Broad pH Stability Research Articles

Novel Thermophilic Bacterial Laccase for the Degradation of Aromatic Organic Pollutants.

We identified a putative laccase from the thermophilic bacterium Geobacillus yumthangensis. The putative laccase was produced recombinantly and its ability to catalyse the degradation of aromatic organic pollutants was investigated. The putative laccase exhibits broad pH and temperature stability, and, notably, it could catalyse the degradation of organic dyes as well as toxic pollutants including bisphenol A, guaiacol and phenol with a redox mediator. Our work further demonstrates the potential of using oxidative enzymes to break down toxic chemicals that possess major threats to human health and the environment.

Heterologous expression and characterization of thermostable chitinase and β-N-acetylhexosaminidase from Caldicellulosiruptor acetigenus and their synergistic action on the bioconversion of chitin into N-acetyl-d-glucosamine

The bioconversion of chitin into N-acetyl-d-glucosamine (GlcNAc) using chitinolytic enzymes is one of the important avenues for chitin valorization. However, industrial applications of chitinolytic enzymes have been limited by their poor thermostability. Therefore, it is necessary to discover thermostable chitinolytic enzymes for GlcNAc production from chitin. In this study, two chitinolytic enzyme-encoding genes CaChiT and CaHex from Caldicellulosiruptor acetigenus were identified and heterologously expressed in Escherichia coli. The purified recombinant CaChiT and CaHex showed optimal activities at 70 °C and 90 °C respectively, and exhibited good thermostability over a range of temperature below 70 °C and broad pH stability at pH range of 3.0-8.0. CaChiT and CaHex were active on colloidal chitin, pNP-(GlcNAc)2, pNP-(GlcNAc)3, and pNP-GlcNAc, pNP-(GlcNAc)2, pNP-(GlcNAc)3, pNP-Glc respectively. Besides, the chitin oligosaccharides and colloidal chitin hydrolysis profiles revealed that CaChiT degraded chitin chains through exo-mode of action. Furthermore, CaChiT and CaHex exhibited a synergistic effect in the degradation of colloidal chitin, reaching 0.60 mg/mL of GlcNAc production after 1 h incubation. These results suggested that a combination of CaChiT and CaHex have great potential for industrial applications in the enzymatic production of GlcNAc from chitin-containing biowastes.

A chlorogenic acid esterase from a metagenomic library with unique substrate specificity and its application in caffeic and ferulic acid production from agricultural byproducts

Soil microbes are an abundant source of enzymes with unique properties that may be useful for industrial applications. As most wild-type strains show low chlorogenic acid esterase expression and activity, and most microbes cannot be cultured in the laboratory, a metagenomic approach provides methods of identifying new enzymes. In this study, a gene encoding a chlorogenic acid esterase, named Tan410, was isolated from a soil metagenomic library and overexpressed in Escherichia coli BL21 (DE3). The recombinant enzyme, with a predicted molecular weight of 54.88 kDa, was purified to homogeneity. The K m and V max values for Tan410 were 1.26 mM and 0.33 mM min–1, respectively, with chlorogenic acid as the substrate. Its optimum temperature and pH for reaction were 30 °C and 7.5, respectively. The enzyme exhibited moderate thermostability and broad pH stability (3.0–10.0). Tan410 was also able to hydrolyse ethyl ferulate, methyl caffeate, propyl gallate, ethyl gallate, methyl vanillate, methyl benzoate, ethyl benzoate, methyl 2,5-dihydroxybenzoate, and methyl 3,5-dihydroxybenzoate, and it released caffeic and ferulic acids from agricultural byproducts (destarched wheat bran and coffee pulp). Tan140 has potential for industrial application in biomass valorization.

