Extracellular Amylase Research Articles

Resin Acid Copper Salt, an Interesting Chemical Pesticide, Controls Rice Bacterial Leaf Blight by Regulating Bacterial Biofilm, Motility, and Extracellular Enzymes.

Bacterial virulence plays an important role in infection. Antibacterial virulence factors are effective for preventing crop bacterial diseases. Resin acid copper salt as an effective inhibitor exhibited excellent anti-Xanthomonas oryzae pv. oryzae (Xoo) activity with an EC50 of 50.0 μg mL-1. Resin acid copper salt (RACS) can reduce extracellular polysaccharides' (EPS's) biosynthesis by down-regulating gumB relative expression. RACS can also effectively inhibit the bio-mass of Xoo biofilm. It can reduce the activity of Xoo extracellular amylase at a concentration of 100 μg mL-1. Meanwhile, the results of virtual computing suggested that RACS is an enzyme inhibitor. RACS displayed good curative activity with a control effect of 38.5%. Furthermore, the result of the phytotoxicity assessment revealed that RACS exhibited slight toxicity compared with the control at a concentration of 200 μg mL-1. The curative effect was increased to 45.0% using an additional antimicrobial agent like orange peel essential oil. RACS markedly inhibited bacterial pathogenicity at a concentration of 100 μg mL-1 in vivo.

Study on Enzyme Activity and Metabolomics during Culture of Liquid Spawn of Floccularia luteovirens.

To comprehensively investigate the physiological characteristics and metabolic processes of the mycelium of Floccularia luteovirens (F. luteovirens), a wild edible fungus unique to the plateau region, we conducted an in-depth analysis of the mycelium enzyme activity and metabolites during different culture periods. The activity of seven enzymes all followed a trend of initially increasing and then decreasing. The intra- and extracellular activity peaks of three hydrolases-amylase, protease, and cellulase-all occurred on the 20th day, except for the extracellular amylase, which peaked on the 15th day. In contrast, the peak activity of laccase occurred on the 10th day. Moreover, three types of oxidoreductases in the mycelium (catalase (CAT), superoxide dismutase (SOD), and 2,3,5-triphenyltetrazolium chloride (TTC)-dehydrogenase (TTC-DH)) also exhibited significant changes in activity. CAT and SOD activity reached their maximum on the 20th day, whereas TTC-DH showed high activity on both the 10th and 20th days. Through a comprehensive assessment of the evolving trends of these physiological parameters, we determined that the optimal cultivation cycle for F. luteovirens liquid spawn is 20 days. An untargeted metabolomic analysis revealed that 3569 metabolites were detected in the F. luteovirens mycelium, including a variety of secondary metabolites and functional components, with terpenoids being particularly abundant, accounting for 148 types. By comparing three different culture stages (10 days, 20 days, and 30 days), 299, 291, and 381 metabolites, respectively, showed different accumulation patterns in the comparison groups of 10d vs. 20d, 20d vs. 30d, and 10d vs. 30d. These differential metabolites were primarily concentrated in carboxylic acids and their derivatives, fatty acyl groups, organic oxygen compounds, and lipid compounds. In addition, there were several amino acids whose abundance continued to grow during culturing. The metabolism of amino acids greatly affects mycelium growth and development. This research delineates the interplay between mycelium growth and metabolism, offering empirical support for a cultivation strategy for liquid F. luteovirens, and an exploration of its metabolites for potential applications.

The interaction between maize resistant starch III and Bifidobacterium adolescentis during in vitro fermentation

As an alternative to traditional dietary fibers with prebiotic effects, the interaction between resistant starch III (RS3) and gut microbiota is worth exploring. In this study, the effects of RS3 on the proliferation of Bifidobacterium adolescentis (B. adolescentis) and their structural changes before and after fermentation were investigated. Autoclaved-debranched resistant starch (ADRS) demonstrated the best proliferative effect for B. adolescentis and the highest roughness (Ra = 21.90 nm; Rq = 16.00 nm). The rough surface of ADRS was the key for B. adolescentis proliferation. B. adolescentis produced an extracellular amylase to assist degradation and showed the highest activity in ADRS. Fermentation disrupted short-range ordered structure and reduced R1047 cm−1/1022 cm−1 by 20.74 % and R995 cm−1/1022 cm−1 by 30.85 %. The extracellular amylase was essential substance for ADRS degradation. These findings help optimize RS3 structure and promote the proliferation of intestinal probiotics.

