Level Of Enzyme Production Research Articles

Effect of Metals on Amaranth Decolorization by White-Rot Fungus Pleurotus eryngi F019

The aim of this study is to evaluate amaranth textile dye decolorization by white-rot fungus Pleurotus eryngi F019 isolated from tropical rain forest. The isolate was able to decolorize an azo dye in wide range (up to 200 mg), at temperature 25°C. The results revealed that the removal azo dye amaranth was rapid at the initial incubation period (15 days) and became slowly with the time (30 days). The maximum decolorization was observed after 30 days incubation. Addition of Cu2+, Mg2+, Mn2+ gave the positive results while decolorization was inhibited by addition of Fe2+. Metal ion also affected the level of enzyme production during decolorization of amaranth. Induction in the activity of laccase and lignin peroxidase was observed during decolorization of amaranth in the culture, which represented their important role in biotransformation. The biodegradation of amaranth dye was monitored by UV-Vis spectrometer and gas chromatography. These promising results suggest the application of Pleurotus eryngi F019 to treat dye containing wastewater having higher concentrations of metals.

ANTIOXIDANT ENZYME LEVELS AS MARKERS FOR TYPE 2 DIABETES MELLITUS

Type 2 diabetes mellitus (T2DM) is a metabolic disorder characterized by increased production of free radicals and oxidative stress. Oxidative stress can be defined as an imbalance between the removal of free radical production and altered activity levels of antioxidant enzymes. The aim of this study was to evaluate the antioxidant enzyme activity such as superoxide dismutase (SOD), catalase (CAT) and glutathione S transferase (GST) in T2DM patients and healthy subjects in a North Indian population. The study was conducted on 474 T2DM and 301 healthy subjects. Blood lysates were prepared and levels of antioxidant enzymes viz. SOD, CAT and GST were measured by spectrophotometric method. Data were analyzed by using Prism software (v 5.01) and expressed as Mean ± SE. The activity levels of SOD and GST were significantly lower in T2DM patients than in healthy subjects (P<0.05) whereas that of catalase was higher in T2DM patients (15.56±1.53 units/mg) than in healthy subjects (12.12±1.212 units/mg). Analysis of data also showed that enzyme activity levels decreased with increasing age both in normal and T2DM conditions. Only GST level showed significant decrease in diabetic males while both GST and SOD decreased in diabetic females. Assay of enzyme activity levels in erythrocytes could be used as markers to identify individuals predisposed to T2DM.

Comparative transcription analysis and toxin production of two fluoroquinolone-resistant mutants of Clostridium perfringens

BackgroundFluoroquinolone use has been listed as a risk factor for the emergence of virulent clinical strains of some bacteria. The aim of our study was to evaluate the effect of fluoroquinolone (gatifloxacin) resistance selection on differential gene expression, including the toxin genes involved in virulence, in two fluoroquinolone-resistant strains of Clostridium perfringens by comparison with their wild-type isogenic strains.ResultsDNA microarray analyses were used to compare the gene transcription of two wild types, NCTR and ATCC 13124, with their gatifloxacin-resistant mutants, NCTRR and 13124R. Transcription of a variety of genes involved in bacterial metabolism was either higher or lower in the mutants than in the wild types. Some genes, including genes for toxins and regulatory genes, were upregulated in NCTRR and downregulated in 13124R. Transcription analysis by quantitative real-time PCR (qRT-PCR) confirmed the altered expression of many of the genes that were affected differently in the fluoroquinolone-resistant mutants and wild types. The levels of gene expression and enzyme production for the toxins phospholipase C, perfringolysin O, collagenase and clostripain had decreased in 13124R and increased in NCTRR in comparison with the wild types. After centrifugation, the cytotoxicity of the supernatants of NCTRR and 13224R cultures for mouse peritoneal macrophages confirmed the increased cytotoxicity of NCTRR and the decreased cytotoxicity of 13124R in comparison with the respective wild types. Fluoroquinolone resistance selection also affected cell shape and colony morphology in both strains.ConclusionOur results indicate that gatifloxacin resistance selection was associated with altered gene expression in two C. perfringens strains and that the effect was strain-specific. This study clearly demonstrates that bacterial exposure to fluoroquinolones may affect virulence (toxin production) in addition to drug resistance.

