Expression and biochemical characterization of a novel thermostable alkaline ß-1,3-1,4-glucanase (lichenase) from an alkaliphilic Bacillus lehensis G1.

New thermostable ß-1,3-1,4-glucanase (lichenase) designated as Blg29 was expressed and purified from a locally isolated alkaliphilic bacteria Bacillus lehensis G1. The genome sequence of B. lehensis predicted an open reading frame of Blg29 with a deduced of 249 amino acids and a molecular weight of 28.99 kDa. The gene encoding for Blg29 was successfully amplified via PCR and subsequently expressed as a recombinant protein using the E. coli expression system. Recombinant Blg29 was produced as a soluble form and further purified via immobilized metal ion affinity chromatography (IMAC). Based on biochemical characterization, recombinant Blg29 showed optimal activity at pH9 and temperature 60 °C respectively. This enzyme was stable for more than 2 h, incubated at 50 °C, and could withstand ∼50 % of its activity at 70 °C for an hour and a half. No significant effect on Blg29 was observed when incubated with metal ions except for a small increase with ion Ca2+. Blg29 showed high substrate activity towards lichenan where Vm, Km, Kcat, and kcat/Km values were 2040.82 µmolmin‾1mg‾1, 4.69 mg/mL, and 986.39 s‾1 and 210.32 mLs‾1mg‾1 respectively. The high thermostability and activity make this enzyme useable for a broad prospect in industry applications.

Purification, and characterization of detergent-compatible serine protease from Bacillussafensis strain PRN1: A sustainable alternative to hazardous chemicals in detergent industry.

Owing to vast therapeutic, commercial, and industrial applications of microbial proteases microorganisms from different sources are being explored. In this regard, the gut microbiota of Monopteruscuchia were isolated and examined for the production of protease. All the isolates were primarily and secondarily screened on skim milk and gelatin agar plates. The protease-positive isolates were characterized morphologically, biochemically, and molecularly. Out of the 20 isolated strains,6 belonging to five different genera viz.Bacillus,Priestia,Aeromonas,Staphylococcus, and Serratia demonstrated proteolytic activity. Bacillussafensis strain PRN1 demonstrated the highest protease production and, thus, the largest hydrolytic clear zones in both skim milk agar (15 ± 1 mm) and gelatin (16 ± 1 mm) plates. The optimized parameters (time, pH, temperature, carbon, nitrogen) for highest protease activity and microbial growth of B.safensis strain PRN1 includes 72 h (OD600 = 0.56,1303 U/mL), pH 8 (OD600 = 0.83, 403.29 U/mL), 40 °C (OD600 = 1.75, 1849.11 U/mL), fructose (OD600 = 1.22, 1502 U/mL), and gelatin (OD600 = 1.88, 1015.33 U/mL). The enzyme was purified to homogeneity using salt-precipitation and gel filtration chromatography. The sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) demonstrated that the purified enzyme was a monomer of a molecular weight of ∼33 kDa. The protease demonstrated optimal activity at pH 8 and 60 °C. It was strongly inhibited by phenylmethylsulfonyl fluoride (PMSF), demonstrating that it belongs to the serine-proteases family. The compatibility of the enzyme with surfactants and commercial detergents demonstrates its potential use in the detergent industry. Furthermore, the purified enzyme showed antibacterial and blood-stain removal properties.

Purification, functional characterization and enhanced production of serratiopeptidase from Serratia marcescens MES-4: An endophyte isolated from Morus rubra.