A Magnetocatalytic Propelled Cobalt–Platinum@Graphene Navigator for Enhanced Tumor Penetration and Theranostics

A Magnetocatalytic Propelled Cobalt–Platinum@Graphene Navigator for Enhanced Tumor Penetration and Theranostics

Expression of an alkaline feruloyl esterases from thermophilic Chaetomium thermophilum and its boosting effect on delignification of pulp

Exploration of feruloyl esterase (FAE) with the resistance to heat and alkali conditions in biobleaching process to improve the separation efficiency of lignocellulose is the key to achieving green papermaking. Herein, we expressed FAEB of C. thermophilum and obtained a thermostable alkaline FAE that can effectively promote the removal of lignin from pulp. The faeB gene was successfully obtained through genomic Blast strategy and high-efficiency expressed under the control of strong alcohol oxidase promoter in Pichia pastoris. The recombinant CtFAEB has an optimal temperature of 65 °C and pH of 7.0. After treated at 65 °C for 1 h, CtFAEB can still retain 63.21 % of its maximum activity, showing a good thermal stability. In addition, the recombinant CtFAEB has broad pH stability and can retain about 56 % of the maximum activity even at pH 11.0. Compared with the effect of mesophilic FAE, pretreatment with thermostable CtFAEB can promote the delignification by laccase and alkaline hydrogen peroxide from the pulp at 70 °C and pH 9.0. Alignment of the protein sequences of CtFAEB and mesophilic FAE suggested that the percentage of amino acids that easily form alpha helix in CtFAEB increases, which enhances its structural rigidity and thereby improves its thermal stability and alkali tolerance. Our study provides an effective method to obtain thermostable and alkaline FAEs, which will promote its application in biobleaching and other biorefining industries.

Degradation of neonicotinoid insecticide acetamiprid by two different nitrile hydratases of Pseudaminobacter salicylatoxidans CGMCC 1.17248

Acetamiprid is a neonicotinoid insecticide used worldwide that has caused environmental pollution and adverse effects on ecosystems. Here, a novel bacterium, Pseudaminobacter salicylatoxidans CGMCC 1.17248, was isolated to rapidly degrade acetamiprid to IM-1-2 via hydration. Gene cloning and overexpression demonstrated that two nitrile hydratases, AnhA and AnhB, converted acetamiprid to IM-1-2. Escherichia coli overexpressing AnhA and AnhB degraded 98.1% and 94.0% of acetamiprid (1.0 mmol L−1) in 5 min and 8 h, respectively. The pure AnhA and AnhB had a Vmax value of 14.12 and 1.20 μmol mg−1 min−1, respectively, and a Km value of 1.02 mmol L−1 and 2.95 mmol L−1, respectively. Compared with AnhA, AnhB had broad pH stability, as well as metal ions and organic solvents tolerance. Expression of AnhA and AnhB was induced by decreasing the nutrient concentration of culture broth and addition of urea and therefore significantly enhanced acetamiprid degradation of CGMCC 1.17248. qPCR indicated that the expression of AnhA and AnhB under the cultured conditions of 1/15 lysogeny broth or 0.5% urea addition was improved by from 2.2 to 5.3 times. The results presented herein will facilitate development of bioremediation agents for acetamiprid pollution and understanding of the functional role of bacterial nitrile hydratase.

Purification, characterization, and mode of action of a novel bacteriocin BM173 from Lactobacillus crustorum MN047 and its effect on biofilm formation of Escherichia coli and Staphylococcus aureus

There is an increasing demand for dairy products, but the presence of food-spoilage bacteria seriously affects the development of the dairy industry. Bacteriocins are considered to be a potential antibacterial or antibiofilm agent that can be applied as a preservative. In this study, bacteriocin BM173 was successfully expressed in the Escherichia coli expression system and purified by a 2-step method. Furthermore, it exhibited a broad-spectrum antibacterial activity, high thermal stability (121°C, 20 min), and broad pH stability (pH 3-11). Moreover, the minimum inhibitory concentration values of BM173 against E. coli ATCC 25922 and Staphylococcus aureus ATCC 25923 were 14.8 μg/mL and 29.6 μg/mL, respectively. Growth and time-kill curves showed that BM173 exhibited antibacterial and bactericidal activity. The results of scanning electron microscopy and transmission electron microscopy demonstrated that BM173 increased membrane permeability, facilitated pore formation, and even promoted cell lysis. The disruption of cell membrane integrity was further verified by propidium iodide uptake and lactic dehydrogenase release. In addition, BM173 exhibited high efficiency in inhibiting biofilm formation. Therefore, BM173 has promising potential as a preservative used in the dairy industry.