Efficient Degradation of Feather by Keratinase ProducingBacillussp.

Keratinase producing microorganisms are being increasingly utilized for degradation and recycling of poultry feather waste. Two native strains BF11 (Bacillus subtilis) and BF21 (Bacillus cereus) degrading keratin completely were characterized. The native strains produced more than 10 KU/mL of enzyme. Strain improvement resulted in isolation of MBF11 and MBF21 from BF11 and BF21 isolates, respectively. Optimization of nutritional and physical parameters of these MBF isolates at laboratory scale increased the overall keratinase activity by 50-fold resulting in a yield of 518–520 KU/mL. Fermentation media designed with starch as carbon source and soya bean meal as nitrogen source supported high levels of enzyme production. The optimum conditions for enzyme production were determined to be pH 8.5 and temperatures of 45–55°C for MBF11 and 37°C for MBF21, respectively. Culture filtrate showed a significant increase in the amounts of cysteine, cystine, methionine, and total free amino acids during the fermentation period. The ratio of organic sulphur concentration was also considerably higher than that of the inorganic sulphate in the culture filtrate suggesting the hydrolysis of disulphide by the isolates.

Study on the Use of Agricultural Wastes for Cellulase Production by Using Aspergillus Niger

It was the goal to investigate the Cellulase enzyme production ability of fungal strains such as Aspergillus niger against the lignocellulosic bio-waste like Rice husks, Millet husks, Maize cobs, Wheat straws and leaves at varying environmental parameters of pH, substrate concentration and dry weight and wet weight. The enzyme production was analyzed individually by Miller's modified method of Dinitrosalicylic acid ( DNSA ). In this study the high level of enzyme production was achieved at pH 3 by using Rice husks, Millet husks, Maize cobs, Wheat straws and leaves and pH 4 by using Maize cobs at varying substrate concentrations. Out of all the five used substrates rice husks gave the higher production of the cellulose enzyme. The importance of cellulase enzyme in industries cannot be over emphasized.

A cellulase-free, thermoactive and alkali xylanase production by terrestrial Streptomyces sp. CA24

Microbial xylanases have attracted much attention because of their applications in industrial processes during the past few decades. Sixty-five terrestrial actinomycetes previously isolated from rhizospheric soils of endemic plants were tested for their ability in production of xylanase. The results revealed that 33 isolates could degrade xylan on both solid and liquid media. Extracellular xylanase production by these isolates was characterized during their growth in liquid medium. Streptomyces sp. CA24, one of the most active strains, was selected for optimization of xylanase production. The enzyme secretion was enhanced when the medium was supplemented with oat spelt xylan (0.5%) and yeast extract (0.5%) at pH 10.0. Optimization of the culture conditions resulted in a 5-fold enhancement in enzyme production (255 U/mL). The most important characteristic of the enzyme is its production over a broad pH range of 6.0-10.0 during all stationary phases. The cellulase-free xylanase was optimally active at 60 °C and almost 90% stable after 4 h at 50 °C. The high levels of enzyme production obtained under alkali batch cultivation conditions, coupled with no production of cellulase, suggests the use of the local terrestrial strain Streptomyces CA24 and/or its enzyme in industrial processes such as pulp and paper production.

