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Background. In view of the worsening forecast for global temperature rise worldwide, it seems relevant to study the effects of abnormal heat waves on systemic regulatory processes in people with chronic diseases, in particular coronary artery disease (CAD).Aims. This study aimed to investigate the effect of hyperthermia on oxidative stress parameters in patients with various severity of CAD and in healthy subjects.Materials and methods. We studied the level of malonic dialdehyde (MDA) and the activity of Cu,Zn-containing superoxide dismutase (Cu,Zn-SOD) in healthy subjects under conditions of 30-day long simulated hyperthermia and in patients with different severity of CAD after the summer heat wavesResults. We revealed signs of oxidative stress in healthy volunteers during model hyperthermia that manifested as an increase in content of MDA in blood plasma. At the same time we observed increasing activity of Cu,Zn-SOD in erythrocytes that utilizes reactive oxygen species. The increase of Cu,Zn-SOD activity started with a certain latency what also can be explained by de novo enzyme biosynthesis induction. We also studied oxidative stress parameters in patients at high and moderate cardiovascular risk according to the SCORE risk chart with uncomplicated CAD course and in patients with complicated CAD with severe coronary damage according to angiography during the summer heat waves. We observed accumulation of MDA in blood plasma and increasing activity of erythrocyte Cu,Zn-SOD in patients with uncomplicated CAD. At the same time we noted that accumulation of MDA in blood plasma was not followed by any increase in activity of red blood cell Cu,Zn-SOD in patients with severe complicated CAD. This fact indicates dysregulation of free radical processes in patients with severe course of CAD during the heat waves.Conclusions. The dysregulation of free-radical processes in patients with a severe clinical course of CAD has been revealed.

Kentucky bluegrass (Poa pratensis L.) is an important species of turfgrass that is commonly planted on golf courses and landscapes all over the world. It is sensitive to salt stress; however, details relating to its molecular mechanisms of salt resistance are not available. We, therefore, analyzed the changes in growth, antioxidant enzyme activity, and microRNA expression in the callus 1 week after treatment with 200 mM NaCl for 12 h, 24 h, 48 h, 96 h, and 144 h. The results demonstrated that callus growth declined and thiobarbituric acid-reactive substance production and cell membrane permeability increased. Treatment with salt increased ascorbate peroxidase, glutathione reductase, superoxide dismutase, catalase, peroxidase, and polyphenol oxidase activity. Changes in the expression levels of microRNAs were observed under salt treatment. The expression of miR162, miR173, miR391, miR408, miR773, and miR857 increased by 70% after 24 h of salt treatment, after which it declined to a level similar to that of the control. The expression level of miR775 and miR827 decreased by 20% after 24 h, and then further decreased by 80% after 144 h. The expression level of miR841 increased by 50% after 24 h of salt treatment, and then stabilized. In contrast, salt treatment increased the expression of the auxin response factors ARF6, ARF8, ARF10, and ARF16 in the callus from 12 to 144 h of salt treatment, during which the expression increased twofold. Gene expression analysis indicated that salt-responsive gene families were regulated by microRNAs in the callus under salinity stress. The activity of antioxidant enzymes is also changing. MiR841 is considered to be a positive regulator of antioxidant enzyme biosynthesis. The present investigation elucidates the manner in which P. pratensis responds to salt stress in the callus, and could be used to inform further studies on the molecular mechanisms of stress tolerance.

The enzyme biosynthesis using agricultural wastes by solid state fermentation (SSF) and the study of their physicochemical properties are meaningful approaches to improve the biomass hydrolysis. Among them, β-glucosidases and β-xylosidases are key enzymes at the lignocellulose depolymerization, which act in the cleavage of oligosaccharides in monosaccharides. In this study, the production of hemicellulases and cellulases by Pleurotus ostreatus and Aspergillus niger monocultures or in consortium was investigated, using raw sugarcane bagasse (SB) and wheat bran (WB) as substrates. The highest enzymatic activities were observed in the crude extract produced by P. ostreatus PLO6 and A. niger SCBM4 consortium with 98.5, 62.9, 3.8, 12.4, 13.3 and 20.2 U/g for β-glucosidase (β-glu), β-xylosidase (Bxyl), filter paper cellulase (FPase), xylanase (Xyl), exoglucanase (Exgl) and endoglucanase (Engl), respectively. The pH and temperature effects on β-glu and β-xyl were characterized. Optimal activities were obtained at pH 4.0 and 45 °C for β-glu and 3.5 and 55 °C for β-xyl. Both enzymes were stable at acid pH and presented thermostability. The results indicated that the enzymatic cocktail demonstrated potential characteristics for future applications in saccharifications. The use of sugarcane bagasse and wheat bran for microbial growth contributed to aggregate value to these byproducts. La biosíntesis de enzimas utilizando desechos agrícolas por fermentación en estado sólido (SSF) y el estudio de sus propiedades fisicoquímicas son enfoques significativos para mejorar la hidrólisis de la biomasa. Entre ellas, las β-glucosidasas y las β-xilosidasas son enzimas clave en la despolimerización de la lignocelulosa, que actúan en la escisión de oligosacáridos en monosacáridos. En este estudio se investigó la producción de hemicelulasas y celulasas por monocultivos de Pleurotus ostreatus y Aspergillus niger o en consorcio, utilizando como sustratos bagazo de caña de azúcar crudo (SB) y salvado de trigo (WB). Las mayores actividades enzimáticas se observaron en el extracto crudo producido por el consorcio P. ostreatus PLO6 y A. niger SCBM4 con 98.5, 62.9, 3.8, 12.4, 13.3 y 20.2 U / g para β-glucosidasa (β-glu), β-xilosidasa (β-xyl), papel de filtro celulasa (FPasa), xilanasa (Xyl), exoglucanasa (Exgl) y endoglucanasa (Engl), respectivamente. Se caracterizaron los efectos del pH y la temperatura sobre β-gl y β-xyl. Las actividades óptimas se obtuvieron a pH 4.0 y 45 °C para β-glu y 3.5 y 55 °C para β-xyl. Ambas enzimas fueron estables a pH ácido y presentaron termoestabilidad. Los resultados indicaron que el cóctel enzimático demostró características potenciales para futuras aplicaciones en sacarificaciones. El uso de bagazo de caña de azúcar y salvado de trigo para el crecimiento microbiano contribuyó al valor agregado de estos subproductos. A biossíntese de enzimas utilizando resíduos agrícolas por fermentação em estado sólido (SSF) e o estudo de suas propriedades físico-químicas são abordagens significativas para melhorar a hidrólise da biomassa. Dentre elas, as β-glicosidases e β-xilosidases são enzimas-chave na despolimerização da lignocelulose, que atuam na clivagem de oligossacarídeos em monossacarídeos. Neste estudo, foi investigada a produção de hemicelulases e celulases por monoculturas de Pleurotus ostreatus e Aspergillus niger ou em consórcio, utilizando bagaço de cana-de-açúcar (SB) e farelo de trigo (WB) como substratos. As maiores atividades enzimáticas foram observadas no extrato bruto produzido pelo consórcio P. ostreatus PLO6 e A. niger SCBM4 com 98,5, 62,9, 3,8, 12,4, 13,3 e 20,2 U/g para β-glucosidase (β-glu), β-xilosidase (β-xyl), celulase de papel de filtro (FPase), xilanase (Xyl), exoglucanase (Exgl) e endoglucanase (Engl), respectivamente. Os efeitos do pH e da temperatura em β-glu e β-xyl foram caracterizados. As atividades ótimas foram obtidas em pH 4,0 e 45 °C para β-glu e 3,5 e 55 °C para β-xyl. Ambas as enzimas foram estáveis em pH ácido e apresentaram termoestabilidade. Os resultados indicaram que o coquetel enzimático demonstrou características potenciais para futuras aplicações em sacarificações. A utilização do bagaço da cana-de-açúcar e do farelo de trigo para o crescimento microbiano contribuiu para agregar valor a esses subprodutos.

