Bioinspired mp20 mimicking uricase in ZIF-8: Metal ion dependent for controllable activity.

Mini protein mimicking uricase (mp20) has shown significant potential as a replacement for natural enzymes in the development of uric acid biosensors. However, the design of mp20 has resulted to an inactive form of peptide, causing of loss their catalytic activity. Herein, this paper delineates the impact of various metal cofactors on the catalytic activity of mp20. The metal ion-binding site prediction and docking (MIB) web server was employed to identify the metal ion binding sites and their affinities towards mp20 residues. Among the tested metal ions, Cu2+ displayed the highest docking score, indicating its preference for interaction with Thr16 and Asp17 residues of mp20. To assess the catalytic activity of mp20 in the presence of metal ions, uric acid assays was monitored using a colorimetric method. The presence of Cu2+ in the assays promotes the activation of mp20, resulting in a color change based on quinoid production. Furthermore, the encapsulation of the mp20 within zeolitic imidazolate framework-8 (ZIF-8) notably improved the stability of the biomolecule. In comparison to the naked mp20, the encapsulated ZIFs biocomposite (mp20@ZIF-8) demonstrates superior stability, selectivity and sensitivity. ZIF's porous shells provides excellent protection, broad detection (3-100 µM) with a low limit (4.4 µM), and optimal function across pH (3.4-11.4) and temperature (20-100°C) ranges. Cost-effective and stable mp20@ZIF-8 surpasses native uricase, marking a significant biosensor technology breakthrough. This integration of metal cofactor optimization and robust encapsulation sets new standards for biosensing applications.

Shifting the pH profiles of Staphylococcus epidermidis lipase (SEL) and Staphylococcus hyicus lipase (SHL) through generating chimeric lipases by DNA shuffling strategy.

Enzymes are often required to function in a particular reaction condition by the industrial procedure. In order to identify critical residues affecting the optimum pH of Staphylococcal lipases, chimeric lipases from homologous lipases were generated via a DNA shuffling strategy. Chimeric 1 included mutations of G166S, K212E, T243A, H271Y. Chimeric 2 consisted of substitutions of K212E, T243A, H271Y. Chimeric 3 contained substitutions of K212E, R359L. From the screening results, the pH profiles for chimeric 1 and 2 lipases were shifted from pH 7 to 6. While the pH of chimeric 3 was shifted to 8. It seems the mutation of K212E in chimeric 1 and 2 decreased the pH to 6 by changing the electrostatic potential surface. Furthermore, chimeric 3 showed 10 ˚C improvement in the optimum temperature due to the rigidification of the catalytic loop through the hydrophobic interaction network. Moreover, the substrate specificity of chimeric 1 and 2 was increased towards the longer carbon length chains due to the mutation of T243A adjacent to the lid region through increasing the flexibility of the lid. Current study illustrated that directed evolution successfully modified lipase properties including optimum pH, temperature and substrate specificity through mutations, especially near catalytic and lid regions.

Features of the rare pathogen Meyerozyma guilliermondii strain SO and comprehensive in silico analyses of its adherence-contributing virulence factor agglutinin-like sequences.

Meyerozyma guilliermondii is a rare yeast pathogen contributing to the deadly invasive candidiasis. M. guilliermondii strain SO, as a promising protein expression host, showed 99% proteome similarity with the clinically isolated ATCC 6260 (type strain) in a recent comparative genomic analysis. However, their in vitro virulence features and in vivo pathogenicity were uncharacterized. This study aimed to characterize the in vitro and in vivo pathogenicity of M. guilliermondii strain SO and analyze its Als proteins (MgAls) via comprehensive bioinformatics approaches. M. guilliermondii strain SO showed lower and higher sensitivity towards ß-mercaptoethanol and lithium, respectively than the avirulent S. cerevisiae but exhibited the same tolerance towards cell wall-perturbing Congo Red with C. albicans. With 7.5× higher biofilm mass, M. guilliermondii strain SO also demonstrated 75% higher mortality rate in the zebrafish embryos with a thicker biofilm layer on the chorion compared to the avirulent S. cerevisiae. Being one of the most important Candida adhesins, sequence and structural analyses of four statistically identified MgAls showed that MgAls1056 was predicted to exhibit the most conserved amyloid-forming regions, tandem repeat domain and peptide binding cavity (PBC) compared to C. albicans Als3. Favoured from the predicted largest ligand binding site and druggable pockets, it showed the highest affinity towards hepta-threonine. Non-PBC druggable pockets in the most potent virulence contributing MgAls1056 provide new insights into developing antifungal drugs targeting non-albicans Candida spp. Virtual screening of available synthetic or natural bioactive compounds and MgAls1056 deletion from the fungal genome should be further performed and validated experimentally.Communicated by Ramaswamy H. Sarma.

