New Insight into Old Bacillus Lipase: Solvent Stable Mesophilic Lipase Demonstrating Enzyme Activity towards Cold

2015 ◽  
Vol 25 (5) ◽  
pp. 340-348 ◽  
Author(s):  
Jyoti Khurana ◽  
Rakesh Kumar ◽  
Arbind Kumar ◽  
Kashmir Singh ◽  
Ranvir Singh ◽  
...  
Keyword(s):  
Enzyme Activity ◽  
Molecular Mass ◽  
Industrial Applications ◽  
Maximum Activity ◽  
Lipase Gene ◽  
Reading Frame ◽  
Wide Range ◽  
Cold Active ◽  
Solvent Stable ◽  
Insight Into

<b><i>Background:</i></b><i>Bacillus</i> lipases are grouped in subfamily 1.4, which are the smallest known lipases having a molecular mass of 19.6 kDa. Lipases active in a wide range of temperatures, specifically at low temperatures, have an advantage under low water conditions for industrial application. <b><i>Methods:</i></b> The lipase gene was cloned and expressed in <i>Escherichia coli</i>. The protein was purified and biochemically characterized in detail. <b><i>Results:</i></b> A lipase gene was cloned from a mesophilic <i>Bacillus</i> isolate. Sequence analysis revealed an open reading frame of 633 bp in length. The predicted molecular mass of protein was 22.6 kDa. The purified enzyme displayed optimal activity at 35°C and pH 8.0. Interestingly, this mesophilic enzyme was also cold active showing retention of 75 and 55% relative enzyme activity at 20 and 10°C, respectively. The purified lipase was stable in various organic solvents (50% v/v) and ionic liquids (5% v/v). The enzyme displayed maximum activity with paranitrophenyl laurate (C<sub>12</sub>). k<sub>cat</sub>/K<sub>m</sub> values for the purified lipase were calculated to be 5.8 ± 0.6 × 10<sup>-6</sup>. <b><i>Conclusions:</i></b> The lipase showed tolerance to various solvents as well as activity at low temperature. Therefore, this lipase might be of great potential to be employed in various industrial applications.

Purification, biochemical and molecular study of lipase producing from a newly thermoalkaliphilic Aeribacillus pallidus for oily wastewater treatment

2019 ◽  
Vol 167 (1) ◽  
pp. 89-99 ◽  
Author(s):  
Ameni Ktata ◽  
Najeh Krayem ◽  
Ahmed Aloulou ◽  
Sofiane Bezzine ◽  
Adel Sayari ◽  
...  
Keyword(s):  
Wastewater Treatment ◽  
Organic Solvents ◽  
Molecular Study ◽  
Maximum Activity ◽  
Oily Wastewater ◽  
Reading Frame ◽  
Oil Biodegradation ◽  
Wide Range ◽  
Oily Wastewater Treatment ◽  
Solvent Tolerant

Abstract Treatment of oily wastewater is constantly a challenge; biological wastewater treatment is an effective, cheap and eco-friendly technology. A newly thermostable, haloalkaline, solvent tolerant and non-induced lipase from Aeribacillus pallidus designated as GPL was purified and characterized of biochemical and molecular study for apply in wastewater treatment. The GPL showed a maximum activity at 65°C and pH 10 after 22 h of incubation, with preference to TC4 substrates. Pure enzyme was picked up after one chromatographic step. It displayed an important resistance at high temperature, pH, NaCl, at the presence of detergents and organic solvents. In fact, GPL exhibited a prominent stability in wide range of organic solvents at 50% (v/v) concentration for 2 h of incubation. The efficiency of the GPL in oil wastewater hydrolysis was established at 50°C for 1 h, the oil removal efficiency was established at 96, 11% and the oil biodegradation was confirmed through fourier transform infrared (FT-IR) spectroscopy. The gene that codes for this lipase was cloned and sequenced and its open reading frame encoded 236 amino acid residues. The deduced amino acids sequence of the GPL shows an important level of identity with Geobacillus lipases.

scholarly journals Characterization of a Novel β-Xylosidase, XylC, fromThermoanaerobacterium saccharolyticumJW/SL-YS485

2010 ◽  
Vol 77 (3) ◽  
pp. 719-726 ◽  
Author(s):  
Weilan Shao ◽  
Yemin Xue ◽  
Ailian Wu ◽  
Irina Kataeva ◽  
Jianjun Pei ◽  
...  
Keyword(s):  
Gel Electrophoresis ◽  
Glycosyl Hydrolase ◽  
Gel Filtration ◽  
Industrial Applications ◽  
Maximum Activity ◽  
Site Directed Mutagenesis ◽  
Stem Loop ◽  
Translational Initiation ◽  
Reading Frame ◽  
Gradient Gel Electrophoresis