Serratiopeptidase, a proteolytic enzyme serves as an important anti-inflammatory and analgesic medication. Present study reports the production and purification of extracellular serratiopeptidase from an endophyte, Serratia marcescens MES-4, isolated from Morus rubra. Purification of the enzyme by Ion exchange chromatography led to the specific activity of 13,030 U/mg protein of serratiopeptidase, showcasing about 3.1 fold enhanced activity. The catalytic domain of the purified serratiopeptidase, composed of Zn coordinated with three histidine residues (His 209, His 213, and His 219), along with glutamate (Glu 210) and tyrosine (Tyr 249). The molecular mass, as determined by SDS-PAGE was ∼51 kDa. The purified serratiopeptidase displayed optimal activity at pH 9.0, temperature 50°C. Kinetic studies revealed Vmax and Km values of 33,333 U/mL and 1.66 mg/mL, respectively. Further, optimized conditions for the production of serratiopeptidase by Taguchi design led to the productivity of 87 U/mL/h with 87.9 fold enhanced production as compared to the previous conditions.

Purification, crystallization and preliminary crystallographic analysis of Chlamydophila pneumoniae AP endonuclease IV.

Base excision is a crucial DNA repair process mediated by endonuclease IV in nucleotide excision. In Chlamydia pneumoniae, CpendoIV is the exclusive AP endonuclease IV, exhibiting DNA replication error-proofreading capabilities, making it a promising target for anti-chlamydial drug development. Predicting the structure of CpendoIV, molecular docking with DNA was performed, analyzing complex binding sites and protein surface electrostatic potential. Comparative structural studies were conducted with E. coli EndoIV and DNA complex containing AP sites.CpendoIV was cloned, expressed in E. coli, and purified via Ni-NTA chelation and size-exclusion chromatography. Low NaCl concentrations induced aggregation during purification, while high concentrations enhanced purity.CpendoIV recognizes and cleaving AP sites on dsDNA, and Zn2+ influences the activity. Crystallization was achieved under 8% (v/v) Tacsimate pH 5.2, 25% (w/v) polyethylene glycol 3350, and 1.91 Å resolution X-ray diffraction data was obtained at 100 K. This research is significant for provides a deeper understanding of CpendoIV involvement in the base excision repair process, offering insights into Chlamydia pneumoniae.

Screening and functional analysis of three Spiroplasma eriocheiris glycosylated protein interactions with Macrobrachium nipponense C-type lectins.

N-glycosylation, one of the main protein posttranslational modifications (PTMs), plays an important role in the pathogenic process of pathogens through binding and invasion of host cells or regulating the internal environment of host cells to benefit their survival. However, N-glycosylation has remained mostly unexplored in Spiroplasma eriocheiris, a novel type of pathogen which has serious adverse effects on aquaculture. In most cases, N-glycoproteins can be detected and analyzed by lectins dependent on sugar recognition domains. In this study, three Macrobrachium nipponense C-type lectins, namely, MnCTLDcp1, MnCTLDcp2 and MnCTLDcp3, were used to screen S. eriocheiris glycosylated proteins. First, qRT-PCR results showed that the expression levels of the three kinds of lectins were all significantly up-regulated in prawn hearts when the host was against S. eriocheiris infection. A bacterial binding assay showed that purified recombinant MnCTLDcp1, MnCTLDcp2 and MnCTLDcp3 could directly bind to S. eriocheiris in vitro. Second, three S. eriocheiris glycosylated proteins, ATP synthase subunit beta (ATP beta), molecular chaperone Dnak (Dnak) and fructose bisphosphate aldolase (FBPA), were screened and identified using the three kinds of full-length C-type lectins. Far-Western blot and coimmunoprecipitation (CO-IP) further demonstrated that there were interactions between the three lectins with ATP beta, Dnak and FBPA. Furthermore, antibody neutralization assay results showed that pretreatment of S. eriocheiris with ATP beta, Dnak and FBPA antibodies could significantly block this pathogen infection. All the above studies showed that the glycosylated protein played a vital role in the process of S. eriocheiris infection.

Characterization of a Novel Alginate Lyase with Two Alginate Lyase Domains from the Marine Bacterium Vibrio sp. C42.

