Stable Enzyme Research Articles

Construction and stabilization of a peptide-based peroxidase mimic for the colorimetric detection of uric acid and sarcosine

Abstract Peptide assembly provides a new approach for the construction of enzyme-like catalysts. However, low stability is still a big challenge for the peptide-based enzyme mimics because the non-covalently driven structures are often destroyed when exposed to environmental changes. Inspired by the catalytic centre of natural horseradish peroxidase (HRP) and the biological function of dityrosine in structural proteins, in this study, we presented the construction of a stable peptide-based HRP mimic (CCA-YH) through introducing covalent bonds. CCA-YH was obtained through the co-assembly of YYHYY and hemin, followed by a ruthenium-mediated photo-crosslinking reaction. Catalytic activity assay demonstrated that CCA-YH displayed enhanced catalytic ability than the non-covalent co-assemblies (CA-YH). Remarkably, CCA-YH exhibited superior long-term stability than CA-YH and natural HRP under different pH, temperature, ion strength and organic medium environments, as well as a favorable reusability. Finally, CCA-YH was applied to the colorimetric detection of uric acid and sarcosine in human urine, which displayed satisfactory sensitivity, specificity and reproducibility. This is the first report the construction of stable peptide-based enzyme mimic based on covalent co-assembly strategy.

Improvement of Laccase Activity Via Covalent Immobilization over Mesoporous Silica Coated Magnetic Multiwalled Carbon Nanotubes for the Discoloration of Synthetic Dyes.

Due to its environmental friendliness and biodegradable ability, the enzymatic decolorization of azo dyes is the best option. However, the free enzyme suffers from various limitations, including poor stability, no repeatable use, and a high expense, which is the key drawback for its practical use. In this analysis, the laccase enzyme was immobilized in mesoporous silica coated magnetic multiwalled carbon nanotubes (Fe3O4-MWCNTs@SiO2) by a glutaraldehyde cross-linker to create an easily separable and stable enzyme. Fourier transform infrared (FTIR) spectroscopy, scanning electron microscopy (SEM), and energy-dispersive X-ray spectroscopy (EDX) were used to characterize the as-synthesized Fe3O4-MWCNTs@SiO2. Laccase immobilized in Fe3O4-MWCNTs@SiO2 showed a good improvement in temperature, pH, and storage stability. Moreover, the operational stability of the biocatalyst was improved, retaining 87% of its original activity even after 10 cycles of 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) oxidation. The biocatalysts were applied for the decolorization of selected azo dyes without a mediator, and up to 99% of Eriochrome Black T (EBT), 98% of Acid Red 88 (AR 88), and 66% of Reactive Black 5 (RB5) were decolorized. Based on these properties, the biocatalysts can be potentially utilized in various environmental and industrial applications.

Stable and Temporary Enzyme Complexes and Metabolons Involved in Energy and Redox Metabolism.

Significance: Alongside well-characterized permanent multimeric enzymes and multienzyme complexes, relatively unstable transient enzyme-enzyme assemblies, including metabolons, provide an important mechanism for the regulation of energy and redox metabolism. Critical Issues: Despite the fact that enzyme-enzyme assemblies have been proposed for many decades and experimentally analyzed for at least 40 years, there are very few pathways for which unequivocal evidence for the presence of metabolite channeling, the most frequently evoked reason for their formation, has been provided. Further, in contrast to the stronger, permanent interactions for which a deep understanding of the subunit interface exists, the mechanism(s) underlying transient enzyme-enzyme interactions remain poorly studied. Recent Advances: The widespread adoption of proteomic and cell biological approaches to characterize protein-protein interaction is defining an ever-increasing number of enzyme-enzyme assemblies as well as enzyme-protein interactions that likely identify factors which stabilize such complexes. Moreover, the use of microfluidic technologies provided compelling support of a role for substrate-specific chemotaxis in complex assemblies. Future Directions: Embracing current and developing technologies should render the delineation of metabolons from other enzyme-enzyme complexes more facile. In parallel, attempts to confirm that the findings reported in microfluidic systems are, indeed, representative of the cellular situation will be critical to understanding the physiological circumstances requiring and evoking dynamic changes in the levels of the various transient enzyme-enzyme assemblies of the cell. Antioxid. Redox Signal. 35, 788-807.