A Strategic Production Improvement of Streptomyces Beta Glucanase Enzymes with Aid of Codon Optimization and Heterologous Expression

β-glucan rich cereals such as barley and oats serve as a raw material in breweries and also used as an animal feed. Digestion of β-glucan is often a major hurdle, thus providing exogenous enzyme β-glucanase serves as an option. The present study takes an effort for the expression and over production of β-glucanase genes from Streptomyces sp in E. coli. The exo-β-1,4-glucanase and endo-β-1,3-glucanase encoding genes were isolated and codon optimized and significant-high expression levels were obtained in E. coli strain. The expressed enzymes showed broad pH stability, good thermostability, and better affinity towards the barley β-glucan substrate. The study implies that heterologous expression with codon optimization strategy enhances the production of Streptomyces origin beta-glucanase enzymes with prominent physio-chemical properties for efficient beta-glucan degradation.

The efficient Δ1-dehydrogenation of a wide spectrum of 3-ketosteroids in a broad pH range by 3-ketosteroid dehydrogenase from Sterolibacterium denitrificans

Cholest-4-en-3-one Δ1-dehydrogenase (AcmB) from Sterolibacterium denitrificans, a key enzyme of the central degradation pathway of cholesterol, is a protein catalyzing Δ1-dehydrogenation of a wide range of 3-ketosteroids. In this study, we demonstrate the application of AcmB in the synthesis of 1-dehydro-3-ketosteroids and investigate the influence of reaction conditions on the catalytic performance of the enzyme. The recombinant AcmB expressed in E. coli BL21(DE3)Magic exhibits a broad pH optimum and pH stability in the range of 6.5 to 9.0. The activity-based pH optimum of AcmB reaction depends on the type of electron acceptor (2,6-dichloroindophenol - DCPIP, phenazine methosulfate - PMS or potassium hexacyanoferrate - K3[Fe(CN)6]) used in the biocatalytic process yielding the best kinetic properties for the reaction with a DCPIP/PMS mixture (kcat/Km = 1.4·105 s-1·M-1 at pH 9.0) followed by DCPIP (kcat/Km = 1.0·105 s-1·M-1 at pH = 6.5) and K3[Fe(CN)6] (kcat/Km = 0.5·102 s-1·M-1 at pH = 8.0). The unique feature of AcmB is its capability to convert both testosterone derivatives (C20–C22) as well as steroids substituted at C17 (C27-C30) such as cholest-4-en-3-one or (25R)-spirost-4-en-3-one (diosgenone). Apparent steady-state kinetic parameters were determined for both groups of AcmB substrates. In a batch reactor synthesis, the solubility of water-insoluble steroids was facilitated by the addition of a solubilizer, 2-hydroxypropyl-β-cyclodextrin, and organic co-solvent, 2-methoxyethanol. Catalytic properties characterization of AcmB was tested in fed-batch reactor set-ups, using 0.81 μM of isolated enzyme, PMS and aerobic atmosphere resulting in >99% conversion of the C17–C20 3-ketosteroids within 2 h. Finally, the whole cell E. coli system with recombinant enzyme was demonstrated as an efficient biocatalyst in the synthesis of 1-dehydro-3-ketosteroids.

A thermostable glucose oxidase from Aspergillus heteromophus CBS 117.55 with broad pH stability and digestive enzyme resistance

In this study, the heterologous expression of an engineered thermostablle glucose oxidase from Aspergillus heteromophus CBS 117.55 was achieved in P. pastoris. This recombinant GoxAh was thermostable, with an optimal temperature range 25 °C-65 °C, and it was capable of retaining greater than 90% of its initial activity following a 10-min incubation at 75 °C. This enzyme had an optimum pH of 6.0, and it could retain above 80% of its initial activity following a 2-h incubation at a broad pH range (2.0–8.0). Moreover, GoxAh displayed excellent pepsin and trypsin resistance, and highly resistant to a range of tested metal ions and chemical reagents. These good properties make GoxAh a promising candidate for feed additive. The Km and kcat/Km values of GoxAh were 187 mM and 1.09/mM/s, which limited its widespread application to some degree. However, due to its excellent characteristics, GoxAh is still of potential economic value for high value-added areas, as well as a good initial enzyme for developing applicable feed enzyme by protein engineering.

Thrombolytic Potential of Novel Thiol-Dependent Fibrinolytic Protease from Bacillus cereus RSA1.