In vitro Detection and Optimization of Streptokinase Production by Two Streptococcal Strains in a Relatively Low Cost Growth Medium

This study sought to demonstrate the optimization of Streptokinase Production. Enzyme production was monitored during the growth of both Streptococcus pyogenes and Streptococcus equisimilis in different media. Adjustment of the pH for culture media of S. pyogenes and S. equisimilis, every 12 hours during incubation, significantly increased the enzyme production levels, when both microbes were grown on Strep-base medium. The best carbon source for streptokinase production was glucose of both S. pyogenes and S. equisimilis, while mannitol and sorbitol were found to be improper carbon sources. Yeast extract, and casein could be used as the primary source of organic nitrogen for streptokinase production, when the microbes were allowed to grow on Strep-base medium. The highest levels of the enzyme production were obtained with 1.5 % (w/v) tryptone and 1.5 % (w/v) casein for S. equisimilis and S. pyogenes, respectively. Detection of streptokinase produced was by the common casein digestion method and by the more sensitive chromozym substrate digestion method. Moreover, the enzyme was assayed electrochemically using the protamine-sensitive electrode to compare different methods of detection. Results obtained from electrochemical method were very close to that obtained with other methods. These results offer alternative and reliable method for streptokinase detection during microbial growth. It provides a faster and less expensive technique for streptokinase determination especially when there is a need to detect the enzyme in turbid media.

Comparison of laccase production levels in Pichia pastoris and Cryptococcus sp. S-2

The heterologous expression of the laccase gene from Trametes versicolor and Gaeumannomyces graminis was evaluated in the yeasts Pichia pastoris and Cryptococcus sp. S-2. The expression levels of both laccase genes in Cryptococcus sp. S-2 were considerably higher than those in P.pastoris. The codon usage of Cryptococcus sp. S-2 as well as the GC content were similar to those of T.versicolor and G.graminis. These results suggest that using a host with a similar codon usage for the expressed gene may improve protein expression. The use of Cryptococcus sp. S-2 as a host may be advantageous for the heterologous expression of genes with high GC content. Moreover, this yeast provides the same advantages as P.pastoris for the production of recombinant proteins, such as growth on minimal medium, capacity for high-density growth during fermentation, and capability for post-translational modifications. Therefore, we propose that Cryptococcus sp. S-2 be used as an expression host to improve enzyme production levels when other hosts have not yielded good results.

Exploring sequence characteristics related to high-level production of secreted proteins in Aspergillus niger.

Protein sequence features are explored in relation to the production of over-expressed extracellular proteins by fungi. Knowledge on features influencing protein production and secretion could be employed to improve enzyme production levels in industrial bioprocesses via protein engineering. A large set, over 600 homologous and nearly 2,000 heterologous fungal genes, were overexpressed in Aspergillus niger using a standardized expression cassette and scored for high versus no production. Subsequently, sequence-based machine learning techniques were applied for identifying relevant DNA and protein sequence features. The amino-acid composition of the protein sequence was found to be most predictive and interpretation revealed that, for both homologous and heterologous gene expression, the same features are important: tyrosine and asparagine composition was found to have a positive correlation with high-level production, whereas for unsuccessful production, contributions were found for methionine and lysine composition. The predictor is available online at http://bioinformatics.tudelft.nl/hipsec. Subsequent work aims at validating these findings by protein engineering as a method for increasing expression levels per gene copy.

A trait‐based approach for modelling microbial litter decomposition

Trait-based models are an emerging tool in ecology with the potential to link community dynamics, environmental responses and ecosystem processes. These models represent complex communities by defining taxa with trait combinations derived from prior distributions that may be constrained by trade-offs. Herein I develop a model that links microbial community composition with physiological and enzymatic traits to predict litter decomposition rates. This approach allows for trade-offs among traits that represent alternative microbial strategies for resource acquisition. The model predicts that optimal strategies depend on the level of enzyme production in the whole community, which determines resource availability and decomposition rates. There is also evidence for facilitation and competition among microbial taxa that co-occur on decomposing litter. These interactions vary with community investment in extracellular enzyme production and the magnitude of trade-offs affecting enzyme biochemical traits. The model accounted for 69% of the variation in decomposition rates of 15 Hawaiian litter types and up to 26% of the variation in enzyme activities. By explicitly representing diversity, trait-based models can predict ecosystem processes based on functional trait distributions in a community. The model developed herein illustrates that traits influencing microbial enzyme production are some of the key controls on litter decomposition rates.