Efficient production of γ-aminobutyric acid by glutamate decarboxylase immobilized on an amphiphilic organic-inorganic hybrid porous material

A novel organic-inorganic hybrid porous material (KCS-2), containing both lipophilic and hydrophilic nanospaces to mimic a lipid bilayer, was utilized as an immobilization support and reaction accelerator for glutamate decarboxylase (GADβ). Upon evaluation of the adsorption of GADβ on KCS-2, the amount of immobilization was found to be approximately four times higher than that on non-porous silica, and a comparable adsorbability to mesoporous silica was observed. Following γ-aminobutyric acid (GABA) production by the decarboxylation of l-glutamic acid using these immobilized enzymes, the enzymatic activity of the GADβ-KCS-2 composite was found to be significantly higher than that of the free enzyme. In contrast, the activity of the more common GADβ-mesoporous silica composite decreased. Furthermore, the enzymatic activity of the GADβ-KCS-2 composite was superior to those of the un-immobilized free enzyme and the amorphous material itself over a wide temperature range. Thereby, these findings suggest that the amphiphilic nanospace of KCS-2 is suitable as a stable enzyme immobilization field and reaction acceleration field under such conditions. In addition, the durability of the immobilized enzyme was examined in terms of GABA production, with approximately 20% activity retention being observed after 10 cycles using KCS-2. Such durability was not observed for the non-porous silica material due to enzyme desorption.

A pilot study to investigate the utility of NAT2 genotype-guided isoniazid monotherapy regimens in NAT2 slow acetylators.

Isoniazid is a therapeutic agent for the treatment of latent tuberculosis infection. Genetic variants in the N-acetyltransferase 2 (NAT2) are associated with the safety and pharmacokinetics of isoniazid. The study aimed to evaluate the safety and pharmacokinetics of a NAT2 genotype-guided regimen of isoniazid monotherapy. A randomized, open-label, parallel-group and multiple-dosing study was performed in healthy subjects. The subjects received isoniazid for 29 days. The NAT2 slow acetylators (NAT2*5/*5, -*5/*6, -*5/*7, -*6/*6, -*6/*7, -*7/*7) randomly received standard dose (300 mg, standard-treatment group) or reduced dose (200 mg, PGx-treatment group) of isoniazid. Also, all the NAT2 rapid acetylators (NAT2*4/*4) received isoniazid 300 mg (reference group). The safety and pharmacokinetics were evaluated during the study. The PGx-treatment group showed a more stable serum liver enzyme profile and a lower incidence of adverse drug reactions (ADRs) than the standard-treatment group. The emergence rates of ADRs were 12.5, 60 and 33.3% in the reference, standard-treatment and PGx-treatment groups, respectively. The PGx-treatment group showed higher plasma isoniazid concentrations than the reference group, although the PGx-treatment group received a reduced dose of isoniazid. Our results showed that a NAT2 genotype-guided regimen may reduce ADRs during isoniazid monotherapy without concern over insufficient drug exposure.

Biocathode design with highly-oriented immobilization of multi-copper oxidase from Pyrobaculum aerophilum onto a single-walled carbon nanotube surface via a carbon nanotube-binding peptide.

Biofuel cells generate electric energy using an enzyme as a catalyst for an electrode but their stability and low battery output pose problems for practical use. To solve these problems, this study aimed to build a long-lasting and high-output biocathode as a catalyst using a highly stable hyperthermophilic archaeal enzyme, multi-copper oxidase, from Pyrobaculum aerophilum (McoP). To increase output, McoP was oriented and immobilized on single-walled carbon nanotubes (SWCNT) with a high specific surface area, and the electrode interface was designed to achieve highly efficient electron transfer between the enzyme and electrode. Type 1 copper (T1Cu), an electron-accepting site in the McoP molecule, is located near the C-terminus. Therefore, McoP was prepared by genetically engineering a CNT-binding peptide with the sequence LLADTTHHRPWT, at the C-terminus of McoP (McoP-CBP). We then constructed an electrode using a complex in which McoP-CBP was aligned and immobilized on SWCNT, and then clarified the effect of CBP. The amounts of immobilized enzymes on McoP-SWCNT and (McoP-CBP)-SWCNT complexes were almost equal. CV measurement of the electrode modified with both complexes showed 5.4 times greater current density in the catalytic reaction of the (McoP-CBP)-SWCNT/GC electrode than in the McoP-SWCNT/GC electrode. This is probably because CBP fusion immobilize the enzyme on SWCNTs in an orientational manner, and T1Cu, the oxidation-reduction site in McoP, is close to the electrode, which improves electron transfer efficiency.

