Extracellular Amylase Research Articles

Effect of copy number of the spoVA2mob operon, sourdough and reutericyclin on ropy bread spoilage caused by Bacillus spp.

Bacillus spp. cause ropy bread spoilage of bread, which is characterized by a rotten fruity odor, followed by discoloration and degradation of the crumb. Bacillus spp. are wheat grain endophytes and form heat resistant endospores, therefore, process hygiene and heating during baking do not prevent ropy spoilage. This study used 8 strains of Bacillus subtilis and Bacillus amyloliquefaciens to determine whether the presence and the copy number of spoVA2mob operon influences survival after baking; in addition, the spoilage phenotype was correlated with the presence of amylolytic enzymes in genomes of Bacillus spp.. The presence and copy number of the spoVA2mob operon had only a minor effect on survival of Bacillus endospores. Strains of B. amyloliquefaciens caused ropy spoilage faster than strains of B. subtilis, this difference correlated to the number and type of extracellular amylases encoded in the genomes of the strains of B. amyloliquefaciens and B. subtilis. The inhibitory effect of sourdough on ropy spoilage was determined by addition of 3–24% sourdough fermented with L. reuteri TMW1.656. Addition of 12% and 24% sourdough, corresponding to a bread pH of 5.93 ± 0.041 and 5.53 ± 0.040, respectively, delayed ropy spoilage for 2 and more than 5 d, respectively. The comparison of addition of 12% sourdough fermented with the reutericyclin producing L. reuteri TMW1.656 and the isogenic reutericyclin-negative strain L. reuteri TMW1.656ΔgtfAΔrtcN demonstrated that reutericyclin produced in sourdough inhibits growth of Bacillus in bread. In conclusion, sourdough inhibits germination of Bacillus spores in bread and the effect of sourdough is enhanced by reutericyclin.

Optimization of Amylase Production From Nocardiopsis sp. DW-4 Isolated From Dwarka, Coastal Region of Gujarat

Halophilic actinomycete Nocardiopsis sp. DW-4 was isolated from Dwarka, the coastal region of Gujarat. The isolate was studied for the extracellular amylase production. The DW-4 strain displayed diversity with respect to growth patterns and production of extracellular amylase. ‘One factor variable at time’ method was used to obtain maximum amylase production. The influence of starch, salt and pH was studied on the amylase production and growth of DW-4. The isolate DW-4 could grow and produce amylase at 5-15% salt and starch 1-3% and produced optimum amylase at 5% NaCl and pH 8. Optimization of the amylase production in conjunction with the growth of the organisms is significant.

Biochemical characterization and structural insights into the high substrate affinity of a dimeric and Ca2+ independent Bacillus subtilis α-amylase.

An extracellular amylase (AmyKS) produced by a newly isolated Bacillus subtilis strain US572 was purified and characterized. AmyKS showed maximal activity at pH 6 and 60°C with a half-life of 10 min at 70°C. It is a Ca2+ independent enzyme and able to hydrolyze soluble starch into oligosaccharides consisting mainly of maltose and maltotriose. When compared to the studied α-amylases, AmyKS presents a high affinity toward soluble starch with a Km value of 0.252 mg ml-1 . Coupled with the size-exclusion chromatography data, MALDI-TOF/MS analysis indicated that the purified amylase is a dimer with a molecular mass of 136,938.18 Da. It is an unusual feature of a non-maltogenic α-amylase. A 3D model and a dimeric model of AmyKS were generated showing the presence of an additional domain suspected to be involved in the dimerization process. This dimer arrangement could explain the high substrate affinity and catalytic efficiency of this enzyme.

