KM Values Research Articles

A new β-galactosidase from Paenibacillus wynnii with potential for industrial applications

Commercial β-galactosidases exhibit undesirable kinetic properties regarding substrate affinity (Michaelis-Menten constant [KM] for lactose) and product inhibition (inhibitor constant [Ki] for galactose). An in silico screening of gene sequences was done and identified a putative β-galactosidase (Paenibacillus wynnii β-galactosidase, BgaPw) from the psychrophilic bacterium Paenibacillus wynnii. The cultivation of the wild-type P. wynnii strain resulted in very low β-galactosidase activities of a maximum of 150 nkat per liter of medium with o-nitrophenyl-β-d-galactopyranoside (oNPGal) as substrate. The recombinant production of BgaPw in Escherichia coli BL21(DE3) increased the yield ∼9,000-fold. Here, a volumetric activity of 1,350.18 ± 11.82 μkatoNPGal/Lculture was achieved in a bioreactor cultivation. The partly purified BgaPw showed a pH optimum at 7.0, a temperature maximum at 40°C, and an excellent stability at 8°C with a half-life of 77 d. Kinetic studies with BgaPw were done in milk or in milk-imitating synthetic buffer (Novo buffer), respectively. Remarkably, the KM value of BgaPw with lactose was as low as 0.63 ± 0.045 mM in milk. It was found that the resulting products of lactose hydrolysis, namely galactose and glucose, did not inhibit the β-galactosidase activity of BgaPw, but instead showed a striking activating effect in both cases (up to 144%). In a comparison study in milk, lactose was completely hydrolyzed by BgaPw in 72 h at 8°C, whereas 2 other known β-galactosidases were less powerful and converted only about 90% of lactose in the same time. Finally, the formation of galactooligosaccharides (GOS) was demonstrated with the new BgaPw, starting with pharma-lactose (400 g/L). A GOS production of about 144 g/L was achieved after 24 h (36.0% yield).

A complex array of factors regulate the activity of Arabidopsis thaliana δ1 -pyrroline-5-carboxylate synthetase isoenzymes to ensure their specific role in plant cell metabolism.

The first and committed step in proline synthesis from glutamate is catalyzed by δ1 -pyrroline-5-carboxylate synthetase (P5CS). Two P5CS genes have been found in most angiosperms, one constitutively expressed to satisfy proline demand for protein synthesis, the other stress-induced. Despite the number of papers to investigate regulation at the transcriptional level, to date, the properties of the enzymes have been subjected to limited study. The isolation of Arabidopsis thaliana P5CS isoenzymes was achieved through heterologous expression and affinity purification. The two proteins were characterized with respect to kinetic and biochemical properties. AtP5CS2 showed KM values in the micro- to millimolar range, and its activity was inhibited by NADP+ , ADP and proline, and by glutamine and arginine at high levels. Mg2+ ions were required for activity, which was further stimulated by K+ and other cations. AtP5CS1 displayed positive cooperativity with glutamate and was almost insensitive to inhibition by proline. In the presence of physiological, nonsaturating concentrations of glutamate, proline was slightly stimulatory, and glutamine strongly increased the catalytic rate. Data suggest that the activity of AtP5CS isoenzymes is differentially regulated by a complex array of factors including the concentrations of proline, glutamate, glutamine, monovalent cationsand pyridine dinucleotides.

Identification and characterization of a novel type of ketohexokinase from the haloarchaeon Haloferax volcanii.

Ketohexokinase (KHK) catalyzes the ATP-dependent phosphorylation of fructose, forming fructose-1-phosphate and ADP. The enzyme is well studied in Eukarya, in particular in humans and other vertebrates, but homologs have not been identified in Bacteria and Archaea. Here we report the identification of a novel type of KHK from the haloarchaeon Haloferax volcanii (HvKHK). The encoding gene khk was identified as HVO_1812. The gene was expressed as a 90-kDa homodimeric protein, catalyzing the phosphorylation of fructose with a Vmax value of 59 U/mg and apparent KM values for ATP and fructose of 0.47 and 1.29 mM, respectively. Homologs of HvKHK were only identified in a few haloarchaea and halophilic Bacteria. The protein showed low sequence identity to characterized KHKs from Eukarya and phylogenetic analyses indicate that haloarchaeal KHKs are largely separated from eukaryal KHKs. This is the first report of the identification of KHKs in prokaryotes that form a novel cluster of sugar kinases within the ribokinase/pfkB superfamily.