The present study demonstrates the production and thrombolytic potential of a novel thermostable thiol-dependent fibrinolytic protease by Bacillus cereus RSA1. Statistical optimization of different parameters was accomplished with Plackett–Burman design and validated further by central composite design with 30.75 U/mL protease production. Precipitation and chromatographic approaches resulted in 33.11% recovery with 2.32-fold purification. The molecular weight of fibrinolytic protease was 40 KDa and it exhibited a broad temperature and pH stability range of 20–80 °C and pH 5–10 with utmost activity at 50 °C and pH 8, respectively. The protease retained its fibrinolytic activity in organic solvents and enhanced the activity in solutions with divalent cations (Mn2+, Zn2+, and Cu2+). The enzyme kinetics revealed Km and Vmax values of 1.093 mg/mL and 52.39 µg/mL/min, respectively, indicating higher affinity of fibrinolytic activity towards fibrin. Also, complete inhibition of fibrinolytic activity with DFP and a 2-fold increase with DTT and β-mercaptoethanol indicates its thiol-dependent serine protease nature. MALDI–TOF analysis showed 56% amino acid sequence homology with Subtilisin NAT OS = Bacillus subtilis subsp. natto. The fibrinolysis activity was compared with a commercial thrombolytic agent for its therapeutic applicability, and fibrinolytic protease was found highly significant with absolute blood clot dissolution within 4 h in in vitro conditions. The isolated fibrinolytic protease of Bacillus cereus RSA1 is novel and different from other known fibrinolytic proteases with high stability and efficacy, which might have wide medicinal and industrial application as a thrombolytic agent and in blood stain removal, respectively.

Expression and characterization of an extremely thermophilic 1,4-α-glucan branching enzyme from Rhodothermus obamensis STB05

A gene encoding 1,4-α-glucan branching enzyme (GBE, EC 2.4.1.18) from the extremely thermophilic bacterium Rhodothermus obamensis STB05 was successfully cloned and expressed in Escherichia coli. Extracellular expression of the recombinant enzyme (R.o-GBE) was achieved with a yield of 1080 mg/L. Then it was purified and further characterized biochemically. R.o-GBE was optimally active at pH 7.0 and 65 °C. It remained stable at temperatures up to 80 °C and had a half-life at 85 °C of approximately 31 min. Far-UV circular dichroism and intrinsic fluorescence analyses revealed that high temperatures reduced its activity by changing the secondary and tertiary structure of R.o-GBE. The enzyme had broad pH stability between pH 3.0 and 11.0 at 4 °C, and preferred weakly acidic conditions at high temperatures. None of the metal ions enhanced the activity of R.o-GBE, but Ca2+ may be required for its activity. Its specific activity with amylopectin was 6651 U/mg, which is much higher than that reported for other GBEs. Its excellent thermostability, broad pH stability, and high specific activity make R.o-GBE highly suitable for industrial applications.

Biological Degradation of Aflatoxin B₁ by Cell-Free Extracts of Bacillus velezensis DY3108 with Broad PH Stability and Excellent Thermostability.

(1) Background: Aflatoxin contamination in food and grain poses serious problems both for economic development and public health protection, thus leading to a focus on an effective approach to control it; (2) Methods: Aflatoxin B1 (AFB1) degrading bacteria were isolated using a medium containing coumarin as the sole carbon source, and the biodegradation of AFB1 by the isolate was examined by high performance liquid chromatography, and liquid chromatography mass spectrometry; (3) Results: a bacterial strain exhibiting strong AFB1 degradation activity (91.5%) was isolated and identified as Bacillus velezensis DY3108. The AFB1 degrading activity was predominantly attributed to the cell-free supernatant of strain DY3108. Besides, it was heat-stable and resistant to proteinase K treatment but sensitive to sodium dodecyl sulfate treatment. The optimal temperature for the maximal degradation of AFB1 was 80 °C. Even more notable, the supernatant showed a high level of activity over a broad pH (4.0 to 11.0) and exhibited the highest degradation (94.70%) at pH 8.0. Cytotoxicity assays indicated that the degradation products displayed significantly (p < 0.05) lower cytotoxic effects than the parent AFB1; (4) Conclusions: B. velezensis DY3108 might be a promising candidate for exploitation in AFB1 detoxification and bioremediation in food and feed matrices.

