Biofilm is a syntrophic association of sessile groups of microbial cells that adhere to biotic and abiotic surfaces with the help of pili and extracellular polymeric substances (EPS). EPSs also prevent penetration of antimicrobials/antibiotics into the sessile groups of cells. Hence, methods and agents to avoid or remove biofilms are urgently needed. Enzymes play important roles in the removal of biofilm in natural environments and may be promising agents for this purpose. As the major component of the EPS is polysaccharide, amylase has inhibited EPS by preventing the adherence of the microbial cells, thus making amylase a suitable antimicrobial agent. On the other hand, salivary amylase binds to amylase-binding protein of plaque-forming Streptococci and initiates the formation of biofilm. This review investigates the contradictory actions and microbe-associated genes of amylases, with emphasis on their structural and functional characteristics.
Polyhydroxybutyrate (PHB) is a natural microbial polyester produced by a variety of bacteria and archaea from renewable resources. PHB resembles some petrochemical plastics but is completely biodegradable. It is desirable to identify suitable microbial strains and develop processes that can directly use starch from agricultural wastes without commercial amylase treatment. Here, PHB production using starch from agricultural waste was developed using a newly isolated strain, Bacillus aryabhattai T34-N4. This strain hydrolyzed cassava pulp and oil palm trunk starch and accumulated up to 17 wt% PHB of the cell dry weight. The α-amylase of this strain, AmyA, showed high activity in the presence of cassava pulp starch (69.72 U) and oil palm trunk starch (70.53 U). High expression of amyA was recorded in the presence of cassava pulp starch, whereas low expression was detected in the presence of glucose. These data suggest that starch saccharification by amyA allows strain T34-N4 to grow and directly produce PHB from waste starch materials such as cassava pulp and oil palm trunk starch, which may be used as low-cost substrates.
Honey is a rich source of natural nutrients. Its production is a slow, natural process with the pace of which varies seasonally. However, based on recent reports, we hypothesize that the long-term storage of processed honey, even under the most appropriate storage conditions, results in a deterioration of its quality. To test our hypothesis, we collected Tualang honey samples harvested during the years 2005, 2008, 2009 and 2010 and tested various parameters including physicochemical properties and also performed comparative analyses of antioxidant capacities to assess its medicinal values. Our results indicate that, upon long-term storage, the quality of honey samples deteriorates, as observed in our TH 2008 and TH 2005 year honey samples, which showed unacceptable quality based on the recommended criteria of free acidity (71 .34±1.31 meq/kg), moisture (27.72%), diastase activity (3.38±0.34 Goth scale) and hydroxymethylfurfural (HMF) (449.89±3.23 mg/kg) by Codex and European Commission Regulation. A significant (p
Amylase is an important and indispensable enzyme that plays a pivotal role in the field of biotechnology. It is produced mainly from microbial sources and is used in many industries. Industrial sectors with top-down and bottom-up approaches are currently focusing on improving microbial amylase production levels by implementing bioengineering technologies. The further support of energy consumption studies, such as those on thermodynamics, pinch technology, and environment-friendly technologies, has hastened the large-scale production of the enzyme. Herein, the importance of microbial (bacteria and fungi) amylase is discussed along with its production methods from the laboratory to industrial scales.
The main purpose of this article is to develop a new and reliable saliva-based clinical diagnostic method for the early detection of oral squamous cell carcinoma (OSCC). This study used an immunoproteomic approach which allowed the detection of immunogenic host proteins in patients' samples using pooled human antibodies. In an attempt to investigate potential biomarkers of OSCC, two-dimensional electrophoresis (2-DE) followed by immunoblotting of saliva from patients and controls were compared. The protein spots of interest were analyzed using 2-DE image analyzer and subsequently subjected to MALDI-TOF/TOF and then matched against NCBI database. The result showed that four protein clusters, namely Human Pancreatic Alpha-amylase (HPA), Human Salivary Amylase (sAA), keratin-10 (K-10), and Ga Module Complexed with Human Serum Albumin (GA-HSA), had exhibited immunoreactivity in western blot. The results are suggestive of the potential use of the differentially expressed saliva protein as tumor biomarkers for the detection of OSCC. However, further studies are recommended to validate this finding.