Nitrogen limitation has been widely reported to affect the growth and development of fungi, and the transcription factor GCN4 (general control nonderepressible 4) is involved in nitrogen restriction. Here, we found that nitrogen limitation highly induced the expression of GCN4 and promoted the synthesis of ganoderic acid (GA), an important secondary metabolite in Ganoderma lucidum. The activated GCN4 is involved in regulating GA biosynthesis. In addition, the accumulation of reactive oxygen species (ROS) also affects the synthesis of GA under nitrogen restrictions. The silencing of the gcn4 gene led to further accumulation of ROS and increased the content of GA. Further studies found that GCN4 activated the transcription of antioxidant enzyme biosynthesis genes gr, gst2, and cat3 (encoding glutathione reductase, glutathione S-transferase, and catalase, respectively) through direct binding to the promoter of these genes to reduce the ROS accumulation. In conclusion, our study found that GCN4 directly interacts with the ROS signaling pathway to negatively regulate GA biosynthesis under nitrogen-limiting conditions. This provides an essential insight into the understanding of GCN4 transcriptional regulation of the ROS signaling pathway and enriches the knowledge of nitrogen regulation mechanisms in fungal secondary metabolism of G. lucidum. IMPORTANCE Nitrogen has been widely reported to regulate secondary metabolism in fungi. Our study assessed the specific nitrogen regulatory mechanisms in Ganoderma lucidum. We found that GCN4 directly interacts with the ROS signaling pathway to negatively regulate GA biosynthesis under nitrogen-limiting conditions. Our research highlights a novel insight that GCN4, the nitrogen utilization regulator, participates in secondary metabolism through ROS signal regulation. In addition, this also provides a theoretical foundation for exploring the regulation of other physiological processes by GCN4 through ROS in fungi.

Background The increasing demand and the continuous depletion in fossil fuels have persuaded researchers to investigate new sources of renewable energy. Bioethanol produced from cellulose could be a cost-effective and a viable alternative to petroleum. It is worth note that β-glucosidase plays a key role in the hydrolysis of cellulose and therefore in the production of bioethanol. This study aims to investigate a simple and standardized method for maximization of extracellular β-glucosidase production from a novel fungal isolate under solid-state fermentation using agro-industrial residues as the sole source of carbon and nitrogen. Furthermore, purification and characterization of β-glucosidase were performed to determine the conditions under which the enzyme displayed the highest performance. Results A fungus identified genetically as a new Aspergillus sp. DHE7 was found to exhibit the highest extracellular β-glucosidase production among the sixty fungal isolates tested. Optimization of culture conditions improved the enzyme biosynthesis by 2.1-fold (174.6 ± 5.8 U/g of dry substrate) when the fungus grown for 72 h at 35 °C on jojoba meal with 60% of initial substrate moisture, pH 6.0, and an inoculum size of 2.54 × 107 spores/mL. The enzyme was purified to homogeneity through a multi-step purification process. The purified β-glucosidase is monomeric with a molecular mass of 135 kDa as revealed by the SDS-PAGE analysis. Optimum activity was observed at 60 °C and pH of 6.0, with a remarkable pH and thermal stability. The enzyme retained about 79% and 53% of its activity, after 1 h at 70 °C and 80 °C, respectively. The purified β-glucosidase hydrolysed a wide range of substrates but displaying its greater activity on p-nitrophenyl-β-D-glucopyranoside and cellobiose. The values of Km and Vmax on p-nitrophenyl β-D-glucopyranoside were 0.4 mM and 232.6 U/mL, respectively. Purified β-glucosidase displayed high catalytic activity (improved by 25%) in solutions contained ethanol up to 15%. Conclusion β-glucosidase characteristics associated with its ability to hydrolyse cellobiose, underscore its utilization in improving the quality of food and beverages. In addition, taking into consideration that the final concentration of ethanol produced by the conventional methods is about 10%, suggests its use in ethanol-containing industrial processes and in the saccharification processes for bioethanol production. Graphical abstract

Seven biological methods were adopted (three bacterial activities of bioluminescence, enzyme, enzyme biosynthetic, algal growth, seed germination, and root and shoot growth) to compare the toxic effects of two different sizes of silver nanoparticles (AgNPs). AgNPs showed a different sensitivity in each bioassay. Overall, the order of inhibitory effects was roughly observed as follows, bacterial bioluminescence activity &asymp, root growth &gt, biosynthetic activity of enzymes &asymp, algal growth &gt, seed germination &asymp, enzymatic activity &gt, shoot growth. For all bacterial activities (bioluminescence, enzyme, and enzyme biosynthesis), the small AgNPs showed statistically significantly higher toxicity than the large ones (p &lt, 0.0036), while no significant differences were observed among other biological activities. The overall effects on the biological activities (except shoot growth) of the small AgNPs were shown to have about 4.3 times lower EC50 (high toxicity) value than the large AgNPs. These results also indicated that the bacterial bioluminescence activity appeared to be an appropriate method among the tested ones in terms of both sensitivity and the discernment of particle sizes of AgNPs.

Bioremediation is a strategy to mitigate environmental impacts of hazardous pollutants from anthropogenic sources. Natural byproducts, including agroindustrial wastes (AW) can be used to induce enzyme biosynthesis, leading up to enhancement of pollutants degradation process. Therefore, this study aimed to evaluate the use of cupuacu, Theobroma grandiflorum AW as Pycnoporus sanguineus Laccase (Lac) inducer in order to promote 17-α-ethinylestradiol (EE2) bioremediation. The macro and micro-nutrients levels of cupuacu AWs were evaluated in order to establish further correlations with enzymatic biosynthesis induction. The fungus was cultivated for 7 days in temperature of 28 ± 2 °C and agitation of 150 rpm. For bioremediation, Lac enzymatic extract was added to EE2 solution (10 µg mL−1) and the percentage of removal was evaluated by HPLC after 1–24 hr of reaction. At optimized conditions, the enzyme extract production was remarkably enhanced by adding only 1% (w/v) of cupuacu AW. Lac activity reached ...