Recent Advances on Filamentous Fungal Biofilms for Industrial Uses

Industrial enzymes are produced by submerged fermentation (SF) and by solid-state fermentation (SSF) to a lesser extent. Although SSF has several advantages, its scale-up is difficult. The role of physiological and genetic properties of microorganisms growing attached to surfaces could explain the advantages of SSF. Filamentous fungi are naturally adapted to growth on surfaces and in these conditions they show a particular physiological behavior which is different from that in SF; thus, they also form biofilms. Fermentation by filamentous fungal biofilms (FFB) is a homogeneous production system within a liquid environment based on the infrastructure of the SF process with the productive efficiency of the SSF. Enzyme production levels of FFB are much higher than those obtained in SF and they are also amenable of mixed fungal cultivation. Transcriptomic and proteomic tools are used to uncover the fundamental biological issues behind FFB. Several genes encoding cellulolytic enzymes are either differentially expressed or overexpressed in FFB. Moreover, our proteomic studies of Aspergillus niger biofilms compared to SF indicate that many intracellular proteins are either differentially expressed or overexpressed. Clinically important fungi like A. fumigatus also form biofilms when they infect lungs and recent studies demonstrate same gene expression features. These results support our hypothesis of cell adhesion and its role in the new schemes for improved fermentative production of industrial enzymes.

Screening and optimization of pectin lyase and polygalacturonase activity from ginseng pathogen Cylindrocarpon Destructans

Cylindrocarpon destructans isolated from ginseng field was found to produce pectinolytic enzymes. A Taguchi's orthogonal array experimental design was applied to optimize the preliminary production of polygalacturonase (PG) and pectin lyase (PL) using submerged culture condition. This method was applied to evaluate the significant parameters for the production of enzymes. The process variables were pH, pectin concentration, incubation time and temperature. Optimization of process parameters resulted in high levels of enzyme (PG and PL) production after ten days of incubation at a pH of 5.0 at 25°C in the presence of 1.5% pectin. Among different nitrogen sources, urea and peptone showed high production of PG and PL, respectively. The enzyme production and mycelial growth seems to have direct influence on the culture conditions; therefore, at stationary state high enzyme production and mycelial growth were obtained than agitation state. Along with this, optimization of enzyme activity was also determined using various physiological parameters like, temperature, incubation time and pH. Taguchi's data was also analyzed using one step ANOVA statistical method.

Screening and optimization of pectin lyase and polygalacturonase activity from ginseng pathogen Cylindrocarpon Destructans

Cylindrocarpon destructans isolated from ginseng field was found to produce pectinolytic enzymes. A Taguchi’s orthogonal array experimental design was applied to optimize the preliminary production of polygalacturonase (PG) and pectin lyase (PL) using submerged culture condition. This method was applied to evaluate the significant parameters for the production of enzymes. The process variables were pH, pectin concentration, incubation time and temperature. Optimization of process parameters resulted in high levels of enzyme (PG and PL) production after ten days of incubation at a pH of 5.0 at 25°C in the presence of 1.5% pectin. Among different nitrogen sources, urea and peptone showed high production of PG and PL, respectively. The enzyme production and mycelial growth seems to have direct influence on the culture conditions; therefore, at stationary state high enzyme production and mycelial growth were obtained than agitation state. Along with this, optimization of enzyme activity was also determined using various physiological parameters like, temperature, incubation time and pH. Taguchi’s data was also analyzed using one step ANOVA statistical method.