Multi-Combilipases: Co-Immobilizing Lipases with Very Different Stabilities Combining Immobilization via Interfacial Activation and Ion Exchange. The Reuse of the Most Stable Co-Immobilized Enzymes after Inactivation of the Least Stable Ones

The lipases A and B from Candida antarctica (CALA and CALB), Thermomyces lanuginosus (TLL) or Rhizomucor miehei (RML), and the commercial and artificial phospholipase Lecitase ultra (LEU) may be co-immobilized on octyl agarose beads. However, LEU and RML became almost fully inactivated under conditions where CALA, CALB and TLL retained full activity. This means that, to have a five components co-immobilized combi-lipase, we should discard 3 fully active and immobilized enzymes when the other two enzymes are inactivated. To solve this situation, CALA, CALB and TLL have been co-immobilized on octyl-vinyl sulfone agarose beads, coated with polyethylenimine (PEI) and the least stable enzymes, RML and LEU have been co-immobilized over these immobilized enzymes. The coating with PEI is even favorable for the activity of the immobilized enzymes. It was checked that RML and LEU could be released from the enzyme-PEI coated biocatalyst, although this also produced some release of the PEI. That way, a protocol was developed to co-immobilize the five enzymes, in a way that the most stable could be reused after the inactivation of the least stable ones. After RML and LEU inactivation, the combi-biocatalysts were incubated in 0.5 M of ammonium sulfate to release the inactivated enzymes, incubated again with PEI and a new RML and LEU batch could be immobilized, maintaining the activity of the three most stable enzymes for at least five cycles of incubation at pH 7.0 and 60 °C for 3 h, incubation on ammonium sulfate, incubation in PEI and co-immobilization of new enzymes. The effect of the order of co-immobilization of the different enzymes on the co-immobilized biocatalyst activity was also investigated using different substrates, finding that when the most active enzyme versus one substrate was immobilized first (nearer to the surface of the particle), the activity was higher than when this enzyme was co-immobilized last (nearer to the particle core).

Molecular identification of cellulase and protease producing Bacillus tequilensis UTMSA14 isolated from the geothermal hot spring in Lau Sidebuk Debuk, North Sumatra, Indonesia

Abstract. Fachrial E, Putri RRJS, Lister INE, Anggraini S, Harmileni, Nugroho TT, Saryono. 2020. Molecular identification of cellulase and protease producing Bacillus tequilensis UTMSA14 isolated from the geothermal hot spring in Lau Sidebuk Debuk, North Sumatra, Indonesia. Biodiversitas 21: 4719-4725. The industrial need for stable microbial enzymes tends to increase every year. The aim of this study was to isolate and identify the protease and cellulase producing thermophilic bacteria isolated from the geothermal spring of Lau Sidebuk Debuk, North Sumatra, Indonesia. The protease activity was determined based on the formation of halo zone on Skim Milk Agar, while cellulase was confirmed using CMC plate agar method. Nine bacterial isolates were successfully obtained from the sediments and water, and both activities were observed in only one isolate, known as UTMSA14. These isolates were characterized biochemically and morphologically. The molecular identification of UTMSA14 was done by 16S rRNA sequencing based on BLAST and phylogenetic analysis using MEGA X. Phylogenetic analysis showed that UTMSA14 clustered together with Bacillus tequilensis strain 10b (accession number NR_104919.1), which ascertained the isolate as Bacillus tequilensis strain 10b (accession number NR_104919.1). This is the first report of a thermophilic bacteria isolated from Lau Sidebuk Debuk hot springs, with the ability to produce cellulase and protease. further research is needed to purify and characterize the resulting enzymes, with a potential for industrial applications.