Alginate is abundant in the cell walls of brown algae. Alginate lyases can degrade alginate, and thus play an important role in the marine carbon cycle and industrial production. Currently, most reported alginate lyases contain only one functional alginate lyase domain. AlyC8 is a putative alginate lyase with two alginate lyase domains (CD1 and CD2) from the marine alginate-degrading strain Vibrio sp. C42. To characterize AlyC8 and its two catalytic domains, AlyC8 and its two catalytic domain-deleted mutants, AlyC8-CD1 and AlyC8-CD2, were expressed in Escherichia coli. All three proteins have noticeable activity toward sodium alginate and exhibit optimal activities at pH 8.0-9.0 and at 30-40 °C, demonstrating that both CD1 and CD2 are functional. However, CD1 and CD2 showed opposite substrate specificity. The differences in substrate specificity and degradation products of alginate between the mutants and AlyC8 demonstrate that CD1 and CD2 can act synergistically to enable AlyC8 to degrade various alginate substrates into smaller oligomeric products. Moreover, kinetic analysis indicated that AlyC8-CD1 plays a major role in the degradation of alginate by AlyC8. These results demonstrate that AlyC8 is a novel alginate lyase with two functional catalytic domains that are synergistic in alginate degradation, which is helpful for a better understanding of alginate lyases and alginate degradation.

Mycobacterial MenG: Partial Purification, Characterization, and Inhibition.

Menaquinone (MK) is an essential component of the electron transport chain (ETC) in the gram-variable Mycobacterium tuberculosis and many Gram-positive pathogens. Three genes in the M. tuberculosis genome were annotated as methyltransferases involved in lipoquinone synthesis in mycobacteria. Heterologous expression of Rv0558 complemented an ubiE (the quinone C-methyltransferase involved in ubiquinone and menaquinone synthesis) deletion in Escherichia coli, and expression in a wild-type E. coli strain increased quinone C-methyltransferase specific activity by threefold. Rv0558 encodes a canonical C-methyltransferase or, more specifically, a S-adenosylmethionine/demethylmenaquinol methyltransferase. Partially purified recombinant protein catalyzed the formation of MK from demethylmenaquinone (DMK), although the activity of the recombinant protein was low and appeared to require a cofactor or intact membrane structure for activity. Membrane preparations from irradiated M. tuberculosis also showed poor activity; however, membrane preparations from wild-type Mycobacterium smegmatis showed robust, substrate-dependent activity. The apparent Km values for demethylmenaquinone and SAM were 14 ± 5.0 and 17 ± 7.0 µM, respectively. Interestingly, addition of dithiothreitol, dithionite, NADH, or other substrates of primary dehydrogenases to reaction mixtures containing membrane preparations stimulated the activity. Thus, these observations strongly suggest that demethylmenaquinol is the actual substrate of MenG. Ro 48-8071, previously reported to inhibit mycobacterial MK synthesis and growth, inhibited Rv0558 activity with an IC50 value of 5.1 ± 0.5 µM, and DG70 (GSK1733953A), first described as a respiration inhibitor in M. tuberculosis, inhibits MenG activity with an IC50 value of 2.6 ± 0.6 µM.

Identification and Characterization of Three Chitinases with Potential in Direct Conversion of Crystalline Chitin into N,N'-diacetylchitobiose.

Chitooligosaccharides (COSs) have been widely used in agriculture, medicine, cosmetics, and foods, which are commonly prepared from chitin with chitinases. So far, while most COSs are prepared from colloidal chitin, chitinases used in preparing COSs directly from natural crystalline chitin are less reported. Here, we characterize three chitinases, which were identified from the marine bacterium Pseudoalteromonas flavipulchra DSM 14401T, with an ability to degrade crystalline chitin into (GlcNAc)2 (N,N'-diacetylchitobiose). Strain DSM 14401 can degrade the crystalline α-chitin in the medium to provide nutrients for growth. Genome and secretome analyses indicate that this strain secretes six chitinolytic enzymes, among which chitinases Chia4287, Chib0431, and Chib0434 have higher abundance than the others, suggesting their importance in crystalline α-chitin degradation. These three chitinases were heterologously expressed, purified, and characterized. They are all active on crystalline α-chitin, with temperature optima of 45-50 °C and pH optima of 7.0-7.5. They are all stable at 40 °C and in the pH range of 5.0-11.0. Moreover, they all have excellent salt tolerance, retaining more than 92% activity after incubation in 5 M NaCl for 10 h at 4 °C. When acting on crystalline α-chitin, the main products of the three chitinases are all (GlcNAc)2, which suggests that chitinases Chia4287, Chib0431, and Chib0434 likely have potential in direct conversion of crystalline chitin into (GlcNAc)2.

