Conditions For Enzyme Production Research Articles

Screening and Optimization of Pathogenic Strains of Pseudomonas sp. for Cholinesterase Enzyme Activity

The identification and optimization of pathogenic strains of Pseudomonas sp. with significant Cholinesterase enzyme activity has promising implications for environmental and medical applications. This study aimed to screen various Pseudomonas strains to identify those exhibiting high cholinesterase activity and to optimize the conditions for maximal enzyme production. Multiple Pseudomonas strains were isolated and characterized from diverse clinical samples, employing selective media and biochemical assays to determine cholinesterase activity. The strains with maximum enzyme activity were further subjected to optimization experiments, including variations in growth conditions such as Temperature, pH and Nutrient availability. The findings revealed sample no.39 among the collected 50 samples had significant enzyme activity of 0.19151 IU is obtained when choline chloride was 0.3999%, Ammonium Chloride was 0.01M, pH was 7.0, Temperature was 27°C and Time of incubation was 24hrs. These results not only advance our understanding of Pseudomonas sp. enzyme capabilities but also offer potential applications in bioremediation, biosensor development and pharmaceutical industries. Future research will focus on the molecular characterization of the cholinesterase enzyme and its applications in various industrial processes.

Production and partial purification of an innovative heat resistant α-keratinase with some remarkable medical and industrial applications

Background and objectives Keratinase has ultimate practical importance in industry, medicine, food industry and waste management fields. Their applications in wool and silk are as good cleaners and in leather industry as the best ever green dehairing agents providing high leather quality, as well they are used as crucial components of sophisticated detergents. Their medical prospective applications are in prion degradation and human callus removal. They convert keratinaceous wastes to valuable products saving the environment from hard keratin waste pollution. Bacteria, fungi and actinomycetes are effective keratinase producers and they are considered the most suitable sources. This study aims to formulate the production medium and to pinpoint the proper physiological conditions for the potent microorganism producing an efficient α-keratinase enzyme. The partial purification of the crude enzyme was successfully performed. The effect of the reaction temperature on both the crude and the partially purified enzyme (PPE) was duly studied with the thermostability of PPE. Some important applications have been implemented on PPE and these include leather dehairing, cloth stain removal, and topical treatment of human callus. Materials and Methods Thirteen recommended microbial strains were screened for effective and applicable α-keratinase productivity. Optimization of the cultural conditions for extracellular enzyme production and also the partial purification of the crude enzyme by ammonium sulphate salting out or by ethanol or acetone precipitation were carried out. The effect of reaction temperature on the enzyme and its thermostability were studied. Finally, the efficiency of the PPE on leather dehairing, stain removal, and human callus treatment was explored. Results and conclusion Among the 13 organisms screened, the fungal strain Trichoderma polysporum HZ-31 was the most potent producer of an influencial α-keratinase. The maximum α-keratinase activity of 58.2 UmL−1 was obtained by the previous-mentioned strain after 5-days fermentation medium containing (%, w/v): whole chicken feathers 0.5, glucose 0.2, peptone 0.5, yeast extract 0.5, K2HPO4 0.1, KH2PO4 0.3, CaCl2 0.1, MgSO4 0.1, and pH 7.0. Acetone fractionation of the crude keratinase was the most proper and offered the most promising keratinase fraction PPE at 80–90% acetone. This fraction had high thermostability and was kept at 55°C for more than 98% of its original activity after 60 min heating and this temperature (55°C) was also the optimum for 2 h enzymatic reaction. Conclusively, the present study succeeded in the achievement of a constitutive extracellular alkaline α-keratinase, which successfully proceeded to complete leather unhairing after 12–16 h at 37°C, afforded high performance to cloth blood stain removal with Arial detergent after 2 h at 50°C and complete degradation of the human callus after 4 h at 50°C.

From agro-food waste to nanoparticles: green synthesis of copper nanoparticles with lignin peroxidase enzyme produced by Anoxybacillus rupiensis using peanut shells.

