Level Of Enzyme Production Research Articles

Enhancement of Catalytic Performance of α-dextranase from Chaetomium gracile Through Optimization and Suitable Shear Force

Dextran causes negative impact on the normal production of sugar industry. Traditional removal methods have many disadvantages. 6-alpha-d-glucan-6-glucanohydrolase (EC 3.2.1.11) from Chaetomium gracile without food safety concern could remove dextran, but it has low level of enzyme production. The present study aimed to improve α-dextranase activity to meet industry demand. Consequently, α-dextranase activity reached 242.8 U/mL when fermentation condition was 29 °C, pH 6.0 and 220 rpm. Meanwhile, ten glass beads were added to fermentation medium when fermentation time was 12 h, which improved enzyme activity by 135.5% with the highest activity. Purified α-dextranase exhibited excellent thermal stability and surfactants tolerance. α-Dextranase from C. gracile could remove dextran by 46.6% in mixed sugarcane juice and 14.1% in clarified sugarcane juice. It had similar dextran removal efficiency with commercial enzymes. These evaluations showed that α-dextranase had great potential in removing dextran in sugar industry.

Enzimas de interés industrial

For many years, industrial enzymes have played an important role in the benefit of our society due to their many useful properties and a wide range of applications. They are key elements in the progress of many industries including foods, beverages, pharmaceuticals, diagnostics, therapy, personal care, animal feed, detergents, pulp and paper, textiles, leather, chemicals and biofuels. During recent decades, microbial enzymes have replaced many plant and animal enzymes. This is because microbial enzymes are widely available and produced economically in short fermentations and inexpensive media. Screening is simple, and strain improvement for increased production has been very successful. The advances in recombinant DNA technology have had a major effect on production levels of enzymes and represent a way to overproduce industrially important microbial, plant and animal enzymes. It has been calculated that 50-60% of the world enzyme market is supplied with recombinant enzymes. Molecular methods, including genomics and metagenomics, are being used for the discovery of new enzymes from microbes. Also, directed evolution has allowed the design of enzyme specificities and better performance.

Control of biofouling by xanthine oxidase on seawater reverse osmosis membranes from a desalination plant: enzyme production and screening of bacterial isolates from the full-scale plant.

Xanthine oxidase, an oxidoreductase enzyme that generates reactive oxygen species, is endogenously produced by many bacterial species. In this study, production of the enzyme by bacterial isolates from a full-scale desalination plant was investigated for potential use as biological control of membrane fouling in seawater desalination. We have previously demonstrated that free radicals generated by a commercially available xanthine oxidase in the presence of a hypoxanthine substrate, effectively dispersed biofilm polysaccharides on industrially fouled membranes. Bacterial xanthine oxidase production in the presence of hypoxanthine may prove to be a cost effective, insitu method for alleviation of fouling.

Production of chitinase from thermophilic Humicola grisea and its application in production of bioactive chitooligosaccharides

A novel thermophilic chitinase producing strain Humicola grisea ITCC 10,360.16 was isolated from soil of semi-arid desert region of Rajasthan. Maximum enzyme production (116±3.45Ul−1) was achieved in submerged fermentation. Nutritional requirement for maximum production of chitinase under submerged condition was optimized using response surface methodology. Among the eight nutritional elements studied, chitin, colloidal chitin, KCl and yeast-extract were identified as the most critical variables for chitinase production by Plackett–Burman design first. Further optimization of these variables was done by four-factor central composite design. The model came out to be significant and statistical analysis of results showed that an appropriate ratio of chitin and colloidal chitin had resulted into enhancement in enzyme production levels. Optimum concentration of the variables for enhanced chitinase production were 7.49, 4.91, 0.19 and 5.50 (gl−1) for chitin, colloidal chitin, KCl and yeast extract, respectively. 1.43 fold enhancement in chitinase titres was attained in shake flasks, when the variables were used at their optimum levels. Thin layer chromatography revealed that enzyme can effectively hydrolyze colloidal chitin to produce chitooligosaccharides. Chitinase production from H. grisea and optimization of economic production medium heighten the employment of enzyme for large scale production of bioactive chitooligosaccharides.

