Maximum Enzyme Activity Research Articles

Cloning, Over-Expression, and Purification of β–Carbonic Anhydrase from an Extremophilic Bacterium: Deinococcus radiodurans

In this study, the cloning, purification and initial characterization of carbonic anhydrase (DrCA) enzyme which we consider to be important in the resistance physiology from extremely radioresistant bacteria Deinococcus radiodurans is performed. In addition, the effect of increased gamma irradiation doses on pH-related DrCA enzyme activity was determined. DrCA activity after radiation treatment showed that the activity continuously increased by 6 fold, up to the first 800 Gy, which a decrease in activity was observed thereafter. The maximum CO2 hydration activity for DrCA enzyme was observed at pH 7.0 and 40°C. DrCA enzyme, homo-dimer complex, is slightly thermostable. The activity of DrCA was significantly enhanced by several metal ions, especially Zn2+, which resulted in 5-fold increases of CO2 hydration activity. Also sulfonamide showed inhibitory effect on the pure enzyme. The apparent Km and Vmax for CO2 as substrate were 8.4 mM and 637 WAU/mg for DrCA respectively. The CO2 hydration assay demonstrated that the specific activity of purified recombinant enzymes (DrCA) was significantly high.

Valorization of corn stover and molasses for enzyme synthesis, lignocellulosic hydrolysis and bioethanol production by Hymenobacter sp. CKS3

During the last few decades, energy demand is increasing rapidly. Concerning this, the use of renewables — lignocellulose biomass, for bioethanol production, as an efficient alternative to replacing fossil fuels, is highly recommended. In this study, valorization of two agricultural wastes was used for various hydrolytic enzyme production by Hymenobacter sp. CKS3, lignocellulosic hydrolysis, and bioethanol production. Conditions for obtaining maximum enzyme production, using agro-industrial waste — molasses and corn stover, were statistically optimized. Under the optimal conditions, in a medium containing 5.0% corn stover, 2.5% molasses, and during 94.55 h (∼4 days) of fermentation, the maximum enzymatic activity was achieved - CMCase 1.11 IU/ml, Avicelase 0.92 IU/ml, and pectinase 3.69 IU/ml. The obtained crude enzyme mixture was further used for enzymatic hydrolysis of non-treated corn stover and bioethanol production. The reducing sugar yield of 3.85 g/l was obtained under optimal conditions (corn stover 6.6% and time of hydrolysis 78.8 h (∼3 days and 7 h)). Scanning electron microscopy revealed structural changes in corn stover samples after enzymatic hydrolysis. Under non-optimized conditions, 0.37% of ethanol was produced by waste brewer’s yeast. The obtained results show that bacteria belonging to the genus Hymenobacter have a still unexplored enzymatic potential that could be used for sustainable production of biotechnologically value-added products including biofuels. Specifically, for the first time, a soil bacterium, classified within the genus Hymenobacter, was used for cellulases (CMCase and Avicelase) and pectinase production.

Optimization of Cultural Conditions for Maximum Production of Fibrinolytic Enzymes from the Local Marine Bacterial Isolates and Evaluation of their Wound Healing and Clot Dissolving Properties

Fibrinolytic enzymes find necessary applications to treat and prevent cardiovascular diseases. In this study, optimal conditions for enhancing the production of fibrinolytic enzyme from local marine bacterial strains were evaluated. The present study also focuses on screening of wound healing efficacy of the isolated fibrinolytic enzymes.Various physical parameters such as temperature, pH, incubation time and medium components viz. inoculum size, substrate (nitrogen and carbon) concentrations were optimized. A cultivation medium was designed using optimized conditions for mass production of fibrinolytic enzyme and specific activity of enzyme was analyzed. The maximum enzyme production was observed at 37 °C temperature, 8.0 pH,substrate concentration with 3 ml inoculum size and 32 h. of incubation time. Among the different carbon sources tested, Mannitol showed maximum enzyme activity i.e 538 U/ml. yeast extract was found to be the best nitrogen source with an enzyme activity of 498 U/ml. The best substrate for the production fibrinolytic enzyme was found to be kernelwith high activity of 1056U/ml. The crude enzyme displayed potent activity and digested blood clot completely in in vitro condition and exhibited potent activity on wound healing property in macrophages.