This study presents a novel approach for the eco-friendly green synthesis of copper nanoparticles (Cu NPs) using enzymatic mediation which is an environmentally benign alternative to conventional methods, offering control over particle size and shape. Anoxybacillus rupiensis BS1 thermophilic bacterium was isolated from Erzurum's Pasinler hot spring and lignin peroxidase enzyme production conditions (incubation time 96h, 40g/L shell amount, pH 8.5, 150rpm, and 60°C temperature) were used in the production of peroxidase enzyme using peanut waste which has been optimized. The characterization of the synthesized Cu NPs was performed using various analytical techniques, including UV-vis spectroscopy, Fourier-transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), and scanning electron microscopy (SEM), confirming the successful production of stable and well-defined nanoparticles. Furthermore, the biological activities of the synthesized Cu NPs were explored, revealing their potential for antimicrobial applications. The antibacterial efficacy of the Cu NPs against some pathogens such as Escherichia coli, Klebsiella pneumoniae, Staphylococcus aureus, Streptococcus pyogenes, and Bacillus cereus was examined. It was determined that Cu NPs were effective on all pathogens and had the highest effectiveness against the S. pyogenes pathogen (19.0mm). This study not only presents an innovative and sustainable approach for the synthesis of Cu NPs but also highlights the multifaceted biological activities of these nanoparticles, opening avenues for diverse applications in the fields of medicine, agriculture, and environmental remediation. The utilization of peanut shell wastes as a substrate for enzyme production adds value to agricultural by-products, contributing to the development of a circular and sustainable economy.

Direct Screening of PET Hydrolase Activity in Culture Medium Based on Turbidity Reduction.

The development of an efficient screening method for the activity of PET-degrading enzymes represents a significant technological advance in the field of enzyme research, with the potential to facilitate the advancement of enzymes for PET recycling. By examining the stable conditions of PET suspension and enzyme production conditions, we developed a method to quantify PET-degrading enzyme activity in E. coli culture medium using turbidity reduction as an indicator. High PET concentration or ionic strength caused aggregation of PET, and the best condition for activity detection was 0.5 mg mL-1 PET in 50 mM sodium phosphate pH 7.0. Preculture of E. coli increased the purity of enzyme secreted in medium. To evaluate the screening method, 720 colonies of the PET2-7M-H229X-F233X mutant library were analyzed and three candidates of high-activity mutants were obtained. The thermostability of the mutants could also be easily measured by measuring the residual activity after heat treatment. The H229T-F233M mutant showed 3.4 times higher degradation rate against PET film than the template enzyme at the initial time. The molecular dynamics simulation implied that the F233M mutation makes space for making an α helix and that the H229T mutation resolved the steric hindrance with Trp199. These mutations were speculated to change the angle of the Trp199 side chain of PET2 to an angle similar to that of the Trp185 of IsPETase, making it suitable for PET binding to the active center. Screening of activity using PET suspensions is compatible with robotic automation and is expected to be useful for validating computationally predicted mutations.

Isolation, identification and characterization of neopullulanase from Thermomonas hydrothermalis GKE 08

The production of neopullulanase from thermophiles, such as Thermomonas hydrothermalis GKE 08, holds paramount significance due to the enzyme's unique thermophilic nature. Harnessing this characteristic ensures enhanced stability and functionality at elevated temperatures, a crucial aspect for industrial processes demanding efficient catalysis under extreme conditions. The investigation into pullulanase from T. hydrothermalis GKE 08 revealed significant findings. Optimal conditions for enzyme production were determined, with peak activity observed in the presence of 1.5% soluble pullulan and 0.5% peptone. The study delved into the pH and temperature dynamics, identifying an optimal pH of 7 and peak activity at 55 °C. Notably, the neopullulanase exhibited time-dependent stability, retaining 72% activity after 1 hour but declining to 50% after 2 hours. Furthermore, the enzyme displayed remarkable thermostability at 60 °C, with 88% activity after 30 minutes. Metal ion studies indicated susceptibility to inhibition by Cu2+, Mg2+, and Zn2+, while Ca2+ stimulated activity up to 138% at higher concentrations. The enzyme's response to specific reagents revealed sensitivity to SDS and EDTA, while urea surprisingly enhanced activity to 85%. The study enhances understanding of pullulanase behavior, offering valuable insights for biotechnological and industrial applications.