Aims To screen and identify a potential extracellular β-d-fructofuranosidase or invertase-producing bacterium from soil, and comparatively evaluate the enzyme biosynthesis under submerged and solid-state fermentation (SSF). Methods and results Extracellular invertase-producing bacteria were screened from soil. Identification of the potent bacterium was performed based on microscopic examinations and 16S rDNA molecular sequencing. Bacillus subtilis LYN12 invertase secretion was surplus with wheat bran humidified with molasses medium (70%), with elevated activity at 48 h and 37°C under SSF, whereas under submerged conditions, increased activity was observed at 24 h and 45°C in the molasses medium. The study revealed a simple fermentative medium for elevated production of extracellular invertase from a fast growing Bacillus strain. Conclusions Bacterial invertases are scarce and limited reports are available. By far, this is the first report on the comparative analysis of optimization of extracellular invertase synthesis from B. subtilis strain by submerged and SSF. The use of agricultural residues increased yields resulting in the development of a cost-effective and stable approach. Significance and impact of the study Bacillus subtilis LYN12 invertase possesses excellent fermenting capability to utilize agro-industrial residues under submerged and solid-state conditions. This could be a beneficial candidate in food and beverage processing industries.

A psychrotrophic yeast strain producing a cold-adapted protease at low temperature was classified as Sporobolomyces roseus. In standard YPG medium, S. roseus LOCK 1119 synthesized an extracellular protease with an activity of approximately 560 U/L. Optimization of medium composition and process temperature considerably enhanced enzyme biosynthesis; an approximate 70% increase in activity (2060 U/L). The native enzyme was purified to homogeneity by cation exchange chromatography followed by a size exclusion step, resulting in a 103-fold increase in specific activity (660 U/mg) with 25% recovery. The enzyme displayed 10%-30% of its maximum activity at 0-25 °C, with the optimum temperature being 50°C. Protease G8 was strongly inactivated by pepstatin A, an aspartic protease inhibitor. The enzyme was used to hydrolyze four natural substrates, and their antioxidant activities were evaluated against 1,1-diphenyl-2-picrylhydrazyl. The highest antioxidant activity (69%) was recorded for beef casein.

Melatonin is a biological hormone that plays crucial roles in stress tolerance. In this study, we investigated the effect of exogenous melatonin on abiotic stress in the tea plant. Under cold, salt and drought stress, increasing malondialdehyde levels and decreasing maximum photochemical efficiency of PSII were observed in tea leaves. Meanwhile, the levels of reactive oxygen species (ROS) increased significantly under abiotic stress. Interestingly, pretreatment with melatonin on leaves alleviated ROS burst, decreased malondialdehyde levels and maintain high photosynthetic efficiency. Moreover, 100 &mu, M melatonin-pretreated tea plants showed high levels of glutathione and ascorbic acid and increased the activities of superoxide dismutase, peroxidase, catalase and ascorbate peroxidase under abiotic stress. Notably, melatonin treatments can positively up-regulate the genes (CsSOD, CsPOD, CsCAT and CsAPX) expression of antioxidant enzyme biosynthesis. Taken together, our results confirmed that melatonin protects tea plants against abiotic stress-induced damages through detoxifying ROS and regulating antioxidant systems.

Laccases have received the attention of researchers in the last few decades due to their ability to degrade phenolic and lignin-related compounds. This study aimed at obtaining the highest possible laccase activity and evaluating the methods of its purification. The crude laccase from bioreactor cultivation of Cerrena unicolor fungus was purified using ultrafiltration, aqueous two-phase extraction (ATPE) and foam fractionation (FF), which allowed for the assessment of these three downstream processing (DSP) methods. The repeated fed-batch cultivation mode applied for the enzyme production resulted in a high laccase specific activity in fermentation broth of 204.1 U/mg. The use of a specially constructed spin filter inside the bioreactor enabled the integration of enzyme biosynthesis and biomass filtration in one apparatus. Other methods of laccase concentration and purification, namely ATPE and FF, proved to be useful for laccase separation; however, the efficiency of FF was rather low (recovery yield of 24.9% and purification fold of 1.4). Surprisingly, the recovery yield after ATPE in a PEG 6000-phosphate system in salt phase was higher (97.4%) than after two-step ultrafiltration (73.7%). Furthermore, it was demonstrated that a simple, two-step purification procedure resulted in separation of two laccase isoforms with specific activity of 2349 and 3374 U/mg. All in all, a compact integrated system for the production, concentration and separation of fungal laccases was proposed.

Many viruses induce oxidative stress and cause S-glutathionylation of Cys residues of the host and viral proteins. Changes in cell functioning during viral infection may be associated with glutathionylation of a number of key proteins including Na,K-ATPase which creates a gradient of sodium and potassium ions. It was found that Na,K-ATPaseα-subunit has a basal glutathionylation which is not abrogated by reducing agent. We have shown that acute hypoxia leads to increase of total glutathionylation level of Na,K-ATPaseα-subunit; however, basal glutathionylation ofα-subunit increases under prolonged hypoxia only. The role of basal glutathionylation in Na,K-ATPase function remains unclear. Understanding significance of basal glutathionylation is complicated by the fact that there are no X-ray structures of Na,K-ATPase with the identified glutathione molecules. We have analyzed all X-ray structures of the Na,K-ATPaseα-subunit from pig kidney and found that there are a number of isolated cavities with unresolved electron density close to the relevant cysteine residues. Analysis of the structures showed that this unresolved density in the structure can be occupied by glutathione associated with cysteine residues. Here, we discuss the role of basal glutathionylation of Na,K-ATPaseα-subunit and provide evidence supporting the view that this modification is cotranslational.

Introduction: Mitochondrial dysfunction results in a wide range of organ disorders through diverse genetic abnormalities. We herein present the detailed clinical course of an infant admitted for extensive, rapidly progressing white matter lesions and hypertrophic cardiomyopathy due to a BOLA3 gene mutation. Case: A 6-month-old girl with no remarkable family or past medical history until 1 month prior presented with developmental regression and feeding impairment. Ultrasound cardiography and brain magnetic resonance imaging (MRI) respectively disclosed the presence of hypertrophic cardiomyopathy and symmetrical deep white matter lesions. She was transferred to our hospital at age 6 months. High lactate levels in her cerebrospinal fluid suggested mitochondrial dysfunction. Despite vitamin supplementation therapy followed by a ketogenic diet, the patient began exhibiting clusters of myoclonic seizures and respiratory failure. Brain and spinal cord MRI revealed rapid progression of the white matter lesions. She died at 10 months of age. Fibroblasts obtained pre-mortem displayed low mitochondrial respiratory chain complex I and II activity. A homozygous H96R (c. 287 A > G) mutation was identified in the BOLA3 gene. Discussion: No reported case of a homozygous BOLA3 gene mutation has survived past 1 year of life. BOLA3 appears to play a critical role in the electron transport system and production of iron-sulfur clusters that are related to lipid metabolism and enzyme biosynthesis.