A multi-functional genetic manipulation system and its use in high-level expression of a β-mannanase mutant with high specific activity in Pichia pastoris.

SummaryTo further extend the practical application of a thermostable and acidic resistance β‐mannanase (ManAK) in animal feed additives, an effective strategy that combined directed evolution and metabolic engineering was developed. Four positive mutants (P191M, P194E, S199G and S268Q) with enhanced specific activity (25.5%–60.9%) were obtained. The S199G mutant exhibited 56.7% enhancement of specific activity at 37°C and good thermostability, and this was selected for high‐level expression in P. pastoris X33. A multi‐functional and scarless genetic manipulation system was proposed and functionally verified (gene deletion, substitution/insertion and point mutation). This was then subjected to Rox1p (an oxygen related transcription regulator) deletion and Vitreoscilla haemoglobin (VHb) co‐expression for high enzyme productivity in P. pastoris X33VIIManAKS199G. An excellent strain, named X33VIIManAKS199G ∆rox1::VHb, was achieved by combining these two factors, and then the maximum enzymatic activity was further increased to 3753 U ml‐1, which was nearly twice as much as the maximum production of ManAK in P. pastoris. This work provides a systematic and effective method to improve the enzymatic yield of β‐mannanase, promotes the application of ManAK in feed additives, and also demonstrated that a scarless genetic manipulation tool is useful in P. pastoris.

Adding value to agro-industrial waste for cellulase and xylanase production via solid-state bioconversion

The lignocellulosic biomass is presenting an enormous potential in biofuel production, which requires a hydrolyzing step in the bioconversion process. The biomass is hydrolyzed using costly enzymes like cellulase and xylanase. These enzymes can be produced by using lignocellulosic biomass as feedstock, and on-site produced crude enzyme can saccharify biomass. The integration of both approaches can lower down the cost of biofuel production. Keeping this in view, the present study was conducted to explore various agro-industrial waste as carbon sources and best-suited conditions for enhanced cellulase and xylanase production by Aspergillus flavus under solid-state fermentation. Among all studied biomass, microwave alkali-pretreated rice straw was found most suitable as a carbon source for enzyme production. Maximum enzyme activities FPase 12.5 IU/gds, CMCase 235 IU/gds, β-glucosidase 190 IU/gds, and xylanase 180 IU/gds were obtained under optimum conditions of initial pH 5.5, temperature 30 °C, substrate to liquid ratio 1:3.5 (w/v), particle size 500–1000 μm, and NaNO3 on the 5th day of the incubation period. Enzyme activities FPase, CMCase, β-glucosidase, and xylanase were enhanced by approximately 1.27-, 1.18-, 1.06-, and 1.09-fold during tray fermentation. The adopted approach utilized low-cost lignocellulosic biomass for enzyme production as an additional avenue to biomass valorization along with solid waste management and its reduction which would otherwise cause environmental pollution problems.

Immobilization of Zymomonas mobilis In situ in Flexible Polyurethane and Potential for Bioconversion in Sodium Maltobionate

Maltobionic acid and its salts are produced by the action of the periplasmic enzymatic complex glucose-fructose oxidoreductase (GFOR), and glicono-δ-lactonase (GL) from Zymomonas mobilis and, for such, cell immobilization is outstanding. Thus, the objective of this work was to immobilize, in situ, Z. mobilis cells containing GFOR/GL in flexible polyurethane foam (PU) in order to produce maltobionic acid. In the immobilization, concentrations of polyurethane and biomass support constituents varied, and the visual aspect of the material immobilized was assessed, its enzymatic activity, immobilization yield, and operational stability. In the maximized immobilization condition, morphological analysis of the support and bioconversion runs were performed. The scanning electron microscopy analysis showed that the microorganism was trapped in the PU (7 g of polyol; 3.5 g of isocyanate; 0.02 g of silicone, and 7 g of biomass). The immobilized material maximum enzymatic activity against glucose was 19.40 U g-1dry cell, with an immobilization yield of 60.29% and 15 reuses. In bioconversion, 491.42 mmol L-1 of sodium maltobionate were produced in 47.56 h, with a conversion factor of 0.88 and mass productivity, specific productivity, and specific product formation speed of 2.58 mmol h-1; 1.81 mmol g-1 h-1 and 6.25 mmol g-1 h-1, respectively. This study's results contribute to the production of aldonic acids because it uses scarce information in the area and application potentials.