Production, characterization and application of polygalacturonase produced by Lentinus tigrinus CCMB 553

Polygalacturonases can be used to improve the processing of fruit juices and wines. This study aimed to produce, characterize and evaluate the potential application of polygalacturonase obtained from Lentinus tigrinus CCMB 553 in mango juice clarification. The fungal was isolated from the northeastern semi-arid region and fermented using Passiflora cincinnata bark as a substrate for solid state fermentation. The optimal conditions for enzyme production were obtained by applying the three-level factorial experimental design and were defined as 9 days and 30 °C for time and temperature, respectively. The optimal pH and temperature were determined by applying the Doehlert design and the results showed higher production between 45 and 60 °C and 4.0 and 5.5 for temperature and pH respectively. Km and Vmax of polygalacturonase from Lentinus tigrinus were 2.7 mg/mL and 12.86 μmol/min. The enzyme maintained 40 % residual activity after 30 min at 80 C. The influence of cations on enzyme activity was studied, and calcium ions seem to have a positive influence on enzyme activity, while Fe2+ ions have a negative influence. K+, Na+ and NH4+ salts showed little influence on the polygalacturonase activity of Lentinus tigrinus. The application in the clarification of mango juice (Mangifera indica) using optimal conditions obtained resulted in a reduction of 45% of the viscosity and the clarification by about 79%. The results show that the polygalacturonase produced by Lentinus tigrinus CCMB 553 was efficiently applied in mango juice, improving the quality of the juice.

The Value of Indigenous <i>Saccharomyces cerevisiae</i> Strains as Useful Enzymes Producers: A Systematic Review

This systematic review has focused on the selection and characterization of indigenous Saccharomyces cerevisiae strains for enzymes' production obtained from the fermentation process. The primary studies assessed have demonstrated that indigenous S. cerevisiae strains produce a wide range of enzymes during fermentation. These strains have shown different characteristics and were isolated from various locations, including those belonging to the main components of the wine production chain. The research methodology applied to find primary studies about the optimal conditions for enzyme production was effective and identified a wide range of enzyme classes. Cultivation media amended with natural or synthetic inducer substrates were found to be suitable carbon and/or nitrogen sources for producing enzymes of interest in plate-based screening. In submerged fermentation studies, the use of agro-industrial residues has been identified as a relevant carbon source for microbe development and metabolism. For the indigenous S. cerevisiae strains, the majority of the enzymes found belong to the glucosidases and hydrolases classes. Due to the activity of these enzymes, yeasts have been traditionally used in classic biotechnological applications, particularly in the process of alcoholic fermentations. Those enzymes are responsible for producing metabolites (such as aromatic compounds) that contribute to the desirable aroma and/or taste during the fermentation of wine, as well as other fermented or distilled beverages. The methods used for enzyme screening were of quantitative and/or qualitative nature and were carried out under acidic pH conditions (3.5-6.5) and at temperatures of 28-35 ℃. The results of this systematic review have shown that the enzymes from indigenous S. cerevisiae are highly relevant in various biotechnological applications, particularly in the food industry. Conclusions and future perspectives of our findings were discussed in terms of potential developments of novel application methods of the indigenous yeasts and their enzymes in agriculture and industry.

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.

Production, purification, and determination of the biochemical properties of β-glucosidase in Trichoderma koningii via solid substrate fermentation.

The β-glucosidase enzyme was obtained from Trichoderma koningii Oudem. NRRL 54330 under optimal conditions by solid substrate fermentation (SSF) using corn cobs as substrate. The enzyme was purified by two-step procedures, ammonium sulphate precipitation and cefarose-4B-l-tyrosine-1-naphthylamine hydrophobic interaction chromatography, followed by biochemical and kinetic characterisation. The β-glucosidase was obtained from T.koningii using ground corn cob as substrate and Na2HPO4, pH 9, as humidification medium. The optimum conditions for enzyme production by SSF were 30 °C and 6days. The purification efficiency of the obtained β-glucosidase was calculated to be 22.56-fold with a yield of 73.51 %. In the determination of β-glucosidase activity, p-nitrophenyl-β-d-glucopyranoside (pNPG) substrate was used, and the optimum pH and temperature values at which β-glucosidase showed high activity were determined to be pH 3.0 and 75 °C. The purity of the enzyme and the presence/number of subunits were checked using two different electrophoretic methods, SDS-PAGE and NATIVE-PAGE electrophoretic methods. The K m and V max values of the purified enzyme were determined to be 0.16 mM and 2000 EU respectively. Itwas also found that d-(+)-glucose and δ-gluconolactone inhibitors exhibited competitive inhibition of β-glucosidase in the presence of pNPG.