Production, purification, characterization and usage of a detergent additive of endoglucanase from isolated halotolerant Amycolatopsis cihanbeyliensis mutated strain Mut43

The Amycolatopsis cihanbeyliensis Mut43, which is obtained by UV radiation, exhibited endoglucanase activity of 5.21 U/mL, which was ∼2.3-fold higher than that of the wild strain (2.04 U/mL). The highest enzyme activity was obtained after 3 days of incubation at 32 °C, pH 7.0, 150 rpm, and 6% NaCl in a liquid medium containing 1.5% (w/v) wheat straw (0.25 mm of particle size) and 0.6% (w/v) yeast extract. Enzyme activity was eluted as a single peak (gel filtration chromatography), and Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) analysis of the corresponding peak revealed a molar mass of 30 kDa. Zymogram analysis confirmed the presence of a single active endoglucanase component. The enzyme was purified to ∼21-fold, and the mean overall yield was ∼6%. The purified endoglucanase was active up to 80 °C and showed a half-life of 214 min at 60 °C in the absence of substrate at pH 8.0. The apparent Km value for the purified endoglucanase was 0.70 mg/mL, while the Vmax value was 6.20 Units/μg. Endoglucanase activity was reduced (25%) by treatment with 30 U of proteinase K/mg. The addition of Mg+2 and Ca+2 (5 mM) enhanced endoglucanase activity. Additionally, endoglucanase activity in the presence of 5 mM SDS or organic solvents was 75 and 50% of maximum activity, respectively. The high levels of enzyme production from A. cihanbeyliensis Mut43 achieved under batch conditions, coupled with the temperature stability, activity over a broad pH range, relatively high stability (70–80%) in the presence of industrial laundry detergents and storage half-lives of 45 days at +4 °C and 75 days at −20 °C signify the suitability of this enzyme for industrial applications as detergent additive.

Pancreatic exocrine insufficiency in malnourished children and those with persistent diarrhoeae.

Persistent diarrhoea, a serious health problem, is closely related to malnutrition. Children with severe malnutrition have a 9-fold risk of death, and children with severe stunting have a 4-fold risk of death. Prolonged mucosal injury from diarrhoea causes reduced secretin and cholecystokinin secretion, which decreases stimulation to the pancreas, and is indicated by faecal elastase-1 levels. This further aggravates persistent diarrhoea and malnutrition because of the low levels of digestive enzyme production. This study evaluated the exocrine function of the pancreas in children with persistent diarrhoea and malnutrition. This study used a cross-sectional design to compare exocrine pancreatic function among children with persistent diarrhoea, children with malnutrition, and apparently healthy children as reference Children aged 6-60 months were selected from the inpatient and outpatient units of various general hospitals in Jakarta. Faecal elastase- 1 levels were used to determine exocrine pancreatic function. The median values of faecal elastase- 1 in children with persistent diarrhoea, children with malnutrition, and reference children were 743 (1-1503) mcg/g, 861 (17-2909) mcg/g, and 1210 (26-3000) mcg/g, respectively. A significant difference was observed in the faecal elastase-1 levels between reference children and those with persistent diarrhoea (p<0.001). However, no differences in the faecal elastase-1 levels were noted between malnourished and reference children (p>0.05). Children with both persistent diarrhoea and malnutrition showed mean FE-1 392.3±206.9 and median 419 (125- 593). Exocrine pancreatic insufficiency is found in children with persistent diarrhoea. Children with combined persistent diarrhoea and malnutrition have the lowest FE-1, to which persistent diarrhea has the most significant contribution.

Screening Cellulolytic Bacteria from the Digestive Tract Snail (Achatina fulica) and Test the Ability of Cellulase Activity