ENZYMATIC AND ANTIMICROBIAL ACTIVITIES OF ACTINOMYCETES SPECIES OBTAINED FROM MAYANUR DAM, TAMIL NADU, INDIA

As understanding the impacts of river impoundment will facilitate conservation initiatives in developing countries in tropical latitudes where development of large impoundments continues to threaten rich biological diversity, the present study was attempted to study the fresh water impoundment, Mayanur Dam, situated in the banks of the River Cauvery in Tamil Nadu. Alterations to species composition or community structure, such as those that follow river impoundment, may disrupt the ability of the ecosystem to provide goods and services. One of the primary goals in bioprospecting studies with microorganisms is to enhance the natural product discovery which can be used in pharmaceutical or other industry sectors. As exploration of potential bacteria is an important approach to discovering novel antibiotics to meet the current needs, the present study was attempted to study antimicrobial activity of selected actinomycetes species obtained from Mayanur fresh water system. All the species under study showed the presence of all enzymatic activities except Actinomyces and Micromonospora which did not record chitinase activity. Among the various actinomycetes species, Streptomyces recorded maximum enzymatic activity for all the enzymes except urease. The maximum urease activity was recorded by Actinopolyspora. Among the various species, Micromonospora recorded minimal enzymatic activities with regard to protease, urease, cellulose, and chitinase. The results clearly indicate that all the actinomycetes species recorded antimicrobial activity but at different levels. Among the various actinomycetes species, Streptomyces recorded the maximum zone of inhibition against all the bacteria except Staphylococcus epidermidis. in addition, Streptomyces was the most efficient actinomycetes species against all the bacteria analysed except for S. epidermis where Actinopolyspora recorded the maximum zone of inhibition. Further, the present study also suggested that Streptomyces and Actino

Cystobasidium psychroaquaticum as a new promising source of valuable bioactive molecules

Screening procedure for the selection of yeasts able to produce bioactive molecules continues to be an important aspect of yeast biotechnology. The aim of the present study was to characterize two oleaginous yeasts from WUT Yeast Collection, WUT71 and WUT117, assigned to Cystobasidium psychroaquaticum. The emphasis was put on their ability to accumulate intracellular carotenoids, a valuable health-promoting molecules and to produce extracellular hydrolases, an important biocatalysts. The best carotenogenesis, 3.146 ± 0.340 mg l−1, was evaluated for C. psychroaquaticum WUT117 at 15 °C, C:N ratio of 20:1 and glucose as the carbon source. Obtained carotenoid extracts were subsequently used in MTT test on HaCaT cell line, showing no cytotoxicity up to 500 mg l−1. Noteworthy, carotenoids derived from C. psychroaquaticum strains have not been analyzed in this respect so far. The maximum enzymatic activities obtained for C. psychroaquaticum WUT71 and WUT117 were 426.81 U ml−1 (at 15 °C) and 346.69 U ml−1 (at 20 °C), respectively. Hence, two new C. psychroaquaticum strains, capable of producing both carotenoids and hydrolases were identified.

Production of Alkaline Proteases using Aspergillus sp. Isolated from Injera: RSM-GA Based Process Optimization and Enzyme Kinetics Aspect.

Alkaline proteases are well known to be significant industrial enzymes. This study focused on isolating the fungus producing proteases from, a typical Ethiopian food, Injera. Further, the process optimization for protease production using response surface methodology (RSM) and the characterization of the acquired protease were investigated. The 18S rDNA gene sequence homology of the fungus isolate revealed that it was Aspergillus sp. Further, it was deposited in NCBI GenBank with accession number MK4262821. Using the isolate, owing to maximize the protease production, the independent process parameters, temperature, pH, and sucrose concentration were optimized using RSM followed by a genetic algorithm (GA). Based on the statistical approach by RSM-GA optimization, maximum enzyme activity (166.4221 U/ml) was found at 30.5°C, pH 8.24, and 0.316% sucrose concentration. Also, the crude cocktail of enzymes acquired from optimal condition was partially purified using ammonium which showed that the increased activity by 1.96-fold. Considerably, the partially purified enzyme exhibited stable performance during the temperature range 30-60°C, pH range 7-10, and NaCl concentration of 0.5-2mM. Also, the antioxidant activity, degree hydrolysis for the protein, Michaelis-Menten (M-M) kinetic parameters, and activation energy were determined for the obtained enzyme cocktail. It showed that the M-M kinetic parameters, Km (5.54mg/ml), and Vmax (24.44mg/ml min) values were observed. Using Arrhenius law, the value of activation energy for the enzyme cocktail was determined as 32.42kJ/mol.