Biochemical Properties of a New Polysaccharide Lyase Family 25 Ulvan Lyase TsUly25B from Marine Bacterium Thalassomonas sp. LD5.

Marine macroalgae, contributing much to the bioeconomy, have inspired tremendous attention as sustainable raw materials. Ulvan, as one of the main structural components of green algae cell walls, can be degraded by ulvan lyase through the ß-elimination mechanism to obtain oligosaccharides exhibiting several good physiological activities. Only a few ulvan lyases have been characterized until now. This thesis explores the properties of a new polysaccharide lyase family 25 ulvan lyase TsUly25B from the marine bacterium Thalassomonas sp. LD5. Its protein molecular weight was 54.54 KDa, and it was most active under the conditions of 60 °C and pH 9.0. The Km and kcat values were 1.01 ± 0.05 mg/mL and 10.52 ± 0.28 s-1, respectively. TsUly25B was salt-tolerant and NaCl can significantly improve its thermal stability. Over 80% of activity can be preserved after being incubated at 30 °C for two days when the concentration of NaCl in the solution is above 1 M, while 60% can be preserved after incubation at 40 °C for 10 h with 2 M NaCl. TsUly25B adopted an endolytic manner to degrade ulvan polysaccharides, and the main end-products were unsaturated ulvan disaccharides and tetrasaccharides. In conclusion, our research enriches the ulvan lyase library and advances the utilization of ulvan lyases in further fundamental research as well as ulvan oligosaccharides production.

Expression, Purification, and Comparative Inhibition of Helicobacter pylori Urease by Regio-Selectively Alkylated Benzimidazole 2-Thione Derivatives.

The urease enzyme has been an important target for the discovery of effective pharmacological and agricultural products. Thirteen regio-selectively alkylated benzimidazole-2-thione derivatives have been designed to carry the essential features of urease inhibitors. The urease enzyme was isolated from Helicobacter pylori as a recombinant urease utilizing the His-tag method. The isolated enzyme was purified and characterized using chromatographic and FPLC techniques showing a maximal activity of 200 mg/mL. Additionally, the commercial Jack bean urease was purchased and included in this study for comparative and mechanistic investigations. The designed compounds were synthesized and screened for their inhibitory activity against the two ureases. Compound 2 inhibited H. pylori and Jack bean ureases with IC50 values of 0.11; and 0.26 mM; respectively. While compound 5 showed IC50 values of 0.01; and 0.29 mM; respectively. Compounds 2 and 5 were docked against Helicobacter pylori urease (PDB ID: 1E9Y; resolution: 3.00 Å) and exhibited correct binding modes with free energy (ΔG) values of -9.74 and -13.82 kcal mol-1; respectively. Further; the in silico ADMET and toxicity properties of 2 and 5 indicated their general safeties and likeness to be used as drugs. Finally, the compounds' safety was authenticated by an in vitro cytotoxicity assay against fibroblast cells.

Conformational trapping of an ABC transporter in polymer lipid nanoparticles.