Escherichia coli has two machineries for the synthesis of FeS clusters, namely Isc (iron–sulfur cluster) and Suf (sulfur formation). The Isc machinery, encoded by the iscRSUA-hscBA-fdx-iscXoperon, plays a crucial role in the biogenesis of FeS clusters for the oxidoreductases of aerobic metabolism. Less is known, however, about the role of ISC in the maturation of key multi-subunit metalloenzymes of anaerobic metabolism. Here, we determined the contribution of each iscoperon gene product towards the functionality of the major anaerobic oxidoreductases in E. coli, including three [NiFe]-hydrogenases (Hyd), two respiratory formate dehydrogenases (FDH) and nitrate reductase (NAR). Mutants lacking the cysteine desulfurase, IscS, lacked activity of all six enzymes, as well as the activity of fumaratereductase, and this was due to deficiencies in enzyme biosynthesis, maturation or FeS cluster insertion into electron-transfer components. Notably, based on anaerobic growth characteristics and metabolite patterns, the activity of the radical-S-adenosylmethionine enzyme pyruvate formate-lyase activase was independent of IscS, suggesting that FeS biogenesis for this ancient enzyme has different requirements. Mutants lacking either the scaffold protein IscU, the ferredoxin Fdx or the chaperones HscA or HscB had similar enzyme phenotypes: five of the oxidoreductases were essentially inactive, with the exception being the Hyd-3 enzyme, which formed part of the H2-producing formate hydrogenlyase (FHL) complex. Neither the frataxin-homologue CyaY nor the IscX protein was essential for synthesis of the three Hyd enzymes. Thus, while IscS is essential for H2 production in E. coli, the other ISC components are non-essential.

The current high cost of lignocellulolytic enzymes is a major bottleneck in the economic bioconversion of lignocellulosic biomass to fuels and chemicals. Fungal lignocellulolytic enzyme systems are secreted at high levels, making them the most promising starting points for further development of highly efficient lignocellulolytic enzyme systems. In this paper, recent advances in improvement of fungal lignocellulolytic enzyme systems are reviewed, with an emphasis on the achievements made using genomic approaches. A general strategy for lignocellulolytic enzyme system development is proposed, including the improvement of the hydrolysis efficiencies and productivities of current enzyme systems. The applications of genomic, transcriptomic and proteomic analysis methods in examining the composition of native enzyme systems, discovery of novel enzymes and synergistic proteins from natural sources, and understanding of regulatory mechanisms for lignocellulolytic enzyme biosynthesis are summarized. By combining systems biology and synthetic biology tools, engineered fungal strains are expected to produce high levels of optimized lignocellulolytic enzyme systems.

We discuss characteristics of the directional formation of samples of heterogeneous biocatalysts based on immobilized cells of different microscopic fungi that are characterized by high productivity of cellulases with different substrate specificities (endoglucanases, exoglucanases, and beta-glucosidases). Samples of immobilized cells characterized by maximum productivity of the enzymes of cellulase complex are selected based on our study of the catalytic and operating characteristics of the designed biocatalysts. It is found that a biocatalyst based on Aspergillus terreus spores immobilized in polyvinyl alcohol cryogel is the best of the ones available. It is shown for the first time that the developed biocatalyst retains a high level of productivity for the full complex of cellulases when using various substrate inducers of enzyme biosynthesis, such as birch and oak sawdust, and rice and wheat straw. We demonstrate the possibility of efficiently using cellulase complexes obtained as a result of the functioning of immobilized cells in the saccharification of various cellulose-containing agricultural wastes and the conversion of the obtained sugars to organic solvents (ethanol, butanol) considered to be promising alternative fuels. The concentrations of organic solvents in media with immobilized cells are considerably higher than those found for free cells of the same microorganisms.

This study reports the optimization of xylanase production under solid state fermentation (SSF) by a thermotolerant Aspergillus fumigatus strain (SCB4) isolated from sugarcane bagasse piles of Brazilian Cerrado. Different combinations of low-cost agricultural byproducts in SSF were evaluated: sugarcane bagasse and wheat bran (1:1), sugarcane bagasse and corn straw (1:1) and only sugarcane bagasse. The enzyme biosynthesis by SSF was carried out at different temperatures (40, 45, 50 and 55 o C). The maximum levels of xylanase activity were obtained after 24 h at 45 °C using a culture medium containing sugarcane bagasse and wheat bran (1:1). Under optimal conditions, the fungal culture produced 574 U g -1 of xylanase (units/g of dry substrate). The crude enzyme showed optimal activity at 60 °C and pH 4.5. It exhibited thermostability up to 55 °C, wide range of pH stability and tolerance to ethanol, xylose and glucose. The physicochemical properties shown by this enzyme are appropriate for its application in hydrolysis of lignocellulosic residues for ethanol production and other bioproducts.

Aim: The purpose of present work was to utilize lignocellulosic forest waste for cost-effective production of commercially important enzymes i.e. cellulase and xylanase by employing co-culture of Aspergillus niger F7 and Fusarium oxysporum F8 in solid state fermentation (SSF). Material and Methods: Fungal strains i.e. A. niger F7 and F. oxysporum F8 isolated from degrading forest litter were used as co-culture and their enzyme biosynthesis on forest waste was noticed. The prominent forest wastes i.e. Toona ciliata, Celtris australis, Cedrus deodara and Pinus roxburghii were procured from local forest and alkali (NaOH) pretreatment was given to these lignocellulosic substrates to simplify the crystalline cellulose present in them for higher yield of enzymes by fungal isolates. Two types of moistening agents i.e. tap water and modified basal salt medium (BSM) were used in the substrate in the ratio of 1:2 to evaluate their effect on enzyme production under SSF. Results: The maximum enzyme titers i.e. 787.89 U/g of cellulase and 669.08 U/g of xylanase were observed in C. australis as compared to untreated biomass which was 450.02 and 398.03 U/g of cellulase and xylanase respectively when tap water was used as moistening agent. Biodegradation of forest biomass under SSF was increased many folds when moistening agent i.e. tap water had been replaced with modified basal salt media (BSM). In BSM mediated degradation of forest waste with co-culture of A. niger and F. oxysporum, extracellular enzyme activity was increased up to 2219.55 U/g of cellulase and 1012.0 U/g of xylanase in pretreated C. australis wood. Conclusion: In untreated biomass enzyme production by fungi was quite low thus emphasizing the need of pretreatment for biodegradation of forest lignocellulosics. Significantly higher production of cellulase and xylanase using pretreated forest waste and modified BSM as moisture are main highlights of the present study which commercially may lead to cost effective production of these industrially important enzymes.