Integration of engineering microbial consortium and boosting extracellular amylase of B. amyloliquefaciens for intensifying bioconversion of food waste into lipopeptides

Lipopeptides possess significant antifungal activity and serve as eco-friendly biopesticides. However, their widespread application is limited by exorbitant production costs and low yields. Food waste emerges as a promising substrate for lipopeptide production. To efficiently harness the starch in food waste during bioconversion it into lipopeptides, the engineered Bacillus amyloliquefaciens HM618, constructed by knocking out multiple extracellular proteolytic enzyme genes (aprE, nprE, vpr, and epr) and overexpressing gene amyA with Phag promoter, capable of producing both high-level extracellular amylase and lipopeptides. It was found that the recombinant B. amyloliquefaciens HM3–5 in the pure culture containing starch exhibited notable amylase activity about 17046.37 U/mL, a total of 2530.72 mg/L lipopeptides, and 80.02 % degradation rate of starch. Considering the crucial roles of fatty acids and amino acids precursors in lipopeptide synthesis, these engineered B. amyloliquefaciens producing higher-level extracellular amylase, Yarrowia lipolytica producing fatty acids, and Corynebacterium glutamicum releasing proline and serine were integrated to construct an artificial consortium for efficiently bio-converting food waste into lipopeptides. After optimized inoculation times and ratios of these engineered strains, the levels of surfactin, fengycin, and iturin A in the four-strains artificial consortium during food waste bioconversion reached 8690.67, 635.83, and 74.19 mg/L, respectively. The total lipopeptides in the four-strain artificial consortium reached 9400.69 mg/L, 2.38-fold increase compared to that in the pure culture of strain HM3–5. Furthermore, the degradation efficiencies of starch and oil in food waste were 96.41 % and 98.35 %, respectively. This work provides a new strategy for efficiently bio-transforming food waste into lipopeptides.

Bioprospecting Antibacterial Properties of Fungi Isolated from Kakinada Sea Coast and Eucalyptus Foliage

Background: Fungi produce a wide range of secondary metabolites with bactericidal or bacteriostatic properties. In search of novel antibacterial compounds recently, many fungi of marine and plant origin have been studied for their antimicrobial properties. Aims: This work aimed to study bio-prospect marine and endophytic fungi for their antibacterial properties. objective: To determine the antibacterial properties of ethyl acetate extracts of fungal isolates. Methods: Intensive microbiological methods were followed for isolation, differential growth, and qualitative screening of enzyme production. The isolates were characterised and identified based on morpho-taxonomy, 18S rRNA gene sequencing, and phylogenetic analysis of the tar-get genes. The antimicrobial activity of fungal ethyl acetate extracts against S. aureus, B. sub-tilis, and E. coli was evaluated using a well-diffusion method, and MIC was determined by the microdilution method. Cell lysis was observed through Transmission Electron Microscopy. Results: A BLAST search of 18S rRNA gene sequences of the marine isolates GSBT S13 and GSBT S14 showed 99.3 % sequence similarity with A. glaucus for both isolates and that of endophyte GSBT E3 showed 99.7 % sequence similarity with B. pinkertoniae. Cellulase pro-duction was comparatively higher in GSBT E3 and lipase from GSBT S13 and GSBT S14. Ethyl acetate extracts of GSBT S14 and GSBT E3 showed a clear zone of inhibition by the well-diffusion method, further confirmed by electron microscopy. HR-TEM showed that the ethyl acetate extracts of the isolates appeared to damage the cell membrane, leading to cell shrinkage and death in E. coli and S. aureus. Conclusion: GSBT S13 and GSBT S14 exhibited extracellular amylase, cellulase, and lipase activities. Ethyl acetate extracts of both GSBT S14 and B. pinkertoniae GSBT E3 showed better antibacterial properties against S. aureus.