Bibliometric analysis and thematic review of Candida pathogenesis: Fundamental omics to applications as potential antifungal drugs and vaccines.

Invasive candidiasis caused by the pathogenic Candida yeast species has resulted in elevating global mortality. The pathogenicity of Candida spp. is not only originated from its primary invasive yeast-to-hyphal transition; virulence factors (transcription factors, adhesins, invasins, and enzymes), biofilm, antifungal drug resistance, stress tolerance, and metabolic adaptation have also contributed to a greater clinical burden. However, the current research theme in fungal pathogenicity could hardly be delineated with the increasing research output. Therefore, our study analysed the research trends in Candida pathogenesis over the past 37 years via a bibliometric approach against the Scopus and Web of Science databases. Based on the 3993 unique documents retrieved, significant international collaborations among researchers were observed, especially between Germany (Bernhard Hube) and the UK (Julian Naglik), whose focuses are on Candida proteinases, adhesins, and candidalysin. The prominent researchers (Neils Gow, Alistair Brown, and Frank Odds) at the University of Exeter and the University of Aberdeen (second top performing affiliation) UK contribute significantly to the mechanisms of Candida adaptation, tolerance, and stress response. However, the science mapping of co-citation analysis performed herein could not identify a hub representative of subsequent work since the clusters were semi-redundant. The co-word analysis that was otherwise adopted, revealed three research clusters; the cluster-based thematic analyses indicated the severeness of Candida biofilm and antifungal resistance as well as the elevating trend on molecular mechanism elucidation for drug screening and repurposing. Importantly, the in vivo pathogen adaptation and interactions with hosts are crucial for potential vaccine development.

International research collaborations have evident its significance in impactful work covering all aspects of Candida pathogenicity. Its current, diverse research was discussed thematically based on the comprehensive scientometric analysis with unidentified hub representatives for subsequent work.

Enhancing uric acid electrochemical detection with copper ion-activated mini protein mimicking uricase within ZIF-8: response surface methodology (RSM) optimization.

This study aims to investigate the influence of copper(II) ions as a cofactor on the electrochemical performance of a biocomposite consisting of a mini protein mimicking uricase (mp20) and zeolitic immidazolate framework-8 (ZIF-8) for the detection of uric acid. A central composite design (CCD) was utilized to optimize the independent investigation, including pH, deposition potential, and deposition time, while the current response resulting from the electrocatalytic oxidation of uric acid was used as the response. The statistical analysis of variance (ANOVA) showed a good correlation between the experimental and predicted data, with a residual standard error percentage (RSE%) of less than 2% for predicting optimal conditions. The synergistic effect of the nanoporous ZIF-8 host, Cu(II)-activated mp20, and reduced graphene oxide (rGO) layer resulted in a highly sensitive biosensor with a limit of detection (LOD) of 0.21 µM and a reproducibility of the response (RSD = 0.63%). The Cu(II)-activated mp20@ZIF-8/rGO/SPCE was highly selective in the presence of common interferents, and the fabricated layer exhibited remarkable stability with signal changes below 4.15% after 60 days. The biosensor's reliable performance was confirmed through real sample analyses of human serum and urine, with comparable recovery values to conventional HPLC.

Enhancement in T1 lipase purification recovery using the novel construct pGEX4T1/His-T1.