The objective of this study was to investigate the impact of mycorrhizal symbiosis on qualitative characteristics of onion (Allium cepa L.). For this reason, five onion cultivars with different scale color and three different strains of arbuscular mycorrhizal fungi (Diversispora versiformis, Rhizophagus intraradices, Funneliformis mosseae) were used. Red cultivars, mainly 'Red Azar-shahr', showed the highest content in vitamin C, flavonols, and antioxidant enzymes. Mycorrhizal inoculation increased total phenolic, pyruvic acid, and vitamin C of onion plants. Considerable increase was observed in quercetin-4'-O-monoglucoside and isorhamnetin-4'-O-monoglucoside content in plants inoculated with Diversispora versiformis, but quercetin-3,4'-O-diglucoside was not significantly influenced. Analyses for phenylalanine ammonia-lyase (PAL) and antioxiodant enzyme activities such as polyphenol oxidase (PPO), catalase (CAT), and peroxidase (POD) revealed that all except PPO were enhanced by mycorrhizal inoculation. Overall, these findings suggested that mycorrhizal inoculation influenced biosynthesis of flavonol glucosides and antioxidant enzymes by increasing nutrient uptake or by induction of the plant defense system.

Twenty-seven fungal isolates recovered on medium containing L-lysine were found to have the potentiality for producing extracellular L-amino acid oxidase (L-AAO). Aspergillus oryzae displayed the highest yield of enzyme (2.6 U/mg protein) and antioxidants (2.3 mg/ml) followed by Aspergillus flavipes and Trichoderma viride. Upon optimization of the fermentation medium, the maximum enzyme yield (4.6 U/mg protein) was obtained on a medium containing L-lysine (80 mM), glucose (0.6%), KH2PO4 (0.1%), KCl (0.05%) and MgSO4⋅7H2O (0.05%), pH 7.0, under submerged conditions. Supplementing the medium with K+, Ni2+ and Cu2+ ions increased enzyme biosynthesis by about twofold compared to the control. The activity of extracellular L-AAO from the submerged cultures of A. oryzae was twofold higher than that of the intracellular L-AAO. Eleven agro-industrial byproducts were tested as substrates under solid state fermentation conditions; of these, rice bran (3.3 U/mg) was the optimum solid substrate for the induction of L-AAO and antioxidants, followed by wheat bran (2.8 U/mg) and cotton seed cake (2.5 U/mg), possibly due to their higher crude protein content than ash and fiber. Glucose (1%) and L-phenylalanine were the best co-metabolic and co-inductive carbon and nitrogen source, respectively, for maximum L-AAO production by A. oryzae under solid state fermentation, at an initial pH of 7.0. The productivity of L-AAO by A. oryzae under solid state fermentation was higher than that of the submerged cultures by about 1.4-fold under optimum conditions, justifying the former fermentation processes for overproduction of this enzyme in terms of both economics and practicality.

The regulatory link between biosynthesis of Bacillus intermedius subtilisin-like serine proteinase and nitrogen metabolism in B. intermedius cells was determined. The level of the enzyme biosynthesis by the recombinant strain of Bacillus subtilis in the medium containing ammonium ions was three- to fivefold less than that in the medium with poorly utilized sodium nitrate. Accumulation of glutamyl endopeptidase in a culture liquid of this microorganism did not depend on the source of nitrogen present in the medium. During cultivation in the rich medium, the productivity of subtilisin-like proteinase in the recombinant B. subtilis strain carrying a mutation in the NrgB sensor protein was demonstrated to increase threefold compared to that of the control strain. In the minimal culture medium, mutation in the nrgB gene abolished the effect of a nitrogen source on the level of the subtilisin-like proteinase gene expression. At the same time, this mutation did not affect glutamyl endopeptidase biosynthesis. Thus, expression of the gene coding for subtilisin-like proteinase from B.intermedius is suggested to be positively regulated by the regulatory system of nitrogen metabolism.

Bacillus brevis-B2 was selected as the most patent lipase(s) producer among nine thermoalkalophilic bacterial isolates isolated from agro industrial wastes at 70oC and pH 10.5. Of the tested nutrient medium, tap water containing 1% fish wastes and 1% yeast extract is the best inducer for lipase synthesis by B. brevis B2. Maximum enzyme biosynthesis could be obtained after 24 hr of incubation. The optimal pH and temperature for enzyme formation are 11.5 and 70oC. Enzyme yield increased in presence of sucrose or lactose in the nutrient medium, also the presence of 100 ppm zinc sulphate and 500 ppm folic acid supported high induction of lipase by B. brevis growing under shaking condition and inocula size of 2ml/100ml culture medium. A very slight increase in enzyme formation occur under exposure of bacterial isolate to 1 KGy of γ-irradiation.

Facile control of targeted intracellular protein degradation has many potential uses in basic science and biotechnology. One promising approach to this goal is to redesign adaptor proteins, which can regulate proteolytic specificity by tethering substrates to energy-dependent AAA+ proteases. Using the ClpXP protease, we have probed the minimal biochemical functions required for adaptor function by designing and characterizing variant substrates, adaptors, and ClpX enzymes. We find that substrate tethering mediated by heterologous interaction domains and a small bridging molecule mimics substrate delivery by the wild-type system. These results show that simple tethering is sufficient for synthetic adaptor function. In our engineered system, tethering and proteolysis depend on the presence of the macrolide rapamycin, providing a foundation for engineering highly specific degradation of target proteins in cells. Importantly, this degradation is regulated by a small molecule without the need for new adaptor or enzyme biosynthesis.