Ancestral sequence reconstruction produces thermally stable enzymes with mesophilic enzyme-like catalytic properties

Enzymes have high catalytic efficiency and low environmental impact, and are therefore potentially useful tools for various industrial processes. Crucially, however, natural enzymes do not always have the properties required for specific processes. It may be necessary, therefore, to design, engineer, and evolve enzymes with properties that are not found in natural enzymes. In particular, the creation of enzymes that are thermally stable and catalytically active at low temperature is desirable for processes involving both high and low temperatures. In the current study, we designed two ancestral sequences of 3-isopropylmalate dehydrogenase by an ancestral sequence reconstruction technique based on a phylogenetic analysis of extant homologous amino acid sequences. Genes encoding the designed sequences were artificially synthesized and expressed in Escherichia coli. The reconstructed enzymes were found to be slightly more thermally stable than the extant thermophilic homologue from Thermus thermophilus. Moreover, they had considerably higher low-temperature catalytic activity as compared with the T. thermophilus enzyme. Detailed analyses of their temperature-dependent specific activities and kinetic properties showed that the reconstructed enzymes have catalytic properties similar to those of mesophilic homologues. Collectively, our study demonstrates that ancestral sequence reconstruction can produce a thermally stable enzyme with catalytic properties adapted to low-temperature reactions.

Mechanisms of CYP450 Inhibition: Understanding Drug-Drug Interactions Due to Mechanism-Based Inhibition in Clinical Practice.

In an ageing society, polypharmacy has become a major public health and economic issue. Overuse of medications, especially in patients with chronic diseases, carries major health risks. One common consequence of polypharmacy is the increased emergence of adverse drug events, mainly from drug–drug interactions. The majority of currently available drugs are metabolized by CYP450 enzymes. Interactions due to shared CYP450-mediated metabolic pathways for two or more drugs are frequent, especially through reversible or irreversible CYP450 inhibition. The magnitude of these interactions depends on several factors, including varying affinity and concentration of substrates, time delay between the administration of the drugs, and mechanisms of CYP450 inhibition. Various types of CYP450 inhibition (competitive, non-competitive, mechanism-based) have been observed clinically, and interactions of these types require a distinct clinical management strategy. This review focuses on mechanism-based inhibition, which occurs when a substrate forms a reactive intermediate, creating a stable enzyme–intermediate complex that irreversibly reduces enzyme activity. This type of inhibition can cause interactions with drugs such as omeprazole, paroxetine, macrolide antibiotics, or mirabegron. A good understanding of mechanism-based inhibition and proper clinical management is needed by clinicians when such drugs are prescribed. It is important to recognize mechanism-based inhibition since it cannot be prevented by separating the time of administration of the interacting drugs. Here, we provide a comprehensive overview of the different types of mechanism-based inhibition, along with illustrative examples of how mechanism-based inhibition might affect prescribing and clinical behaviors.

Impacts of Culture Conditions on Ligninolytic Enzymes (LIP, MNP, and Lac) Activity of Five Bacterial Strains

In this study, with the aim of determining and assessing the influence of several culture conditions on the ligninolytic enzyme (LiP, MnP, and Lac) activity of bacteria, five lignin-degrading bacteria strains were isolated from two different soil samples and cultured on minimum salt medium agar containing alkaline lignin (MSML agar). Among the five isolated strains, DL1 and X3 expressed strong and stable ligninase enzyme activity at various temperature levels (30°C, 37°C, 50°C, and 60°C) and were selected for further study. Notably, at 60°C, the ligninase activity of both strains lasted until the seventh day before decreasing. The effects of the culture medium conditions, namely, carbohydrate sources, nitrogen sources, and pH, on the ligninolytic system illustrated that both X3 and DL1 were able to generate good enzymatic activity at a pH range of 3.0 to 7.0. These strains could use various sources of carbohydrates and nitrogen, derived from glucose, lactose, peptone, meat extract, and yeast extract. In addition, the analyses of biochemical characteristics revealed that X3 was capable of hydrolyzing starch and cellulose, while DL1 was not. Therefore, the results of this study suggested the potential of applying selected lignin-degrading bacterial strains on lignin treatments of agricultural wastes.