Molecular purification, and kinetic study of the enzyme glutathione peroxidase in patients with cardiovascular disease

This study was conducted to evaluate serum glutathione peroxidase activity and partial purification of the enzyme from the serum of heart attack patients using a precipitant or what is called salting out, ion exchange dialysis using DEAE-Cellulose, and gel filtration using Sephadex G100 gel. This study was conducted on 90 samples, (60) patients whose ages ranged between (35-75) years and control (30) individuals whose ages ranged between (23-55). It showed that the number of purification times was (4.64), and the enzyme yield was (40%). The specific activity was (1.3 U/mg). Likewise, kinetic studies were carried out in which values of Km and Vmax of glutathione peroxidase were (16.66 mM) of Vmax, and (0.909 IU/L) of Km, and the optimal concentration of the base material was (100 mmol/L). The optimal temperature for enzyme action was (37°C), while the optimal pH was (7.2), and the molecular weight of glutathione peroxidase was (23 kDa). The study also studied the effect of carvedilol as an inhibitor of the enzyme glutathione peroxidase, and the results showed a decrease in enzyme activity, and the type of inhibition was uncompetitive.

The alternative coproporphyrinogen III oxidase (CgoN) catalyzes the oxygen-independent conversion of coproporphyrinogen III into coproporphyrin III.

Nature utilizes three distinct pathways to synthesize the essential enzyme cofactor heme. The coproporphyrin III-dependent pathway, predominantly present in Bacillaceae, employs an oxygen-dependent coproporphyrinogen III oxidase (CgoX) that converts coproporphyrinogen III into coproporphyrin III. In this study, we report the bioinformatic-based identification of a gene called ytpQ, encoding a putative oxygen-independent counterpart, which we propose to term CgoN, from Priestia (Bacillus) megaterium. The recombinantly produced, purified, and monomeric YtpQ (CgoN) protein is shown to catalyze the oxygen-independent conversion of coproporphyrinogen III into coproporphyrin III. Minimal non-enzymatic conversion of coproporphyrinogen III was observed under the anaerobic test conditions employed in this study. FAD was identified as a cofactor, and menadione served as an artificial acceptor for the six abstracted electrons, with a KM value of 3.95 μmol/L and a kcat of 0.63 per min for the substrate. The resulting coproporphyrin III, in turn, acts as an effective substrate for the subsequent enzyme of the pathway, the coproporphyrin III ferrochelatase (CpfC). Under aerobic conditions, oxygen directly serves as an electron acceptor, but is replaced by the more efficient action of menadione. An AlphaFold2 model of the enzyme suggests that YtpQ adopts a compact triangular shape consisting of three domains. The N-terminal domain appears to be flexible with respect to the rest of the structure, potentially creating a ligand binding site that opens and closes during the catalytic cycle. A catalytic mechanism similar to the oxygen-independent protoporphyrinogen IX oxidase PgoH1 (HemG), based on the flavin-dependent abstraction of six electrons from coproporphyrinogen III and their potential quinone-dependent transfer to a membrane-localized electron transport chain, is proposed.

Immobilization and biochemical characterization of purified L-Asparaginase produced by Brevibacillus borstelensis ML12

Purified L-Asparaginase from Brevibacillus borstelensis ML12 was immobilized, and sequentially studied for biochemical characterization and thermodynamic feasibility. Immobilization has long been used as a way to enhance enzyme stability, facilitate recovery, and multiple re-uses in industries. It is preferable to utilize natural nontoxic carriers and crosslinking agents to immobilize enzymes. L-Asparaginase is an important industrial enzyme due to its significant role in the pharmaceutical and food sectors. In this study, immobilization of purified L-Asparaginase from a novel bacterium Brevibacillus borstelensis ML12 was performed using different carrier matrices and concentration of the suitable matrix was varied to obtain maximum enzyme production. Biochemical parameters like effect of pH, temperature, surfactants, organic reagents, bile salt were also studied. Arrhenius plot and thermal stability of immobilized enzyme were obtained from thermodynamic studies. ΔH, ΔG and ΔS values were also determined. Operational and storage stability studies revealed that the enzyme was able to retain 80% of its activity after 3 cycles and relative activity falls by 45% after 48 hours under refrigerated conditions and by 50% at normal room temperature. Vmax and KM values were calculated by using Lineweaver-Burk plot and the values came out to be 121.654 µmole ml-1 min-1 and 0.310 mM respectively.