The soils of the Antarctic dry valleys are exposed to extremely dry and cold conditions. Nevertheless, they contain small communities of micro-organisms that contribute to the biogeochemical transformations of the bioelements, albeit at slow rates. We have determined the dehydrogenase, β-glucosidase, acid and alkaline phosphatase and arylsulphatase activities and the rates of respiration (CO 2 production) in laboratory assays of soils collected from a field experiment in an Antarctic dry valley. The objective of the field experiment was to test the responses of the soil microbial community to additions of C and N in simple (glucose and NH 4 Cl) and complex forms (glycine and lacustrine detritus from the adjacent lake comprising principally cyanobacterial necromass). The soil samples were taken 3 years after the experimental treatments had been applied. In unamended soil, all enzyme activities and respiration were detected indicating that the enzymatic capacity to mineralize organic C, P and S compounds existed in the soil, despite the very low organic matter content. Relative to the control (unamended soil), respiration was significantly increased by all the experimental additions of C and N except the smallest NH 4 Cl addition (1 mg N g −1 soil) and the smallest detritus addition (1.5 mg C g −1 soil and 0.13 mg N g −1 soil). The activities of all enzymes except dehydrogenase were increased by C and combined large C (10 mg C g −1 soil) and N additions, but either unchanged or diminished by addition of either N only or N (up to 10 mg N g −1 soil) with only small C (1 mg C g −1 soil) additions in the form of glucose and NH 4 Cl. This suggests that in the presence of a large amount of N, the C supply for enzyme biosynthesis was limited. When normalized with respect to soil respiration, only arylsulphatase per unit of respiration showed a significant increase with C and N additions as glucose and NH 4 Cl, consistent with S limitation when C and N limitations have been alleviated. Based on the positive responses of enzyme activity, detritus appeared to provide either conditions or resources which led to a larger biological response than a similar amount of C and more N added in the form of defined compounds (glucose, NH 4 Cl or glycine). Assessment of the soil microbial community by ester-linked fatty acid (ELFA) analysis provided no evidence of changes in the community structure as a result of the C and N supplementation treatments. Thus the respiration and enzyme activity responses to supplementation occurred in an apparently structurally stable or unresponsive microbial community.

The gene encoding for Bacillus intermedius serine proteinase was cloned and the complete nucleotide sequence was determined. Gene expression was explored in the protease-deficient strain Bacillus subtilis AJ73 during different stages of growth. Catabolite repression involved in control of proteinase expression during transition state and onset of sporulation was not efficient at the late stationary phase. Salt stress leads to induction of serine proteinase production during B. subtilis AJ73(pCS9) post-exponential growth. Expression of proteinase in B. subtilis deg-mutants may be controlled by DegU regulator. B. subtilis spo0-mutants failed to accomplish B. intermedius proteinase production. These data suggest complex network regulation of B. intermedius serine proteinase expression, including the action of spo0, degU, catabolite repression and demonstrate changes in control of enzyme biosynthesis at different stages of growth.

The influence of the cultivation conditions on Bacillus pumilus KMM 62 growth and effectiveness of the production of a subtilisin-like serine proteinase were investigated. Enzyme accumulation in the culture fluid reached the maximum value after 32 and 46-48 h of growth; it depends on the composition of the nutrient medium. The ratio of the concentrations of two main components of the medium, peptone and inorganic phosphate, which was optimal for enzyme biosynthesis was determined by multifactor experiments. Ammonium salts, when introduced as an additional nitrogen source, had different effects on the proteinase biosynthesis at different growth stages: they suppress enzyme production at the early stationary growth phase and stimulate the biosynthesis of the enzyme after 46-48 h of growth. Complex organic substrates (albumin, casein, hemoglobin, and gelatin) have a repressive effect on the biosynthesis of the enzyme. The effect of amino acids on culture growth and enzyme biosynthesis during the early and late stationary growth phase is different. Hydrophilic amino acids, glutamine, and glutamic acid exhibit the most pronounced repressive action on biosynthesis. The activity of different regulatory mechanisms for the synthesis of this proteinase is assumed at the early and late stationary stages of growth.

The gene encoding for B. intermedius glutamyl endopeptidase (gseBi) has previously been cloned and its nucleotide sequence analyzed. In this study, the expression of this gene was explored in protease-deficient strain B. subtilis AJ73 during stationary phase of bacterial growth. We found that catabolite repression usually involved in control of endopeptidase expression during vegetative growth was not efficient at the late stationary phase. Testing of B. intermedius glutamyl endopeptidase gene expression with B. subtilis spo0-mutants revealed slight effect of these mutations on endopeptidase expression. Activity of glutamyl endopeptidase was partly left in B. subtilis ger-mutants. Probably, gseBi expression was not connected with sporulation. This enzyme might be involved in outgrowth of the spore, when germinating endospore converts into the vegetative cell. These data suggest complex regulation of B. intermedius glutamyl endopeptidase gene expression with contribution of several regulatory systems and demonstrate changes in control of enzyme biosynthesis at different stages of growth.

In this review, I compare the development of Monod's intellectual leadership in two fields, the regulation of enzyme biosynthesis and the control of enzymatic activity. I characterize the comings and goings between his scrupulous analysis of a given model system, his ability to compare the outcome with very distant experimental results, his audacity in formulating, then a physical interpretation of this convergence through a unifying mechanism. Finally, I briefly discuss how his attitude has durably impacted the whole field of molecular biology.

The effect of certain nutrients on the growth and production of the Bacillus intermedius subtilisin-like serine proteinase by the recombinant strain Bacillus subtilis AJ73(pCS9) was studied. Glucose was found to inhibit the synthesis of proteinase in the early (28 h of growth) but not in the late stationary phase (48 h of growth). The inhibitory effect of the other mono-and disaccharides studied was less pronounced. Casamino acids added to the medium at concentrations of 0.1–1% as an additional carbon and nitrogen source stimulated enzyme biosynthesis. Individual amino acids (cysteine, asparagine, glutamine, tryptophan, histidine, and glutamate) also stimulated enzyme biosynthesis in the early stationary phase by 25–30%, whereas other amino acids (valine, leucine, alanine, and aspartate) were ineffective or even slightly inhibitory to enzyme production. The stimulatory effect of the first group of amino acids on the synthesis of proteinase in the late stationary phase was negligible. In contrast, the bivalent ions Ca2+, Mg2+, and Mn2+ stimulated biosynthesis of proteinase in the late stationary phase (by 20–60%) and not in the early stationary phase. The data indicate that there are differences in the biosyntheses of proteinase by the recombinant B. subtilis strain during the early and late periods of the stationary phases.

The ability of metabolites contained in culture liquid of 62 strains of marine fungi to affect the activity of two digestive enzymes of marine mollusks—endo-1,3-β-D-glucanase of Spisula sachalinensis and β-D-glucosidase of Littorina kurila—was studied. It was found that 66 and 71% of specimens activated, 18 and 7% inhibited, and 16 and 22% did not affect the activity of endo-1,3-β-D-glucanase and β-D-glucosidase, respectively. It is demonstrated that the metabolites of brown algae and marine sponges can be used for a targeted regulation of enzyme biosynthesis by marine fungi. The protein inhibitor of endo-1,3-β -D-glucanases isolated from the brown alga Laminaria cichorioides blocked the biosynthesis of almost all O-glycosylhydrolases in five strains of marine fungi studied. The presence in culture medium of halistanol sulfate from the marine sponge of the family Halichondriidae either did not affect or activated the biosynthesis of enzymes involved in carbohydrate metabolism by marine fungi.