L-arabinose isomerase from Lactobacillus parabuchneri and its whole cell biocatalytic application in D-tagatose biosynthesis from D-galactose

l-arabinose isomerases (L-AIS) are enzymes that mediate the conversion of l-arabinose into l-ribulose and D-galactose into D-tagatose. The potential of L-AI from Lactobacillus parabuchneri CICC 6004 (LPAI) in D-galactose biotransformation using E. coli whole-cells was studied. The parameters investigated in this study were 1) the optimum pH, temperature, and cofactor specificity for maximum enzyme activity; 2) the D-tagatose biosynthesis from D-galactose using whole-cells containing LPAI. The results showed that with D-galactose, the purified LPAI showed the highest activity, 15.52 U/mg at pH 6.7, 45 °C, and necessitates 3 mM Ca2+ as its cofactor. The Km, Vmax and Kcat/Km values of LPAI for D-galactose were 5.4 mM, 35.5 U/mg and 2.8 mM−1min−1, respectively, while the respective values for l-arabinose were found to be 4.3 mM, 63.1 U/mg and 5.9 mM−1min−1.The applicability of LPAI for D-tagatose synthesis was shown by using whole-cell biocatalyst approach. Under the optimum conditions, during one-stage biotransformation of D-galactose by E. coli whole cells, 39.0 g/L of D-tagatose was produced from 140 g/L of D-galactose after 50 h, which corresponded to the yield and productivity of 0.28 g/g and 0.78 g/L/h, respectively. Further addition of new cells into the reaction mixture in the second stage produced 54.0 g/L of D-tagatose after 100 h. The D-tagatose total yield and productivity were 0.39 g/g and 0.54 g/L/h, respectively. The results attained in this study showed LPAI as a potential candidate for the industrial production of D-tagatose.

The degradation activities for three seaweed polysaccharides of Shewanella sp. WPAGA9 isolated from deep-sea sediments.

Seaweed oligosaccharides possess great bioactivities. However, different microbial strains are required to degrade multiple polysaccharides due to their limited biodegradability, thereby increasing the cost and complexity of production. Shewanella sp. WPAGA9 was isolated from deep-sea sediments in this study. According to the genomic and biochemical analyses, the extracellular fermentation broth of WPAGA9 had versatile degradation abilities for three typical seaweed polysaccharides including agar, carrageenan, and alginate. The maximum enzyme activities of the extracellular fermentation broth of WPAGA9 were 71.63, 76.4, and 735.13 U/ml for the degradation of agar, alginate, and carrageenan, respectively. Moreover, multiple seaweed oligosaccharides can be produced by the extracellular fermentation broth of WPAGA9 under similar optimum conditions. Therefore, WPAGA9 can simultaneously degrade three types of seaweed polysaccharides under similar conditions, thereby greatly reducing the production cost of seaweed oligosaccharides. This finding indicates that Shewanella sp. WPAGA9 is an ideal biochemical tool for producing multiple active seaweed oligosaccharides at low costs and is also an important participant in the carbon cycle process of the deep-sea environment.