Applicability of the sigmoid model to estimate heavy metal uptake in maize and sorghum as affected by organic acids.

Although assisted phytoremediation using chemical treatments is a suitable technique for the removal of heavy metals (HMs), the estimation of this process using simple models is also crucial. For this purpose, a greenhouse trial was designed to evaluate the effectiveness of citric, oxalic, and tartaric acid on Cd, Pb, Ni, and Zn phytoremediation by maize and sorghum and to estimate this process using sigmoid HMs uptake model. Results showed that mean values of root and shoot dry weight and metals uptake, translocation factor (TF) of Pb and Zn, and uptake efficiency (UE) of Cd in maize were higher than sorghum but the TF of Cd and the phytoextraction efficiency (PEE) and UE of Pb in sorghum were higher than maize. Citric, oxalic, and tartaric acid significantly increased the UE of Pb by 17.7%, 22.5%, and 32.5%, respectively. Tartaric acid significantly increased the mean values of shoot dry weight, shoot Cd, Pb, and Ni uptake, and PEE of Pb and Ni, but decreased TF of Zn. The R2, NRMSE, and KM values indicated the ability of sigmoid HM uptake model in estimating HMs uptake in maize and sorghum treated with organic acids. Thus, tartaric acid was more effective than citric and oxalic acids to enhance phytoremediation potential. Sigmoid HM uptake model is suitable to estimate the HMs uptake in plants treated with organic acids at different growth stages.

A revised model of energy transactions and body composition in sheep.

A mechanistic, dynamic model was developed to calculate body composition in growing lambs by calculating heat production (HP) internally from energy transactions within the body. The model has a fat pool (f) and three protein pools: visceral (v), nonvisceral (m), and wool (w). Heat production is calculated as the sum of fasting heat production, heat of product formation (HrE), and heat associated with feeding (HAF). Fasting heat production is represented as a function of visceral and nonvisceral protein mass. Heat associated with feeding (HAF) is calculated as ((1 - km) x MEI), where km is partial efficiency of ME use for maintenance, and MEI = metabolizable energy intake) applies at all levels above and below maintenance. The value of km derived from data where lambs were fed above maintenance was 0.7. Protein change (dp/dt) is the sum of change in the m, v, and w pools, and change in fat is equal to net energy available for gain minus dp/dt. Heat associated with a change in body composition (HrE) is calculated from the change in protein and fat with estimated partial efficiencies of energy use of 0.4 and 0.7 for protein and fat, respectively. The model allows for individuals to gain protein while losing fat or vice versa. When evaluated with independent data, the model performed better than the current Australian feeding standards (Freer etal., 2007) for predicting protein gain in the empty body but did not perform as well as for gain of fat and fleece-free empty body weight. Models performed similarly for predicting clean wool growth. By explicit representation of the major energy using processes in the body, and through simplification of the way body composition is computed in growing animals, the model is more transparent than current feeding systems while achieving similar performance. An advantage of this approach is that the model has the potential for wider applicability across different growth trajectories and can explicitly account for the effects of systematic changes on energy transactions, such as the effects of selective breeding, growth manipulation, or environmental changes.

Inhibition effect of Gly-Arg-Gly-Asp-Ser (GRGDS) and Arg-Gly-Asp (RGD) Bioactive Peptides on Angiotensin-Converting Enzyme Activity Purified from Human Serum