Cordyceps militaris mycelium produced mainly Cu, Zn containing superoxide dismutase (Cu, Zn-SOD). Cu, Zn-SOD activity was detectable in the culture filtrates, and intracellular Cu, Zn-SOD activity as a proportion protein was highest in early log phase culture. The effects of Cu 2+ , Zn 2+ , Mn 2+ and Fe 2+ on enzyme biosynthesis were studied. The Cu, Zn-SOD was isolated and purified to homogeneity from C. militaris mycelium and partially characterized. The purification was performed through four steps: (NH 4 ) 2 SO 4 precipitation, DEAE-sepharose™ fast flow anion-exchange chromatography, CM-650 cation-exchange chromatography, and Sephadex G-100 gel filtration chromatography. The purified enzyme had a molecular weight of 35070 ± 400 Da and consisted of two equal-sized subunits each having a Cu and Zn element. Isoelectric point value of 7.0 was obtained for the purified enzyme. The N-terminal amino acid sequence of the purified enzyme was determined for 12 amino acid residues and the sequences was compared with other Cu, Zn-SODs. The optimum pH of the purified enzyme was obtained to be 8.2–8.8. The purified enzyme remained stable at pH 5.8–9.8, 25 °C and up to 50 °C at pH 7.8 for 1.5 h incubation. The purified enzyme was sensitive to H 2 O 2 , KCN. 2.5 mM NaN 3 , PMSF, Triton X-100, β-mercaptoethanol and DTT showed no significant inhibition effect on the purified enzyme within 5 h incubation period.

Among the ten strains of marine fungi studied, the mycelial fungus Phoma glomerata showed maximum potency in producing N-acetyl-beta-D-glucosaminidase. The conditions for fungal growth and enzyme biosynthesis were evaluated. N-acetyl-beta-D-hexosaminidase was isolated from the culture liquid of Phoma glomerata by ion-exchange chromatography (on DEAE-cellulose and DEAE-Sephacell) and gel filtration (on Toyopearl HW-55) with a yield of 35%; the enzyme, purified 36.4-fold, had a molecular weight of 20 kDa. The homogeneity of the enzyme was confirmed by gel filtration and SDS-PAGE. Transglycosylation reactions catalyzed by the enzyme produced N-acetyl-D-glucosamine and N-acetyl-D-galactosamine with respective yields of 38 and 46%.

The present study describes the biosynthesis of proteases by a locally isolated mould culture of Rhizopus oligosporus IHS13 in a low-cost medium by solid-state fermentation technique. The fermentation was carried out in a low-cost medium such as sunflower meal, wheat bran and rice bran. Sunflower meal and wheat bran in a ratio of 1:1 and moistened with distilled water was found to be the best substrate for protease synthesis. All the three substrates are very cheap agricultural by-products found in Pakistan. The production of proteases in sunflower meal and rice bran was also investigated but the results were unsatisfactory. Different cultural conditions such as rate of fermentation, effect of incubation temperature, effect of pH and depth of the fermentation medium were also optimized. The maximum enzyme synthesis was found after 72 h of fermentation at a temperature of 30 degrees C. The optimum pH and depth of the medium for protease synthesis were found to be 5.0 and 20 mm respectively. The maximum enzyme biosynthesis found during the course of present studies was 7.0 U ml(-1).

The main parameters of growth and glucose oxidase production by the mutant Penicillium funiculosum strains BIM F-15.3, NMM95.132, and 46.1 were studied. The synthesis of extracellular glucose oxidase by these strains was constitutive and occurred following the phase of exponential growth. The mutant strains also synthesized extracellular invertase and cell-associated catalase and glucose oxidase. The syntheses of invertase, the cell-associated enzymes, and extracellular glucose oxidase were found to be maximum between 14 and 18 h, between 48 and 52 h, and by the 96th hour of cultivation, respectively. Among the mutants studied, P. funiculosum 46.1 showed the maximal rates of growth and glucose oxidase synthesis.

Two enzyme assays, namely β-galactosidase in Escherichia coli (E.C.3.2.1.23) and α-amylase in Bacillus subtilis (E.C.3.2.1.1), were compared following exposure to various concentrations of heavy metals and organic pesticides. It was shown that α-amylase biosynthesis in the Gram-positive bacteria was more sensitive to heavy metals, particularly to mercury as low as 0.1 ppm, compared to β-galactosidase in the Gram-negative bacteria. The results of organic pesticide treatment were more clear-cut, when comparing both assays. Alpha-amylase test system can detect the inhibition by the organometallic fungicides Maneb and Mancozeb at as low as 0.1 ppm, while no inhibition with β-galactosidase system up to as high as 100 ppm. The effect of organic insecticide endosulfan on α-amylase was also seen at very low concentrations (0.048 ppm). The electrophoretic analysis also revealed the inhibition of enzyme biosynthesis at the tested toxicant concentrations.

Effects of various conditions of nitrogen and carbon nutrition on the biosynthesis of proteolytic enzymes in a selected culture of Aspergillus awamori 21/96 were studied. This strain was shown to produce proteolytic enzymes constitutively. In the presence of mineral sources of nitrogen, the synthesis of the enzymes under study was not induced by proteinaceous substrates. Optimum conditions of the enzyme biosynthesis were achieved with casein as a source of nitrogen and starch or dulcitol as a source of carbon (which increased the production of the enzymes 1.7 and 8 times, respectively). When the cells were grown on starch, their specific activity exceeded control levels 18 times.

The patC gene encoding the cystathionine beta-lyase (CBL) of Lactobacillus delbrueckii subsp. bulgaricus NCDO 1489 was cloned and expressed in Escherichia coli. Overexpression of CBL complemented the methionine auxotrophy of an E. coli metC mutant, demonstrating in vivo that this enzyme functions as a CBL. However, PatC is distinguishable from the MetC CBLs by a low identity in amino acid sequence, a sensitivity to iodoacetic acid, greater thermostability and a lower substrate affinity. Homologues of patC were detected in the 13 Lb. delbrueckii strains studied, but only seven of them showed CBL activity. In constrast to CBL(+) strains, all CBL-deficient strains analysed were auxotrophic for methionine. This supports the hypothesis that CBLs from lactobacilli are probably involved in methionine biosynthesis. Moreover, the results of this study suggest that post-transcriptional mechanisms account for the differences in CBL activities observed between strains of Lb. delbrueckii.