ATP-binding cassette (ABC) proteins play important roles in cells as importers and exporters but as membrane proteins they are subject to well-known challenges of isolating pure and stable samples for study. One solution to this problem is to use styrene-maleic acid lipid particles (SMALPs). Styrene-maleic acid (SMA) can be added directly to membranes, forming stable nanoparticles incorporating membrane proteins and lipids. Here we use Sav1866, a well-characterised bacterial protein, as a proxy for ABC proteins in general. We show that stable and monodispersed Sav1866 can be purified at high yield using SMA. This protein can be used for biophysical characterisations showing that its overall structure is consistent with existing evidence. However, like other ABC proteins in SMALPs it does not hydrolyse ATP. The lack of ATPase activity in ABC-SMALPs may result from conformational trapping of the proteins in SMALPs. Undertaken in a controlled manner, conformational trapping is a useful tool to stabilise protein samples into a single conformation for structural studies. Due to their inability to hydrolyse ATP, the conformation of Sav1866-SMALPs cannot be altered using ATP and vanadate after purification. To achieve controlled trapping of Sav1866-SMALPs we show that Sav1866 in crude membranes can be incubated with ATP, magnesium and sodium orthovanadate. Subsequent solubilisation and purification with SMA produces a sample of Sav1866-SMALPs with enhanced stability, and in a single conformational state. This method may be generally applicable to vanadate-sensitive ABC proteins and overcomes a limitation of the SMALP system for the study of this protein family.

Anti-mycobacterial activity of heat and pH stable high molecular weight protein(s) secreted by a bacterial laboratory contaminant.

BACKGROUND: Tuberculosis currently stands as the second leading cause of deaths worldwide due to single  infectious agent after Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). The current challenges of drug resistance in tuberculosis highlight an urgent need to develop newer anti-mycobacterial compounds. In the present study, we report the serendipitous discovery of a bacterial laboratory contaminant (LC-1) exhibiting a zone of growth inhibition on an agar plate seeded with Mycobacterium tuberculosis. RESULTS: We utilized microbiological, biochemical and biophysical approaches to characterize LC-1 and anti-mycobacterial compound(s) in its secretome. Based on 16S rRNA sequencing and BIOLOG analysis, LC-1 was identified as Staphylococcus hominis, a human bacterial commensal. Anti-mycobacterial activity was initially found in 30 kDa retentate that was obtained by ultrafiltration of culture filtrate (CF). SDS-PAGE analysis of peak fractions obtained by size exclusion chromatography of 30 kDa retentate confirmed the presence of high molecular weight (≥ 30 kDa) proteins. Peak fraction-1 (F-1) exhibited inhibitory activity against M. bovis BCG, but not against M. smegmatis, E. coli and S. aureus. The active fraction F-1 was inactivated by treatment with Proteinase K and α-chymotrypsin. However, it retained its anti-mycobacterial activity over a wide range of heat and pH treatment. The anti-mycobacterial activity of F-1 was found to be maintained even after a long storage (~12 months) at - 20 °C. Mass spectrometry analysis revealed that the identified peptide masses do not match with any previously known bacteriocins. CONCLUSIONS: The present study highlights the anti-mycobacterial activity of high molecular weight protein(s) present in culture filtrate of LC-1, which may be tested further to target M. tuberculosis. The heat and pH stability of these proteins add to their characteristics as therapeutic proteins and may contribute to their long shelf life. LC-1 being a human commensal can be tested in future for its potential as a probiotic to treat tuberculosis.

Discovery of a novel antibacterial protein CB6-C to target methicillin-resistant Staphylococcus aureus.