An amylase enzyme from pitaya peel was purified 234.2-folds with 72.1% recovery using ammonium sulphate precipitation, gel filtration and ion exchange chromatography. Gel filtration chromatography and SDS-PAGE revealed that the enzyme is monomeric with a molecular weight of 42.1kDa. The apparent Km and Vmax of the amylase were 2.7 mg/ml and 34.30 u/min/mg of protein, respectively. The enzyme was highly active and stable over a wide pH range from pH 3 to pH 11.0, with optimum activity being observed at pH 5.0. The enzyme was highly selective for soluble starch, amylopectin, glycogen and pulullan. The purified amylase did not require calcium and displayed extreme stability with regard to surfactants and oxidising agents. EDTA, a powerful chelating agent, did not have any significant effect on the stability of the enzyme. Such characteristics have not been previously reported for this type of enzyme from fruit peel. This enzyme, which possesses unique properties, could be widely used in different types of industries, especially in food and biotechnological applications.
Aspergillus flavus produced approximately 50 U/mL of amylolytic activity when grown in liquid medium with raw low-grade tapioca starch as substrate. Electrophoretic analysis of the culture filtrate showed the presence of only one amylolytic enzyme, identified as an alpha-amylase as evidenced by (i) rapid loss of color in iodine-stained starch and (ii) production of a mixture of glucose, maltose, maltotriose and maltotetraose as starch digestion products. The enzyme was purified by ammonium sulfate precipitation and ion-exchange chromatography and was found to be homogeneous on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified enzyme had a molar mass of 52.5 +/- 2.5 kDa with an isoelectric point at pH 3.5. The enzyme was found to have maximum activity at pH 6.0 and was stable in a pH range from 5.0 to 8.5. The optimum temperature for the enzyme was 55 degrees C and it was stable for 1 h up to 50 degrees C. The Km and V for gelatinized tapioca starch were 0.5 g/L and 108.67 mumol reducing sugars per mg protein per min, respectively.
The influence of light regulation on the growth and enzyme production of three endolichenic fungal isolates, i.e. Pseudopestalotiopsis theae (EF13), Fusarium solani (EF5), and Xylaria venustula (PH22), was determined. The isolates were exposed to blue, red, green, yellow, white fluorescent light (12 h light-12 h dark photoperiod) (test), and 24 h dark (control) conditions. Results revealed that the alternating light-dark conditions resulted in the formation of dark rings in most fungal isolates but was absent in PH22. Red light induced sporulation while yellow light elicited higher biomass in all isolates (0.19 ± 0.01 g, 0.07 ± 0.00 g, and 0.11 ± 0.00 g, for EF13, PH22, and EF5, respectively) as compared to incubation in the dark. Results also showed that blue light induced higher amylase activity in PH22 (15.31 ± 0.45 U/mL) and L-asparaginase activity in all isolates (0.45 ± 0.01 U/mL, 0.55 ± 0.39 U/mL, and 0.38 ± 0.01 U/mL, for EF13, PH22, and EF5, respectively) compared to both control conditions. Green light enhanced the production of xylanase (6.57 ± 0.42 U/mL, 10.64 ± 0.12 U/mL, and 7.55 ± 0.56 U/mL for EF13, PH22, and EF5, respectively) and cellulase (6.49 ± 0.48 U/mL, 9.57 ± 0.25 U/mL, and 7.28 ± 0.63 U/mL, for EF13, PH22, and EF5, respectively). In contrast, red light was the least effective light treatment as production of enzymes was the least, with lower levels of amylase, cellulase, xylanase, and L-asparaginase detected. To conclude, all three endolichenic fungi are light-responsive, with fungal growth regulated with the use of red light and yellow light, and manipulation of enzyme production via blue and green light.