Enzymes and Bioconversions of Industrial, Pharmaceutical, and Biotechnological Significance

Enzymes are an important part of industry due to their many favorable properties. The development of industrial enzymes has depended heavily on the use of microbial sources. Microbes are useful because they can be produced economically in short fermentations and inexpensive media. Screening is simple, and strain improvement for increased production has been very successful. In the 1980s and 1990s, microbial enzymes replaced many plant and animal enzymes. They have found use in many industries including food, detergents, textiles, leather, pulp and paper, diagnostics, and therapy. The development of recombinant DNA technology had a major effect on production levels of enzymes as their genes were transferred from native species into industrial strains. Over 50% of the enzyme market is provided by recombinant enzymes. In many cases, enzymes have also been used to carry out conversions that were synthetic in previous years. Accompanying this trend is the use of whole cells to carry out bioconversions. Bioconv...

Penicillium oxalicum SAE M-51: a mutagenised strain for enhanced production of chitin deacetylase for bioconversion to chitosan

A novel chitin deacetylase (CDA) producing strain Penicillium oxalicum ITCC 6965 was isolated from residual materials of sea food processing industries. Strain following mutagenesis using ethidium bromide (EtBr) and microwave irradiation had resulted into a mutant P. oxalicum SAE(M)-51 having improved levels of chitin deacetylase (210.71 ± 1.65 Ul(-1)) as compared to the wild type strain (108.26 ± 1.98 Ul(-1)). Maximum enzyme production was achieved in submerged fermentation following 144 hours of incubation with notably improved productivity of 1.46 ± 0.82 Ul(-1) h(-1) as compared to the wild type strain (0.75 ± 0.53 Ul(-1)h(-1)). Scanning electron micrographs of mutant and wild type strains had revealed distinct morphological features. Evaluation of kinetic parameters viz. Q(s), Q(p), Y(p/x), Y(p/s), q(p), q(s) had denoted that strain P. oxalicum SAE(M)-51 is a hyper producer of chitin deacetylase. Glucose as compared to chitin or colloidal chitin had resulted in increased levels of enzyme production. However, replacement of glucose with chitinous substrates had prolonged the duration for enzyme production. The mutant strain had two pH optima that is 6.0 and 8.0 and had an optimum temperature of 30 °C for growth and enzyme production.

Extracellular ligninolytic enzymes by Lentinus polychrous Lév. under solid-state fermentation of potential agro-industrial wastes and their effectiveness in decolorization of synthetic dyes

Six agro-industrial wastes were evaluated as a support for ligninolytic enzyme production by the white-rot fungus Lentinus polychrous Lev. under solid-state fermentation. Enzyme production was markedly different according to the substrate used. Rice bran (RB) yielded the highest laccase activity of 1,449 U/L (after 21 days of culture) with specific activity of 4.4 U/g substrate. Rice bran supplemented with rice husk (RH) (2:1 by wt) showed high laccase activity of 1,425 U/L with specific activity of 10.0 U/g substrate (after 17 days of culture). The crude enzyme of the RH-RB culture also contained manganese peroxidase (MnP) and manganese-independent peroxidase (MIP) activities in relative proportions of 1.9:1.4:1 of laccase:MnP:MIP, respectively. Zymogram studies showed the same isoenzyme pattern with these ligninolytic enzymes. The high enzyme production level and low substrate cost of SSF-L. polychrous Lev. suggest that it has potential for industrial applications. Our studies showed that the crude enzyme from this culture exhibited in vitro decolorization of Indigo Carmine. The highest efficiency of dye decolorization was observed under alkaline conditions (pH 9.0) at an initial dye concentration of 10 mg/L. The rather high pH conditions and high efficiency in Indigo Carmine decolorization make the enzyme further interest for the applications in treatment of waste water from the textile industry, which contains synthetic dyes.

Optimization of culture conditions for the production of thermostable polygalacturonase by Penicillium SPC-F 20

Penicillium strain isolated from citrus fruit was found to produce thermostable polygalacturonases. Optimization of process parameters resulted in high levels of enzyme production after 3 days of incubation at a pH of 5.0 at 30 degrees C in the presence of 1% pectin. The optimum temperature for enzyme activity was 60 degrees C and a pH of 5.5 was found to be the optimal pH. The enzyme showed a high level of thermostability in the presence of substrate with a residual activity of 48% after 2 h of incubation at 60 degrees C. A thermostable nature with a high pH range for activity makes it an industrially important enzyme.