The Geobacillus zalihae strain T1 produces a thermostable T1 lipase that could be used for industrial purposes. Previously, the GST-T1 lipase was purified through two chromatographic steps: affinity and ion exchange (IEX) but the recovery yield was only 33%. To improve the recovery yield to over 80%, the GST tag from the pGEX system was replaced with a poly-histidine at the N-terminal of the T1 lipase sequence. The novel construct of pGEX/His-T1 lipase was developed by site-directed mutagenesis, where the XbaI restriction site was introduced upstream of the GST tag, allowing the removal of tag via double digestion using XbaI and EcoRI (existing cutting site in the pGEX system). Fragment of 6 × His-T1 lipase fusion was synthesized, cloned into the pGEX4T1 system, and expressed in Escherichia coli BL21 (DE3) pLysS, resulting in lipase-specific activity at 236 U/mg. The single purification step of His-T1 lipase was successfully achieved using nickel Sepharose 6FF with an optimized concentration of 5 mM imidazole for binding, yielding the recovery of 98%, 1,353 U/mg lipase activity, and a 5.7-fold increase in purification fold. His-T1 lipase was characterized and was found to be stable at pH 5-9, active at 70 °C, and optimal at pH 9.

Rational design of mini protein mimicking uricase: Encapsulation in ZIF-8 for uric acid detection.

Five mini proteins mimicking uricase comprising 20, 40, 60, 80, and 100 amino acids were designed based on the conserved active site residues within the same dimer, using the crystal structure of tetrameric uricase from Arthrobacter globiformis (PDB ID: 2yzb) as a template. Based on molecular docking analysis, the smallest mini protein, mp20, shared similar residues to that of native uricase that formed hydrogen bonds with uric acid and was chosen for further studies. Although purified recombinant mp20 did not exhibit uricase activity, it showed specific binding towards uric acid and evinced excellent thermotolerance and structural stability at temperatures ranging from 10°C to 100°C, emulating its natural origin. To explore the potential of mp20 as a bioreceptor in uric acid sensing, mp20 was encapsulated within zeolitic imidazolate framework-8 (mp20@ZIF-8) followed by the modification on rGO-screen printed electrode (rGO/SPCE) to maintain the structural stability. An irreversible anodic peak and increased semicircular arcs of the Nyquist plot with an increase of the analyte concentrations were observed by utilizing cyclic voltammetry and electrochemical impedance spectroscopy (EIS), suggesting the detection of uric acid occurred, which is based on substrate-mp20 interaction.

In silico structural exploration of serine protease from a CTG-clade yeast Meyerozyma guilliermondii strain SO.

In eukaryotes, serine proteases are cellular localized hydrolases reported to regulate essential biological reactions. Improved industrial applications of proteins are aided by prediction and analysis of their 3-dimensional structures (3D). A serine protease was identified from CTG-clade yeast Meyerozyma guilliermondii strain SO and its 3D structure as well as its catalytic attributes have not been fully understood yet, thus we seek to report on the catalytic mechanism of M. guilliermondii strain SO MgPRB1 using substrate PMSF via in silico docking as well as its stability by way of disulfide bonds formation. Herein, bioinformatics tools and techniques were used to predict, validate and analyze the possible changes of CUG ambiguity (if any) in strain SO using template PDB ID: 3F7O. Structural assessments confirmed the classic catalytic triad Asp305, His337, and Ser499. Superimposition of MgPRB1 and template 3F7O structures revealed the unlinked cysteine residues between Cys341, Cys440, Cys471 and Cys506 of MgPRB1 compared to template 3F7O with two disulfide bonds formation, which confers structural stability. In conclusion, serine protease structure from strain SO was successfully predicted and studies towards understanding at the molecular level may be undertaken for its potential applications in the degradation of peptide bonds.

Enolase in Meyerozyma guilliermondii strain SO: Sequential and structural insights of MgEno4581 as a putative virulence factor and host-fungal interactions through comprehensive in silico approaches.