ABSTRACTThe 1,914-bp open reading frame ofxylCfromThermoanaerobacterium saccharolyticumJW/SL-YS485 encodes a calculated 73-kDa β-xylosidase, XylC, different from any glycosyl hydrolase in the database and representing a novel glycohydrolase family. Hydrolysis occurred under retention of the anomeric configuration, and transglycosylation occurred in the presence of alcohols as acceptors. With the use of vector pHsh, expression of XylC, the third β-xylosidase in this bacterium, increased approximately 4-fold when a loop within the translational initiation region in the mRNA was removed by site-directed mutagenesis. The increased expression ofxylCmis due to removal of a stem-loop structure without a change of the amino acid sequence of the heterologously expressed enzyme (XylCrec). When gel filtration was applied, purified XylC had molecular masses of 210 kDa and 265 kDa using native gradient gel electrophoresis. The protein consisted of 78-kDa subunits based on SDS gel electrophoresis and contained 6% carbohydrates. XylC and XylCrecexhibited maximum activity at 65°C and pH65°C6.0, a 1-h half-life at 67°C, aKmforp-nitrophenyl-β-d-xyloside of 28 mM, and aVmaxof 276 U/mg and retained 70% activity in the presence of 200 mM xylose, suggesting potential for industrial applications.

Cold-active extracellular alkaline protease from an alkaliphilicStenotrophomonas maltophilia: production of enzyme and its industrial applications

2009 ◽  
Vol 55 (11) ◽  
pp. 1294-1301 ◽  
Author(s):  
Mohammed Kuddus ◽  
Pramod W. Ramteke
Keyword(s):  
High Efficiency ◽  
Western Himalaya ◽  
Industrial Applications ◽  
Culture Conditions ◽  
Maximum Activity ◽  
Exchange Chromatography ◽  
Protease Production ◽  
Cold Active ◽  
Metalloprotease Inhibitors ◽  
Potential Component

A novel psychro-tolerant bacterium Stenotrophomonas maltophilia (MTCC 7528) with an ability to produce extracellular, cold-active, alkaline, and detergent-stable protease was isolated from soil samples obtained from Gangotri glacier, Western Himalaya, India. The culture conditions for higher protease production were optimized with respect to incubation time, agitation, substrate, pH, and temperature. Maximum protease production of 56.2 U·mL–1was achieved in the medium at 20 °C and pH 9.0 after 120 h incubation. The protease was partially purified by ion-exchange chromatography and approximately 55-fold purification was achieved. The purified enzyme was a 75 kDa protease with maximum activity and stability at pH 10 and 20 °C. The activity of enzyme is stimulated by Mn2+and inhibited completely by metalloprotease inhibitors, indicating that it is a metalloprotease. The protease showed excellent stability and compatibility with commercial detergents and exhibited high efficiency for the removal of different types of protein-containing stains at low temperature. The wash performance analysis of blood and grass stains on cotton fabric showed an increase in reflectance by 26% and 23%, respectively, after treatment with enzyme in comparison to detergent only. These results indicate that it may be a potential component to use as a detergent additive for cold washing and in environmental bioremediation in cold regions.

scholarly journals Functional and biochemical characterization of a recombinant Arabidopsis thaliana 3-deoxy-D-manno-octulosonate 8-phosphate synthase

2004 ◽  
Vol 381 (1) ◽  
pp. 185-193 ◽  
Author(s):  
Jing WU ◽  
Mayur A. PATEL ◽  
Appavu K. SUNDARAM ◽  
Ronald W. WOODARD
Keyword(s):  
Arabidopsis Thaliana ◽  
Molecular Mass ◽  
Biochemical Characterization ◽  
Dipicolinic Acid ◽  
Kinetic Studies ◽  
Maximum Activity ◽  
Reading Frame ◽  
Sequential Mechanism ◽  
Phosphate Synthase