Angiotensin-converting enzyme (ACE, EC 3.4.15.1) is a physiological target for researching new antihypertensive drugs, as it is a substantial enzyme in the regulation of blood pressure. Herein, ACE was purified from human serum with affinity chromatography. Vmax and KM values were found as 60.98 (µmol/min)/mL and 0.34 mM, respectively. The effects of Gly-Arg-Gly-Asp-Ser (GRGDS) and Arg-Gly-Asp (RGD) bioactive peptides on purified ACE were researched. Also, captopril, a specific ACE inhibitory, was used as a reference inhibitor. Bioactive peptides, GRGDS and RGD, demonstrated the inhibitory effect on purified ACE with IC50 values of 46.39 µM and 456.46 µM, respectively. Ki values and kind of inhibition for GRGDS and RGD by the Lineweaver-Burk chart were found. The kind of inhibitory for these bioactive peptides was calculated as reversible-competitive inhibitory. Ki values for GRGDS and RGD were obtained as 93.28 µM and 435.67 µM, respectively. The IC50 value of captopril was designated as 1.57 nM. The inhibitory kind of captopril was designated as reversible non-competitive inhibitory and the Ki value was 0.99 nM. In this study, it was concluded that RGD and GRGDS bioactive peptides have the potential to be utilized as ACE inhibitors.

Comprehensive evaluation of recombinant lactate dehydrogenase production from inclusion bodies

A broad application spectrum ranging from clinical diagnostics to biosensors in a variety of sectors, makes the enzyme Lactate dehydrogenase (LDH) highly interesting for recombinant protein production. Expression of recombinant LDH is currently mainly carried out in uncontrolled shake-flask cultivations leading to protein that is mostly produced in its soluble form, however in rather low yields. Inclusion body (IB) processes have gathered a lot of attention due to several benefits like increased space-time yields and high purity of the target product. Thus, to investigate the suitability of this processing strategy for ldhL1 production, a fed-batch fermentation steering the production of IBs rather than soluble product formation was developed. It was shown that the space-time-yield of the fermentation could be increased almost 3-fold by increasing qs to 0.25 g g−1 h−1 which corresponds to 21% of qs,max, and keeping the temperature at 37°C after induction. Solubilization and refolding unit operations were developed to regain full bioactivity of the ldhL1. The systematic approach in screening for solubilization and refolding conditions revealed buffer compositions and processing strategies that ultimately resulted in 50% product recovery in the refolding step, revealing major optimization potential in the downstream processing chain. The recovered ldhL1 showed an optimal activity at pH 5.5 and 30∘C with a high catalytic activity and KM values of 0.46 mM and 0.18 mM for pyruvate and NADH, respectively. These features, show that the here produced LDH is a valuable source for various commercial applications, especially considering low pH-environments.

A Dark Siren Measurement of the Hubble Constant with the LIGO/Virgo Gravitational Wave Event GW190412 and DESI Galaxies

We present a measurement of the Hubble Constant H 0 using the gravitational wave event GW190412, an asymmetric binary black hole merger detected by LIGO/Virgo, as a dark standard siren. This event does not have an electromagnetic counterpart, so we use the statistical standard siren method and marginalize over potential host galaxies from the Dark Energy Spectroscopic Instrument (DESI) survey. GW190412 is well-localized to 12 deg2 (90% credible interval), so it is promising for a dark siren analysis. The dark siren value for km s−1 Mpc−1, with a posterior shape that is consistent with redshift overdensities. When combined with the bright standard siren measurement from GW170817 we recover km s−1 Mpc−1, consistent with both early and late-time Universe measurements of H 0. This work represents the first standard siren analysis performed with DESI data, and includes the most complete spectroscopic sample used in a dark siren analysis to date.

Enzyme-triggered approach to reduce water bodies' contamination using peroxidase-immobilized ZnO/SnO2/alginate nanocomposite