The effect of spent coffee ground (SCG) loading, matrix ratio and biological treatment of SCG on poly(hydroxybutyrate) (PHB)/poly(lactic acid) (PLA) polymer blend

This study investigates the effects of SCG embedded into biodegradable polymer blends and aimed to formulate and characterise biomass-reinforced biocomposites using spent coffee ground (SCG) as reinforcement in PHB/PLA polymer blend. The effect of SCG filler loading and varying PHB/PLA ratios on the tensile properties and morphological characteristics of the biocomposites were examined. The results indicated that tensile properties reduction could be due to its incompatibility with the PHB/PLA matrixSCG aggregation at 40 wt% content resulted in higher void formation compared to lower content at 10 wt%. A PHB/PLA ratio of 50/50 with SCG loading 20 wt% was chosen for biocomposites with treated SCG. Biological treatment of SCG using Phanerochaete chrysosporium CK01 and Aspergillus niger DWA8 indicated P. chrysosporium CK01 necessitated a higher moisture content for optimum growth and enzyme production, whereas the optimal conditions for enzyme production (50–55 %, w/w) differed from those promoting A. niger DWA8 growth (40 %, w/w). SEM micrographs highlighted uniform distribution and effective wetting of treated SCG, resulting in improvements of tensile strength and modulus of biocomposites, respectively. The study demonstrated the effectiveness of sustainable fungal treatment in enhancing the interfacial adhesion between treated SCG and the PHB/PLA matrix.

Molecular identification of protease producer ORSK-4 strain and determination of optimum enzyme production conditions

A microorganism resistant to high temperatures and producing alkaline proteases was isolated from soil samples from a protein-rich region (Kırıkkale/Yahşihan). This isolate, with high protein production, was identified as ORSK-4 by determining its morphological and biochemical properties using the 16s rRNA molecular approach and the Amplified ribosomal DNA restriction analysis (ARDRA) technique employed in strain differentiation. The optimum enzyme production conditions of the strain ORSK-4 were found to be the enzyme media, 3 days of incubation, 27.0 °C, and pH 7.0. Different components were utilized to determine the effect of changing the medium content on enzyme activity. Under the optimal production conditions determined in this way, the enzyme activity of ORSK-4 was found to be higher than that of some ATCC reference Bacillus species. To purify the extracellular protease of ORSK-4, precipitation with ammonium sulfate (30% and 80%), dialysis, and DEAE ion exchange chromatography were performed. SDS-PAGE analysis determined the molecular weight of the purified enzyme as approximately 30 kDa. Although the enzyme showed activity at various pH ranges, it showed its maximum activity when increased up to pH 9.0. In conclusion, the stability of the obtained alkaline protease enzyme under different conditions shows that it can be used in industrial and environmental applications.

PRODUCTION OF CELLULASE FROM NATURAL BACTERIAL ISOLATES FROM SOIL SAMPLE OF DISTRICT JAUNPUR, U.P., INDIA

The present study aims at isolating, screening, characterizing and optimizing cellulolytic bacteria isolated from soil samples. Different soil samples were collected from different locations of district Jaunpur, Uttar Pradesh, India. The morphological, biochemical and molecular characterization of the isolates were carried out for their identification. A total number of 23 isolates were screened at preliminary level for cellulolytic activity out of which three potent cellulase producers were selected for further study. On the basis of 16S rDNA sequencing these three isolates were identified as Bacillus velezensis PS-14, Bacillus velezensis PS-09 and Bacillus paramycoides PS-05. The optimal conditions for cellulase enzyme production was also determined and maximum activity was obtained at pH 7 and 40 0 C temperature, while 2% inoculum level and 48 hr incubation period were found to be optimum for cellulase production by all the strains. Glucose and Carboxymethyl cellulose was proved to be the best carbon sources, while ammonium choride and peptone was observed to be the most favourable nitrogen source for all three bacterial isolates.

