Optimal Enzyme Activity Research Articles

Lipase and Water in a Deep Eutectic Solvent: Molecular Dynamics and Experimental Studies of the Effects of Water-In-Deep Eutectic Solvents on Lipase Stability

Given the accumulated evidence on the effects of water-in-deep eutectic solvents (DESs) on the solvent nanostructure and the yield of lipase reactions, here we have used molecular dynamics (MD) simulations to delineate the structure and dynamics of thermoalkalophilic lipases in choline chloride/urea-based DES (reline) with varying hydration levels. Results indicated that pure reline almost froze the lipase backbone, while hydrated reline that showed a less ordered nanostructure than the pure form introduced some fluctuations to lipase structures, particularly to the lid domain. Although none of the solvents led to unfolding, solvation by 8 M urea or water when accompanied with elevated temperature caused the most significant loss of secondary structure. Experimental results indicated that lipase incubation in slightly hydrated reline [5% (v/v)] led to the highest level of residual activity, implying interfacial activation. Overall, we report that slightly hydrated reline activates thermoalkalophilic lipases, consistent with the particular MD observation showing enhanced mobility of the lid domain. These insights provided by this study contribute to designing efficient lipase applications in DES-based reaction media, giving cues for customizing water-in-DESs for optimal enzyme stability and activity.

Adding value in barley malt rootlets as a source of 5’-phosphodiesterase

A thermostable 5’-phosphodiesterase (5’-PDE, EC 3.1.4.1) was extracted from barley (Hordeum distichum var. Rex) malt rootlets. The purification procedure comprised acetone precipitation, S-Sepharose cation-exchange and DEAE-Sepharose anion-exchange chromatography. The enzyme was purified 101-fold with a recovery of 22% and a specific activity of 81.9 U mg-1 protein, Optimum enzyme activity was obtained at 70 °C, and pH 8.9. The SDS-PAGE profiling of the purified protein exhibited molecular weight of 116 kDa and revealed three sub-unit fractions of 26, 43, and 56 kDa making up its active configuration. The kinetic constants Km and Vmax were determined as 0.25 mM and 0.816 mmol min-1, respectively. Thermodynamic studies showed that the thermal inactivation of purified barley malt rootlets 5’-PDE followed the first-order kinetics, indicating inactivation energy (Ed) of 134 kJ mol-1. The half-life (t1/2) at 70 °C was estimated as 169 min. Thermodynamic parameters ΔH*, ΔS* and ΔG* were determined as a function of temperature and were 131.15 kJ mol-1, 37.01 kJ mol-1 K-1 and 118.4 kJ mol-1, respectively. The purified enzyme has long half-life with 11 days at 0 °C, 37 hours at 4 °C and 11 hours at room temperature. These results provide useful information about the factors that affects the activity of barley malt rootlets 5’-PDE and suggests a good indication for application of this enzyme in pharmaceutical and food industry.

Tailor-Made Fluorinated Ionic Liquids for Protein Delivery.

Nowadays, pharmaceutical companies are facing several challenges with the development and approval of new biological products. The unique properties of several fluorinated ionic liquids (FILs), such as their high surfactant power in aqueous solutions, their chemical and biological stability, and low toxicity, favor their application in the pharmaceutical industry. Furthermore, the numerous combinations between cations and anions, in the FILs design, enlarge the possibilities to construct a successful delivery system. Several FILs also proved to not affect the activity, stability, and secondary structure of the therapeutic protein lysozyme. This work aims to study the aggregation behavior of distinct FILs in the protein suitable medium, in the presence or absence of lysozyme. Besides, different incubation conditions were tested to guarantee the optimal enzymatic activity of the protein at more stable delivery systems. Following the optimization of the incubation conditions, the quantification of the encapsulated lysozyme was performed to evaluate the encapsulation efficiency of each FIL-based system. The release of the protein was tested applying variables such as time, temperature, and ultrasound frequency. The experimental results suggest that the aggregation behavior of FILs is not significantly influenced by the protein and/or protein buffer and supports their application for the design of delivery systems with high encapsulation efficiencies, maintaining the biological activity of either encapsulated and released protein.