Enzyme immobilization on solid support offers advantages over free enzymes by overcoming characteristic limitations. To synthesize new stable and hyperactive nano-biocatalysts (co-precipitation method), ginger peroxidase (GP) was surface immobilized (adsorption) on ZnO/SnO2 and ZnO/SnO2/SA nanocomposite with immobilization efficacy of 94 % and 99 %, respectively. Thereafter, catalytic and biochemical characteristics of free and immobilized GP were investigated by deploying various techniques, i.e., FTIR, PXRD, SEM, and PL. Diffraction peaks emerged at 2θ values of 26°, 33°, 37°, 51°, 31°, 34°, 36°, 56°, indicating the formation of SnO2 and ZnO. The OH stretching of the H2O molecules was attributed to broad peaks between 3200 and 3500 cm−1, whereas ZnO/SnO2 spikes occurred in the 1626–1637 cm−1 range. SnO stretching mode and ZnO terminal vibrational patterns have been verified at corresponding wavelengths of 625 cm−1 and 560 cm−1. Enzyme entrapment onto substrate was verified via interactions between GP and ZnO/SnO2/SA as corroborated by signals beneath 1100 cm−1. GP-immobilized fractions were optimally active at pH 5, 50 °C, and retained maximum activity after storage of 4 weeks at −4 °C. Kinetic parameters were determined by using a Lineweaver-Burk plot and Vmax for free GP, ZnO/SnO2/GP and ZnO/SnO2/SA/GP with guaiacol as a substrate, were found to be 322.58, 49.01 and 11.45 (μM/min) respectively. A decrease in values of Vmax and KM indicates strong adsorption of peroxidase on support and maximum affinity between nano support and enzyme, respectively. For environmental remediation, free ginger peroxidase (GP), ZnO/SnO2/GP and ZnO/SnO2/SA/GP fractions effectively eradicated highly intricate dye. Multiple scavengers had a significant impact on the depletion of the dye. In conclusion, ZnO/SnO2 and ZnO/SnO2/SA nanostructures comprise an ecologically acceptable and intriguing carrier for enzyme immobilization.

Higher precision in initial rates may be achievable: A test of a pseudo-statistical method

There has been a concerted effort at establishing the best method for the measurement of initial rates for various purposes, including the calculation of kinetic parameters, the maximum velocity (Vmax), and the Michaelis-Menten constant (KM). The objectives of this research are: 1) to derive equations without KM for the determination of the Vmax in particular and vice versa; 2) to determine the KM and Vmax with other equations other than the Michaelian equation; and 3) to subject the calculated and extrapolated kinetic parameters to pseudo-statistical remediation where necessary as a test of their viability and usefulness. The study was experimental and theoretical. It is supported by the Bernfeld method of enzyme assay. By graphical means, the Vmax and KM values for galactosidase respectively range between 163 and 185 mM/min and between 2.07 and 2.77 mg/L; the range by calculations is 177 and 214 mM/min and 2.45 and 3.311 mg/L, subject to pseudo-statistical remediation. Overall, the ranges of Vmax and KM values for alpha-amylase from both the graphical method and calculation are, respectively, 1.095 to 1.018 mM/min and 18.15 to 20.554 g/L. The equations for the determination of the KM and Vmax, which are respectively invariant with respect to each other, were rederived. The initial rates must not be characterised by a mixture of conditions that validate different quasi-steady-state assumptions if the true KM and Vmax are of interest. The new pseudo-statistical method for the remediation of error in all measurements, if necessary, is viable, useful, and robust.

Response surface methodology use in construction of polianiline-coated carbon paste electrode-based biosensor: Modification and characterization.

In this study, the effect of amperometric glucose biosensor construction and using conditions on the current response was investigated in detail applying experimental design. Polyaniline (PANI) was synthesized on the carbon paste electrode (CPE) surface using the cyclic voltammetry technique in sodium oxalate (NaOx ) electrolyte medium, and an amperometric biosensor was constructed by immobilizing glucose oxidase (GOD). Biosensor preparation (aniline, GOD and NaOx concentrations, and scan rate) and operating conditions (pH and applied potential) were optimized by Box-Behnken and optimal designs, respectively, via State Ease Design Expert 7.0.1.1 software. ANOVA analyses showed that among the biosensor preparation parameters, the NaOx concentration has the highest effect on the current measured in the presence of glucose, whereas in the optimization of pH and potential parameters applied in current measurement studies, it has been revealed that pH has a very high effect on the measured current. Several compounds, such as MWCNT, two different ionic liquids and two different organic molecules were added to carbon paste, and, among them, 2-cyanoethylpyrrole (CPy) enhanced the efficacy highly, most probably due to its polymerization in the paste and increasing the electron transfer rate of the CPE. Sucrose- and lactose-sensitive biosensors were also constructed by co-immobilizing GOD with invertase (INV) or β-galactosidase, respectively, onto modified CPE, and sensitivities to their substrates were shown by cyclic voltammetry and impedance analysis. CPy modification caused an increase in the current values, and also Imax /KM values increased approximately 11.8, 7.83, and 2.56 times for glucose-, sucrose-, and lactose-sensitive CPEs, respectively.