Beauveria Bassiana Amylase-Polygalacturonase Production Using Lignocellulosic Biomass and Application in Juice Processing.

The search for sources of industrial biocatalysts, which are non-pathogenic and can utilise cheap nutrient sources, has been a continuous endeavour in the ~ 7 billion USD enzyme industry. Beauveria bassiana, an endophytic fungal entomopathogen, is non-pathogenic and possesses the potential to secrete various bioproducts while utilising readily available lignocellulosic biomass. This study investigated the optimised production of two glycosyl hydrolases, amylase and polygalacturonase, by B. bassiana while utilising readily available agricultural residues. Subsequently, the industrial potential of the enzymes in the clarification of fruit juice was evaluated. Initially, seven agro residues were screened for the concomitant production of amylase and polygalacturonase by B. bassiana SAN01. Subsequently, statistical optimisation tools, Plackett Burman Design (PBD) and Central Composite Design (CCD), were employed for the optimisation of enzyme production. The enzyme mixture was partially purified and applied in the clarification of pineapple juice. The production of B. bassiana SAN01 amylase and polygalacturonase was found to be maximal while utilising wheat bran. Subsequent to PBD and CCD optimisation, the optimal conditions for enzyme production were identified to be at 30 °C, pH 6.0 and wheat bran concentration of ~40 g.L-1. Under these optimised conditions, heightened production levels of 34.82 and 51.05 U.mL-1 were recorded for amylase and polygalacturonase, respectively, which were 179% and 187% of the initial unoptimised levels. In addition, the most effective clarification of the juice (~90%) was observed at 35 °C after an incubation time of 120 min with no significant effect on the pH and total dissolved solids. B. bassiana, a well-known biocontrol agent, was shown to produce amylase and polygalacturonase using readily available agricultural residues for the first time. These enzyme production levels are the highest for these enzymes from any known endophytic fungal entomopathogen. This study further demonstrates the potential applicability of B. bassiana in other industrial processes besides its widespread use as a biopesticide.

Identification and Growth Characteristics of a Gluten-Degrading Bacterium from Wheat Grains for Gluten-Degrading Enzyme Production.

Immunogenic peptides from wheat gluten can be produced during digestion, which are difficult to digest by gastrointestinal proteases and negatively affect immune responses in humans. Gluten intolerance is a problem in countries where wheat is a staple food, and a gluten-free diet is commonly recommended for its treatment and prevention. Enzyme approaches for degradation of the peptides can be considered as a strategy for its prevention. Here, we isolated a gluten-degrading bacterium, Bacillus amyloliquefaciens subsp. plantarum, from wheat grains. The culture conditions for enzyme production or microbial use were considered based on gluten decomposition patterns. Additionally, the pH range for the activity of the crude enzyme was investigated. The bacterium production of gluten-degrading enzymes was temperature-dependent within 25 °C to 45 °C, and the production time decreased with increasing culture temperature. However, it was markedly decreased with increasing biofilm formation. The bacterium decomposed high-molecular-weight glutenin proteins first, followed by gliadin proteins, regardless of the culture temperature. Western blotting with an anti-gliadin antibody revealed that the bacterium decomposed immunogenic proteins related to α/β-gliadins. The crude enzyme was active in the pH ranges of 5 to 8, and enzyme production was increased by adding gliadin into the culture medium. In this study, the potential of the B. amyloliquefaciens subsp. plantarum for gluten-degrading enzyme production was demonstrated. If further studies for purification of the enzyme specific to the immunogenic peptides and its characteristics are conducted, it may contribute as a strategy for prevention of gluten intolerance.

Optimization of xylanase production by Pichia kudriavzevii and Candida tropicalis isolated from the wood product workshop.