Production and Optimization of Extracellular α-amylase From Halophilic Bacteria Cytobacillus oceanisediminis Isolated From Wadi-El-Natrun, Egypt

The current study aimed to maximize α-amylase enzyme production by halophilic bacteria Cytobacillus oceanisediminis isolated from soil of Wadi-El-Natrun, Egypt. The results showed that of 21 halophilic bacterial isolates recovered from saline soil samples, eight isolates were high amylase producers. Interestingly, the bacterial isolate AHB6 exhibited the highest extracellular amylase production and was selected for further studies. This isolate was molecularly identified based on the 16srRNA gene sequencing as Cytobacillus oceanisediminis and has the accession number OM759999. The optimum temperature for α-amylase enzyme was 45 °C and exhibited thermostability at 45 °C for 1 h and retained more than 80% of its original activity. The α-amylase enzyme was active in almost all tested pHs and reached its highest activity at pH 9. Also, the optimum culture conditions for α-amylase enzyme production were wheat bran as carbon source, 2 g/l K2HPO4 as potassium source, 6 g/l MgSO4 as magnesium source and 0.5 g/l KNO3 as nitrogen source. These findings suggest the applicability of bacterial isolate Cytobacillus oceanisediminis as a potent producer of α-amylase for industrial purposes. Keywords: Amylase; Optimization; Cytobacillus oceanisediminis; Starch hydrolysis; Thermo-halophilic bacteria.

PURIFICATION AND CHARACTERIZATION OF AMYLASE PRODUCED FROM PROBIOTIC LACTOBACILLUS PLANTARUM CS FOR INDUSTRIAL APPLICATIONS

Previous studies have demonstrated that probiotic Lactobacillus plantarum CS was able to generate an appreciable amount of extracellular amylase, hence the need to purify and characterize it. The aim of the study was to purify and characterize crude amylase from probiotic Lactobacillus plantarum CS for its industrial applications Three purification steps including ammonium sulphate precipitation, ion exchange chromatography on carboxymethyl sephadex and gel filtration on Sephadex G-75 were utilized. The homogeneity of the purified enzyme was confirmed using sodium deodocyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). The purified amylase was characterized on different parameters including substrates hydrolyses, pH and temperature activity and stability profiles. The general purification elution profile revealed two different peaks of amylase activities with outstanding one having a molecular weight of 59.7kDa. Its purification fold was 4.0 with specific activity of 16.44U/mg protein and enzyme yield of 3%. Temperature optimal activity and stability was at 400C and 7.5 for pH activity and stability. Mangenese (Mn2+) (135.17%), tween 80 (128.30%) and some food condiments garlic, thyme, ginger, and tumeric) significantly (p> 0.05) enhanced amylase activity (≥262.40%). However, selenium (Se4+) and hydrogen peroxide (H2O2) were observed to have greatest inhibiting effect (≥30.9%) on the enzyme. Substrate hydrolysis profiles showed that the amylase hydrolyzed all the test starchy substrates with the highest hydrolytic potential on indigenous sweet potato starch (Km value/ Vmax of 1.33mg/ml/ 7.89ml). The rate of hydrolysis of other test substrates had yam> rice>cassava>corn with km values ≤ 4.0mg/ml and Vmax ≤ 25ml. The obtained results gave an insight that amylase produced from Lactobacillus plantarum CS met with the possessed properties suitable for any industrial application especially in food

Programmed cell death in Xanthomonas axonopodis pv. glycines is associated with modulation of gene expression resulting in altered states of motility, biofilm and virulence

One of the foremost report of apoptosis-like programmed cell death (PCD) came from Xanthomonas axonopodis pv. glycines (Xag), which displayed rapid post-exponential cell death in PCD inducing media (PIM) but not in a non-inducing media (PNIM). The current study aims to decipher for the first time, the advantages of the existence of PCD in this phytopathogenic microorganism. Analysis of RNA-seq under inducing and non-inducing conditions, revealed differential expression of a number of genes related to key physiology of Xag, such as, motility, xanthan biosynthesis and export as well as virulence. A PCD negative mutant Xag M42 displayed diminished virulence and a contrasting transcriptome pattern. In vitro experiments revealed that under PCD inducing condition, Xag produced negligible xanthan gum as well as extracellular amylase, displayed enhanced swarming motility, released copious e-DNA and formed scanty biofilm. Lack of ‘diffusible signalling factor’ production was eliminated as possible reason for PCD-induction. Altogether, it appears that, in planta existence of the pathogen metabolically resembles PNIM, and on being transferred to PIM, the cells experience oxidative stress and circumvents it by adopting PCD as an altruistic response. Survival of the remaining population is encouraged by upregulating motility, detachment from the fragile biofilm to achieve dispersal.