Multi-copy expression and fed-batch production of Rhodotorula araucariae epoxide hydrolase in Yarrowia lipolytica

Epoxide hydrolases (EHs) of fungal origin have the ability to catalyze the enantioselective hydrolysis of epoxides to their corresponding diols. However, wild type fungal EHs are limited in substrate range and enantioselectivity. Additionally, the production of fungal epoxide hydrolase (EH) by wild-type strains is typically very low. In the present study, the EH-encoding gene from Rhodotorula araucariae was functionally expressed in Yarrowia lipolytica, under the control of a growth phase inducible hp4d promoter, in a multi-copy expression cassette. The transformation experiments yielded a positive transformant, with a final EH activity of 220 U/g dw in shake-flask cultures. Evaluation of this transformant in batch fermentations resulted in approximately 7-fold improvement in EH activity over the flask scale. Different constant specific feed rates were tested in fed-batch fermentations, resulting in an EH activity of 1,750 U/g dw at a specific feed rate of approximately 0.1 g/g/h, in comparison to enzyme production levels of 0.3 U/g dw for the wild type R. araucariae and 52 U/g dw for an Escherichia coli recombinant strain expressing the same gene. The expression of EH in Y. lipolytica using a multi-copy cassette demonstrates potential for commercial application.

A Thermostable α-Amylase Producing Natural Variant of Bacillus spp. Isolated From Soil in Iran

Thermophilic processes appear more stable, rapid and less expensive and facilitate reactant activity and product recovery. Amylases have a quarter of the world enzyme market and thermostable a-amylases possess extensive commercial applications. Since little work has been done on strain isolation, growth and enzyme yield optimization, the level of thermophilic enzyme production remains relatively low. Therefore, large scale exploitation of thermophiles requires further intensive and integrated work. The present study describes isolation of an α-amylase producing bacillus from soil. The isolated bacillus was identified and named as Bacillus licheniformis Shahed-07. The strain was cultured in liquid media to produce α-amylase. The enzyme production conditions of the newly isolated bacillus revealed that the maximum enzyme production after 26 h of cultivation at pH 7.0 and 50°C. 0.5% tryptophan in production medium enhanced the enzyme productivity to two fold whereas peptone and lysin at 0.5% level showed a strong repression. Crude α-amylase characterization revealed that optimum activity was at pH 7.5 and 70°C. The crude enzyme was stable for 24 h at pH range of 6-7 at 70°C. Enzyme activity increased with temperature within the range of 40-70°C. The Bacillus licheniformis Shahed-07 strain produced thermostable α-amylase with characteristics suitable for application in starch processing and food industries.

Colony morphology mutants of Trichoderma harzianum with increased β -1,4- N -acetyl-glucosaminidase production

A total of 36 UV-induced mutants with altered colony morphology were isolated from strain Trichoderma harzianum T334, a potential biocontrol agent against plant pathogenic fungi with the ability to produce constitutively low levels of chitinases. The level of constitutive beta-1,4-N-acetyl-glucosaminidase production in standing and shaken cultures under non-inductive conditions was tested in mutants and compared to that of the parental strain. About 30% of the mutants showed significantly increased levels of enzyme production, with strain T334 col26a being the best producer. This mutant and the parental strain were subjected to in vitro confrontation assays with plant pathogenic Fusarium culmorum, Pythium debaryanum and Rhizoctonia solani strains. The mutant derivative could be characterized by significantly higher biocontrol index values than the parental strain in each experiment, suggesting, that mutants with improved constitutive extracellular chitinase secretion could be applied for biocontrol purposes against fungal plant pathogens.