Enzymatic compounds can be found abundantly and provide numerous advantages in microbial organisms. Xylanases are used in various pharmaceutical, food, livestock, poultry, and paper industries. This study aimed to investigate xylanase-producing yeasts, xylose concentration curve and their enzymatic activity under various factors including carbon and nitrogen sources, temperature, and pH. Enzyme activity was evaluated under different conditions before, during, and after purification. The yeast strains were obtained from the wood product workshop and were subsequently cultivated on YPD (yeast extract peptone dextrose) medium. Additionally, the growth curve of the yeast and its molecular identification were conducted. The optimization and design process of xylan isolated from corn wood involved the use of Taguchi software to test different parameters like carbon and nitrogen sources, temperature, and pH, with the goal of determining the most optimal conditions for enzyme production. In addition, the Taguchi method was utilized to conduct a multifactorial optimization of xylanase enzyme activity. The isolated species were partially purified using ammonium sulfate precipitation and dialysis bag techniques. The results indicated that 3 species (8S, 18S, and 16W) after molecular identification based on 18S rRNA gene sequencing were identified as Candida tropicalis SBN-IAUF-1, Candida tropicalis SBN-IAUF-3, and Pichia kudriavzevii SBN-IAUF-2, respectively. The optimal parameters for wheat carbon source and peptone nitrogen source were found at 50°C and pH 9.0 through single-factor optimization. By using the Taguchi approach, the best combination for highest activity was rice-derived carbon source and peptone nitrogen source at 50°C and pH 6.0. The best conditions for xylanase enzyme production in single-factor optimization of wheat bran were 2135.6 U/mL, peptone 4475.25 U/mL, temperature 50°C 1868 U/mL, and pH 9.0 2002.4 U/mL. Among the tested yeast, Candida tropicalis strain SBN-IAUF-1 to the access number MZ816946.1 in NCBI was found to be the best xylanase product. The highest ratio of enzyme production at the end of the delayed phase and the beginning of the logarithmic phase was concluded by comparing the growth ratio of 8S, 16W, and 18S yeasts with the level of enzymatic activity. This is the first report on the production of xylan polymer with a relative purity of 80% in Iran. The extracellular xylanases purified from the yeast species of C. tropicalis were introduced as a desirable biocatalyst due to their high enzymatic activity for the degradation of xylan polymers.

Temperature and pH Optimization for Protease Production Fermented by Yarrowia lipolytica from Agro-Industrial Waste

The use of yeasts for the production of proteases has increased in demand in recent years. Y. lipolytica has been reported as a strain with high yields of protease production. This work aimed to evaluate the impact of pH and temperature on the production of proteases using Y. lipolytica in solid-state fermentation (SSF). Soybean, canola meal, cottonseed meal, and sesame meal wastes were used as nutrient sources at seven pH levels (4, 5, 6, 7, 8, 9, 10) and five temperatures (25, 30, 35, 40, 45 °C). The waste source and optimal conditions for maximum enzyme production (EP) were obtained by Box–Benhken design. The results revealed that at pH of 7, temperature of 30 °C, and for 48 h cultivation period, canola meal showed the best EP with 188.75 U/L, followed by soybean with 117.07 U/L, cottonseed meal with 66.71 U/L, and sesame with the lowest production, reaching 88.5 U/L up to 35 °C. The temperature factor exhibited the greatest effect on protease production. The biotechnological and economic potential of canola meal in the production of enzymes is highlighted.

Screening, Identification, and Optimization of Enzyme-Producing Conditions for Cellulose-Degrading Bacteria in Distillery Lees

Cellulose-degrading bacteria were identified from distillery lees, and the strains were optimized for fermentation and enzyme production, providing effective strains for the resource utilization of distillery lees and developing cellulase. Based on the univariate test, the response surface test was used to optimize enzyme production conditions for fermentation. The screened strain JZ2 had a clear circle-to-colony diameter ratio of 2.0. The enzyme activities of exoglucanase, endoglucanase, and β-glucosidase were 4.341 ± 0.05 U/mL, 1.874 ± 0.04 U/mL, and 0.739 ± 0.02 U/mL, respectively. The bacterial colonies were large, and the bacterial cells’ morphology was rod-shaped. We identified Bacillus cereus (JZ2) from 16S rDNA sequence homology and phylogenetic tree analysis as belonging to the genus Bacillus in the thick-walled Bacillus phylum. The optimum production conditions of cellulase from strain JZ2 were a fermentation time of 2 d, a rotation rate of 180× g, and a temperature of 26 °C using the response surface method. The enzyme activity of JZ2 was 4.625 U/mL under optimal enzyme production conditions. In addition to good activity, the cellulase from the JZ2 strain may have the potential to convert distillery lees cellulose into useful compounds.