Characteristics of Raw-Starch Degrading Amylase Bacteria from Natar Hot Spring Lampung

Indonesia has a diversity of hot spring as a habitat of bacteria. One of the hot springs is Natar hot spring, Lampung. This study is to report the characteristics of a bacterium called Nat1 isolate that produces amylase to degrade raw starch from Natar hot spring. Water samples were taken from hot springs with a temperature of 45°C and a pH of 7.0. Nat1 was isolated by screening on the medium of Starch-Luria Bertani at 37°C. Its amylase-producing bacteria showed an optimum amylolytic activity of a crude enzyme of Nat1 isolate in soluble starch was 267.2774 U/mL at 60°C. Genotypic identification results using the 16S rRNA gene showed that the Nat1 isolate is identified as Panninobacter phragmatetus. A crude enzyme of Nat1 isolate showed a novel amylase ability and could degrade the raw starch substrates, such as corn and sago, with the amount of reducing sugar for each raw starch, 37.0688 µmol/mg, and 24.2697 µmol/mg. In conclusion, Nat1 amylase is potentially used in industry for its ability to degrade raw starch directly.

A lytic polysaccharide monooxygenase from Myceliophthora thermophila C1 and its characterization in cleavage of glycosidic chain of cellulose

Lytic polysaccharide monooxygenase (LPMO), a kind of copper-dependent oxidases, has been widely used for the efficient enzymatic hydrolysis of lignocellulosic biomass. In this study, a gene encoding the LPMO from Myceliophthora thermophila C1 (MtC1LPMO) was expressed in Pichia pastoris GS115. A high secretion level of recombinant MtC1LPMO protein was obtained in the culture medium using its native signal peptide. Consequently, MtC1LPMO with a purity of more than 95 % and yield of 46 mg/L was obtained. The optimal enzyme activity was observed at pH 7.5 and 85 °C, and the enzyme displayed remarkable thermostability at 40–60 °C. MtC1LPMO exhibited a synergistic activity with cellulase in the degradation of microcrystalline cellulose, and the reducing sugar content increased by 29 % compared to cellulase alone. Additionally, the results of scanning electron microscopy revealed that MtC1LPMO would cleave the glycosidic chain, leading to subsequent disruption to the surface of microcrystalline cellulose. Finally, the interaction model of MtC1LPMO and cellohexaose was explored using molecular docking simulations, which showed that the residues H1, H68, Y153, H142, Y67, V143, S146, G149, Q151 and P69 of MtC1LPMO played an important role in interacting with cellohexaose.

Molecular, physiological, and biochemical characterization of extracellular lipase production by Aspergillus niger using submerged fermentation.

BackgroundExtracellular production of fungal lipases especially the lipases obtained from the Aspergilli has gained immense interest in recent years due to its diverse biotechnological applications. In this study, we focused on determining the fermentation parameters required for the optimal lipase production.MethodsA total of 256 fungal isolates were obtained from oil seeds. From each genus, one isolate was selected to evaluate lipase production using phenol red and tributyrin plate assays. Lipase activity was estimated using the spectrophotometric pNPP hydrolysis assay. The highest lipase producer isolates were identified using 18S ribosomal RNA gene sequencing. The genetic variability was determined by random amplified polymorphic DNA (RAPD) analysis and the dendrogram was constructed using the unweighted pair group method with arithmetic averages method. The isolates were examined in a submerged fermentation culture (Smf) to measure the effect of temperature, pH, incubation time, carbon source, nitrogen source, inoculum volume, and lipid source on lipase production.ResultsEleven isolates belonging to the genus Aspergillus were analyzed for lipase production where they were found to be the highest lipase producers among various fungal genera. All the tested isolates were identified as A. niger using 18s rRNA sequencing. Genetic diversity was evaluated among all of the studied A. niger isolates using RAPD primers. The RAPD primers were used to amplify 285 loci, of which five were polymorphic (1.75%) and seven were monomorphic (2.45%). Thus, a high level of genetic diversity was observed among all isolates. The tributyrin test and the lipase activity assay identified five strains of A. niger as high lipase producers, and their optimal enzyme activities were 709.74, 532.54, 735.64, 794.62, and 787.69 U/ml. The optimal conditions for lipase production were as follows: 40 °C, pH 7.5, 1% fructose as the carbon source, 1% yeast extract as the nitrogen source, 2% palm oil, 2.5 × 107 spores/ml suspension, and 3 days of incubation.ConclusionsThe current study provides a comprehensive characterization of the optimal conditions, which are essential to enhance lipase production in five A. niger isolates.