Screening, Isolation, and Enzyme Kinetics of Bacterial Amylase collected from Rhizosphere soil

Rhizosphere is a region where microbial communities are in complex association with the roots of plants where the activity of microbes and their enzymes are greatly influenced by root exudates. Amylase enzyme has great importance in biotechnology, with enormous utilization in food, fermentation, textile, and paper industries. They are produced intracellularly and extracellularly by different life forms including microorganisms. Microbial amylases are preferred over other sources because of their vast availability and it also meets the growing needs of industry. The present investigation deals with the isolation, screening, and enzyme kinetics of bacterial amylase from the rhizosphere soil samples collected from a fertile field. Soil samples were collected from the rhizosphere and amylase-producing bacteria screening was carried out by using a starch agar plate. Extracellular amylase was extracted from fermentation broth followed by quantification by starch-iodine assay. Bacterial amylase enzyme kinetics were determined by changing enzyme/substrate concentrations and incubation time. We successfully screened and isolated out starch hydrolyzing colonies from the rhizosphere soil samples. Studies on enzyme kinetics indicate that the activity of amylase increased initially as substrate and enzyme concentrations increased. If we kept enzyme concentration constant, to a certain point, there was no change in enzyme activity as the enzyme was saturated and no more enzyme was available to react with the excess substrate. Initially, enzyme activity increased as enzyme volume increased, but since substrate concentration was kept constant, higher volumes of the enzyme could not speed up the reaction. Further, under a prolonged incubation period, less amount of substrate was available at the end of a reaction. Therefore, it is concluded that the reaction velocity increases.

Isolation, identification, and enzyme screening of fungi infested various vegetables in the local markets of Visakhapatnam, Andhra Pradesh

This study aimed to examine the fungi present in fruits and vegetables obtained from the local vegetable markets, Visakhapatnam and assess their enzymatic activity. seven fungal pathogens responsible for vegetable spoilage were isolated using sterilization and spread plate techniques on potato dextrose agar (PDA) medium. The isolated fungi were identified based on macroscopic and microscopic characteristics. The identified fungal strains included Aspergillus, Curvularia, Alternaria, Cladosporium, Fusarium, sclerotinia, Mucor, Penicillium, and Rhizopus. Only three selected strains were tested for extracellular amylase, pectinase and cellulase activity, which were showing 15mm,20mm,30mm clear zone capability. These findings provide essential information about the degradative properties of filamentous fungi and their secretion of extracellular enzymes. The study also highlights the presence of toxigenic fungi in local vegetables, emphasizing the importance of removing spoiled fruits to prevent the accumulation of toxins associated with fungal growth.

Purification and Characterization of an Amylase from a Newly Isolated Geobacillus thermoleovorans TTIO4 from the Hot Spring of Tuwa‐Timba

AbstractA thermophilic amylase produced by a newly isolated Geobacillus thermoleovorans TTIO4 from a hot spring in India is investigated. The isolate TTIO4 optimally produces extracellular amylase at 55 °C, pH 7, and 24 h of growth. The amylase is purified by 42 fold with a yield of 9.7%. The enzyme has a molecular weight of 40 kDa. The purified amylase catalyzes the reaction optimally at 60 °C and pH 7. Besides, it displays maximum thermal stability at 50 °C for 18 h. While the catalyst is improved by Ca+2, it is wholly inhibited by Hg+2. Furthermore, the chelators, inhibitors and solvents adversely affect the enzyme activity. The amylase is stabilized by SDS, while other surfactants negatively affect the enzyme. Km, Vmax, Kcat, and Kcat/Km of the amylase are 0.26 mg mL−1, 192.3 µg mL−1 min−1, 1.92 s−1, and 7.383 s−1 mg−1, respectively. The deactivation constant, half‐life, and change in Gibb's free energy, enthalpy, and entropy are determined at 50–70 °C, which supports the thermal stability and substrate affinity of the amylase. The washing quality of commercial detergents is distinctly enhanced when supplemented with this enzyme. Therefore, this amylase possesses significant thermal and surfactant stability has significant commercial potential.