Given a serious threat of multidrug-resistant bacterial pathogens to global healthcare, there is an urgent need to find effective antibacterial compounds to treat drug-resistant bacterial infections. In our previous studies, Bacillus velezensis CB6 with broad-spectrum antibacterial activity was obtained from the soil of Changbaishan, China. In this study, with methicillin-resistant Staphylococcus aureus as an indicator bacterium, an antibacterial protein was purified by ammonium sulfate precipitation, Sephadex G-75 column, QAE-Sephadex A 25 column and RP-HPLC, which demonstrated a molecular weight of 31.405 kDa by SDS-PAGE. LC-MS/MS analysis indicated that the compound was an antibacterial protein CB6-C, which had 88.5% identity with chitosanase (Csn) produced by Bacillus subtilis 168. An antibacterial protein CB6-C showed an effective antimicrobial activity against gram-positive bacteria (in particular, the MIC for MRSA was 16 µg/mL), low toxicity, thermostability, stability in different organic reagents and pH values, and an additive effect with conventionally used antibiotics. Mechanistic studies showed that an antibacterial protein CB6-C exerted anti-MRSA activity through destruction of lipoteichoic acid (LTA) on the cell wall. In addition, an antibacterial protein CB6-C was efficient in preventing MRSA infections in in vivo models. In conclusion, this protein CB6-C is a newly discovered antibacterial protein and has the potential to become an effective antibacterial agent due to its high therapeutic index, safety, nontoxicity and great stability.

Solid-state fermentation production and characterization of an alkaline lipase from a newly isolated Burkholderia gladioli strain.

The newly isolated Burkholderia gladioli BRM58833 strain was shown to secrete an alkaline lipase highly active and stable in organic solvents. Lipase production was optimized through the cultivation of the strain by solid-state fermentation in wheat bran. The lipase extraction conditions were also optimized. The low-cost extract obtained has shown a high hydrolytic activity of 1096.7 ± 39.3 U·gds-1 (units per gram of dry solids) against pNPP and 374.2 ± 20.4 U·gds-1 against triolein. Proteomic analysis revealed the optimized extract is composed of two esterases and three true lipases, showing a preference for long-chain substrates. The highest activity was obtained at 50 °C and pH 9. However, the extract maintained more than 50% of its maximum activity between pH 8.0 and 10.0 and throughout the whole temperature range evaluated (32-70 °C). The enzymes were inhibited by SDS, EDTA, ZnSO4 and FeCl3 and activated by FeSO4, MgCl2 and BaCl2. The lipases conserved their activity when incubated in solvents as acetonitrile, diethyl ether, n-heptane n-hexane, toluene, methanol and t-butanol. The resistance of these lipases to solvents and expressive thermostability when compared to other lipases, reveal their potential both in hydrolysis reactions and in synthesis of esters.

Expression, purification and preliminary characterisation of the choline transporter LicB from opportunistic bacterial pathogens.

Many opportunistic bacteria that infect the upper respiratory tract decorate their cell surface with phosphorylcholine to support colonisation and outgrowth. These surface modifications require the active import of choline from the host environment, a process thought to be mediated by a family of dedicated integral membrane proteins that act as choline permeases. Here, we present the expression and purification of the archetype of these choline transporters, LicB from Haemophilus influenzae. We show that LicB can be recombinantly produced in Escherichia coli and purified to homogeneity in a stable, folded state using the detergent n-dodecyl-ß-d-maltopyranoside. Equilibrium binding studies with the fluorescent ligand dansylcholine suggest that LicB is selective towards choline, with reduced affinity for acetylcholine and no apparent activity towards other small molecules including glycine, carnitine and betaine. We also identify a conserved sequence motif within the LicB family and show that mutations within this motif compromise protein structure and function. Our results are consistent with previous observations that LicB is a specific high-affinity choline transporter, and provide an experimental platform for further studies of this permease family.

Characterization of a Novel Shewanella algae Arginine Decarboxylase Expressed in Escherichia coli.