Glycogenosis type II (GSDII, Pompe disease) is an autosomal recessive lysosomal storage disease caused by a deficiency of acid alpha-glucosidase (acid maltase, GAA). The enzyme degrades alpha -1,4 and alpha -1,6 linkages in glycogen, maltose, and isomaltose. Deficiency of the enzyme results in accumulation of glycogen within lysosomes and in cytoplasm eventually leading to tissue destruction. The discovery of the acid a-glucosidase gene has led to rapid progress in understanding the molecular basis of glycogenosis type II and the biological properties of the GAA protein. The last decade has seen several developments: 1) extensive mutational analysis in patients with different forms of the disease, 2) characterization of the enzyme biosynthesis, processing, and lysosomal targeting, 3) generation of knockout mouse models, 4) development of viral vectors for gene replacement therapy, 5) the production of recombinant human enzyme, and 6) a shift in the enzyme replacement therapy approach from theory to practice. It is anticipated that the enzyme replacement therapy will be widely available for human use in the near future. Several recent reviews (including the most comprehensive one by R. Hirschhorn and A. Reuser [1]), address clinical, biochemical and genetic aspects of the disease, as well as development of new therapies for GSDII [2, 3, 4]. In this article we will review recent findings in the area including rapidly accumulating molecular genetic data (more than 20 mutations need to be added to the list), transcriptional control of gene expression, studies in mouse models, and new approaches to gene therapy. We will also highlight some emerging questions following the introduction of enzyme replacement therapy.

cDNAs encoding acyl-CoA:diacylglycerol acyltransferase (DGAT, EC 2.3.1.20), designated BnDGAT1 and BnDGAT2, were obtained from a microspore-derived cell suspension culture of oilseed rape (Brassica napus L. cv Jet Neuf). BnDGAT2 shares a very high level of identity with BnDGAT1, but is a smaller protein lacking the relatively hydrophilic N-terminal segment found in BnDGAT1. Both transcripts were produced in the cell suspension cultures and the cDNAs were functionally expressed in transformed yeast (Pichia pastoris) cells. Sucrose-mediated changes in triacylglycerol (TAG) metabolism and expression of BnDGAT1 were examined in the cell suspension cultures following transfer of cells from media containing 6% (w/v) sucrose to media containing 14% sucrose. TAG content and DGAT activity of the cells increased transiently within the first 12 h after transfer (HAT). The rapid decline in TAG content observed at 12 HAT was inversely related to an increase in TAG lipase (EC 3.1.1.3) activity. The transient increases in TAG content and DGAT activity correlated with the elevated amounts of BnDGAT1 polypeptide. Transcript levels were also induced, but levels of mRNA encoding BnDGAT1 were not tightly correlated with DGAT activity and amount of polypeptide suggesting some control of expression at the post-transcriptional level. In general, the rapid changes in TAG content were closely associated with the changes in the activity of TAG-metabolizing enzymes and expression of BnDGAT1.

The biological effects of ribonucleases (RNases), such as the control of the blood vessels growth, the toxicity towards tumour cells and antiviral activity, require a detailed explanation. One of the most intriguing properties of RNases which can contribute to their biological effects is the ability to form dimers, which facilitates efficient RNA hydrolysis and the evasion of ribonuclease inhibitor. Dimeric forms of microbial RNase binase secreted by Bacillus pumilus (former B. intermedius) have only been found in crystals to date. Our study is the first report directly confirming the existence of binase dimers in solution and under natural conditions of enzyme biosynthesis and secretion by bacilli. Using different variants of gel electrophoresis, immunoblotting, size-exclusion chromatography and mass-spectrometry, we revealed that binase is a stable natural dimer with high catalytic activity.

Bu çalışmada çeşitli topraklardan izole edilen 30 bakteri izolatının ekstraselüler lipaz üretim kapasiteleri test edilmiştir. En iyi lipaz üreticisi olarak B5 izolatı seçilmiştir. İzolatın tanılaması 16S rRNA gen bölgesine göre yapılmış ve Gen Bankası'ndaki baz dizisi çakışmasına göre Burkholderia cenocepacia olarak belirlenmiştir. Kantitatif ekstraselüler lipaz aktivitesi spektrofotometrik yöntemle ölçülmüştür ve 24 saat inkübasyon sonunda lipaz aktivitesi 7.59 U/mL, spesifik aktivitesi ise 263.16 U/mg olarak bulunmuştur. B. cenocepacia B5 lipazının molekül kütlesi yaklaşık 30 kDa olarak tespit edilmiştir. Enzimin Michaelis-Menten kinetik sabitleri olan Km ve Vmaks değerleri sırasıyla 0.072 mM ve 263.16 U/mg olarak tespit edilmiştir. Enzimin 30-60 °C ve pH 5.0-10.0 arasında aktif olduğu, optimum aktiviteyi 50 °C ve pH 10.0'da gösterdiği tespit edilmiştir. Enzim, en yüksek aktiviteyi substrat olarak p-nitrofenil palmitatta göstermiştir. Metanol, etanol, 2-propanol, asetonitril ve aseton gibi organik çözücüler lipaz aktivitesinde artışa, DMSO ise azalmaya neden olmuştur. Bazı metal ve kimyasalların aktiviteyi arttırdığı (Al3+, EDTA) görülürken bazılarının (Tween 80, Cr3+) ise aktiviteyi azalttığı görülmüştür. B5 lipazının zeytinyağını yağ asitlerine parçaladığı ince tabaka kromotografisinde gösterilmiştir. B. cenocepacia B5 lipazının özellikleri incelendiğinde bu enzimin organik çözücülerde aktivitesinin iyi olması onun biyoteknolojik uygulamalar ve biyodizel üretiminde kullanılabileceğini göstermektedir. In this study, extracellular lipase production capacity of 30 bacteria, isolated from various soils, were tested. Isolate B5 was selected as the most lipase producer. Identification of the microorganism was done by its 16S rRNA gene sequencing and it was determined Burkholderia cenocepacia according to the GeneBank sequence comparison. Quantitative extracellular lipase activity was determined spectrophotomertrically and the maximum lipase activity was observed with 7.59 U/mL at 24 hours and the spesific activity was found 263.16 U/mg.The molecular mass of lipase enzyme was observed approximately 30 kDa with SDS-PAGE. Michaelis-Menten kinetic constant Km and Vmax values of B. cenocepacia B5 lipase were determined as 0.072 mM and 263.16 U/mg, respectively. It was observed the lipase was active at pH range of 5.0-10.0 and temperature on 30-60 °C, the optimum pH of the lipase was 10.00 and optimum temperature was 50 °C. The enzyme showed the highest lipase activity when p-nitrophenyl palmitate used as a substrate. Organic solvents such as methanol, ethanol, 2-propanol, acetonitrile, and acetone cause an increase in lipase activity but DMSO cause decrease. Some metals and chemicals increased activity (Al3+, EDTA), whereas some were found to inhibit the activity (Tween 80, Cr3+). It was displayed that lipase enzyme broke down the olive oil into fatty acids by thin-layer chromatography. It was concluded that the enzyme can be used in biotechnological applications and biodiesel production due to good activity in organic solvents when the characteristics of B.cenocepacia B5 lipase was investigated. 73