Meyerozyma guilliermondii is a rare opportunistic fungal pathogen that causes deadly invasive candidiasis in human. M. guilliermondii strain SO is a local yeast isolate that possesses huge industrial interests but also pathogenic towards zebrafish embryos. Enolases that bind to human extracellular matrix (ECM) proteins are among the fungal virulence factors. To understand its pathogenicity mechanism down to molecular level, especially in the rare M. guilliermondii, this study aimed to identify and characterize the potentially virulence-associated enolase in M. guilliermondii strain SO using bioinformatics approaches. Profile Hidden-Markov model was implemented to identify enolase-related sequences in the fungal proteome. Sequence analysis deciphered only one (MgEno4581) out of nine sequences exhibited potent virulence traits observed similarly in the pathogenic Candida albicans. MgEno4581 structure that was predicted via SWISS-MODEL using C. albicans enolase (CaEno1; PDB ID: 7vrd) as the homology modeling template portrayed a highly identical motif with CaEno1 that facilitates ECM proteins binding. Amino acid substitutions (D234K, K235A, Y238H, K239D, G243K, V248C and Y254F) in ECM-binding motif of Saccharomyces cerevisiae enolase (ScEno) compared to MgEno4581 and CaEno1 caused changes in motif's surface charges. Protein-protein docking indicated F253 in ScEno only interacted hydrophobically with human plasminogen (HPG). Hydrogen linkages were observed for both MgEno4581 and CaEno1, suggesting a stronger interaction with HPG in the hydrophilic host microenvironments. Thus, our in silico characterizations on MgEno4581 provided new perspectives on its potential roles in candidiasis (fungal-host interactions) caused by M. guilliermondii, especially M. guilliermondii strain SO on zebrafish embryos that mimic the immunocompromised individuals as previously evident.

Designed Mini Protein 20 Mimicking Uricase Encapsulated in ZIF-8 as Nanozyme Biosensor for Uric Acid Detection.

This work presents the use of encapsulated mini protein 20 mimicking uricase (mp20)-zeolitic imidazolate framework-8 (ZIF-8) as a bioreceptor for the development of a nanozyme-based electrochemical biosensor for uric acid detection. The electrochemical performance of the biofunctionalized mp20@ZIF-8 on the reduced graphene oxide/screen-printed carbon electrode (rGO/SPCE) was investigated by optimizing operating parameters such as pH, deposition potential, and deposition time using a central composite design-response surface methodology (CCD-RSM). The quadratic regression model was developed to correlate the combination of each variable to the oxidation current density as a response. A significant effect on current response was observed under optimized conditions of pH of 7.4 at −0.35 V deposition potential and 56.56 s deposition time, with p < 0.05 for each interacted factor. The obtained coefficient of determination (R2) value of 0.9992 indicated good agreement with the experimental finding. The developed nanozyme biosensor (mp20@ZIF-8/rGO/SPCE) exhibited high selectivity in the presence of the same fold concentration of interfering species with a detection limit of 0.27 µM, over a concentration range of 1 to 34 µM. The practicality of the tailored biosensor in monitoring uric acid in human serum and urine samples was validated with high-performance liquid chromatography (HPLC) and a commercial uric acid meter. Hence, nanozyme-based is a promising platform that offers a rapid, sensitive, selective, and low-cost biosensor for the non-enzymatic detection of uric acid in biological samples.

Opportunistic yeast pathogen Candida spp.: Secreted and membrane-bound virulence factors.

Candidiasis is a fungal infection caused by Candida spp. especially Candida albicans, C. glabrata, C. parapsilosis and C. tropicalis. Although the medicinal therapeutic strategies have rapidly improved, the mortality rate as candidiasis has continuously increased. The secreted and membrane-bound virulence factors (VFs) are responsible for fungal invasion, damage and translocation through the host enterocytes besides the evasion from host immune system. VFs such as agglutinin-like sequences (Als), heat shock protein 70, phospholipases, secreted aspartyl proteinases (Sap), lipases, enolases and phytases are mostly hydrolases which degrade or interact with the enterocyte membrane components. Candidalysin, however, acts as a peptide toxin to induce necrotic cell lysis. To date, structural studies of the VFs remain underexplored, hindering their functional analyses. Among the VFs, only Sap and Als have their structures deposited in Protein Data Bank (PDB). Therefore, this review scrutinizes the mechanisms of these VFs by discussing the VF-deficient studies of several Candida spp. and their abilities to produce these VFs. Nonetheless, their latest reported sequential and structural analyses are discussed to impart a wider perception of the host-pathogen interactions and potential vaccine or antifungal drug targets. This review signifies that more VFs structural investigations and mining in the emerging Candida spp. are required to decipher their pathogenicity and virulence mechanisms compared to the prominent C. albicans. LAY SUMMARY: Candida virulence factors (VFs) including mainly enzymes and proteins play vital roles in breaching the human intestinal barrier and causing deadly invasive candidiasis. Limited VFs' structural studies hinder deeper comprehension of their mechanisms and thus the design of vaccines and antifungal drugs against fungal infections.