Arginine decarboxylase (ADC) catalyzes the decarboxylation of arginine to form agmatine, an important physiological and pharmacological amine, and attracts attention to the enzymatic production of agmatine. In this study, we for the first time overexpressed and characterized the marine Shewanella algae ADC (SaADC) in Escherichia coli. The recombinant SaADC showed the maximum activity at pH 7.5 and 40 °C. The SaADC displayed previously unreported substrate inhibition when the substrate concentration was higher than 50 mM, which was the upper limit of testing condition in other reports. In the range of 1-80 mM L-arginine, the SaADC showed the Km, kcat, Ki, and kcat/Km values of 72.99 ± 6.45 mM, 42.88 ± 2.63 s-1, 20.56 ± 2.18 mM, and 0.59 s/mM, respectively, which were much higher than the Km (14.55 ± 1.45 mM) and kcat (12.62 ± 0.68 s-1) value obtained by assaying at 1-50 mM L-arginine without considering substrate inhibition. Both the kcat values of SaADC with and without substrate inhibition are the highest ones to the best of our knowledge. This provides a reference for the study of substrate inhibition of ADCs.

Identification of FtpA, a Dps-Like Protein Involved in Anti-Oxidative Stress and Virulence in Actinobacillus pleuropneumoniae.

Bacteria have evolved a variety of enzymes to eliminate endogenous or host-derived oxidative stress factors. The Dps protein, first identified in Escherichia coli, contains a ferroxidase center, and protects bacteria from reactive oxygen species damage. Little is known of the role of Dps-like proteins in bacterial pathogenesis. Actinobacillus pleuropneumoniae causes pleuropneumonia, a respiratory disease of swine. The A. pleuropneumoniae ftpA gene is upregulated during shifts to anaerobiosis, in biofilms and, as found in this study, in the presence of H2O2. An A. pleuropneumoniae ftpA deletion mutant (ΔftpA) had increased H2O2 sensitivity, decreased intracellular viability in macrophages, and decreased virulence in a mouse infection model. Expression of ftpA in an E. coli dps mutant restored wild-type H2O2 resistance. FtpA possesses a conserved ferritin domain containing a ferroxidase site. Recombinant rFtpA bound and oxidized Fe2+ reversibly. Under aerobic conditions, the viability of an ΔftpA mutant was reduced compared with the wild-type strain after extended culture, upon transition from anaerobic to aerobic conditions, and upon supplementation with Fenton reaction substrates. Under anaerobic conditions, the addition of H2O2 resulted in a more severe growth defect of ΔftpA than it did under aerobic conditions. Therefore, by oxidizing and mineralizing Fe2+, FtpA alleviates the oxidative damage mediated by intracellular Fenton reactions. Furthermore, by mutational analysis, two residues were confirmed to be critical for Fe2+ binding and oxidization, as well as for A. pleuropneumoniae H2O2 resistance. Taken together, the results of this study demonstrate that A. pleuropneumoniae FtpA is a Dps-like protein, playing critical roles in oxidative stress resistance and virulence. IMPORTANCE As a ferroxidase, Dps of Escherichia coli can protect bacteria from reactive oxygen species damage, but its role in bacterial pathogenesis has received little attention. In this study, FtpA of the swine respiratory pathogen A. pleuropneumoniae was identified as a new Dps-like protein. It facilitated A. pleuropneumoniae resistance to H2O2, survival in macrophages, and infection in vivo. FtpA could bind and oxidize Fe2+ through two important residues in its ferroxidase site and protected the bacteria from oxidative damage mediated by the intracellular Fenton reaction. These findings provide new insights into the role of the FtpA-based antioxidant system in the pathogenesis of A. pleuropneumoniae, and the conserved Fe2+ binding ligands in Dps/FtpA provide novel drug target candidates for disease prevention.

Artificial Neural Networks and Response Surface Methodology Approach for Optimization of an Eco-Friendly and Detergent-Stable Lipase Production from Actinomadura Keratinilytica Strain Cpt29.