Sustainable production, biochemical and molecular characterization of thermo-and-solvent stable alkaline serine keratinase from novel Bacillus pumilus AR57 for promising poultry solid waste management

The increasing amount of recalcitrant keratinous wastes generated from the poultry industry poses a serious threat to the environment. Keratinase have gained much attention to convert these wastes into valuable products. Ever since primitive feathers first appeared on dinosaurs, microorganisms have evolved to degrade this most recalcitrant keratin. In this study, we identified a promising keratinolytic bacterial strain for bioconversion of poultry solid wastes. A true keratinolytic bacterium was isolated from the slaughterhouse soil and was identified and designated as Bacillus pumilus AR57 by 16S rRNA sequencing. For enhanced keratinase production and rapid keratin degradation, the media components and substrate concentration were optimized through shake flask culture. White chicken feather (1% w/v) was found to be the good substrate concentration for high keratinase production when supplemented with simple medium ingredients. The biochemical characterization reveals astounding results which makes the B. pumilus AR57 keratinase as a novel and unique protease. Optimum activity of the crude enzyme was exhibited at pH 9 and 45 °C. The crude extracellular keratinase was characterized as thermo-and-solvent (DMSO) stable serine keratinase. Bacillus pumilus AR57 showed complete degradation (100%) of white chicken feather (1% w/v) within 18 h when incubated in modified minimal medium supplemented with DMSO (1% v/v) at 150 rpm at 37 °C. Keratinase from modified minimal medium supplemented with DMSO exhibits a half-life of 4 days. Whereas, keratinase from the modified minimal medium fortified with white chicken feather (1% w/v) was stable for 3 h only. Feather meal produced by B. pumilus AR57 was found to be rich in essential amino acids. Hence, we proposed B. pumilus AR57 as a potential candidate for the future application in eco-friendly bioconversion of poultry waste and the keratinase could play a pivotal role in the detergent industry. While feather meal may serve as an alternative to produce animal feed and biofertilizers.

Cloning, characterization, and enzymatic identification of a new tryptophan decarboxylase from Ophiorrhiza pumila

Tryptophan decarboxylase (TDC, EC 4.1.1.28) catalyzes tryptophan decarboxylation to form tryptamine through the cofactor pyridoxal-5'-phosphate (PLP), a crucial stage in the production of the terpenoid indole alkaloids like camptothecin (CPT). A new gene encoding TDC was identified from the CPT-producing plant Ophiorrhiza pumila by transcriptome analysis, termed OpTDC2. It contained a 1,536bp open reading frame that encodes a 511 amino acid protein with a molecular mass of 57.01kDa and an isoelectric point of 6.39. Multiple sequence alignment and phylogenetic tree analysis showed the closest similarity (85%) with the TDC from Mitragyna speciosa. Moreover, the highest expression of OpTDC2 was observed in the O. pumila root. To achieve high-efficiency expression of OpTDC2 in Escherichia coli, we fused the TF tag onto the N-terminal of the OpTDC2. Optimum enzymatic activity was observed at 45°C, pH 8 and cofactor concentration of 0.1mM. The catalytic reaction was strongly inhibited by metal ions of Cu2+ , Zn2+ , and Fe2+ . The l-tryptophan was particularly catalyzed compared with d-tryptophan. Besides, the Km and kcat of the OpTDC2 were 1.08mM and 0.78Sec-1 , respectively. The results provided information on new functional OpTDC2 that might be used in synthetic biology for the enhanced biosynthesis of CPT in O. pumila.