Process desired functional attributes of an endoxylanase of GH10 family from a new strain of Aspergillus terreus S9

Huge industrial application potential of xylanases is stalled due to lack of process suitable characteristics like thermostability, broad range pH stability, and high catalytic efficiency in the available enzymes. Current study presents the first ever report of a pH stable (pH 6–11) and thermostable (80-100 °C) xylanase from a novel strain of Aspergillus terreus S9. The xylanase was purified to homogeneity (6.67-fold) by ammonium sulphate precipitation, ion exchange chromatography, and molecular exclusion chromatography. SDS-PAGE analysis revealed an estimated molecular mass of ~33 kDa for the xylanase. Metal ions and surfactants such as K+, Ca2+, Mn2+, Mg2+, CTAB and Tween-80 enhanced the xylanase activity while Cu2+ and Hg2+ strongly inhibited the activity. Kinetic parameters i.e. Km, Vmax,Kcat and Kcat/Km of A. terreus S9 xylanase were 2.94 mg/ml, 285.71 μmol/min/mg, 1587.28 s−1and 539.89 ml/mg/s, respectively. The substrate specificity confirmed the true endoxylanolytic nature of xylanase. The conserved domain analysis, and Blastn and Blastx results showed that the xylanase belonged to GH10 family. A. terreus S9 xylanase may be used as model system for understanding the molecular basis of robust nature of enzymes, and the knowledge generated may help designing novel enzymes that are suitable for industrial applications.

The hydrogen-bond network around Glu160 contributes to the structural stability of chitosanase CsnA from Renibacterium sp. QD1

CsnA, a chitosanase from Renibacterium sp. QD1, has great potential for industrial applications due to its high yield and broad pH stability. In this study, a specific Glu160 in CsnA was identified by sequence alignment, and structural analysis and MD simulation predicted that Glu160 formed a hydrogen-bond network with Lys163 and Thr114. To evaluate the effect of the network, we constructed four mutants, including E160A, E160Q, K163A, and T114A, which partially or completely destroy this network. Characterization of these mutants demonstrated that the disruption of the network significantly decreased the enzyme thermostability. The underlying mechanisms responsible for the change of thermostability analyzed by circular dichroism spectroscopy revealed that the hydrogen-bond network conferred the structural stability of CsnA. Moreover, the length of the side chain of residue at 160 impacted conformational stability of the enzyme. Taken together, the hydrogen-bond network around Glu160 plays important roles in stabilization of CsnA.

The purification and characterization of a novel alkali-stable pectate lyase produced by Bacillus subtilis PB1.

Pectinase is an important kind of enzyme with many industrial applications, among which pectinases produced by bacteria were scarce compared with fungal sources. In this study, a novel bacterium which produced extracellular pectinase was firstly isolated from flue-cured tobacco leaves and identified as Bacillus subtilis PB1 according to its 16S rRNA gene. The pectinolytic enzyme was purified by ammonium sulfate precipitation, ion-exchange and gel filtration chromatography, after which molecular weight was determined as 43.1 ± 0.5kDa by SDS-PAGE. Peptide mass fingerprinting of the pectinase by MALDI-TOF MS showed that the purified enzyme shared homology with pectate lyase and wasdesignated as BsPel-PB1. The optimal temperature for BsPel-PB1 was 50°C. The optimal pH was pH 9.5 for BsPel-PB1 while it had a broad pH stability from 5 to 11. The values of K m and V max were 0.312mg/mL and 1248 U/mL, respectively. Accordingly, the BsPel-PB1 was a novel alkaline pectate lyase which could find potential application as a commercial candidate in the pectinolytic related industries.