Studies on a thermostable nitrilase from Staphylococcus Sp and its In-silico characterisation

An inducible and heterocyclic thermostable nitrilase was obtained from Staphylococcus sp. KP789135.1. The whole cell nitrilase was active at 50 °C temperature and neutral pH with a half-life of 4 h at 50 °C and 2.5 h at 60 °C. Metal ions such as Fe3+, Mg2+, and Ca2+ enhanced the biocatalytic activity while Ag2+, Hg2+, and Cu2+ completely inhibited biocatalysis, which confirmed the presence of thiol group in the catalytic site. Staphylococcus sp. KP789135.1 has broad hydrolytic activity towards heterocyclic nitriles and few amides. It showed high tolerance against 3-cyanopyridine (5 mM - 20 mM). The calculated Km and Vmax were 1.33 mM and 0.98 U, lower km values demonstrated the highest affinity towards 3-cyanopyridine. In silico study revealed 99% similarity with Staphylococcus strain. The 3D structures were deduced from I-TASSER server and validated from PROCHECK, found relatively low % of amino acid residues has phi/psi angles in the disallowed region that suggest the acceptability of Ramachandran plot in the present work. Docking study revealed 3-cyanopyridine has lowest potential energy (potential energy OPLS -7.00745) and thus showed more stable enzyme ligand interaction. This is the first report on biotransformation of 3-cyanopyridine to nicotinic acid using thermostable nitrilase from Staphylococcus sp.

Bioconversion of chitin and concomitant production of chitinase and N-acetylglucosamine by novel Achromobacter xylosoxidans isolated from shrimp waste disposal area

Marine pollution is a significant issue in recent decades, with the increase in industries and their waste harming the environment and ecosystems. Notably, the rise in shellfish industries contributes to tons of shellfish waste composed of up to 58% chitin. Chitin, the second most ample polymer next to cellulose, is insoluble and resistant to degradation. It requires chemical-based treatment or enzymatic hydrolysis to cleave the chitin polymers. The chemical-based treatment can lead to environmental pollution, so to solve this problem, enzymatic hydrolysis is the best option. Moreover, the resulting biopolymer by-products can be used to boost the fish immune system and also as drug delivery agents. Many marine microbial strains have chitinase producing ability. Nevertheless, we still lack an economical and highly stable chitinase enzyme for use in the industrial sector. So we isolate a novel marine bacterial strain Achromobacter xylosoxidans from the shrimp waste disposal site using chitin minimal medium. Placket–Burman and central composite design statistical models for culture condition optimisation predicted a 464.2 U/ml of chitinase production. The culture conditions were optimised for maximum chitinase production recording up to 467 U/ml. This chitinase from the A. xylosoxidans was 100% active at an optimum temperature of 45 °C (withstand up to 55 °C) and pH 8 with 80% stability. The HPLC analysis of chitinase degraded shellfish waste reveals a major amino acid profile composition—arginine, lysine, aspartic acid, alanine, threonine and low levels of isoleucine and methionine. These chitinase degraded products and by-products can be used as supplements in the aquaculture industry.

Immobilization of the Highly Active UDP-Glucose Pyrophosphorylase From Thermocrispum agreste Provides a Highly Efficient Biocatalyst for the Production of UDP-Glucose.

Biocatalysis that produces economically interesting compounds can be carried out by using free enzymes or microbial cells. However, often the cell metabolism does not allow the overproduction or secretion of activated sugars and thus downstream processing of these sugars is complicated. Here enzyme immobilization comes into focus in order to stabilize the enzyme as well as to make the overall process economically feasible. Besides a robust immobilization method, a highly active and stable enzyme is needed to efficiently produce the product of choice. Herein, we report on the identification, gene expression, biochemical characterization as well as immobilization of the uridine-5′-diphosphate-glucose (UDP-glucose) pyrophosphorylase originating from the thermostable soil actinobacterium Thermocrispum agreste DSM 44070 (TaGalU). The enzyme immobilization was performed on organically modified mesostructured cellular foams (MCF) via epoxy and amino group to provide a stable and active biocatalyst. The soluble and highly active TaGalU revealed a Vmax of 1698 U mg–1 (uridine-5′-triphosphate, UTP) and a Km of 0.15 mM (UTP). The optimum reaction temperature was determined to be 50°C. TaGalU was stable at this temperature for up to 30 min with a maximum loss of activity of 65%. Interestingly, immobilized TaGalU was stable at 50°C for at least 120 min without a significant loss of activity, which makes this enzyme an interesting biocatalyst for the production of UDP-glucose.