&lt;p&gt;On the research of enzyme production levels observed cellulase produced by bacteria in the digestive tract of the isolation of the Snail (&lt;em&gt;Achatina fulica&lt;/em&gt;). Isolation of bacteria based on the ability of bacteria to grow on CMC media. The purpose of this study was to determine cellulase activity by cellulolytic bacteria. Some bacterial isolates were identified as cellulolytic bacteria, they were KE-B1, KE-B2, KE-B3, KE-B4, KE-B5, and KE-B6. Isolates KE-B6 was the best isolates. Furthermore KE-B6 isolates were grown on media production to determine the pattern of growth and enzyme activity. Measurement of cell growth was conducted by inoculating starter aged 22 hours at CMC production of liquid medium. Cellulase enzyme activity measurements was performed by the DNS method. The results showed that the highest activity by new isolate bacteria KE-B6 and its value of the activity of 0.4539 U/mL, growth rate (µ) 0.377/hour and generation time (g) 1.84 hour. This research expected cellulase of producing bacteria were easy, inexpensive and efficient. This enzyme can be used as an enzyme biolytic once expected to replace expensive commercial enzyme. The biotylic enzyme can be applied to strains improvement (protoplast fusion).&lt;/p&gt;&lt;p&gt;&lt;strong&gt;How to Cite&lt;/strong&gt;&lt;/p&gt;&lt;p&gt;Wijanarka, W., Kusdiyantini, E. &amp;amp; Parman, S. (2016). Screening Cellulolytic Bacteria from the Digestive Tract Snail (&lt;em&gt;Achatina fulica&lt;/em&gt;) and Test the Ability of Cellulase Activity. &lt;em&gt;Biosaintifika: Journal of Biology &amp;amp; Biology Education&lt;/em&gt;, 8(3), 386-392. &lt;/p&gt;

β-galactosidase Production by Aspergillus niger ATCC 9142 Using Inexpensive Substrates in Solid-State Fermentation: Optimization by Orthogonal Arrays Design.

Background:Enzymatic hydrolysis of lactose is one of the most important biotechnological processes in the food industry, which is accomplished by enzyme β-galactosidase (β-gal, β-D-galactoside galactohydrolase, EC 3.2.1.23), trivial called lactase. Orthogonal arrays design is an appropriate option for the optimization of biotechnological processes for the production of microbial enzymes.Methods:Design of experimental (DOE) methodology using Taguchi orthogonal array (OA) was employed to screen the most significant levels of parameters, including the solid substrates (wheat straw, rice straw, and peanut pod), the carbon/nitrogen (C/N) ratios, the incubation time, and the inducer. The level of β-gal production was measured by a photometric enzyme activity assay using the artificial substrate ortho-Nitrophenyl-β-D-galactopyranoside.Results:The results showed that C/N ratio (0.2% [w/v], incubation time (144 hour), and solid substrate (wheat straw) were the best conditions determined by the design of experiments using the Taguchi approach.Conclusion:Our finding showed that the use of rice straw and peanut pod, as solid-state substrates, led to 2.041-folds increase in the production of the enzyme, as compared to rice straw. In addition, the presence of an inducer did not have any significant impact on the enzyme production levels.

A novel high molecular weight thermo-acidoactive β-glucosidase from Beauveria bassiana

An extracellular high molecular weight β-glucosidase was secreted by a local strain P1 of Beauveria bassiana. The enzyme was produced in the presence of various carbon sources, namely glucose, maltose, lactose, glycerol, starch, wheat bran and gruel. The highest level of β-glucosidase activity was produced with wheat bran at the concentration of 3%. Glucose caused a repressor effect on the β-glucosidase expression in a dose-dependent manner. The highest enzyme production level was obtained at initial pH of 6.0 and 7.0 in the culture medium. The zymography analysis revealed that B. bassiana secreted a β-glucosidase with high molecular weight between 400 and 600 kDa. The enzymatic preparation was characterized and showed temperature and pH optima of 55°C and 5.0, respectively. The enzyme was stable at 40 and 50°C but its stability declined at 60°C. Interestingly, this β-glucosidase had high stability at acid and basic pH saving its initial activity after 24 h incubation at pH from 3.0 to 11.0. It was stable also in presence of monovalent Na+ and K+ ions saving 60% of its initial activity at 2 M salts. Bivalent metal ions preserved totally or partially the enzymatic activity; in addition, Ba2+ was revealed as an activator. This is the first report that focuses on the production and the biochemical characterization of a β-glucosidase from the entomopathogenic fungus, B. bassiana.

Assessment of hemolytic activity, enzyme production and bacteriocin characterization of Bacillus subtilis LR1 isolated from the gastrointestinal tract of fish.