An open reading frame, encoding for KDOPS (3-deoxy-D-manno-octulosonate 8-phosphate synthase), from Arabidopsis thaliana was cloned into a T7-driven expression vector. The protein was overexpressed in Escherichia coli and purified to homogeneity. Recombinant A. thaliana KDOPS, in solution, displays an apparent molecular mass of 76 kDa and a subunit molecular mass of 31.519 kDa. Unlike previously studied bacterial KDOPSs, which are tetrameric, A. thaliana KDOPS appears to be a dimer in solution. The optimum temperature of the enzyme is 65 °C and the optimum pH is 7.5, with a broad peak between pH 6.5 and 9.5 showing 90% of maximum activity. The enzyme cannot be inactivated by EDTA or dipicolinic acid treatment, nor it can be activated by a series of bivalent metal ions, suggesting that it is a non-metallo-enzyme, as opposed to the initial prediction that it would be a metallo-enzyme. Kinetic studies showed that the enzyme follows a sequential mechanism with Km=3.6 μM for phosphoenolpyruvate and 3.8 μM for D-arabinose 5-phosphate and kcat=5.9 s−1 at 37 °C. On the basis of the characterization of A. thaliana KDOPS and phylogenetic analysis, plant KDOPSs may represent a new, distinct class of KDOPSs.

scholarly journals Metaproteomic Discovery and Characterization of a Novel Lipolytic Enzyme From an Indian Hot Spring

2021 ◽  
Vol 12 ◽  
Author(s):  
Dennis Sander ◽  
Yanfei Yu ◽  
Premankur Sukul ◽  
Sina Schäkermann ◽  
Julia E. Bandow ◽  
...  
Keyword(s):  
Chain Length ◽  
Prolonged Exposure ◽  
Hot Spring ◽  
Hydrolytic Activity ◽  
Industrial Applications ◽  
Maximum Activity ◽  
Lipolytic Enzyme ◽  
Lipolytic Enzymes ◽  
Optimum Activity ◽  
Solvent Stable

Lipolytic enzymes are produced by animals, plants and microorganisms. With their chemo-, regio-, and enantio-specific characteristics, lipolytic enzymes are important biocatalysts useful in several industrial applications. They are widely used in the processing of fats and oils, detergents, food processing, paper and cosmetics production. In this work, we used a new functional metaproteomics approach to screen sediment samples of the Indian Bakreshwar hot spring for novel thermo- and solvent-stable lipolytic enzymes. We were able to identify an enzyme showing favorable characteristics. DS-007 showed high hydrolytic activity with substrates with shorter chain length (&lt;C8) with the maximum activity observed against p-nitrophenyl butyrate (C4). For substrates with a chain length &gt;C10, significantly less hydrolytic activity was observed. A preference for short chain acyl groups is characteristic for esterases, suggesting that DS-007 is an esterase. Consistent with the high temperature at its site of isolation, DS-007 showed a temperature optimum at 55°C and retained 80% activity even after prolonged exposure to temperatures as high as 60°C. The enzyme showed optimum activity at pH 9.5, with more than 50% of its optimum activity between pH 8.0 and pH 9.5. DS-007 also exhibited tolerance toward organic solvents at a concentration of 1% (v/v). One percent of methanol increased the activity of DS-007 by 40% in comparison to the optimum conditions without solvent. In the presence of 10% methanol, DMSO or isopropanol DS-007 still showed around 50% activity. This data indicates that DS-007 is a temperature- and solvent-stable thermophilic enzyme with reasonable activity even at lower temperatures as well as a catalyst that can be used at a broad range of pH values with an optimum in the alkaline range, showing the adaptation to the habitat’s temperature and alkaline pH.

Unsteady Heat Conduction in Granular Materials

2000 ◽  
Vol 627 ◽  
Author(s):  
Watson L. Vargas ◽  
Joseph J. McCarthy
Keyword(s):  
Heat Conduction ◽  
Granular Materials ◽  
Thermal Response ◽  
Transient Heat Conduction ◽  
Packed Bed ◽  
Industrial Applications ◽  
Multiphase Reactors ◽  
Wide Range ◽  
Unsteady Heat Conduction ◽  
Insight Into

ABSTRACTHeat transfer in granular materials impacts a variety of industrial applications, such as calcination, drying kilns, packed bed and multiphase reactors, etc. and may yield insight into the thermal response of some porous materials (in combustion synthesis or sintering, for example). In a dense bed of granular material, conduction occurs almost exclusively through the particle-particle contacts over a wide range of conditions. We have developed a novel Thermal Particle Dynamics (TPD) Simulation technique which incorporates both contact mechanics and contact conductance theories in order to model the dynamics of flow and heat conduction through granular materials. This model is uniquely suited to studying the effects of microstructure and flow on the dynamics of heat conduction in particulate materials. In this paper, we present experimental as well as numerical results of transient heat conduction through a bed of cylinders.

scholarly journals Characterization of β-Galactosidase from Lactose Utilizing Yeast Isolated from the Dairy Sample