Stabilization of immobilized l-arabinose isomerase for the production of d-tagatose from d-galactose.

The aim of this work was to develop a stable immobilized enzyme biocatalyst for the isomerization of d-galactose to d-tagatose at high temperature. l-Arabinose isomerase from the hyperthermophilic bacterium Thermotoga maritima (TMAI) was produced as a (His)6 -tagged protein, immobilized on a copper-chelate epoxy support and subjected to several postimmobilization treatments aimed at increasing its operational and structural stability. Treatment with glutaraldehyde and ethylenediamine resulted in a more than twofold increase in the operational stability and in all enzyme subunits linked, directly or indirectly, to the support via covalent bonds. A postimmobilization treatment of the immobilized derivatives with mercaptoethanol for the removal of any remaining copper ions, determined a further increase of the operational biocatalytic activity. Immobilized derivatives subjected to both treatments were used for the bioconversion of 18 g/L d-galactose to d-tagatose at 80°C in a packed bed reactor in three repeated cycles and showed a better operational stability compared with the literature data. This study shows that a postimmobilization stabilization treatment with glutaraldehyde and ethylenediamine can stabilize the multi-subunit structure of an enzyme immobilized on a metal-chelate epoxy support with an increase of its operational stability, results that are not easily achievable with the sole immobilization on epoxy or metal chelate-epoxy supports in the case of complex multimeric enzymes with geometric incongruence with the support.

Catalytic Foldamers: When the Structure Guides the Function

Enzymes are predominantly proteins able to effectively and selectively catalyze highly complex biochemical reactions in mild reaction conditions. Nevertheless, they are limited to the arsenal of reactions that have emerged during natural evolution in compliance with their intrinsic nature, three-dimensional structures and dynamics. They optimally work in physiological conditions for a limited range of reactions, and thus exhibit a low tolerance for solvent and temperature conditions. The de novo design of synthetic highly stable enzymes able to catalyze a broad range of chemical reactions in variable conditions is a great challenge, which requires the development of programmable and finely tunable artificial tools. Interestingly, over the last two decades, chemists developed protein secondary structure mimics to achieve some desirable features of proteins, which are able to interfere with the biological processes. Such non-natural oligomers, so called foldamers, can adopt highly stable and predictable architectures and have extensively demonstrated their attractiveness for widespread applications in fields from biomedical to material science. Foldamer science was more recently considered to provide original solutions to the de novo design of artificial enzymes. This review covers recent developments related to peptidomimetic foldamers with catalytic properties and the principles that have guided their design.

A new brilliantly blue-emitting luciferin-luciferase system from Orfelia fultoni and Keroplatinae (Diptera)

Larvae of O. fultoni (Keroplatidae: Keroplatinae), which occur along river banks in the Appalachian Mountains in Eastern United States, produce the bluest bioluminescence among insects from translucent areas associated to black bodies, which are located mainly in the anterior and posterior parts of the body. Although closely related to Arachnocampa spp (Keroplatidae: Arachnocampininae), O.fultoni has a morphologically and biochemically distinct bioluminescent system which evolved independently, requiring a luciferase enzyme, a luciferin, a substrate binding fraction (SBF) that releases luciferin in the presence of mild reducing agents, molecular oxygen, and no additional cofactors. Similarly, the closely related Neoceroplatus spp, shares the same kind of luciferin-luciferase system of Orfelia fultoni. However, the molecular properties, identities and functions of luciferases, SBF and luciferin of Orfelia fultoni and other luminescent members of the Keroplatinae subfamily still remain to be fully elucidated. Using O. fultoni as a source of luciferase, and the recently discovered non-luminescent cave worm Neoditomiya sp as the main source of luciferin and SBF, we isolated and initially characterized these compounds. The luciferase of O. fultoni is a stable enzyme active as an apparent trimer (220 kDa) composed of ~70 kDa monomers, with an optimum pH of 7.8. The SBF, which is found in the black bodies in Orfelia fultoni and in smaller dark granules in Neoditomiya sp, consists of a high molecular weight complex of luciferin and proteins, apparently associated to mitochondria. The luciferin, partially purified from hot extracts by a combination of anion exchange chromatography and TLC, is a very polar and weakly fluorescent compound, whereas its oxidized product displays blue fluorescence with an emission spectrum matching the bioluminescence spectrum (~460 nm), indicating that it is oxyluciferin. The widespread occurrence of luciferin and SBF in both luminescent and non-luminescent Keroplatinae larvae indicate an additional important biological function for the substrate, and therefore the name keroplatin.