This work mainly focused on the production of an efficient, economical, and eco-friendly lipase (AKL29) from Actinomadura keratinilytica strain Cpt29 isolated from poultry compost in north east of Algeria, for use in detergent industries. AKL29 shows a significant lipase activity (45 U/mL) towards hydrolyzed triacylglycerols, indicating that it is a true lipase. For maximum lipase production the modeling and optimization of potential culture parameters such as incubation temperature, cultivation time, and Tween 80 (v/v) were built using RSM and ANN approaches. The results show that both the two models provided good quality predictions, yet the ANN showed a clear superiority over RSM for both data fitting and estimation capabilities. A 4.1-fold increase in lipase production was recorded under the following optimal condition: incubation temperature (37.9 °C), cultivation time (111 h), and Tween 80 (3.27%, v/v). Furthermore, the partially purified lipase showed good stability, high compatibility, and significant wash performance with various commercial laundry detergents, making this novel lipase a promising potential candidate for detergent industries.

Highly Active Thermophilic L-Asparaginase from Melioribacter roseus Represents a Novel Large Group of Type II Bacterial L-Asparaginases from Chlorobi-Ignavibacteriae-Bacteroidetes Clade.

L-asparaginase (L-ASNase) is a biotechnologically relevant enzyme for the pharmaceutical, biosensor and food industries. Efforts to discover new promising L-ASNases for different fields of biotechnology have turned this group of enzymes into a growing family with amazing diversity. Here, we report that thermophile Melioribacter roseus from Ignavibacteriae of the Bacteroidetes/Chlorobi group possesses two L-ASNases-bacterial type II (MrAII) and plant-type (MrAIII). The current study is focused on a novel L-ASNase MrAII that was expressed in Escherichia coli, purified and characterized. The enzyme is optimally active at 70 °C and pH 9.3, with a high L-asparaginase activity of 1530 U/mg and L-glutaminase activity ~19% of the activity compared with L-asparagine. The kinetic parameters KM and Vmax for the enzyme were 1.4 mM and 5573 µM/min, respectively. The change in MrAII activity was not significant in the presence of 10 mM Ni2+, Mg2+ or EDTA, but increased with the addition of Cu2+ and Ca2+ by 56% and 77%, respectively, and was completely inhibited by Zn2+, Fe3+ or urea solutions 2-8 M. MrAII displays differential cytotoxic activity: cancer cell lines K562, Jurkat, LnCap, and SCOV-3 were more sensitive to MrAII treatment, compared with normal cells. MrAII represents the first described enzyme of a large group of uncharacterized counterparts from the Chlorobi-Ignavibacteriae-Bacteroidetes clade.

A novel protein fusion partner, carbohydrate-binding module family 66, to enhance heterologous protein expression in Escherichia coli.

BACKGROUND: Proteins with novel functions or advanced activities developed by various protein engineering techniques must have sufficient solubility to retain their bioactivity. However, inactive protein aggregates are frequently produced during heterologous protein expression in Escherichia coli. To prevent the formation of inclusion bodies, fusion tag technology has been commonly employed, owing to its good performance in soluble expression of target proteins, ease of application, and purification feasibility. Thus, researchers have continuously developed novel fusion tags to expand the expression capacity of high-value proteins in E. coli. RESULTS: A novel fusion tag comprising carbohydrate-binding module 66 (CBM66) was developed for the soluble expression of heterologous proteins in E. coli. The target protein solubilization capacity of the CBM66 tag was verified using seven proteins that are poorly expressed or form inclusion bodies in E. coli: four human-derived signaling polypeptides and three microbial enzymes. Compared to native proteins, CBM66-fused proteins exhibited improved solubility and high production titer. The protein-solubilizing effect of the CBM66 tag was compared with that of two commercial tags, maltose-binding protein and glutathione-S-transferase, using poly(ethylene terephthalate) hydrolase (PETase) as a model protein; CBM66 fusion resulted in a 3.7-fold higher expression amount of soluble PETase (approximately 370 mg/L) compared to fusion with the other commercial tags. The intact PETase was purified from the fusion protein upon serial treatment with enterokinase and affinity chromatography using levan-agarose resin. The bioactivity of the three proteins assessed was maintained even when the CBM66 tag was fused. CONCLUSIONS: The use of the CBM66 tag to improve soluble protein expression facilitates the easy and economic production of high-value proteins in E. coli.