In the present investigation, probiotic potential (antagonistic activity, enzyme production, hemolytic activity, biosafety, antibiotic sensitivity and bile tolerance level) of Bacillus subtilis LR1 was evaluated. Bacteriocin produced by the bacterial strain B. subtilis LR1 isolated from the gastrointestinal tract of Labeo rohita was purified and characterized. The molecular weight of the purified bacteriocin was ~50kDa in 12% Native PAGE and showed inhibitory activity against four fish pathogens such as Bacillus mycoides, Aeromonas salmonicida, Pseudomonas fluorescens and Aeromonas hydrophila. The purified bacteriocin was maximally active at temperature 40°C and pH 7.0, while none of the tested surfactants affect the bacteriocin activity. Extracellular enzyme activity of the selected bacterial strain was also evaluated. Amylase activity was estimated to be highest (38.23±1.15µg of maltose liberated mg-1proteinml-1 of culture filtrate) followed by cellulase and protease activity. The selected bacterium was sensitive to most of the antibiotics used in this experiment, can tolerate 0.25% bile salt and non-hemolytic in nature. Finally, the efficiency of the proposed probiotic candidate was evaluated in in vivo condition. It was detected that the bacterial strain can effectively reduce bacterial pathogenicity in Indian major carps.

New sources of halophilic lipases: Isolation of bacteria from Spanish and Turkish saltworks

High-salinity biotopes in Turkey and Spain have been demonstrated to be a suitable source of lipolytic enzyme-producing halophilic microorganisms. Three strains turned out to grow at high NaCl concentration (greater than 15%) and their ability to synthesize lipolytic enzymes was demonstrated on tributyrin and olive oil-containing plates by the formation of transparent or orangish halos around the colonies. The three isolates were cultivated in liquid medium and one of them was cherry-picked in terms of the enzyme production levels. It was subsequently genetically identified as Halomonas sp. LM1C. Response Surface Methodology (RSM) based on central composite design was used to optimize the culture conditions for lipase production, concluding that the maximum levels of lipase biosynthesis were obtained at 21.6°C and pH 6.9. The impact of NaCl on the lipolytic activity of the obtained halophilic crude enzyme revealed an improved resistance against deactivation, when compared with a commercial lipase from Candida rugosa. The optimum reaction conditions (obtained by RSM) for the crude enzyme-biocatalyzed hydrolysis of p-nitrophenyl laurate turned out to be neutral pH and 29°C. The process at optimum conditions was kinetically characterized by means of logistic equations, obtaining maximum activities near to 250U/L and the use of a Luedeking & Piret type model allowed elucidating the metabolic characteristics of the synthesized lipolytic enzymes. The present investigation lays the foundation for implementing a biotechnological process to produce highly resistant lipolytic enzymes able to perform its biocatalytic function under extreme salt conditions.

An age-dependent model to analyse the evolutionary stability of bacterial quorum sensing

Bacterial communication is enabled through the collective release and sensing of signalling molecules in a process called quorum sensing. Cooperative processes can easily be destabilized by the appearance of cheaters, who contribute little or nothing at all to the production of common goods. This especially applies for planktonic cultures. In this study, we analyse the dynamics of bacterial quorum sensing and its evolutionary stability under two levels of cooperation, namely signal and enzyme production. The model accounts for mutation rates and switches between planktonic and biofilm state of growth. We present a mathematical approach to model these dynamics using age-dependent colony models. We explore the conditions under which cooperation is stable and find that spatial structuring can lead to long-term scenarios such as coexistence or bistability, depending on the non-linear combination of different parameters like death rates and production costs.

Copper exposure induces toxicity to the antioxidant system via the destruction of Nrf2/ARE signaling and caspase-3-regulated DNA damage in fish muscle: Amelioration by myo-inositol