This research aimed to determine the biofunctional properties of wheat flour (WF) protein fractions and modifications to the antioxidant, anti-α-amylase and anti-angiotensin-I converting enzyme (ACE) activities induced by the action of digestive endopeptidases in vitro. A molecular characterization of the most abundant protein fractions, i.e., albumins, glutelins-1, glutelins-2 and prolamins, showed that low- and high-MW polypeptides rich in cysteine, glutamic acid and leucine were present in albumins and glutelins, whereas low-MW subunits with a high proportion of polar amino acids prevailed in prolamins. Prolamins exhibited the second-highest water holding capacity (54%) after WF (84%), while albumins provided superior foam stability (76%). Prolamins, glutenins-1 and globulins demonstrated the highest antioxidant activity (up to 95%, 68% and 59%, respectively) both before and after hydrolysis with pepsin (P-H) or trypsin-chymotrypsin (TC-H). Prolamins, globulins and WF strongly inhibited α-amylase (>90%) before and after TC-H, and before P-H (55-71%). Moreover, P-H significantly increased α-amylase inhibition by albumins from 53 to 74%. The fractions with strong ACE inhibitory activity (70-89%) included prolamins and globulins after TC-H or P-H, as well as globulins before TC-H and WF before P-H. This novel evidence indicates that WF protein fractions and their peptide-enriched P and TC hydrolysates are excellent sources of multifunctional bioactives with antioxidant, antihyperglycemic and antihypertensive potential.
Amylase is one of the most important enzymes in the world due to its wide application in various industries and biotechnological processes. In this study, amylase enzyme from Hylocereus polyrhizus was encapsulated for the first time in an Arabic gum-chitosan matrix using freeze drying. The encapsulated amylase retained complete biocatalytic activity and exhibited a shift in the optimum temperature and considerable increase in the pH and temperature stabilities compared to the free enzyme. Encapsulation of the enzyme protected the activity in the presence of ionic and non-ionic surfactants and oxidizing agents (H₂O₂) and enhanced the shelf life. The storage stability of amylase is found to markedly increase after immobilization and the freeze dried amylase exhibited maximum encapsulation efficiency value (96.2%) after the encapsulation process. Therefore, the present study demonstrated that the encapsulation of the enzyme in a coating agent using freeze drying is an efficient method to keep the enzyme active and stable until required in industry.
Bacillus licheniformis α-amylase (BLA) was chemically modified using 100-fold molar excess of succinic anhydride over protein or 0.66 M potassium cyanate to obtain 42 % succinylated and 81 % carbamylated BLAs. Size and charge homogeneity of modified preparations was established by Sephacryl S-200 HR gel chromatography and polyacrylamide gel electrophoresis. Conformational alteration in these preparations was evident by the larger Stokes radii (3.40 nm for carbamylated and 3.34 nm for succinylated BLAs) compared to 2.43 nm obtained for native BLA. Urea denaturation results using mean residue ellipticity (MRE) as a probe also showed conformational destabilization based on the early start of transition as well as ΔG(D)(H(2)O) values obtained for both modified derivatives and Ca-depleted BLA. Decrease in ΔG(D)(H(2)O) value from 5,930 cal/mol (for native BLA) to 3,957 cal/mol (for succinylated BLA), 3,336 cal/mol (for carbamylated BLA) and 3,430 cal/mol for Ca-depleted BLA suggested reduced conformational stability upon modification of amino groups of BLA or depletion of calcium. Since both succinylation and carbamylation reactions abolish the positive charge on amino groups (both α- and ε- amino), the decrease in conformational stability can be ascribed to the disruption of salt bridges present in the protein which might have released the intrinsic calcium from its binding site.