Molecular cloning, mRNA expression and functional characterization of a catalase from Chinese black sleeper (Bostrychus sinensis)

In this study, the catalase gene of Chinese black sleeper Bostrychus sinensis (termed as BsCat) was sequenced and characterized. The BsCat, which encodes 525 amino acids, contains a catalase proximal active site signature domain (64FDRERIPERVVHAKGAG80) and a catalase proximal heme-ligand signature domain (354RLFAYPDTH362). The BsCat exhibits high sequence similarity with Cat of other species. Phylogenetic tree reconstruction revealed a close evolutionary relationship of BsCat to catalase genes of other fishes. The results of Real-time PCR showed that the BsCat gene was constitutively expressed in most organs of B. sinensis, with predominant expression detected in liver, followed by peripheral blood and spleen. Moreover, the BsCat gene was significantly changed after either poly (I:C) stimulation or Vibrio parahemolyticus infection in peripheral blood, head kidney, liver and spleen. The enzymatic activity of purified recombinant BsCat (rBsCat) was 2261 ± 96 U/mg. The rBsCat exhibits optimum enzymatic activity at 15 °C and pH 7.0. Our results suggested that the BsCat is involved in the antioxidant defense and host immune response of Chinese black sleeper during pathogen invasion.

A novel thermostable aspartic protease from Talaromyces leycettanus and its specific autocatalytic activation through an intermediate transition state.

Aspartic proteases exhibit optimum enzyme activity under acidic conditions and have been extensively used in food, fermentation, and leather industries. In this study, a novel aspartic protease precursor (proTlAPA1) from Talaromyces leycettanus was identified and successfully expressed in Pichia pastoris. Subsequently, the auto-activation processing of the zymogen proTlAPA1 was studied by SDS-PAGE and N-terminal sequencing, under different processing conditions. TlAPA1 shared the highest identity of 70.3% with the aspartic endopeptidase from Byssochlamys spectabilis (GAD91729) and was classified into a new subgroup of the aspartic protease A1 family, based on evolutionary analysis. Mature TlAPA1 protein displayed an optimal activity at 60°C and remained stable at temperatures of 55°C and below, indicating the thermostable nature of TlAPA1 aspartic protease. During the auto-activation processing of proTlAPA1, a 45-kDa intermediate was identified that divided the processing mechanism into two steps: formation of intermediates and activation of the mature protein (TlAPA1). The former step can be processed without proteolytic activity, while the latter process depended on protease activity completely. The discovery of the novel aspartic protease TlAPA1 and the study of its activation process will contribute to a better understanding of the mechanism of aspartic protease auto-activation.

Characterization and synthetic biology of lipase from Bacillus amyloliquefaciens strain.

Lipases with high tolerance to temperature play a significant role in industry from food manufacturing to waste management systems. Thus, there is a need to investigate these enzymes from different geographical areas to look out for a more thermo-stable one. Characterization of lipases through experimental approaches is time consuming process and sometimes the results are ambiguous due to errors. However, integration of computational technologies is quite useful for prediction of optimized conditions. Such technologies can be applied as synthetic biology, which has many major applications in engineered biological approaches for accurate prediction of effects of different physical and chemical parameters on the system. In this study, cloning and expression of a lipase gene from Bacillus amyloliquefaciens, isolated from a novel geographical region of Pakistan, in Escherichia coli DH5α cells followed by sequencing was carried out. To isolate thermostable lipase producing strains, all the samples were kept at 50 °C. Genomic DNA was isolated and signal peptide (1-32 residues) sequence was chopped (ΔSPLipase). The ΔSPLipase was amplified and expressed in Linearized p15TV-L vector. The purified lipase appeared as single band of approximately 26kDa. Suitable conditions of factors required for maximum lipase activity such as temperature, pH, substrate, organic solvent, detergents and metal ions were predicted through synthetic biology approach and further confirmed in wet lab. The predicted suitable factors for enzyme were almost similar to those determined experimentally. The optimum enzyme activity was recorded at pH 8 and 50 °C temperature. Interestingly, the activity of enzyme was found on a number of solvents, metal ions, detergents, and surfactants. The predicted optimum values and their experimental confirmations highlights the importance of integrated synthetic biology approaches in wet lab experiments. The characterized lipase of B. amyloliquefaciens at molecular level from Pakistani strains displayed good activity on a range of factors that implies this strain to be used for application in industrial level production.