Production of extracellular amylase by Aspergillus niger under submerged fermentation using jack fruit rag as the carbon source

Production of extracellular amylase by Aspergillus niger was studied under submerged fermentation using jackfruit rag as the carbon source. Different parameters, such as incubation period, pH of the culture broth and level of substrate were changed to optimise the conditions for amylase production. Maximum enzyme production ~ 8400 units/g was obtained in 5 days old cultures, grown at pH, 6.5 and 30°C with substrate level 20 gL -1 . As nitrogen sources NH 4 Cl, KNO 3 casein, peptone and beef extract were tested. Except NH 4 Cl all other sources enhanced the amylase production. Study on the kinetics of extracellular and intracellular amylase production revealed that extracellular amylase production was always higher than that in intracellular. Crude amylase obtained from culture broth was partially purified by ammonium sulphate fractionation followed by DEAE Cellulose chromatography. Partially purified enzyme exhibited optimum pH and incubation temperature at pH 6 and 60 ° C respectively and higher thermal and pH stability at 50-60°C and pH 5-7 respectively and enhanced activity with Ca 2+ . These unique features of the enzyme indicates its suitability for various industrial applications. Shorter incubation period and lower substrate cost offer the potential for inexpensive production of amylase, making the process industrially and economically feasible.

Characterization of amylase produced by cold-adapted bacteria from Antarctic samples

Abstract The Antarctic continent is characterized by extreme conditions and encompasses peculiar environments which represent promising habitats for the recovery of well-adapted microorganisms with unique metabolic capabilities. Enzymatic activity combined to salinity and pH tolerance of bacteria that thrive in low temperature environment have attracted attention of researchers in recent years and, as scarce information on that topic is available, the aim of this study was to prospect bacteria isolated from Antarctic samples for extracellular amylase activity and to determine their salinity and pH tolerances. Ninety-one bacteria isolated from Antarctic environmental samples, including marine sponges and invertebrates, sediments and soil biofilm, were screened. Fourty-two isolates were selected as putative amylase producers based on their ability to produce clearing zones in agar plates. Eighty-six and forty-four isolates were able to grow at 5 and 10% NaCl concentrations, respectively. The best enzymatic activities for isolates 226 and 227 were 768.5 U dL−1 (20 °C) and 1410,16 U dL−1 (20 °C), respectively, both influenced by the temperature. All variables assayed showed statistically significant differences for isolates 227 and 226, demonstrating that enzyme activities respond differently to the applied conditions. This is the first report that describes the amylase activity from Carnobacterium and Psychrobacter strains isolated from Antarctic samples. These results encourage subsequent investigations of enzymes produced by microorganisms adapted to low temperatures and high osmolarity conditions, aiming future biotechnological application in pharmaceutical, textile or food industries.

Sponge-associated fungi from a mangrove habitat in Indonesia: species composition, antimicrobial activity, enzyme screening and bioactive profiling

There is no report of diversity, biological properties and bioactive compounds of sponge-associated fungi from Indonesia’s mangrove ecosystem to date. This study was designed to isolate sponge-associated fungi from a mangrove ecosystem in Mangkang, to screen the antimicrobial and extracellular enzyme properties of the isolates, characterize the biologically promising isolates using molecular approaches, and profile the secondary metabolites using phytochemical and thin-layer chromatography (TLC) analyses. An unidentified sponge that lived in association with mangrove roots was collected from Mangkang. Total of eight associated fungi were isolated from the sponge. Among all isolates, only two fungi SPMKF 1 and SPMKF 6 produced extracellular amylase, another two fungi SPMKF 4 and 5 showed antibacterial activity against MRSA, and only one fungus SPMKF 8 was able to produce extracellular amylase and show antimicrobial activity against ESBL E. coli, Salmonella enterica ser. Typhi strain MDR and C. albicans, while SPMKF 2, SPMKF 3 and SPMKF 9 did not show any biological properties. The result of genetic characterization proved that SPMKF 1 was Cladosporium tenuissimum, SPMKF 4 was Eutypella sp., SPMKF 5 was Lasiodiplodia theobromae, SPMKF 6 was Fusarium keratoplasticum and SPMKF 8 was F. solani. Furthermore, an amylase gene was detected in fungi SPMKF 1, 6 and 8 while among the BGC, only NRPS genes were detected in SPMKF 4, 5 and 8. Interestingly, several same metabolites indicating the same retention factor (Rf) values in TLC were detected in the fungal crude extracts by TLC.