2020 ◽  
Vol 7 ◽  
pp. 133-141
Author(s):  
Bivek Dahal ◽  
Sujan Karki ◽  
Nabaraj Adhikari ◽  
Upendra Thapa Shrestha
Keyword(s):  
Enzyme Activity ◽  
Maximum Activity ◽  
Commercial Application ◽  
Local Market ◽  
Time Duration ◽  
Optimum Time ◽  
Crude Enzyme Extract ◽  
Single Strain ◽  
Wide Range ◽  
Menten Constant

Objectives: The objective of the study was to isolate lactose positive yeasts from dairy samples collected from local markets of Kathmandu, to extract crude β-galactosidase from the lactose positive yeast and to characterize the enzyme for optimum time duration, pH, temperature, Michaelis-Menten constant (Km) and maximum activity (Vmax). Methods: Four lactose positive yeast strains were isolated from dairy samples collected from local market of Kathmandu by pour plate method. Single strain having maximum lactose positive activity was selected for the study. The mass culture of the lactose positive yeast strain was lysed by 2% Chloroform and the yeast cell lysate containing β-galactosidase (i.e. crude enzyme extract) was characterized by using ONPG (Ortho-Nitrophenyl-β-D-galactopyranoside) as substrate. ONPG is a colorless substrate for the enzyme assay which is hydrolyzed by the enzyme into yellow colored product ONP (Ortho-Nitrophenol). The concentration of product formed was monitored spectrophotometrically at 420 nm to determine the enzyme activity and to characterize the enzyme. Results: The enzyme had wide range of working temperature from 0-50ºC, with optimal temperature of 37ºC. However, greater than 50% hydrolyzing ability was maintained in the range of 14-40ºC. Optimum time of reaction was 70 min. The enzyme had maximum activity in the near neutral pH of 6.8. Michaelis-Menten constant of the enzyme was found to be 2.23 mM of ONPG and Vmax was 58.82 nmol/min/ml. Enzyme activity was 27.88 nmol/min/ml, Specific enzyme activity was 59.97 nmol/ min/mg and total enzyme activity was 3346.33 nmol/min. Conclusion: The activity over a wide range of temperature 0-50ºC with low Km value shows that the enzyme has a commercial application in clearance of lactose pollution in waste water in different environmental conditions.

scholarly journals Rational Engineering of a Cold-Adapted α-Amylase from the Antarctic Ciliate Euplotes focardii for Simultaneous Improvement of Thermostability and Catalytic Activity

2017 ◽  
Vol 83 (13) ◽  
Author(s):  
Guang Yang ◽  
Hua Yao ◽  
Matteo Mozzicafreddo ◽  
Patrizia Ballarini ◽  
Sandra Pucciarelli ◽  
...  
Keyword(s):  
Catalytic Activity ◽  
Catalytic Efficiency ◽  
Industrial Applications ◽  
Enzyme Engineering ◽  
Content Type ◽  
Cold Adapted ◽  
Wide Range ◽  
Cold Active ◽  
Surface Loops ◽  
The Antarctic

ABSTRACT The α-amylases are endo-acting enzymes that hydrolyze starch by randomly cleaving the 1,4-α-d-glucosidic linkages between the adjacent glucose units in a linear amylose chain. They have significant advantages in a wide range of applications, particularly in the food industry. The eukaryotic α-amylase isolated from the Antarctic ciliated protozoon Euplotes focardii (EfAmy) is an alkaline enzyme, different from most of the α-amylases characterized so far. Furthermore, EfAmy has the characteristics of a psychrophilic α-amylase, such as the highest hydrolytic activity at a low temperature and high thermolability, which is the major drawback of cold-active enzymes in industrial applications. In this work, we applied site-directed mutagenesis combined with rational design to generate a cold-active EfAmy with improved thermostability and catalytic efficiency at low temperatures. We engineered two EfAmy mutants. In one mutant, we introduced Pro residues on the A and B domains in surface loops. In the second mutant, we changed Val residues to Thr close to the catalytic site. The aim of these substitutions was to rigidify the molecular structure of the enzyme. Furthermore, we also analyzed mutants containing these combined substitutions. Biochemical enzymatic assays of engineered versions of EfAmy revealed that the combination of mutations at the surface loops increased the thermostability and catalytic efficiency of the enzyme. The possible mechanisms responsible for the changes in the biochemical properties are discussed by analyzing the three-dimensional structural model. IMPORTANCE Cold-adapted enzymes have high specific activity at low and moderate temperatures, a property that can be extremely useful in various applications as it implies a reduction in energy consumption during the catalyzed reaction. However, the concurrent high thermolability of cold-adapted enzymes often limits their applications in industrial processes. The α-amylase from the psychrophilic Antarctic ciliate Euplotes focardii (named EfAmy) is a cold-adapted enzyme with optimal catalytic activity in an alkaline environment. These unique features distinguish it from most α-amylases characterized so far. In this work, we engineered a novel EfAmy with improved thermostability, substrate binding affinity, and catalytic efficiency to various extents, without impacting its pH preference. These characteristics can be considered important properties for use in the food, detergent, and textile industries and in other industrial applications. The enzyme engineering strategy developed in this study may also provide useful knowledge for future optimization of molecules to be used in particular industrial applications.