The importance and development of industrial biotechnology processing has led to the utilisation of microbial enzymes in various applications. One of the important enzymes is amylase, which hydrolyses starch to glucose. In Malaysia, the use of sago starch has been increasing, and it is presently being used for the production of glucose. Sago starch represents an alternative cheap carbon source for fermentation processes that is attractive out of both economic and geographical considerations. Production of fermentable sugars from the hydrolysis of starches is normally carried out by an enzymatic processes that involves two reaction steps, liquefaction and saccharification, each of which has different temperature and pH optima with respect to the maximum reaction rate. This method of starch hydrolysis requires the use of an expensive temperature control system and a complex mixing device. Our laboratory has investigated the possibility of using amylolytic enzyme-producing microorganisms in the continuous single-step biological hydrolysis of sago flour for the production of a generic fermentation medium. The ability of a novel DNA-recombinated yeast, Saccharomyces cerevisiae strain YKU 107 (expressing alpha-amylase production) to hydrolyse gelatinised sago starch production has been studied with the aim of further utilizing sago starch to obtain value-added products.
Effects of metsulphuron-methyl on the activities of amylase, invertase and xylanase in loamy sand and clay were evaluated for up to 28 days under laboratory conditions. Metsulphuron-methyl at 1.0 microg/g caused a significant reduction in amylase, invertase and xylanase activities for the entire period of study, especially at 28 days incubation in both soils. The lowest activities of the three enzymes were observed in the presence of 5.0 microg/g at 28 days incubation.
A therapeutic approach for treating diabetes is to decrease thepost-prandial hyperglycaemia. This is done by retarding the absorption of glucose through the inhibition of carbohydrate hydrolyzing enzymes, α-amylaseand α-glucosidase, in the digestive tract. Inhibition of both enzymes helpsto reduce the glucose level in the blood of a diabetic patient. This study was aimed to investigate the production of α-glucosidase and α-amylase inhibitors from local fruit wastes (honeydew skin, banana peel, and pineapple skin) using solid state fermentation. Each of the fruit wastes was fermented with three different types of white rot fungus Phenarochaete chrysosporium(PC), Panus tigrinusM609RQY(M6) andRO209RQY(RO2)for 7 days. Sampling was carried out starting from day 4 to day 7 to determine the enzyme inhibition activity. The samples were extracted using water prior to enzyme analysis. Most of the fruit samples showed varying degree of percentage inhibition activity depending on the sampling time. Extract of fermented banana peels with RO2 on day 4 showed the higherα-glucosidase inhibition (56.57±0.32%), followed byhoneydew extract fermented with the same fungus on the same day (39.68±0.05%). Extracts of each fruit wastesample fermented with PCshowed the least α-glucosidase inhibition (below 15%). Meanwhile for α-amylase inhibition activity, the extract from fermented honeydew skins with PCon day7 showed the highest inhibition activity i.e.98.29±0.63%. The least inhibition activity (43.37±0.54%) was observed in the extract from honeydew skins fermented withM6 on day 5. All positive resultsshowed that fruit wastes could be the alternative sourcesfor antidiabetic agent especially for α-amylase and α-glucosidase inhibitors.