Recombinant expression and characterization of Oryctolagus cuniculus chymosin in Komagataella phaffii (Pichia pastoris)

This study was conducted for investigating expression and enzymatic characteristics of recombinant Oryctolagus cuniculus chymosin (ROCC) expressed in Pichia pastoris. SDS-PAGE of partially purified supernatant displayed two distinct molecular bands approximately at the sizes of 40 kDa and 45 kDa corresponding to chymosin and partially glycosylated chymosin, respectively. Proteolysis assay demonstrated that rabbit chymosin was more specific compared to bovine and camel chymosins when it comes to hydrolyzing α, β, and κ-casein. Rabbit chymosin kept its stability in a wide pH range (3.0–6.0) at 37 °C for 8 h. Active chymosin exhibited maximum enzymatic activity at 40 °C and pH 4.0 with the addition of 75 mM CaCl2. The ROCC clotting activity on donkey, cow, goat, lamb, camel milk was determined as 40, 10, 5.7, 3.07, and 2.66 IMCU/mL, respectively. These results revealed that ROCC might possess a potential for incorporation into cheese manufacture technology as a milk-clotting enzyme.

Antioxidants, lysosomes and elements status during the life cycle of sea trout Salmo trutta m. trutta L.

The aim of our study was to elucidate the effects of both development stages (parr, smolt, adult, spawner), and kelt as a survival form and sex (male, female) on the functional stability of the lysosomal complex, biomarkers of oxidative stress, and element contents in the muscle tissue of the sea trout (Salmo trutta m. trutta L.) sampled in the Pomerania region (northern Poland). We have evaluated the maximal activities of lysosomal enzymes (alanyl aminopeptidase, leucyl aminopeptidase, β-N-acetylglucosaminidase, and acid phosphatase), lipid peroxidation level, and protein carbonyl derivatives as indices of muscle tissue degradation. The relationship between lysosomal activity and oxidative stress biomarkers estimated by the lipid peroxidation level and protein carbonyl derivatives was also assessed, as well as the relationships between element levels and oxidative stress biomarkers. Trends of the main effects (i.e., the development stages and sex alone, the interaction of the sex and development stage simultaneously) on oxidative stress biomarkers, lysosomal functioning, and element contents in the muscle tissue were evaluated. The study has shown sex-related relationships between the pro- and antioxidant balance and the tissue type in the adult stage as well as modifications in the lysosomal functioning induced by long-term environmental stress associated with changing the habitats from freshwater to seawater and intense migrations. The highest level of toxic products generated in oxidative reactions and oxidative modification of proteins was noted in both the spawner stage and the kelt form. The holistic model of analysis of all parameters of antioxidant defense in all development stages and sex demonstrated the following dependencies for the level of lipid peroxidation, oxidative modification of proteins, lysosomal activities, and element contents: TBARS > OMP KD > OMP AD > TAC, AcP > NAG > LAP > AAP and Cu > Fe > Ca > Mn > Zn > Mg, respectively.

An implication of biotransformation in detoxification of mercury contamination by Morganella sp. strain IITISM23.

The contamination of soil by heavy metals such as Hg is growing immensely nowadays. The drawbacks of physicochemical methods in the decontamination of polluted soils resulted in the search for an eco-friendly and cost-effective means in this regard. In this study, a potential Hg-resistant bacterial (IITISM23) strain was investigated for their removal potential of Hg, isolated from Hg-contaminated soil. IITISM23 strain was identified as Morganella sp. (MT062474.1) as it showed 99% similarity to genus Morganella of Gammaproteobacteria based on 16S rRNA gene sequencing. The toxicity experiment confirmed that the strain showed high resistance toward Hg. In low nutrient medium, EC50 (effective concentration) values were 6.8 ppm and minimum effective concentration (MIC) was 7.3 ppm, and in a nutrient-rich medium, EC50 value was 32.29 ppm and MIC value was 34.92 ppm, respectively. In in vitro conditions, IITISM23 showed the removal efficiency (81%) of Hg (II) by the volatilization method in Luria-Bertani (LB) broth. The changes in surface morphology of bacteria upon the supplementation of Hg (II) in broth media were determined by SEM-EDX studies, while the changes in functional groups were studied by FT-IR spectroscopy. The mercury reductase activity was determined by a crude extract of the bacterial strain. The optimal pH and temperature for maximum enzyme activity were 8 and 30oC, with Km of 3.5 μmol/l and Vmax of 0.88 μmol/min, respectively. Also, strain IITISM23 showed resistance toward various antibiotics and other heavy metals like cadmium, lead, arsenic, and zinc. Hence, the application of microbes can be an effective measure in the decontamination of Hg from polluted soils.