Physicochemical Characteristics of Fungal Xylanases and their Potential for Biobleaching of Kraft and Non-wood Pulps

Xylanases are enzymes with a wide variety of biotechnological applications, such as in the bioconversion of lignocellulosic materials, improvement of feed digestibility, and bleaching kraft pulps to increase pulp brightness. Many studies have been conducted and published over the years on cellulose pulp bleaching due to the need to search for more sustainable tools and thus reduce environmental pollution. Thus, in this review, we focus on analyzing the biochemical properties of xylanases produced by mesophilic and thermophilic fungi that have been used in the bleaching processes of kraft and non-wood pulps. Eucalyptus kraft pulp is still one of the most used raw materials in the production of pulp and paper, while straw and bagasse are alternative sources of non-wood pulps. Thermophilic fungal xylanases show optimum enzymatic activity at high temperatures and a shorter treatment period when compared to mesophilic xylanases in the bleaching step. However, mesophilic fungal xylanases exhibit a longer period of enzymatic treatment but achieve a satisfactory brightness and kappa number. Hence, these approaches will contribute to future applications of these xylanolytic enzymes in pulp and paper industries.

Schistosoma mansoni glyceraldehyde-3-phosphate dehydrogenase enhances formation of the blood-clot lysis protein plasmin.

ABSTRACTSchistosomes are intravascular blood flukes that cause the parasitic disease schistosomiasis. In agreement with Schistosoma mansoni (Sm) proteomic analysis, we show here that the normally intracellular glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is also found at the parasite surface; live worms from all intravascular life stages display GAPDH activity. Suppressing GAPDH gene expression using RNA interference significantly lowers this live worm surface activity. Medium in which the worms are cultured overnight displays essentially no activity, showing that the enzyme is not shed or excreted but remains associated with the worm surface. Immunolocalization experiments confirm that the enzyme is highly expressed in the parasite tegument (skin). Surface activity in schistosomula amounts to ∼8% of that displayed by equivalent parasite lysates. To address the functional role of SmGAPDH, we purified the protein following its expression in Escherichia coli strain DS113. The recombinant protein displays optimal enzymatic activity at pH 9.2, shows robust activity at the temperature of the parasite's hosts, and has a Michaelis–Menten constant for glyceraldehyde-3-phosphate (GAP) of 1.4 mM±0.24. We show that recombinant SmGAPDH binds plasminogen (PLMG) and promotes PLMG conversion to its active form (plasmin) in a dose response in the presence of tissue plasminogen activator. Since plasmin is a key mediator of thrombolysis, our results support the hypothesis that SmGAPDH, a host-interactive tegumental protein that can enhance PLMG activation, could help degrade blood clots around the worms in the vascular microenvironment and thus promote parasite survival in vivo.This article has an associated First Person interview with the first author of the paper.

Reliability of <i>in silico</i> Modeling Based on Restriction Patterns for the Study of the Carotenogenic Gene <i>pds</i> of <i>Haematococcus</i> sp

Phytoeno desaturase (pds gene) is an enzyme involved in the biosynthesis of carotenoids such as β-carotene and astaxanthin in microalgae and some autotrophic organisms. Carotenoids have antioxidant, anti-inflammatory and anti-carcinogenic properties. Prior to biotechnological studies it is necessary to carry out the integral analysis of this carotenogenic gene and its molecules for future industrial applications. In silico modeling is an important bioinformatics tool that allows to adjust, summarize and organize experimental information, test theories and generally understand the interaction of molecules and biological systems. In this work, specific primers were used to amplify the phytoene desaturase (pds) gene of Haematococcus sp. (PM015). NEBcutter V2.0 and BioEdit provided the simulation design of the pds restriction patterns together with the restriction enzymes FsPI, AlwI, HincII, BlpI and MluCI. The amplification product resulted in two fragments one of 1200 bp and one of 1700 bp. The in silico design was reproduced and tested experimentally, in this way the identification of the pds gene of Haematococcus sp. (PM015) and the effectiveness of bioinformatics programs as an important alternative for in silico modeling of molecules and/or biological systems, which mapping predict reliable results, saving time and reducing costs in the experimental stage, in addition to allowing the selection of restriction enzymes with optimal enzymatic activity to digest DNA.