The muscle is the main portion of fish that is consumed by humans. Copper (Cu) can induce oxidative damage in fish muscle. However, the effects of Cu exposure on the muscle antioxidant system and molecular patterns and preventive measures against these effects remain unclear. In this study, ROS production, enzymatic and mRNA levels of antioxidant enzymes and NF-E2-related factor 2 (Nrf2) signaling-related molecules, antioxidant response element (ARE) binding ability, DNA fragmentation and caspase-3 activities were analyzed in fish muscle following Cu exposure or myo-inositol (MI) pre-administration. The results indicated that contamination due to copper exposure caused an approximately three-fold increase in ROS production, induced lipid peroxidation and protein oxidation, and resulted in depletion of the glutathione (GSH) content of fish muscle. Moreover, Cu exposure caused decreases in the activities of total superoxide dismutase (T-SOD), CuZnSOD, and glutathione peroxidase (GPx) that were accompanied by decreases in CuZnSOD, GPx1a, GPx1b and signaling factor protein kinase C delta mRNA levels. The decreases in the antioxidant enzyme gene mRNA levels were confirmed to be partly due to the reduced nuclear Nrf2 protein levels, poor ARE binding ability and increased caspase-3 signaling-modulated DNA fragmentation in the fish muscle. Interestingly, MI pre-treatment prevented fish muscle from Cu-induced oxidative damages mainly through increasing the GSH content, and increasing the CuZnSOD and GPx activities and corresponding mRNA levels and ARE binding ability. Taken together, our results show for the first time that Cu exposure caused oxidative damage to the muscle by decreasing the antioxidant enzyme activities via the down-regulation of the expression of genes related to the disruption of the Nrf2/ARE signaling, and this down-regulation was partially caused by caspase-3-regulated DNA fragmentation. Finally, MI protects fish against Cu toxicity.

Degradation of intact chicken feathers by Thermoactinomyces sp. CDF and characterization of its keratinolytic protease.

Thermoactinomyces is known for its resistance to extreme environmental conditions and its ability to digest a wide range of hard-to-degrade compounds. Here, Thermoactinomyces sp. strain CDF isolated from soil was found to completely degrade intact chicken feathers at 55 °C, with the resulting degradation products sufficient to support growth as the primary source of both carbon and nitrogen. Although feathers were not essential for the expression of keratinase, the use of this substrate led to a further 50-300 % increase in enzyme production level under different nutrition conditions, with extracellular keratinolytic activity reaching its highest level (∼400 U/mL) during the late-log phase. Full degradation of feathers required the presence of living cells, which are thought to supply reducing agents necessary for the cleavage of keratin disulfide bonds. Direct contact between the hyphae and substrate may enhance the reducing power and protease concentrations present in the local microenvironment, thereby facilitating keratin degradation. The gene encoding the major keratinolytic protease (protease C2) of strain CDF was cloned, revealing an amino acid sequence identical to that of subtilisin-like E79 protease from Thermoactinomyces sp. E79, albeit with significant differences in the upstream flanking region. Exogenous expression of protease C2 in Escherichia coli resulted in the production of inclusion bodies with proteolytic activity, which could be solubilized to an alkaline solution to produce mature protease C2. Purified protease C2 was able to efficiently hydrolyze α- and β-keratins at 60-80 °C and pH 11.0, representing a promising candidate for enzymatic processing of hard-to-degrade proteins such as keratinous wastes.

When, where and how does microbial community composition matter?

When, where and how does microbial community composition matter?

Protein redesign by learning from data.

Protein redesign methods aim to improve a desired property by carefully selecting mutations in relevant regions guided by protein structure. However, often protein structural requirements underlying biological characteristics are not well understood. Here, we introduce a methodology that learns relevant mutations from a set of proteins that have the desired property and demonstrate it by successfully improving production levels of two enzymes by Aspergillus niger, a relevant host organism for industrial enzyme production. We validated our method on two enzymes, an esterase and an inulinase, creating four redesigns with 5-45 mutations. Up to 10-fold increase in production was obtained with preserved enzyme activity for small numbers of mutations, whereas production levels and activities dropped for too aggressive redesigns. Our results demonstrate the feasibility of protein redesign by learning. Such an approach has great potential for improving production levels of many industrial enzymes and could potentially be employed for other design goals.

Improvement of cellulase and xylanase production by solid‐state fermentation of Stachybotrys microspora

The current study investigated the production of cellulases and xylanases from the rare fungus Stachybotrys microspora under solid-state fermentation (SSF) on wheat bran (WB). A comparison of both activities was first performed in submerged cultures using various concentrations of WB, glucose, and cellulose as substrates. The maximal activity of β-glucosidases and xylanases was obtained with 2% and 4% WB, respectively, whereas cellulose yielded the highest endoglucanase production. The SSF conditions were therefore consequently optimized. A moisture content of 70% gave the most significant levels of enzyme production. Inoculation by spores led to better results than by preculture, with 10(5) spores per gram of dried matter as the best inoculum dose for all activities tested. Interestingly, the WB-based medium need not to be supplemented by an exogeneous nitrogen source. Considering the richness of S. microspora secreted proteins as lytic hydrolases, the crude extracellular enzyme extracts were successfully tested in two different biotechnological fields: protoplasting of fungi and subsequent extraction of their DNA, paper pulp hydrolysis to produce fermentable sugars.

