que permitiu a separação de duas frações protéicas, analisadas por eletroforese SDS-PAGE e eletroforese bidimensional (2D). Os polipeptídeos identificados no gel 2D apresentaram massa molecular semelhante entre 22 a 27 kDa, e os pontos isoelétricos entre 4,8 e 7,3.
Maca (Lepidium meyenii Walpers) is a biennial herbaceous plant from Brassicae family, grown mainly in the Andes of South America. The underground part has been consumed for a long time due to its nutritional value and energy, but is best known in the Peruvian and international market for alleged therapeutic properties. This root has up to 76% carbohydrates, of which 30% is starch. This work aimed to study: the physico-chemical properties of isolated starch, the enzymatic parameters during storage and partial purification of amylases. In relation to the properties of starch, the amylose content showed a 20% value similar to those found in roots and tubers alike. The turbidity of starch suspensions was stable during storage. The gelatinization temperature and viscosity of the paste were 45.7 ° and 46 ° C, respectively. Based on data obtained from the starch of litter would be given to foods that require moderate temperatures in processing and is not suitable for use in frozen foods. The enzymatic parameters measured such as total starch content, soluble sugars, total amylolytic activity, activity of α and β amylases, showed no significant differences between the measures over a storage period of 16 days. Electronic microscopy (SEM) of starch granules showed grains with smooth surfaces, with some depressions around the granules which could indicate the beginning of enzymatic attack, or fractures in the purification. Regarding the purification of amylases was possible to separate an active fraction with carboxymethylcellulose (CMC) followed by high-resolution liquid chromatography (HPLC) which allowed the separation of two protein fractions, analyzed by SDS-PAGE and two-dimensional electrophoresis (2D ). The polypeptides had a molecular mass between 22 and 27 kDa and isoelectric points ranging from 4.8 to 7.3.

碩士
國立師範大學
生物學研究所
82
A.espejiana Bal 31 野生型菌株及其突變株之澱粉水解酵素純化及物理， 化學性質探討為本研究之主題。利用硫酸銨沉澱，膠體過濾層析和親和性 管柱層析等方法將釋出於培養基的澱粉水解酵素加以純化分離。得到野生 型菌株澱粉水解酵素的純化倍率為 18 倍，突變株澱粉水解酵素則為 7 倍。兩者之分子量差異不大約為 80 KD。依酵素對可溶性澱粉之水解產物 分析結果推論，兩者皆為"liquefying".alpha. 澱粉水解酵素。 且其作 用最適溫度與最適 PH 值分別為攝氏 40 度及 7.0。所純化酵素對熱十分 敏感，突變株之澱粉水解酵素於攝氏 45 度作用 10 分鐘幾乎完全喪失活 性，野生型菌株澱粉水解酵素於攝氏45 度反應 10 分鐘，活性僅剩下約百 分之四十；若於 55 度作用 15 分鐘後即測不到活性。兩者作用的活化能 都有偏低的趨勢，二者分別為每莫耳 3.52 仟卡（ 野生型 ）及 4.01 仟 卡（ 突變株 ）。鈣離子可增加兩者對熱的耐受性，而鈉離子和氯離子對 突變株澱粉水解酵素之 耐熱性有提高的作用；若兩種離子同時添加則效果 更好，但野生型菌株澱粉水解酵素則否。然而兩者的活性均會受到 SDS、 尿素、papain 及 EDTA 的抑制，唯突變株澱粉水解酵素之耐受性均比野生 型菌株澱粉水解酵素高。Triton X 100及一般常見之金屬離子對酵素活性 並不具有明顯的抑制作用。兩者胺基酸的組成中，小分子厭水性胺基酸所 佔比例很高且均缺少 cysteine 和 arginine；這些特殊的胺基酸組成可能 可以增加蛋白質分子結構之彈性，使其得以適應溫度較低的海洋環境。 The properties of secreted amylolytic enzyme from A. espejiana Bal 31 and its mutant were investigated。The enzymes in caltured medium were purified by ammonium sulfate precipitation, gel filtration chromatography and affinity chromatography.The molecular weight of both enzymes were close and estimated to be about 80KD by SDS-PAGE. According to the substrate specificity and the hydrolytic productsanalysis, we interfer both enzymes are deduced to be the liquefyingtype of α-amylase. Both anzymes exhibit the same optimum pH andtemperature of 7.0 and 40℃ respectively. Relatived lower activationenergy and heatlabile of the enzymes were observed. Addition of calciumcould increase the thermostability of enzymes, but sodium and cholride ions showed more effective to the mutant's enzyme on the protection against heat. The tolerances of enzymes to the inactivators, such as urea,SDS,papain and EDTA were also different. However,triton X 100 and metal ions in our testing concentrations showed no obvious effects to enzyme activity. The amino acid composition of the enzymes displayed the unusual fraction of hydrophobic amino acids and the lacking of cysteine and arginine residues. This characteristic constitution might enhance the structural flexibility of enzyme protein to adapt to the marine enviroment.