ACTIVITY AND ISOZYME COMPOSITION OF PEROXIDASE IN SCOTS PINE (PINUS SYLVESTRIS L.) NEEDLES EFFECTED BY TECHNOGENIC EMISSIONS FROM VARIOUS ENTERPRISES AND VEHICLES

Background. The technogenic pollution leads to excessive production of reactive oxygen species (ROS) in plants which are highly reactive and toxic and cause damage to biomolecules. Plants have a complex antioxidant defense system that protects cells from the ROS and maintain homeostasis. The most important link this system is enzymes, in particular, peroxidase. It was of interest to determine the expression of the protective properties of one of the sensitive species of coniferous plants under the influence of technogenic emissions from various enterprises and vehicles.
 Purpose. Investigation the activity and isoenzyme composition of peroxidase in the needles of Pinus sylvestris L. under the influence of technogenic emissions of different compositions in the Baikal region.
 Materials and methods. The pine needles were collected on sample plots located near an aluminum plant, thermal power plant, chemical plant, coal mining enterprise, and the highway. The activity of soluble guaiacol-dependent peroxidases was defined by spectrophotometry in a reaction mixture with citrate-phosphate buffer, hydrogen peroxide, and guaiacol. Native polyacrylamide gel electrophoresis was used for determination of peroxidase isoforms.
 Results. It was shown that an increase in the total guaiacol-dependent peroxidase activity ranged from 6 to 22 times in the pine needles in polluted areas. Maximum enzyme activity was found in needle samples collected near the aluminum smelter, whose emissions are characterized by large amounts of fluorides and polycyclic aromatic hydrocarbons. The high variability of peroxidase isoform composition in Scots pine needles under industrial pollution was revealed. It was expressed in the emergence of new isoforms in the zone of fast-moving (Rf from 61 to 100) and medium-moving (Rf from 31 to 60) items. The maximum number of isoforms (nine) was found in pine needles near the aluminum smelter with only two ones detected in the background area.
 Conclusion. Peroxidase activity and the number of its newly formed isoforms can adequately reflect the degree of technogenic pollution and trees decline. The indicators can also be used in monitoring of coniferous forests condition.

Vermicomposting Process to Endosulfan Lactone Removal in Solid Substrate Using Eisenia fetida

Pesticide by-products found in soil are usually more toxic and persistent than the pesticides themselves. For example, Endosulfan lactone (EL) (a by-product of the organochloride pesticide endosulfan). EL is created by the enzymatic activity (and related oxidative processes) of microorganisms in the soil. A sustainable method of EL removal is the introduction of Eisenia fetida earthworm. In this paper, it will be demonstrated the impact of vermicomposting process related to Eisenia fetida earthworm on EL by measuring initial and final concentrations of the compound and overall enzymatic activity in sterile and non-sterile solid substrate over 56 days. As a baseline, it be observed there were higher EL removals in non-sterile solid substrate (90.86%) at day 5 than in sterile solid substrate (83.86%) at day 14. In samples with Eisenia fetida, the presence of EL in non-sterile solid substrate was 36%, however in sterile solid substrate it was only 18% at day 1 and 7, with a maximum enzyme activity of 0.4659 mmol/mg protein per min at day 7. The evidence found in this study suggests that EL removal in a non-sterile solid substrate is higher when a vermicomposting is present and that the influence of microorganisms from the solid substrate with the earthworm, increases removal.

Producción y caracterización de una esterasa extracelular termoestable de Aspergillus niger

A strain of Aspergillus niger NRRL 337 degrade agro-industrial waste i.e wheat bran in solid state fermentation and produce esterase enzyme. Maximum enzyme activity 15.2 U/ml was produced after 72 h of incubation at 30˚C. The protein was partially purified by performing ammonium sulfate precipitation and immobilized metal ion chromatography (IMAC) technique. SDS-PAGE analysis showed molecular weight of esterase was 70 kDa. Effects of different nitrogrn and carbon sources with different concentrations were also studied. Partially purified enzyme was active at pH of 6.0 for up to 3 h and at temperature of 70˚C for 1 h. Ca2+, Mg2+, Na+1, and K+1 play an important role in increasing enzyme activity. The residual activity of esterase was decreased in the presence of organic solvents. SDS, β-mercaptoethanol, and DMSO are good inhibitors for esterase enzyme. Under these conditions, partially purified esterase with specific activity of 114.20 U/mg and 47.58 purification fold was obtained. The data achieved from this work are important in terms of heat stability and pH of esterase and giving a new enzyme source for industrial applications.