Prediction of the allergic response of extracellular amylase producing bacteria through in-silico method

Allergies intolerance is a common problem worldwide. The major difficulties are related to the correct diagnosis of causes which is associated with amino acid sequences present in the epitope region of allergen. So there is a need to find out the factors causing allergies and allergens themselves. In the present study a bioinformatics tool is used to predict amino acid sequence and mast cell association with different integrated approaches. Internet databases for amylase producing bacteria were used in In-silico method to check the allergy for microorganism producing the extracellular enzyme. Amylase is an extracellular enzyme isolated from soil bacteria Salmonella species and Proteus vulgaris. It is very important in the pharmaceutical industry to check the allergenicity of any drug, protein or enzyme that be used in the treatment of diseases or food industries for various purpose. The aim of the present study is isolation and characterisation of extracellular enzyme produced from soil bacteria and to analyzed allergic response through AlgPred tool of bioinformatics. From results, it was concluded that the protein sequence of amylase did not contain any epitope, no hits for mast and blast which proved that it was not an allergen. So, bacterial isolates from the industrial soil are a good alternative source of enzyme production and may be used as an industrial level. Thus, from the results, it may be concluded that microbes from soil sample can be a good source of industrially important enzymes without any allergy.

Characterization of a novel extracellular α-amylase from Ruminococcus bromii ATCC 27255 with neopullulanase-like activity.

Ruminococcus is one of the keystone bacteria of the human colonic microbiota and is highly specific for utilization of resistant starch via formation of amylosomes. Here, we present the characteristics of an extracellular amylase, Rbamy5, in Ruminococcus bromii. In an in silico study, it showed low homology with any other known amylases, but it was evolutionarily classified as a GH13_36 subfamily intermediary amylase. Recombinant Rbamy5 exhibited maximum activity toward amylose (384 ± 26 U·mg-1) over soluble starch (254 ± 3 U·mg-1), amylopectin (46.1 ± 2.6 U·mg-1) and pullulan (72.5 ± 0.41 U·mg-1) at 45 °C and pH 5.0. It was also able to degrade small substrates such as maltotriose (G3), maltotetraose (G4), and maltopentaose (G5) into maltose (G2). Despite lacking a specific N-terminal domain, Rbamy5 opened the cyclodextrin ring, which resembles those of neopullulanase. Moreover, it accumulated short α-(1 → 6)-branched maltooligosaccharides from soluble starch and maltosyl-β-cyclodextrin (G2-β-CD), while panose was solely derived from pullulan. These results suggest that Rbamy5 may have a role to provide branched maltooligosaccharides to stimulate the growth of beneficial microorganisms in the human intestine.

Deleting mrdA and mrcB to significantly improve extracellular recombinant protein production in Escherichia coli

In this work, two key genes (mrdA and mrcB) involved in cell wall biosynthesis were deleted (singly and doubly) in Escherichia coli, and the effects on extracellular recombinant protein production were investigated. The mrdA and mrcB genes encode penicillin-binding protein (PBP) 2 and PBP1B, respectively. The growth of deletion mutants was not significantly inhibited compared with control cells, but cell morphology was altered. The concentration of intracellular soluble peptidoglycan was increased in deletion mutants, especially the double deletion mutant. Extracellular protein production was significantly improved in deletion mutants compared with controls, in the order double deletion > single deletion of mrcB > single deletion of mrdA. Extracellular green fluorescent protein production by the double deletion mutant BL21 ΔmrdA/ΔmrcB::pET28a-gfp was most significantly enhanced compared with control cells (2.6-fold). Extracellular production of recombinant fibroblast growth factor receptor 2 and collagen E4 was also improved in deletion mutants compared with control cells. Extracellular amylase activity of the double deletion mutant BL21 ΔmrdA/ΔmrcB::pET28a-amyk was increased 5.2-fold relative to control cells, but activity in BL21 ΔmrdA::pET28a-amyk and BL21 ΔmrcB::pET28a-amyk single deletion mutants was only increased 1.7- and 4.3-fold, respectively. Extracellular α-galactosidase activity of deletion mutants was also improved, especially the double deletion mutant (2.6-fold). Membrane permeability of deletion mutants was improved compared with control cells, which might increase extracellular recombinant protein production.