Defining the molecular architecture, metal dependence, and distribution of metal-dependent class II sulfofructose-1-phosphate aldolases

Sulfoquinovose (SQ, 6-deoxy-6-sulfoglucose) is a sulfosugar that is the anionic head group of plant, algal, and cyanobacterial sulfolipids: sulfoquinovosyl diacylglycerols. SQ is produced within photosynthetic tissues, forms a major terrestrial reservoir of biosulfur, and is an important species within the biogeochemical sulfur cycle. A major pathway for SQ breakdown is the sulfoglycolytic Embden-Meyerhof-Parnas pathway, which involves cleavage of the 6-carbon chain of the intermediate sulfofructose-1-phosphate (SFP) into dihydroxyacetone and sulfolactaldehyde, catalyzed by class I or II SFP aldolases. While the molecular basis of catalysis is understood for class I SFP aldolases, comparatively little is known about class II SFP aldolases. Here, we report the molecular architecture and biochemical basis of catalysis of two metal-dependent class II SFP aldolases from Hafnia paralvei and Yersinia aldovae. 3D X-ray structures of complexes with substrate SFP and product dihydroxyacetone phosphate reveal a dimer-of-dimers (tetrameric) assembly, the sulfonate-binding pocket, two metal-binding sites, and flexible loops that are implicated in catalysis. Both enzymes were metal-dependent and exhibited high KM values for SFP, consistent with their role in a unidirectional nutrient acquisition pathway. Bioinformatic analysis identified a range of sulfoglycolytic Embden-Meyerhof-Parnas gene clusters containing class I/II SFP aldolases. The class I and II SFP aldolases have mututally exclusive occurrence within Actinobacteria and Firmicutes phyla, respectively, while both classes of enzyme occur within Proteobacteria. This work emphasizes the importance of SQ as a nutrient for diverse bacterial phyla and the different chemical strategies they use to harvest carbon from this sulfosugar.

Immobilization of Aspergillus fumigatus α-Amylase via Adsorption onto Bentonite/Chitosan for Stability Enhancement

Stability enhancement attempted in this study demonstrated that significant improvement in the stability of the α-amylase isolated from Aspergillus fumigatus was achieved by immobilizing the enzyme on a bentonite/chitosan hybrid matrix using the adsorption method. Centrifugation was used to isolate the α-amylase, which was then refined using (NH4)2SO4 salt precipitation and dialysis. The purity of the α-amylase improved 19.40 times when compared to that of the crude extract. The optimal temperature for free α-amylase is 50˚C, while the optimum temperature for α-amylase/bentonite/chitosan is 60˚C. The KM value of α-amylase/bentonite/chitosan was 1.69 ± 0.08 mg mL-1 substrate and the Vmax value was 52.32 ± 3.29 µmol mL-1 min-1, whereas for free α-amylase, the KM value of 2.56 ± 0.09 mg mL-1 substrate and the Vmax value of 3.78 ± 0.09 µmol mL-1 min-1 were obtained. The ΔGi value of free α-amylase is 102.68 ± 0.30 kJ mol-1 and the t½ is 21.23 ± 0.23 min, whereas the ΔGi value of α-amylase/bentonite/chitosan is 104.43 ± 0.00 kJ mol-1 and the t½ is 94.29 ± 0.91 min. The higher values of ΔGi and t½demonstrated that α-amylase/bentonite/chitosan has better stability than that of free α-amylase. Another important finding is that α-amylase /bentonite/chitosan was able to retain their activity as high as 47.61 ± 0.53% after six recycles, indicating that the enzyme has the potential to be used in industry. Doi: 10.28991/ESJ-2023-07-05-023 Full Text: PDF

Unraveling the kinetics and pharmacology of human PepT1 using solid supported membrane-based electrophysiology

The human Peptide Transporter 1 (hPepT1) is known for its broad substrate specificity and its ability to transport (pro-)drugs. Here, we present an in-depth comprehensive study of hPepT1 and its interactions with various substrates via solid supported membrane-based electrophysiology (SSME). Using hPepT1-containing vesicles, we could not identify any peptide induced pre-steady-state currents, indicating that the recorded peak currents reflect steady-state transport. Electrogenic co-transport of H+/glycylglycine (GlyGly) was observed across a pH range of 5.0 to 9.0. The pH dependence is described by a bell-shaped activity curve and two pK values. KM and relative Vmax values of various canonical and non-canonical peptide substrates were contextualized with current mechanistic understandings of hPepT1. Finally, specific inhibition was observed for various inhibitors in a high throughput format, and IC50 values are reported. Taken together, these findings contribute to promoting the design and analysis of pharmacologically relevant substances.