A Novel Halo-Acid-Alkali-Tolerant and Surfactant Stable Amylase Secreted from Halophile Bacillus siamensis F2 and Its Application in Waste Valorization by Bioethanol Production and Food Industry.

The extracellular amylase production level by the moderate halophile Bacillus siamensis F2 was optimized, and the enzyme was biochemically characterized. The culture parameters for NaCl, carbon, nitrogen, pH, and temperature were optimized for high titers of amylase production. Growing B. siamensis F2 cultures in Great Salt Lake-2 medium with additions of (in g/L) NaCl (100), starch (30), yeast extract (2), KNO3 (2), and MgSO4 (1) at pH 8, 30 °C resulted in the maximum amylase production (4.2 U/ml). The amylase was active across a wide range of salinities (0 to 30% NaCl), pH (5.0-10.0), and temperatures (20-70 °C) and showed good stability with surfactants (sodium dodecyl sulfate (SDS) and Triton X-100); hence, it was identified as halo-acid-alkali-tolerant and surfactant stable. Temperature, pH, and salinity were optimal for amylase activity at 50 °C, pH 7, and 5% NaCl, respectively. It also generates amylase by utilizing agricultural wastes like sugarcane bagasse, sweet potato peel, and rice husk. Based on the performance of B. siamensis F2 using agricultural wastes and synthesizing amylase, the current study attempted to produce bioethanol by coculturing with baker's yeast using sugarcane bagasse and sweet potato peel as a substrate, which yielded 47 and 57 g/L of bioethanol, respectively. Besides bioethanol production, amylase secreted by F2 was also employed for juice clarification for better yield and clarity and for softening dough to produce better-quality buns. This novel amylase may have many potential applications in waste valorization, biorefinery sectors, and food industries.

Efficacy of the Fruit and Vegetable Peels as Substrates for the Growth and Production of α-Amylases in Marine Actinobacteria.

Enzymes from haloalkaliphilic microorganisms have recently focused attention on their potential and suitability in various applications. In this study, the growth and production of extracellular amylases in the marine actinomycetes, using kitchen waste as the raw starch source, have been investigated. Actinobacteria were isolated from the seawater of the Kachhighadi Coast near Dwarika, Gujarat. Seven Actinobacterial isolates of pre-monsoon, monsoon, and post-monsoon seasons belonging to different strains of Nocardiopsis genera were screened and selected for amylase production. The amylase production was initially assessed on the solid media supplemented with the extracts of different fruits and vegetable peels as a substrate by agar plate assay. The strains Kh-2(13), Kh-2(1), and Kh-3(12) produced maximum amylase with potato peel as a substrate, while no significant differences were found with the media containing other peels. Nevertheless, all strains produced amylases at a significant level with other raw substrates as well. For the optimization of the growth and enzyme production, the selected two isolates Kh-2(13) and Kh-3(12) of the monsoon and winter seasons were cultivated in a liquid medium under the submerged fermentation conditions, with potato peel as a substrate. In both organisms, the optimum amylase production was observed in the stationary phase of growth. For amylase production, the effect of different physical and chemical parameters was evaluated. The optimum growth and amylase production was achieved in 2% inoculum size, at pH 8.0, 28℃, and 5% salt concentration. On the basis of the amylase production index (API) (a ratio of the amylase units and cell growth), both isolates produced significant amylase with the only extract of potato peels, without any other supplements. The trends further indicated that while additional complex sources, such as yeast extract and peptone can enhance the cell growth of the actinobacteria, the amylase production remained unaltered. The study projects the significance of waste raw materials for the production of enzymes in extremophilic microorganisms.

Bioinspired Trichogenic Silver Nanoparticles and Their Antifungal Activity Against Plant Pathogenic Fungi Sclerotinia sclerotiorum MTCC 8785

There is a pressing need for new nanomaterials for multipurpose functions. The biological synthesis of nanoparticles is environment-friendly, least toxic, and cost-effective. An experiment was designed to use extracellular amylases in the cell-free filtrate (CFF) for the biosynthesis of silver nanoparticles (AgNPs) from the Trichoderma harzianum MTCC 801 strain. Potato dextrose broth (PDB) as general-purpose growth media and amylase production media (APM) as enzyme-specific production media have been used for sub-merged fungal cultivation and nanoparticle synthesis. AgNPs synthesized in the CFF of PDB were compared with AgNPs synthesized from the CFF using APM. The cell-free filtrate obtained upon enzyme stimulation has contributed to the reduction and capping process of nanosilver. The synthesized AgNPs showed a spectral peak at 420 nm, a characteristic feature of AgNPs. The particles were monodispersed, 50 nm in size, and spherical in shape as well as have shown an antifungal effect (100% inhibition) against Sclerotinia sclerotiorum MTCC8785. This is the first report to synthesize trichogenic AgNPs using extracellular amylases against the phytopathogen Sclerotinia strain.