Bacteriological and ecfX- gene specific PCR based identification of Pseudomonas aeruginosa isolated from Chittagong industrial area and characterization of its extracellular amylase

Amylases are the starch degrading enzymes with applications ranging from food, fermentation, textiles to paper industries. In this study, a soil bacterium from the Chittagong industrial area was screened primarily for amylase activity using starch agar media. An attempt has been made to isolate and identify this amylolytic bacterium, optimization of its amylase production and characterization of the crude enzyme. The isolate was identified as Pseudomonas aeruginosa based on microscopic and biochemical tests. Beside this a speciesspecific gene ecfX (146 bp) for P. aeruginosa was amplified by PCR to confirm the identification of the isolate. A broad range of parameters for the optimization of enzyme production based on selection of best carbon and nitrogen sources, optimum pH, temperature, incubation period and effect of additional metal ions on culture media were investigated. Best enzyme activity (343.60 U/mL) was found in the presence of glucose as carbon source with yeast extract as nitrogen source at temperature 37°C after 24h at pH 9 in addition of Mg++ in submerged fermentation broth culture. The Mg++, SDS and Ca++ increased the amylase activity whereas EDTA and Zn++ were found as the inhibitory agents for amylase.
 Jahangirnagar University J. Biol. Sci. 7(2): 99-114, 2018 (December)

Aspergillus oryzae S2 AmyA amylase expression in Pichia pastoris: production, purification and novel properties.

A synthetic cDNA-AmyA gene was cloned and successfully expressed in Pichia pastoris as a His-tagged enzyme under the methanol inducible AOX1 promoter. High level of extracellular amylase production of 72 U/mL was obtained after a 72h induction by methanol. As expected, the recombinant strain produced only the AmyA isoform since the host is a protease deficient strain. Besides, the purified r-AmyA showed a molecular mass of 54kDa, the same pH optimum equal to 5.6 but a higher thermoactivity of 60°C against 50°C for the native enzyme. Unlike AmyA which maintained 50% of its activity after a 10-min incubation at 60°C, r-AmyA reached 45min. The higher thermoactivity and thermostability could be related to the N-glycosylation. The r-AmyA activity was enhanced by 46% and 45% respectively in the presence of 4mM Fe2+ and Mg2+ ions. This enzyme was more efficient in bread-making since such ions were reported to have a positive impact on the nutriment quality and the rheological characteristics of the wheat flour dough. The thermoactivity/thermostability as well as the iron and magnesium activations could also be ascribed to the presence of an additional C-terminal loop containing the His tag.

Extracellular Amylases Produced by Bacillus thuringiensis

Extracellular Amylases Produced by Bacillus thuringiensis

Purification and characterization of \u03b1-amylase from Trichoderma pseudokoningii