Discovery of new inhibitor for the protein arginine deiminase type 4 (PAD4) by rational design of α-enolase-derived peptides.

Rheumatoid arthritis (RA) is an inflammatory autoimmune disease affecting about 0.24 % of the world population. Protein arginine deiminase type 4 (PAD4) is believed to be responsible for the occurrence of RA by catalyzing citrullination of proteins. The citrullinated proteins act as autoantigens by stimulating an immune response. Citrullinated α-enolase has been identified as one of the autoantigens for RA. Hence, α-enolase serves as a suitable template for design of potential peptide inhibitors against PAD4. The binding affinity of α-enolase-derived peptides and PAD4 was virtually determined using PatchDock and HADDOCK docking programs. Synthesis of the designed peptides was performed using a solid phase peptide synthesis method. The inhibitory potential of each peptide was determined experimentally by PAD4 inhibition assay and IC50 measurement. PAD4 assay data show that the N-P2 peptide is the most favourable substrate among all peptides. Further modification of N-P2 by changing the Arg residue to canavanine [P2 (Cav)] rendered it an inhibitor against PAD4 by reducing the PAD4 activity to 35 % with IC50 1.39 mM. We conclude that P2 (Cav) is a potential inhibitor against PAD4 and can serve as a starting point for the development of even more potent inhibitors.

Enhancing the stability of Geobacillus zalihae T1 lipase in organic solvents and insights into the structural stability of its variants.

Critical to the applications of proteins in non-aqueous enzymatic processes is their structural dynamics in relation to solvent polarity. A pool of mutants derived from Geobacillus zalihae T1 lipase was screened in organic solvents (methanol, ethanol, propanol, butanol and pentanol) resulting in the selection of six mutants at initial screening (A83D/K251E, R21C, G35D/S195 N, K84R/R103C/M121I/T272 M and R106H/G327S). Site-directed mutagenesis further yielded quadruple mutants A83D/M121I/K251E/G327S and A83D/M121I/S195 N/T272 M, both of which had improved activity after incubation in methanol. The km and kcat values of these mutants vary marginally with the wild-type enzyme in the methanol/substrate mixture. Thermally induced unfolding of mutants was accompanied with some loss of secondary structure content. The root mean square deviations (RMSD) and B-factors revealed that changes in the structural organization are intertwined with an interplay of the protein backbone with organic solvents. Spatially exposed charged residues showed correlations between the solvation dynamics of the methanol solvent and the hydrophobicity of the residues. The short distances of the radial distribution function provided the required distances for hydrogen bond formation and hydrophobic interactions. These dynamic changes demonstrate newly formed structural interactions could be targeted and incorporated experimentally on the basis of solvent mobility and mutant residues.

The use of response surface methodology for enhanced production of a thermostable bacterial lipase in a novel yeast system.

A thermostable bacterial lipase from Geobacillus zalihae was expressed in a novel yeast Pichia sp. strain SO. The preliminary expression was too low and discourages industrial production. This study sought to investigate the optimum conditions for T1 lipase production in Pichia sp. strain SO. Seven medium conditions were investigated and optimized using Response Surface Methodology (RSM). Five responding conditions namely; temperature, inoculum size, incubation time, culture volume and agitation speed observed through Plackett-Burman Design (PBD) method had a significant effect on T1 lipase production. The medium conditions were optimized using Box-Behnken Design (BBD). Investigations reveal that the optimum conditions for T1 lipase production and Biomass concentration (OD600) were; Temperature 31.76 °C, incubation time 39.33 h, culture volume 132.19 mL, inoculum size 3.64%, and agitation speed of 288.2 rpm with a 95% PI low as; 12.41 U/mL and 95% PI high of 13.65 U/mL with an OD600 of; 95% PI low as; 19.62 and 95% PI high as; 22.62 as generated by the software was also validated. These predicted parameters were investigated experimentally and the experimental result for lipase activity observed was 13.72 U/mL with an OD600 of 24.5. At these optimum conditions, there was a 3-fold increase on T1 lipase activity. This study is the first to develop a statistical model for T1 lipase production and biomass concentration in Pichia sp. Strain SO. The optimized production of T1 lipase presents a choice for its industrial application.