Penentuan Tipe Inhibisi Senyawa Analog Kurkumin CA2 terhadap Enzim α-Glukosidase dari Beras Lapuk

Diabetes is a health problem that exists throughout the world, especially in Indonesia. Based on data compiled from the International Diabetes Federation (IDF) in 2017, people with diabetes in Indonesia reached 10.3 million people, and if not handled properly it is suspected that there will be an increase to 21.3 million people in 2030. Curcumin analog compounds can used for the treatment of diabetes by inhibiting the enzyme α-glucosidase. The curcumin analog compound used in this study was synthesized from 4 methoxybenzaldehyde with cyclopentanone which was then called the CA2 compound. The α-glucosidase enzyme used in this study was obtained through extraction of weathered rice with an optimum pH of 4.5 and the enzyme extract fraction used was fraction II which had the highest activity compared to fractions I and III. The results showed that the values of the kinetic parameters Km and Vmax of the extracted α-glucosidase enzyme were 1.53 mM and 0.03 U/mL, respectively. The Km kinetic parameter value changed to 1.66 Mm while the Vmax value did not change when the α-glucosidase enzyme was inhibited by CA2 compound, so that the type of inhibition shown by CA2 compound was competitive.

Identification and characterization of a novel sativene synthase from Fischerella thermalis

Sesquiterpene synthases (TPS) determine the structural diversity of terpenoids, which are species specific. In this study, we report a TPS from Fischerella thermalis (named as FtTPS), recombinantly expressed as a soluble protein in Escherichia coli BL21(DE3) strain. The FtTPS protein could catalyze the conversion of farnesyl pyrophosphate (FPP) to sativene, a kind of tricyclic sesquiterpene. The optimal pH and temperature of FtTPS were 7.5 and 30 °C, respectively. The KM and Vmax values of FtTPS for FPP were 1.846 μM and 0.372 μM/min, respectively. By constructing an engineered E. coli strain carrying the FtTPS and the heterologous mevalonate (MVA) pathway genes, sativene could be detected and its yield reached 24 mg/L after 96 h cultivation. The highest yield of sativene was obtained when E.coli BL21 Star was used as the host with SBMSN medium. These results exhibited the biosynthesis of sativene for the first time.

Bioinspired synthesis of micelle-templated ultrathin silica-layered mesoporous nanoparticles with enhanced mass transfer and stability for biocatalysis

Bioinspired enzyme encapsulation technologies have received increasing attention in sustainable development owing to the enzyme protection from external stressors while mimicking the cellular environment and structure. In this study, we developed a diatom-inspired synthesis of ultrathin silica-layered nanoparticles directed by silica-forming R5 peptide- and carbonic anhydrase (CA)-functionalized micelles. Each CA and R5 peptide was covalently conjugated with N-hydroxysuccinimide (NHS)-ester-modified hydrophilic ends of the triblock copolymer F127 (F127–CA and F127–R5). F127–CA/R5 micelles were prepared by controlling the molar ratio of F127–CA and F127–R5. F127–CA/R5 micelle@silica nanoparticles (SiNPs) were synthesized through R5 peptide-catalyzed silicification of the F127–CA/R5 micelle in a two-phase system. F127–CA/R5 micelle@SiNPs exhibited uniform and monodisperse particles with a size of 17 nm, indicating the formation of a ∼1.5-nm ultrathin and mesoporous silica layer compared with F127–CA/R5 micelle. F127–CA/R5 micelle@SiNPs exhibited almost identical KM and kcat values in CO2 hydration activity compared with the free enzyme. In addition, F127–CA/R5 micelle@SiNPs also showed enhanced stability compared to free ngCA under the operation condition of CO2 capture and sequestration with good storage stability. These results indicated that an ultrathin and mesoporous silica layer on the micelle could protect enzymes from external environments while minimizing limited mass transfer. Thus, our strategy could offer a new direction for enzyme-based green chemistry in practical applications, providing enhanced mass transfer and stability.