Over-expression of α-amylase enzyme causes hyperglycemia which lead to many physiological complications including oxidative stress, one of the most commonly associated problem with diabetes mellitus. Marketed α-amylase inhibitors such as acarbose, voglibose, and miglitol used to treat type-II diabetes mellitus, but also linked to several harmful effects. Therefore, it is essential to explore new and nontoxic antidiabetic agents with additional antioxidant properties. In this connection, a series of new N-sulfonohydrazide substituted indazoles 1-19 were synthesized by multistep reaction scheme and assessed for in vitro α-amylase inhibitory and radical (DPPH and ABTS) scavenging properties. All compounds were fully characterized by different spectroscopic techniques including 1H, 13C NMR, EI-MS, HREI-MS, ESI-MS, and HRESI-MS. Compounds showed promising α-amylase inhibitory activities (IC50 = 1.23 ± 0.06-4.5 ± 0.03 µM) as compared to the standard acarbose (IC50 1.20 ± 0.09 µM). In addition to that all derivatives were found good to moderate scavengers of DPPH (IC50 2.01 ± 0.13-5.3 ± 0.11) and ABTS (IC50 = 2.34 ± 0.07-5.5 ± 0.07 µM) radicals, in comparison with standard ascorbic acid having scavenging activities with IC50 = 1.99 ± 0.09 µM, and IC50 2.03 ± 0.11 µM for DPPH and ABTS radicals. In silico molecular docking study was conducted to rationalize the binding interaction of α-amylase enzyme with ligands. Compounds were observed as mixed type inhibitors in enzyme kinetic characterization.
Five improved Nigerian barley cultivars (ESCOBA, ASE – 2, ALOE, GOB – 2, SUMBARD) were obtained from Lake Chad Research Institute Maiduguri, Nigeria, and their physicochemical, malting and biochemical properties investigated employing standard procedures. Data were analyzed by means of ANOVA [at 95% significant level] and correlations using SPSS 14 software. Results showed GOB-2 grain and malt recording the highest kernel weight (47.50 g) and kernel volume (41.21 ml); whereas ALOE grain had the longest kernel length (13.40 mm) and GOB-2 the shortest (9.40 mm). GOB-2 had the largest major diameter (3.39 mm) and SUMBARD had the least (2.86 mm). ESCOBA, SUMBARD and ASE-2 cultivars had the highest protein values (as %N) of 14.90%, 13.90% and 13.69% respectively, while ALOE, ASE-2 and GOB-2 had the highest total carbohydrates of 69.97, 69.39 and 68.90% respectively. All the cultivars had good germinative capacities (> 90%), with GOB-2 and ASE-2 having the highest germinative energy values of 96.65% and 95.00%. No significant (p > 0.05) changes
in the dimension of the kernels after malting. SUMBARD recorded the highest malt yields (88.55%) followed by ASE-2 (83.45%) and ALOE (82.00%). The highest α-amylase activities of 105.34 and 96.23 unit/mg protein/min were recorded by ASE-2 and ALOE, respectively, with corresponding diastatic powers of 81.92 and 76.23oL. Thousand kernel weight correlated positively with protein (r = 0.500, P < 0.05) and with total soluble solids (r = 0.435, p < 0.05) but negatively with α-amylase (r = -0.869, p < 0.05) and with diastatic power (r = -0.838, p < 0.05). This study showed that the cultivars have good potentials for use as malting materials in beverage making.
Kinetic analysis of solid-state fermentation (SSF) of fruit peels with Phanerochaete chrysosporium and Schizophyllum commune mixed culture was studied in flask and 7 kg capacity reactor. Modified Monod kinetic model suggested by Haldane sufficiently described microbial growth with co-efficient of determination (R2) reaching 0.908 at increased substrate concentration than the classical Monod model (R2 = 0.932). Leudeking-Piret model adequately described product synthesis in non-growth-dependent manner (R2 = 0.989), while substrate consumption by P. chrysosporium and S. commune fungal mixed culture was growth-dependent (R2 = 0.938). Hanes-Woolf model sufficiently represented α-amylase and cellulase enzymes synthesis (R2 = 0.911 and 0.988); α-amylase had enzyme maximum velocity (Vmax) of 25.19 IU/gds/day and rate constant (Km) of 11.55 IU/gds/day, while cellulase enzyme had Vmax of 3.05 IU/gds/day and Km of 57.47 IU/gds/day. Product yield in the reactor increased to 32.65 mg/g/day compared with 28.15 mg/g/day in shake flask. 2.5 cm media thickness was adequate for product formation within a 6 day SSF in the tray reactor.