Design and Characterisation of Inhibitory Peptides against Bleg1_2478, an Evolutionary Divergent B3 Metallo-ß-lactamase.

Previously, a hypothetical protein (HP) termed Bleg1_2437 (currently named Bleg1_2478) from Bacillus lehensis G1 was discovered to be an evolutionary divergent B3 subclass metallo-ß-lactamase (MBL). Due to the scarcity of clinical inhibitors for B3 MBLs and the divergent nature of Bleg1_2478, this study aimed to design and characterise peptides as inhibitors against Bleg1_2478. Through in silico docking, RSWPWH and SSWWDR peptides with comparable binding energy to ampicillin were obtained. In vitro assay results showed RSWPWH and SSWWDR inhibited the activity of Bleg1_2478 by 50% at concentrations as low as 0.90 µM and 0.50 µM, respectively. At 10 µM of RSWPWH and 20 µM of SSWWDR, the activity of Bleg1_2478 was almost completely inhibited. Isothermal titration calorimetry (ITC) analyses showed slightly improved binding properties of the peptides compared to ampicillin. Docked peptide-protein complexes revealed that RSWPWH bound near the vicinity of the Bleg1_2478 active site while SSWWDR bound at the center of the active site itself. We postulate that the peptides caused the inhibition of Bleg1_2478 by reducing or blocking the accessibility of its active site from ampicillin, thus hampering its catalytic function.

A Host-Vector System for the Expression of a Thermostable Bacterial Lipase in a Locally Isolated Meyerozyma guilliermondii SMB.

Screening for a new yeast as an alternative host is expected to solve the limitations in the present yeast expression system. A yeast sample which was isolated from the traditional food starter 'ragi' from Malaysia was identified to contain Meyerozyma guilliermondii strain SMB. This yeast-like fungus strain SMB was characterized to assess its suitability as an expression host. Lipase activity was absent in this host (when assayed at 30 °C and 70 °C) and Hygromycin B (50 µg/mL) was found to be its best selection marker. Then, the hyg gene (Hygromycin B) was used to replace the sh ble gene (Zeocin) expression cassette in a Komagataella phaffii expression vector (designated as pFLDhα). A gene encoding the mature thermostable lipase from Bacillus sp. L2 was cloned into pFLDhα, followed by transformation into strain SMB. The optimal expression of L2 lipase was achieved using YPTM (Yeast Extract-Peptone-Tryptic-Methanol) medium after 48 h with 0.5% (v/v) methanol induction, which was 3 times faster than another K. phaffii expression system. In conclusion, a new host-vector system was established as a platform to express L2 lipase under the regulation of PFLD1. It could also be promising to express other recombinant proteins without inducers.

The Influence of Calcium toward Order/Disorder Conformation of Repeat-in-Toxin (RTX) Structure of Family I.3 Lipase from Pseudomonas fluorescens AMS8.

Calcium-binding plays a decisive role in the folding and stabilization of many RTX proteins, especially for the RTX domain. Although many studies have been conducted to prove the contribution of Ca2+ ion toward the folding and stabilization of RTX proteins, its functional dynamics and conformational structural changes remain elusive. Here, molecular docking and molecular dynamics (MD) simulations were performed to analyze the contribution of Ca2+ ion toward the folding and stabilization of the RTX lipase (AMS8 lipase) structure. AMS8 lipase contains six Ca2+ ions (Ca1-Ca6). Three Ca2+ ions (Ca3, Ca4, and Ca5) were bound to the RTX parallel ß-roll motif repeat structure (RTX domain). The metal ion (Ca2+) docking analysis gives a high binding energy, especially for Ca4 and Ca5 which are tightly bound to the RTX domain. The function of each Ca2+ ion is further analyzed using the MD simulation. The removal of Ca3, Ca4, and Ca5 caused the AMS8 lipase structure to become unstable and unfolded. The results suggested that Ca3, Ca4, and Ca5 stabilized the RTX domain. In conclusion, Ca3, Ca4, and Ca5 play a crucial role in the folding and stabilization of the RTX domain, which sustain the integrity of the overall AMS8 lipase structure.