BackgroundPrevious studies have demonstrated that members of Trichoderma are able to generate appreciable amount of extracellular amylase and glucoamylase on soluble potato starch. In this study the α-amylase was purified and characterized from Trichoderma pseudokoningii grown on orange peel under solid state fermentation (SSF).ResultsFive α-amylases A1-A5 from Trichodrma pseudokoningii were separated on DEAE-Sepharose column. The homogeneity of α-amylase A4 was detected after chromatography on Sephacryl S-200. α-Amylase A4 had molecular weight of 30 kDa by Sephacryl S-200 and SDS-PAGE. The enzyme had a broad pH optimum ranged from 4.5 to 8.5. The optimum temperature of A4 was 50 °C with high retention of its activity from 30 to 80 °C. The thermal stability of A4 was detected up to 50 °C and the enzyme was highly stable till 80 °C after 1 h incubation. All substrate analogues tested had amylase activity toward A4 ranged from 12 to 100% of its initial activity. The Km and Vmax values of A4 were 4 mg starch/ml and 0.74 μmol reducing sugar, respectively. The most of metals tested caused moderate inhibitory effect, except of Ca2+ and Mg2+ enhanced the activity. Hg2+ and Cd+ 2 strongly inhibited the activity of A4. EDTA as metal chelator caused strong inhibitory effect.ConclusionsThe properties of the purified α-amylase A4 from T. pseudokoningii meet the prerequisites needed for several applications.

Fermentative production of extracellular amylase from novel amylase producer, Tuber maculatum mycelium, and its characterization

Truffles are symbiotic hypogeous edible fungi (form of mushroom) that form filamentous mycelia in their initial phase of the growth cycle as well as a symbiotic association with host plant roots. In the present study, Tuber maculatum mycelia were isolated and tested for extracellular amylase production at different pH on solid agar medium. Furthermore, the mycelium was subjected to submerged fermentation for amylase production under different culture conditions such as variable carbon sources and their concentrations, initial medium pH, and incubation time. The optimized conditions after the experiments included soluble starch (0.5% w/v), initial medium pH of 7.0, and incubation time of 7 days, at room temperature (22 ± 2 °C) under static conditions which resulted in 1.41 U/mL of amylase. The amylase thus obtained was further characterized for its biocatalytic properties and found to have an optimum activity at pH 5.0 and a temperature of 50 °C. The enzyme showed good thermostability at 50 °C by retaining 98% of the maximal activity after 100 min of incubation. The amylase activity was marginally enhanced in presence of Cu2+ and Na+ and slightly reduced by K+, Ca2+, Fe2+, Mg2+, Co2+, Zn2+, and Mn2+ ions at 1 mM concentration.

Media optimization for extracellular amylase production by Pseudomonas balearica vitps19 using response surface methodology

In this study, we optimized the process for enhancing amylase production from Pseudomonas balearica VITPS19 isolated from agricultural lands in Kolathur, India. Process optimization for enhancing amylase production from the isolate was carried out by Response Surface Methodology (RSM) with optimized chemical and physical sources using Design expert v.7.0. A central composite design was used to evaluate the interaction between parameters. Interaction between four factors–maltose (C-source), malt extract (Nsource), pH, and CaCl2 was studied. The factors pH and CaCl2 concentration were found to affect amylase production. Validation of the experiment showed a nearly twofold increase in alpha amylase production. Amylase production was thus optimized and increased yield was achieved.

RECONSTITUTION OF DIFFERENT SIGNAL PEPTIDES FOR ENHANCED THERMOSTABLE ALPHA AMYLASE SECRETION IN Bacillus subtilis

Three signal peptides of alpha amylase genes of three isolated strains that were Bacillus licheniformis DA23, Bacillus subtilis D5-2, Bacillus cereus CN1-5 were successfully sequenced. Three predicted “Sec – type” signal peptides have a length varying from 27 (CN1-5) to 33 residues (D5-2). The secretion of alpha amylase of the recombination B. subtilis 168MPgrac strain (pHV33–PgracAmy3BT2) with 71.4U/ml was larger than that of 168MPamy with 53.2U/ml. Base on analyzed rerults of PAGE and zymogram about molecular weight, alpha amylases in both strains were the same size, nearly 58kDa. The extracellular amylase activity of signal peptide SsubtilisD5.2 in 168M was highest with 76.4±4.3 U/ml in four signal peptide targets.