Cathepsin characterization from crude extract of yellow pike (Congresox talabon)

Fish is a highly perishable food due to internal and external factors. Cathepsin, a proteolytic enzyme available in animal tissues, affects quality deterioration of fish by hydrolizing muscle proteins. Yellow pike fish have an edible portion that is relatively high but has not been used optimally. The aim of the study was to extract the cathepsin enzyme from yellow pike meat. The method used extraction, characterization, and precipitation of ammonium sulfate (NH4)2SO4 fraction at concentrations of 40-50% to 70-80%. The extraction results showed the crude extract activity of cathepsin from yellow pike fish which was 0.19 U/mL. The optimum activity of the cathepsin enzyme at a pH 5 (0.24 U/mL), temperature of 50°C (0.10 U/mL) and metal ions increased cathepsin activity, one of which was CuCl2 increased cathepsin activity to 0.92 U/mL and FeCl3 increased activity to 1.41 U/mL. The best precipitate at 40-50% ammonium sulfate concentration with specific activity 1.15 U/mg.

Nitrogen source and pH interact and modulate lipase secretion in a non-clinical strain of Candida parapsilosis

Lipases (E.C. 3.1.1.3) are serine-hydrolases, and act on long chain fatty acid ester bonds. They exhibit specific and enantioselective activities, which are desirable for many industrial applications. This study aimed at screening and optimizing the production of lipases by wild yeast strains from a variety of substrates, as well as characterizing the enzyme. An initial selection was made in oxygenated oil-supplemented minimum medium, and the enzymatic activity of the supernatant was tested over p- nitrophenyl palmitate. One-hundred and twenty-four yeast strains from different substrates were tested, and twenty-three showed significantly higher lipolytic activity (p <0.01). One yeast in particular, QU110, showed best lipase production and therefore was selected for the optimization and characterization processes. This yeast exhibits enzyme secretion in initial pH 6.0, with olive oil and tryptone as carbon and nitrogen sources, respectively. There was a strong interaction between nitrogen source and initial pH, and pH 9.0 seems to inhibit enzyme secretion. The crude enzyme (cell-free supernatant) shows stability in surfactants and n-hexane, but not in ethanol or methanol. A Response Surface Model was created and optimal enzyme activity conditions were observed at 36°C and pH 8.0. The lipase is appropriate for transesterification reactions, as the enzyme is more stable in strong apolar solvents than moderately apolar ones. Also, secretion by pH was not reported elsewhere, which should be further investigated and contribute for other yeast bioprocesses as well.

Biodegradation of lignin and the associated degradation pathway by psychrotrophic Arthrobacter sp. C2 from the cold region of China

Degradation of most of the lignocellulose-rich agricultural residue in the cold regions of China is limited due to the cold climate. Lignin is the main component of lignocellulose, and the effective degradation of lignin is one of the most crucial processes in degrading lignocellulose. Psychrotrophic lignin-degrading bacteria and cold adapted ligninolytic enzymes have promising potential for the degradation and transformation of lignin, which are conducive to the resource utilization of lignocelluloses and energy-saving production under cold conditions. In this study, a newly psychrotrophic bacterial strain, Arthrobacter sp. C2, was isolated. The optimal enzyme activity conditions and lignin degradation pathways of C2 were investigated using sodium lignin sulfonate as substrate. The optimal conditions for enzyme activity included an initial pH of 6.74, a temperature of 14.9 °C, an incubation time of 6.87 days, and an inoculum size of 2.24%. Under the optimal conditions, the lignin peroxidase and manganese peroxidase activities and the degradation rate reached 29.8 U/L, 56.4 U/L and 40.1%, respectively. The biodegradation products including acids, phenols, aldehydes and alcohols were analyzed by gas chromatography–mass spectrometry and Fourier transform infrared spectroscopy. Further, the potential degradation pathways were proposed according to the results obtained in this study and those presented in the relevant literature. This study not only provides valuable psychrotrophic strain resources for the sustainable utilization of lignocellulose in cold regions, but also supplies potential application options for energy-saving production of useful chemicals using cold adapted enzymes.