Lactose inducible fermentation in Escherichia coli for improved production of recombinant urate oxidase: Optimization by statistical experimental designs

Abstract The enzyme urate oxidase (UOX) is used as a drug for preventing and treatment of hyperuricemia. This study deals with the statistical optimization of lactose inducible fermentation for production of soluble recombinant Aspergillus flavus UOX. 10 independent variables were investigated by Plackett–Burman design (PBD), and the most significant factors were further optimized by central composite design (CCD). PBD results indicated that glycerol, yeast extract, tryptone, and lactose affected UOX activity significantly. The CCD results showed that the maximum enzyme activity (19.34 U/ml) and biomass concentration (44.0 g/L) can be achieved under the optimum conditions of glycerol 0.87 g/L, yeast extract 9.11 g/L, tryptone 10.29 g/L, K2HPO4 1.81 g/L, and lactose 12.79 g/L. When the same induction strategy was tested at shake flask, 19.34 U/mL of UOX activity was obtained, which was 12.5 folds higher than IPTG induction protocol. Furthermore, the lower total cost (€0.7 vs. €13.5) was additional feature that confirmed the suitability of the lactose induction method. Collectively, our results showed that design of experiment methodology can be applied as a suitable tool for improved production of UOX enzyme using lactose as the inducer.

Optimization of the cultivation conditions of Bacillus licheniformis BCLLNF-01 for cellulase production

The objective of this study was to optimize the production of CMCase by Bacillus licheniformis BCLLNF-01, a strain associated with the mucus of the zoanthid Palythoa caribaeorum (Cnidaria, Anthozoa). Production of total cellulase and CMCase was investigated in the supernatant, intracellular content and wall content. Cultivation was carried out in BLM medium supplemented with 1.5 % (w/v) CMC, 5.5 % (v/v) inoculum, 40 °C, pH 6.5, 500 rpm for 72 h, and the highest activity was recorded in the supernatant. A Rotational Central Composite Design (RCCD) 2³ was used to investigate the influence of the carbon source concentration (CMC-0.5 to 1.5 % w/v), inoculum concentration (1–10 % v/v) and temperature (35–45 °C) on CMCase production. The maximum enzyme activity was achieved for a CMC concentration of 1.5 % w/v at 40 °C, attaining 0.493 IU/mL after 96 h of cultivation.

Statistical factorial designs for optimum production of thermostable α-amylase by the degradative bacterium Parageobacillus thermoglucosidasius Pharon1 isolated from Sinai, Egypt

BackgroundThis study aimed to isolate potent thermophilic and amylolytic bacteria from a hot spring of Pharaoh’s bath, Sinai, Egypt, and screen its degradative activity. The amylolytic activity was further optimized using a statistical full factorial design followed by response surface methodology. ResultsA thermophilic bacterium was isolated from the hot spring of Pharaoh’s Bath, Sinai, Egypt. The isolate produced amylase, cellulase, and caseinase and was identified by 16S rRNA gene sequencing as Parageobacillus thermoglucosidasius Pharon1 (MG965879). A growth medium containing 1% soluble starch was found to optimize the amylase production. Dinitrosalycalic acid method (DNS) was used to estimate the amount of reducing sugar produced. Statistical full factorial and response surface designs were employed to optimize physical variables affecting the α-amylase production and determine the significant interactions of the studied variables during the fermentation process. According to the results obtained by the response optimizer, the maximum amylase activity reached 76.07 U/mL/ min at 54.1°C, pH 5.6 after 98.5 h incubation under aerobic conditions. Moreover, the produced enzyme was thermostable and retained most of its activity when exposed to a high temperature of 100°C for 120 min. Maximum enzyme activity was attained when the enzyme was incubated at 70°C for 30 min. ConclusionsThis is the first report of the production of thermostable α-amylase by the potent thermophilic Parageobacillus thermoglucosidasius. The enzyme endured extreme conditions of temperature and pH which are important criteria for commercial and industrial applications.