ISOLATION, IDENTIFICATION, AND MEDIA OPTIMIZATION OF HIGH-LEVEL CELLULASE PRODUCTION BY Bacillus sp. BCCS A3, IN A FERMENTATION SYSTEM USING RESPONSE SURFACE METHODOLOGY

Cellulases are important glycosyl hydrolase enzymes, which break down cellulose to β-glucose. They have been used widely in biotechnological processing such as bioethanol production. In this work we studied maximizing cellulase production by Bacillus sp. BCCS A3 using response surface methodology (RSM). A good result was attained with these conditions (% w/v): tryptone 0.1, Na2PO4 0.25, (NH4)2SO4 0.2, MgSO4 · 7H2O 0.005, CaCl2 0.005, KH2PO4 0.1, NaCl 0.1, sodium carboxymethylcellulose (CMC) 0.75, and pH 9. The cellulase activity in optimized medium was 49.80 U/ml. Moreover, high level of enzyme production was obtained by using fermentor system (50.30 U/ml). Thus, according to the obtained results, this statistical method provided quick identification and integration of key medium details for Bacillus sp. BCCS A3, leading to more cellulase production.

Expression of organophosphorus hydrolase in Escherichia coli for use as whole-cell biocatalyst

Among the various removal strategies against neurotoxic organophosphorus (OP) compounds, a live catalyst using whole-cell microorganisms has been considered as a useful scavenger with the virtue of cost-effectiveness and elimination efficiency. To over the catalytic activity by wild-type isolates, the expression of organophosphorus hydrolase (OPH) has been attempted in Escherichia coli for the extended detoxification efficacy to OP chemicals. However, early studies had unsatisfactory results, like low enzyme production levels due to the formation of insoluble inclusion bodies from the recombinant enzyme produced within the cells, and a bottleneck caused by the cell membrane of gram-negative strain E. coli acting as a permeability barrier to substrate diffusions. To date, various approaches have been suggested as effective solutions for overcoming these limits. This review will outline current studies, and their critical findings, on the successful expression of OPH in E. coli for the effective recombinant E. coli whole-cell biocatalysts.

Optimization and partial characterization of culture conditions for the production of alkaline protease from Bacillus licheniformis P003

Proteolytic enzymes have occupied a pivotal position for their practical applications. The present study was carried out under shake flask conditions for the production of alkaline protease from Bacillus licheniformis P003 in basal medium containing glucose, peptone, K2HPO4, MgSO4 and Na2CO3 at pH 10. The effect of culture conditions and medium components for maximum production of alkaline protease was investigated using one factor constant at a time method along with its characterization. Maximum level of enzyme production was obtained after 48h of incubation with 2% inoculum size at 42°C, under continuous agitation at 150 rpm, in growth medium of pH 9. Highest enzyme production was obtained using 1% rice flour as carbon source and 0.8% beef extract as organic nitrogen source. Results indicated that single organic nitrogen source alone was more suitable than using in combinations and there was no significant positive effect of adding inorganic nitrogen sources in basal medium. After optimization of the parameters, enzyme production was increased about 20 fold than that of in basal medium. The crude enzyme was highly active at pH 10 and stable from pH 7–11. The enzyme showed highest activity (100%) at 50°C, and retained 78% relative activity at 70°C. Stability studies showed that the enzyme retained 75% of its initial activity after heating at 60°C for 1h. The enzyme retained about 66% and 46% of its initial activity after 28 days of storage at 4°C and room temperature (25°C) respectively. Mn2+ and Mg2+ increased the residual activity of the enzyme, whereas Fe2+ moderately inhibited its residual activity. When pre-incubated with Tween-20, Tween-80, SDS and H2O2, each at 0.5% concentration, the enzyme showed increased residual activity. These characteristics may make the enzyme suitable for several industrial applications, especially in leather industries.