Modeling and in-vivo evaluation of fibrinolytic enzyme produced by Bacillus subtilis Egy under solid state fermentation

Blood clot formation increases cases of myocardial infarction (AMI) and stroke, thus urges directing much research works for treatment and prevention of the causes. One of these directions is the microbial production of fibrinolytic enzymes as thrombolytic agents. In the current work, Bacillus subtilis Egy has been used for enzyme production under solid state fermentation. Among twelve nutrient meals in addition to wheat bran as a control fodder yeast yielded the highest enzyme activity reaching 114U/g. Applying statistical model for optimization of enzyme production revealed that 3.6%, fodder yeast; 40%, moisture content; 6 days, incubation period and 2%, inoculum size were the optimum conditions for maximum fibrinolytic enzyme production (141.02 U/g) by Bacillus subtilis Egy under solid-state fermentation The model was significant and data were experimentally validated. The produced fibrinolytic enzyme was evaluated for in vitro and in vivo cytotoxicity. In-vivo examination of the enzyme resulted in no mortality during the first 24 h after treatment. After 14 days, the results revealed no significant changes detected in hematological parameters (RBCs, MCV, hemoglobin except WBCs which showed an increase for both sexes. Histopathological examination of liver and kidney of rats received oral and subcutaneous treatments showed normal architecture. The data showed the applicability of the produced enzyme for the treatment of blood clot with no significant effect on living cells or on physiological functions.

A glucose tolerant β-glucosidase from a newly isolated Neofusicoccum parvum strain F7: production, purification, and characterization

Cellulase-producing microorganisms produce low titres of β-glucosidases with low tolerance to glucose. This study aimed to improve production, purify, and characterize a β-glucosidase from a newly isolated Neofusicoccum parvum strain F7. β-Glucosidase production was significantly enhanced by a sequential statistical modelling approach from 1.5-fold in Plackett–Burman design to 2.5 U/ml in the Box–Behnken design compared to the preliminary one variable at a time experiments (1.6 U/ml). The optimal conditions for enzyme production by BBD were 12 days of fermentation at 20 °C, 175 rpm, 0.5% glycerol and 1.5% casein in pH 6.0 buffer. Three β-glucosidase isoforms referred to as Bgl1, Bgl2, Bgl3 were purified and characterized from the optimized crude extract displaying IC50 values of 2.6, 22.6 and 319.5 mM for glucose, respectively. Bgl3 with a molecular mass of approximately 65 kDa demonstrated the highest tolerance to glucose among the isoforms. The optimum activity and stability for Bgl3 was observed at pH 4.0 in 50 mM sodium acetate buffer with 80% β-glucosidase residual activity retained for three hours. This isoform also retained 60% residual activity at 65 °C for one hour which was then reduced to 40% which remained stable for another 90 min. The β-glucosidase activity of Bgl3 was not enhanced after the addition of metal ions in assay buffers. The Km and vmax for 4-nitrophenyl-β-d-glucopyranoside were 1.18 mM and 28.08 µmol/min, respectively indicating high affinity for the substrate. The ability to withstand the presence of glucose in conjunction with its thermophilic nature indicates promise for this enzyme in industrial application.

Screening of Dominant Cellulose-Degrading Microbe in Humus and Optimisation of its Enzyme Producing Conditions and Application Optimization of Enzyme Production Conditions and Evaluation of Efficient Straw Degradation by Penicillium oxalicum

Screening of Dominant Cellulose-Degrading Microbe in Humus and Optimisation of its Enzyme Producing Conditions and Application Optimization of Enzyme Production Conditions and Evaluation of Efficient Straw Degradation by <i>Penicillium oxalicum</i>