Purification and Production Optimization of Alpha Amylases from Bacillus Species

Amylases are the digestive enzymes belong to the family of glycoside hydrolase capable of hydrolysing starch and polysaccharides. These are the recent breakthroughs in food, feed, pulp and pharma industries. Our aim of this study is to purify amylases from Bacillus subtilis and Bacillus megaterium and optimize the growth conditions to achieve the cost-effective production of amylases from bacillus species. Extensive research on amylases from different sources is essential in producing enzymes in large scale and in optimizing the growth conditions and minimize the production cost to meet the increased demand of these industrially important enzymes. Amylases were produced in the current study using submerged fermentation and purified the extracellular enzymes from bacillus species using ultracentrifugation, Ammonium sulphate precipitation and gel filtration chromatography. There was no effect on amylase production from bacillus species when the initial substrate concentration is increased from 1% to 5%. Increase in inoculum size from 1% to 5% and the addition of yeast extract to broth had significantly increased the amylase production. The study observed significantly higher levels of amylase production using rice starch when compared with wheat starch also higher levels of amylase production observed with B. megaterium when compared with B. subtilis. The study concludes 1% rice starch and 5% B. megaterium inoculum can be used for cost effective production of amylases. In the current study, cost effective amylase production was evaluated and able to optimize the yield of extracellular amylases from Bacillus megaterium to a concentration level of 120 IU/ml of fermentation broth.

Glycogen-Degrading Activities of Catalytic Domains of α-Amylase and α-Amylase-Pullulanase Enzymes Conserved in Gardnerella spp. from the Vaginal Microbiome.

Gardnerella spp. are associated with bacterial vaginosis in which normally dominant lactobacilli are replaced with facultative and anaerobic bacteria, including Gardnerella spp. Co-occurrence of multiple species of Gardnerella is common in the vagina, and competition for nutrients such as glycogen likely contributes to the differential abundances of Gardnerella spp. Glycogen must be digested into smaller components for uptake, a process that depends on the combined action of glycogen-degrading enzymes. In this study, the ability of culture supernatants of 15 isolates of Gardnerella spp. to produce glucose, maltose, maltotriose, and maltotetraose from glycogen was demonstrated. Carbohydrate-active enzymes (CAZymes) were identified bioinformatically in Gardnerella proteomes using dbCAN2. Identified proteins included a single-domain α-amylase (EC 3.2.1.1) (encoded by all 15 isolates) and an α-amylase-pullulanase (EC 3.2.1.41) containing amylase, carbohydrate binding modules, and pullulanase domains (14/15 isolates). To verify the sequence-based functional predictions, the amylase and pullulanase domains of the α-amylase-pullulanase and the single-domain α-amylase were each produced in Escherichia coli. The α-amylase domain from the α-amylase-pullulanase released maltose, maltotriose, and maltotetraose from glycogen, and the pullulanase domain released maltotriose from pullulan and maltose from glycogen, demonstrating that the Gardnerella α-amylase-pullulanase is capable of hydrolyzing α-1,4 and α-1,6 glycosidic bonds. Similarly, the single-domain α-amylase protein also produced maltose, maltotriose, and maltotetraose from glycogen. Our findings show that Gardnerella spp. produce extracellular amylase enzymes as "public goods" that can digest glycogen into maltose, maltotriose, and maltotetraose that can be used by the vaginal microbiota. IMPORTANCE Increased abundance of Gardnerella spp. is a diagnostic characteristic of bacterial vaginosis, an imbalance in the human vaginal microbiome associated with troubling symptoms, and negative reproductive health outcomes, including increased transmission of sexually transmitted infections and preterm birth. Competition for nutrients is likely an important factor in causing dramatic shifts in the vaginal microbial community, but little is known about the contribution of bacterial enzymes to the metabolism of glycogen, a major food source available to vaginal bacteria. The significance of our research is characterizing the activity of enzymes conserved in Gardnerella species that contribute to the ability of these bacteria to utilize glycogen.