Characterization of recombinant glutathione reductase from Antarctic yeast Rhodotorula mucilaginosa

Glutathione reductase (GR) catalyzes the reduction of glutathione disulfide, which helps to maintain a cellular reducing environment during stress in organisms. However, GR of polar yeast has not been well-characterized so far. To fully understand the molecular and enzymatic properties of GR from extreme area and broaden its knowledge, a cDNA-encoding GR from Antarctic sea-ice yeast Rhodotorula mucilaginosa (designated as RmGR) was cloned and expressed in Escherichia coli. The open reading frame of RmGR was 1500 bp, encoding an enzyme of 499 amino acids with a predicted pI of 6.07 and molecular weight of 54.8 kDa. SDS-PAGE and gel filtration analysis results showed that the RmGR was a homodimer. Conserved sequence analysis revealed that RmGR behaved typical characteristics of GR, containing a pyridine nucleotide-disulfide oxidoreductase active site, a flavin adenine dinucleotide (FAD), and reduced nicotinamide adenine dinucleotide phosphate (NADPH) binding motifs and two glutathione oxidized binding motifs. The recombinant enzyme displayed relatively high stability at pH 3.0–6.0 and 20–40 °C with optimal enzymatic activity at 30 °C and pH 7.5. Real-time quantitative PCR data showed that the expression of RmGR gene was upregulated after copper treatment of yeast cells. Moreover, RmGR-transformed E. coli exhibited stronger growth profiles than cells with an empty vector after copper and cadmium treatment. Our results demonstrated the possible function of RmGR in adaptation to heavy metals and its potential application in heterologous expression systems.

Using design of experiment to optimize enzyme activity assays

This laboratory exercise teaches students the application of design of experiments (DOE) for optimizing a trypsin activity assay using the artificial substrate Nα-benzoyl-l-arginine-p-nitroanilide (BAPNA). The response surface modeling (RSM) approach is exploited to design and evaluate the combined effects of relevant factors on the inhibition of trypsin by the competitive inhibitor benzamidine. RSM matrices and statistics are generated using commercial software. Experimental data are compared with Michaelis–Menten theory. The predicted values are in good agreement with the experimental data. The comprehensive analysis of data provides deep insight into the relationship between substrate, inhibitor concentration, and assay sensitivity in the context of high-throughput drug screening. Data analysis reveals also unexpected but explainable interactions between the substrate and the pH value in the applied enzyme activity assay. The combined design, performance, and analysis of practical experiments demonstrates that DOE is an excellent tool to explore the relationship between several parameters affecting the enzyme activity and to optimize functional enzyme activity assays in a variety of applications.

Karakterisasi α-Amilase Bacillus firmus KH.9.4 Alkalotoleran dari Limbah Cair Tapioka

Alkalotolerant bacteria identified as Bacillus firmus KH.9.4 was isolated from tapioca liquid waste from Kedung Halang, Bogor. The isolate showed optimum a-amylase activity after 15 hours of cultivation on liquid media. Optimum enzyme activity occurred at pH 6.0 and temperature 800C. Upon heat treatment at pH 6.0 and 800C the enzyme was still 100% active after 6 hours incubation without substrate. The enzyme activity was enhanced by supplement 10 mM MgSO4.7H2O. The presence of 1, 5, and 10 mM ethylenediaminetetraacetic acid demonstrated the inhibitory effect to this enzyme. Zymogram analysis revealed one translucent zone corresponding to soluble starch activities after 60 minutes of incubation at 400C in the 0.05 M phoshate buffer pH 7.0. Whereas SDS-PAGE analysis showed that there are 5 bands of proteins.