Calcium-Induced Activity and Folding of a Repeat in Toxin Lipase from Antarctic Pseudomonas fluorescens Strain AMS8.

It is hypothesized that the Ca2+ ions were involved in the activity, folding and stabilization of many protein structures. Many of these proteins contain repeat in toxin (RTX) motifs. AMS8 lipase from Antarctic Pseudomonas fluorescens strain AMS8 was found to have three RTX motifs. So, this research aimed to examine the influence of Ca2+ ion towards the activity and folding of AMS8 lipase through various biophysical characterizations. The results showed that CaCl2 increased lipase activity. The far-UV circular dichroism (CD) and Fourier-transform infrared (FTIR) analysis suggested that the secondary structure content was improved with the addition of CaCl2. Fluorescence spectroscopy analysis showed that the presence of CaCl2 increased protein folding and compactness. Dynamic light scattering (DLS) analysis suggested that AMS8 lipase became aggregated at a high concentration of CaCl2.The binding constant (Kd) value from the isothermal titration calorimetry (ITC) analysis proved that the Ca2+ ion was tightly bound to the AMS8 lipase. In conclusion, Ca2+ ions play crucial roles in the activity and folding of the AMS8 lipase. Calcium binding to RTX nonapeptide repeats sequences will induced the formation and folding of the RTX parallel ß-roll motif repeat structure.

New Recombinant Cold-Adapted and Organic Solvent Tolerant Lipase from Psychrophilic Pseudomonas sp. LSK25, Isolated from Signy Island Antarctica.

In recent years, studies on psychrophilic lipases have become an emerging area of research in the field of enzymology. The study described here focuses on the cold-adapted organic solvent tolerant lipase strain Pseudomonas sp. LSK25 isolated from Signy Station, South Orkney Islands, maritime Antarctic. Strain LSK25 lipase was successfully cloned, sequenced, and over-expressed in an Escherichia coli system. Sequence analysis revealed that the lipase gene of Pseudomonas sp. LSK25 consists of 1432 bp, lacks an N-terminal signal peptide and encodes a mature protein consisting of 476 amino acids. The recombinant LSK25 lipase was purified by single-step purification using Ni-Sepharose affinity chromatography and had a molecular mass of approximately 65 kDa. The final recovery and purification fold were 44% and 1.3, respectively. The LSK25 lipase was optimally active at 30 °C and at pH 6. Stable lipolytic activity was reported between temperatures of 5⁻30 °C and at pH 6⁻8. A significant enhancement of lipolytic activity was observed in the presence of Ca2+ ions, the organic lipids of rice bran oil and coconut oil, a synthetic C12 ester and a wide range of water immiscible organic solvents. Overall, lipase strain LSK25 is a potentially desirable candidate for biotechnological application, due to its stability at low temperatures, across a range of pH and in organic solvents.

A Novel Mini Protein Design of Haloalkane Dehalogenase.

The application of native enzymes may not be economical owing to the stability factor. A smaller protein molecule may be less susceptible to external stresses. Haloalkane dehalogenases (HLDs) that act on toxic haloalkanes may be incorporated as bioreceptors to detect haloalkane contaminants. Therefore, this study aims to develop mini proteins of HLD as an alternative bioreceptor which was able to withstand extreme conditions. Initially, the mini proteins were designed through computer modeling. Based on the results, five designed mini proteins were deemed to be viable stable mini proteins. They were then validated through experimental study. The smallest mini protein (model 5) was chosen for subsequent analysis as it was expressed in soluble form. No dehalogenase activity was detected, thus the specific binding interaction of between 1,3-dibromopropane with mini protein was investigated using isothermal titration calorimetry. Higher binding affinity between 1,3-dibromopropane and mini protein was obtained than the native. Thermal stability study with circular dichroism had proven that the mini protein possessed two times higher Tm value at 83.73 °C than the native at 43.97 °C. In conclusion, a stable mini protein was successfully designed and may be used as bioreceptors in the haloalkane sensor that is suitable for industrial application.