Sentence-Based Experience Logging in New Hearing Aid Users

2020 ◽  
Vol 29 (3S) ◽  
pp. 631-637
Author(s):  
Katja Lund ◽  
Rodrigo Ordoñez ◽  
Jens Bo Nielsen ◽  
Dorte Hammershøi
Keyword(s):  
Hearing Aid ◽  
Patient Experiences ◽  
Online Tool ◽  
Rehabilitation Process ◽  
Hearing Rehabilitation ◽  
Clinical Observations ◽  
Hearing Aid Use ◽  
Wide Range ◽  
Insight Into ◽  
Shed Light

Purpose The aim of this study was to develop a tool to gain insight into the daily experiences of new hearing aid users and to shed light on aspects of aided performance that may not be unveiled through standard questionnaires. Method The tool is developed based on clinical observations, patient experiences, expert involvement, and existing validated hearing rehabilitation questionnaires. Results An online tool for collecting data related to hearing aid use was developed. The tool is based on 453 prefabricated sentences representing experiences within 13 categories related to hearing aid use. Conclusions The tool has the potential to reflect a wide range of individual experiences with hearing aid use, including auditory and nonauditory aspects. These experiences may hold important knowledge for both the patient and the professional in the hearing rehabilitation process.

Substituent Effects on the Thermal Decomposition of Phosphate Esters on Ferrous Surfaces

2019 ◽  
Author(s):  
James Ewen ◽  
Carlos Ayestaran Latorre ◽  
Arash Khajeh ◽  
Joshua Moore ◽  
Joseph Remias ◽  
...  
Keyword(s):  
Thermal Decomposition ◽  
Film Formation ◽  
Substituent Effects ◽  
Vapour Phase ◽  
Industrial Applications ◽  
Phosphate Esters ◽  
Antiwear Additives ◽  
Formation Mechanisms ◽  
Phosphate Ester ◽  
Wide Range

<p>Phosphate esters have a wide range of industrial applications, for example in tribology where they are used as vapour phase lubricants and antiwear additives. To rationally design phosphate esters with improved tribological performance, an atomic-level understanding of their film formation mechanisms is required. One important aspect is the thermal decomposition of phosphate esters on steel surfaces, since this initiates film formation. In this study, ReaxFF molecular dynamics simulations are used to study the thermal decomposition of phosphate esters with different substituents on several ferrous surfaces. On Fe<sub>3</sub>O<sub>4</sub>(001) and α-Fe(110), chemisorption interactions between the phosphate esters and the surfaces occur even at room temperature, and the number of molecule-surface bonds increases as the temperature is increased from 300 to 1000 K. Conversely, on hydroxylated, amorphous Fe<sub>3</sub>O<sub>4</sub>, most of the molecules are physisorbed, even at high temperature. Thermal decomposition rates were much higher on Fe<sub>3</sub>O<sub>4</sub>(001) and particularly α-Fe(110) compared to hydroxylated, amorphous Fe<sub>3</sub>O<sub>4</sub>. This suggests that water passivates ferrous surfaces and inhibits phosphate ester chemisorption, decomposition, and ultimately film formation. On Fe<sub>3</sub>O<sub>4</sub>(001), thermal decomposition proceeds mainly through C-O cleavage (to form surface alkyl and aryl groups) and C-H cleavage (to form surface hydroxyls). The onset temperature for C-O cleavage on Fe<sub>3</sub>O<sub>4</sub>(001) increases in the order: tertiary alkyl < secondary alkyl < primary linear alkyl ≈ primary branched alkyl < aryl. This order is in agreement with experimental observations for the thermal stability of antiwear additives with similar substituents. The results highlight surface and substituent effects on the thermal decomposition of phosphate esters which should be helpful for the design of new molecules with improved performance.</p>

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