Characterization of extracellular amylase from Bacillus sp. strain RU1

Optimization production of thermo active levansucrase from Bacillus subtilis Natto CCT 7712Bruna Caroline Marques Gonçalves, Janaina Mantovan, Mara Lúcia Luis Ribeiro, Dionísio Borsato, Maria Antonia Pedrine Colabone Celligoi

Complete signal peptide of Tk1884, an α-amylase from Thermococcus kodakarensis, is not necessary for extracellular secretion of the enzyme by Escherichia coli

AbstractIn order to elucidate if Escherichia coli secretion system recognizes the N-terminally truncated signal sequence of an archaeal α-amylase from Thermococcus kodakarensis (Tk1884) and secretes the recombinant protein to the extracellular medium, we have cloned Tk1884 with the deletion of the sixteen N-terminal amino acids and produced the recombinant protein Tk1884Δ16 in E. coli. Analysis of the intracellular, membranous and extracellular fractions demonstrated the presence of Tk1884Δ16 in all the three fractions. The intracellular α-amylase activity, similar to the membranous fraction, increased with the passage of time till 8 h of induction and then decreased. In contrast, the extracellular α-amylase activity slowly increased with the passage of time after induction. The extracellular amylase activity was purified and determination of the molecular mass by electrospray ionization mass spectrometry demonstrated that Tk1884Δ16 was secreted to the extracellular medium without cleavage of the signal peptide. To the best of our knowledge, this is the first report on recognition of N-terminally truncated signal peptide of archaeal origin by E. coli.

A study to evaluate the effect of phyto silver nano-particles synthesized using Azadiracta indica leaf extract on extracellular fungal amylase and cellulase

In the present study, an attempt has been made to evaluate the effect of plant mediated silver nano-particles against two enzymes i.e. Amylase and Cellulase produced by Aspergillus niger through submerged fermentation under optimum condition and the crude enzyme thus obtained was incubated with the respective substrate in presence of phyto silver nano-particles. The enzyme activity was determined with different concentration of nano-particles. Silver nano-particles were fabricated using leaf extract of Azadiracta indica. The bio-reduction was monitored by UV-Vis spectrophotometer, the possible biomolecules responsible for bioreduction, capping and efficient stabilization were identified using Fourier transform infrared spectroscopy, while the size and shape of the synthesized silver nano-particles was determined using scanning electron microscopy (SEM). The synthesized silver nano-particles were found to be potentially active in enhancing the activity of fungal amylases and cellulase. The study thereby concludes utilization of plant material for a rapid, simple, cost-effective and ecofriendly alternative for nano-particles synthesis and these silver nano-particles may have potential application in processes that requires amylolytic and cellulolytic activity at commercial or industrial levels. The “green nanotechnology” thus aims to exploit plant materials to generate products and processes that are energy efficient as well as economically and environmentally sustainable.

Enhanced Production of α-Amylase from <I>Bacillus licheniformis</I> SKB 4

The effect of some inductive factors including incubation temperature, aeration, inorganic phosphate source, supplementation of amino acids and vitamins on production of extracellular amylase by Bacillus licheniformis SKB 4 was optimized in a submerged fermentation. The maximum production of enzyme was obtained in highly aerobic condition at 42°C, pH 6.5 and 24 h incubation period. 1%, v/v inoculumn concentration exerts best result for growth and enzyme production. Several chemical factors such as inorganic phosphate (KH 2 PO 4 ; 0.3%), amino acids (0.5 mg %; glutamic acid, cysteine, isoleucine), vitamins (biotin, 50μg/ml; pyridoxine, 50μg/ml; riboflavin, 20μg/ml), metallic salt MgSO 4 (1 mM) and surfactant Tween 80 (0.075% (v/v) have positive effect on amylase production. Among the raw food grains used for amylase production, soya bean seed favoured enzyme production and rate production is much higher (2.5 fold) than the normal culture medium, whereas cereals and other pulses have no effect on enzyme production within normal incubation time. Use of raw food grain as culture medium and optimization of certain factors are the most important part for production of amylase in industrial sector.