Overexpression of a Thermostable α-Amylase through Genome Integration in Bacillus subtilis

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.

DETERMINATION AND OPTIMIZATION OF CRUDE EXTRACELLULAR AMYLASE EXTRACTED FROM DIFFERENT BACILLUS SPECIES


 
 
 Amylases are starch hydrolyzing enzymes and have commercial applications in different industries, such as food, fermentation, detergent and pharmaceuticals. In the current study amylases were extracted from bacteria found in kitchen wastes and rice fields. Isolates with maximum starch hydrolysis determined by the clear zones formation against blue background were selected and examined for further extracellular amylase activity. 16S rRNA gene sequencing confirmed these isolates as Bacillus subtilis, Bacillus sp. and Lysinibacillus sp. Highest dextrizing activity (83%)f and starch hydrolysis ratio (2.8) were shown by Bacillus subtilis G3. Furthermore optimum amylase activity was also checked at different temperatures, pH, media composition and substrate concentration. Optimum reaction temperature and pH for crude amylase activity from Bacillus subtilis G3 was 60°C -75°C and 9pH in minimal media supplemented with 0.1% soluble starch. Thin layer chromatography and high performance liquid chromatography were carried out to investigate the starch hydrolyzed products (glucose, maltose and maltosaccharides) produced by amylolytic action of amylase. In addition to it molecular weight of amylase was found to be about 68kDa as revealed by SDS PAGE analysis after acetone precipitation. These bacteria could be genetically manipulated for large scale enzyme production which can be ultimately used for commercial purposes.
 
 

Temperature and pH Profiling of Extracellular Amylase from Antarctic and Arctic Soil Microfungi

While diversity studies and screening for enzyme activities are important elements of understanding fungal roles in the soil ecosystem, extracting and purifying the target enzyme from the fungal cellular system is also required to characterize the enzyme. This is, in particular, necessary before developing the enzyme for industrial-scale production. In the present study, partially purified α-amylase was obtained from strains of Pseudogymnoascus sp. obtained from Antarctic and Arctic locations. Partially purified α-amylases from these polar fungi exhibited very similar characteristics, including being active at 15 °C, although having a small difference in optimum pH. Both fungal taxa are good candidates for the potential application of cold-active enzymes in biotechnological industries, and further purification and characterization steps are now required. The α-amylases from polar fungi are attractive in terms of industrial development because they are active at lower temperatures and acidic pH, thus potentially creating energy and cost savings. Furthermore, they prevent the production of maltulose, which is an undesirable by-product often formed under alkaline conditions. Psychrophilic amylases from the polar Pseudogymnoascus sp. investigated in the present study could provide a valuable future contribution to biotechnological applications.

Beer fermentation performance and sugar uptake of Saccharomycopsis fibuligera-A novel option for low-alcohol beer.

There is a growing trend for beers with novel flavor profiles, as consumers demand a more diversified product range. Such beers can be produced by using non-Saccharomyces yeasts. The yeast species Saccharomycopsis fibuligera is known to produce exceptionally pleasant plum and berry flavors during brewer’s wort fermentation while its mycelia growth is most likely a technological challenge in industrial-scale brewing. To better understand and optimize the physiological properties of this yeast species during the brewing process, maltose and maltotriose uptake activity trials were performed. These revealed the existence of active transmembrane transporters for maltose in addition to the known extracellular amylase system. Furthermore, a single cell isolate of S. fibuligera was cultured, which showed significantly less mycelial growth during propagation and fermentation compared to the mother culture and would therefore be much more suitable for application on an industrial scale due to its better flocculation and clarification properties. Genetic differences between the two cultures could not be detected in a (GTG)5 rep-PCR fingerprint and there was hardly any difference in the fermentation process, sugar utilization and flavor profiles of the beers. Accordingly, the characteristic plum and berry flavor could also be perceived by using the culture from the single cell isolate, which was complemented by a dried fruit flavor. A fermentation temperature of 20°C at an original gravity of 10 °P proved to be optimal for producing a low-alcohol beer at around 0.8% (v/v) by applying the S. fibuligera yeast culture from the single cell isolate.