Comparative modelling of a novel enzyme: Mus musculus leucine decarboxylase.

Leucine decarboxylase (LDC) is a recently proposed enzyme with no official enzyme commission number yet. It is encoded by the Mus musculus gene Gm853 which is expressed at kidneys, generating isopentylamine, an alkylmonoamine that has not been described to be formed by any metazoan enzyme yet. Although the relevance of LDC in mammalian physiology has not been fully determined, isopentylamine is a potential modulator which may have effects on insulin secretion and healthy gut microbiota formation. The LDC is a stable enzyme that specifically decarboxylates L-leucine but does not decarboxylate ornithine or lysine as its paralogues ornithine decarboxylase (ODC; EC: 4.1.1.17) and lysine decarboxylase (KDC; EC: 4.1.1.18) do. It does not act as an antizyme inhibitor and does not decarboxylate branched amino acids such as valine and isoleucine as it is another paralogue valine decarboxylase (VDC; EC: 4.1.1.14). The crystal structure of the enzyme has not been determined yet but there are homologous structures with complete coverage in Protein Data Bank (PDB) which makes LDC a good candidate for comparative modelling.In this study, homology models of LDC were generated and used in cofactor and substrate docking to understand the structure/function relationship underlying the unique selectivity of LDC enzyme.

Enhancing thermostability of xylanase from rumen microbiota by molecular cyclization

The capacity for thermal tolerance is critical for industrial enzyme. In the past decade, great efforts have been made to endow wild-type enzymes with higher catalytic activity or thermostability using gene engineering and protein engineering strategies. In this study, a recently developed SpyTag/SpyCatcher system, mediated by isopeptide bond-ligation, was used to modify a rumen microbiota-derived xylanase XYN11-6 as cyclized and stable enzyme C-XYN11-6. After incubation at 60, 70 or 80 ℃ for 10 min, the residual activities of C-XYN11-6 were 81.53%, 73.98% or 64.41%, which were 1.48, 2.92 or 3.98-fold of linear enzyme L-XYN11-6, respectively. After exposure to 60-90°C for 10 min, the C-XYN11-6 remained as soluble in suspension, while L-XYN11-6 showed severely aggregation. Intrinsic and 8-anilino-1-naphthalenesulfonic acid (ANS)-binding fluorescence analysis revealed that C-XYN11-6 was more capable of maintaining its conformation during heat challenge, compared with L-XYN11-6. Interestingly, molecular cyclization also conferred C-XYN11-6 with improved resilience to 0.1-50 mmol/L Ca²⁺ or 0.1 mmol/L Cu²⁺ treatment. In summary, we generated a thermal- and ion-stable cyclized enzyme using SpyTag/SpyCatcher system, which will be of particular interest in engineering of enzymes for industrial application.

Preparation of thermally stable and digestive enzyme resistant flour directly from Japonica broken rice by combination of steam infusion, enzymatic debranching and heat moisture treatment

Through a series of gelatinization, enzymatic debranching and heat moisture treatment, unmilled japonica broken rice was applied to prepare thermally stable and digestive enzyme resistant flour. The optimal conditions of gelatinization temperature, enzyme/substrate ratio, and debranching duration were determined by the three-factor three-level response surface methodology (RSM). Raw flour and products processed in each step were collected to test and compare the hydration ability, reheated solubility, pasting property, starch classification (RDS, SDS, RS) and in vitro digestibility. The results of RSM showed that E/S ratio was the main factor influencing the RS formation, while influence aroused by hydrolysis time and gelatinization temperature were minor. Gelatinization broke down the intermolecular bonds of starch molecules, allowed the hydrogen bonding sites to engage more water, and improved the availability of digestion. However, debranching provided an opportunity for the alignment of short-chain and long-chain amylose molecules. Therefore, RS increased to 42%, and digestibility decreased accordingly. HMT could further enhance the molecular rearrangement, while the RS was increased to as high as 55% and the final product met the low GI criteria.