Expression and characterization of an enhanced recombinant heparinase I with chitin binding domain

Heparinase I (Hep I) can efficiently depolymerize heparin and heparin sulfate to oligosaccharides or unsaturated disaccharides, which resulted in loss of physiological function such as blood coagulation. In order to realize the immobilization of Hep I on chitin carriers, we cloned Hep I with the chitin binding domain (ChBD) as a chitin-affinity tag, and the Small Ubiquitin-like MOdifier (SUMO) linker as a solvation enhancer in different fusion sequence. DNA and protein gels suggested that 4 kinds of recombinants were successfully constructed and expressed in Escherichia coli (E. coli). And the triple functional heparinases isolated from cell lysate could be efficiently purified by chitin beads. After optimizing fermentation conditions, it gave the specific enzyme activities of 1.88±0.11, 3.69±0.45, 3.44±0.38, and 2.73±0.29IU/mg total proteins for ChBD-Hep I, ChBD-SUMO-Hep I, SUMO-ChBD-Hep I, and ChBD-Hep I-SUMO, respectively, with unfractionated heparin as substrate. The optimal reaction temperature and pH were determined to be 30°C and 7.0 for all the fusion enzymes. ChBD-SUMO-Hep I exhibited the maximum half-life (48min) at 30°C and best thermo-stability under 15–50°C. All the fusion enzymes showed broad pH-stability in the range of 5.4–9.0.

Biochemical and Computational Insights on a Novel Acid-Resistant and Thermal-Stable Glucose 1-Dehydrogenase.

Due to the dual cofactor specificity, glucose 1-dehydrogenase (GDH) has been considered as a promising alternative for coenzyme regeneration in biocatalysis. To mine for potential GDHs for practical applications, several genes encoding for GDH had been heterogeneously expressed in Escherichia coli BL21 (DE3) for primary screening. Of all the candidates, GDH from Bacillus sp. ZJ (BzGDH) was one of the most robust enzymes. BzGDH was then purified to homogeneity by immobilized metal affinity chromatography and characterized biochemically. It displayed maximum activity at 45 °C and pH 9.0, and was stable at temperatures below 50 °C. BzGDH also exhibited a broad pH stability, especially in the acidic region, which could maintain around 80% of its initial activity at the pH range of 4.0–8.5 after incubating for 1 hour. Molecular dynamics simulation was conducted for better understanding the stability feature of BzGDH against the structural context. The in-silico simulation shows that BzGDH is stable and can maintain its overall structure against heat during the simulation at 323 K, which is consistent with the biochemical studies. In brief, the robust stability of BzGDH made it an attractive participant for cofactor regeneration on practical applications, especially for the catalysis implemented in acidic pH and high temperature.

Biological response of HeLa cells to gold nanoparticles coated with organic molecules

In this work, gold nanospheres functionalized with low weight organic molecules (4-aminothiphenol and cysteamine) were synthesized in a one-step method for their in vitro cytotoxic evaluation on HeLa cells. To enhance the biocompatibility of the cysteamine-capped GNPs, BSA was used due to its broad PH stability and high binding affinity to gold nanoparticles. Besides, the widely reported silica coated gold nanorods were tested here to contrast their toxic response against our nanoparticles coated with organic molecules. Our results shown, the viability measured at 1.9×10−5M did not show significant differences against negative controls for all the samples; however, the metabolic activity of HeLa cells dropped when they were exposed to silica gold nanorods in the range of concentrations from 2.9×10−7M to 3.0×10−4M, while in the cases of gold nanospheres, we found that only at concentrations below 1.9×10−5M metabolic activity was normal. Our preliminary results did not indicate any perceivable harmful toxicity to cell membrane, cytoskeleton or nucleus due to our nanospheres at 1.9×10−5M. Additional test should be conducted in order to ensure a safe use of them for biological applications, and to determine the extent of possible damage.

Heterologous production of a temperature and pH-stable laccase from Bacillus vallismortis fmb-103 in Escherichia coli and its application

Abstract To enhance laccase yield, the laccase gene from Bacillus vallismortis fmb-103 was cloned and heterologously expressed in Escherichia coli BL21 (DE3) cells. The auto-induction strategy was applied during fermentation, and the process was controlled, as follows: Cu2+ was added when the optical density at 600 nm (OD600) was 0.3, the fermentation temperature was adjusted to 16 °C when the OD600 was 0.9, and fermentation was stopped after 50 h. The yield of recombinant laccase was up to 3420 U/L, as assayed by 2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid). Recombinant laccase was purified 4.47-fold by heating for 10 min at 70 °C and dialyzing against 50–60% ammonium sulfate, retained more than 50% activity after 10 h at 70 °C, and demonstrated broad pH stability. Malachite green was efficiently degraded by recombinant laccase, especially in combination with mediators. These results provided a basis for the future application of recombinant laccase to malachite green degradation.