Recombinant production, characterization and industrial application testing of a novel acidic exo/endo-chitinase from Rasamsonia emersonii.

An acid-active exo/endo-chitinase; comprising a GH18 catalytic domain and substrate insertion domain; originating from the thermophilic filamentous fungus Rasamsonia emersonii, was expressed in Pichia pastoris. In silico analysis including phylogenetic analysis, and recombinant production, purification, biochemical characterisation, and industrial application testing, was carried out. The expressed protein was identified by SDS-PAGE as a smear from 56.3 to 125.1 kDa, which sharpens into bands at 46.0 kDa, 48.4 kDa and a smear above 60 kDa when treated with PNGase F. The acid-active chitinase was primarily a chitobiosidase but displayed some endo-chitinase and acetyl-glucosamidase activity. The enzyme was optimally active at 50 °C, and markedly low pH of 2.8. As far as the authors are aware, this is the lowest pH optima reported for any fungal chitinase. The acid-active chitinase likely plays a role in chitin degradation for cell uptake in its native environment, perhaps in conjunction with a chitin deacetylase. Comparative studies with other R. emersonii chitinases indicate that they may play a synergistic role in this. The acid-active chitinase displayed some efficacy against non-treated substrates; fungal chitin and chitin from shrimp. Thus, it may be suited to industrial chitin hydrolysis reactions for extraction of glucosamine and chitobiose at low pH.

Production, characteristics and applications of microbial heparinases.

Heparinases are enzymes that selectively cleave heparin and heparan sulfate chains, via cleavage of the glycosidic linkage between hexosamines and uronic acids, producing disaccharide and oligosaccharide products. While heparin is well known as an anti-coagulant drug, heparin and heparan sulfate are also involved in biological processes such as inflammation, cancer and angiogenesis and viral and bacterial infections and are of growing interest for their therapeutic potential. Recently, potential roles of heparin and heparan sulfate in relation to COVID-19 infection have been highlighted. The ability of heparinases to selectively cleave heparin chains has been exploited industrially to produce low molecular weight heparin, which has replaced heparin in several clinical applications. Other applications of heparinases include heparin and heparan sulfate structural analysis, neutralisation of heparin in blood and removal of the inhibitory effect of heparin on various enzymes. Heparinases are known to inhibit neovascularization and heparinase III is of interest for treating cancer and inhibiting tumour cell growth. Heparinase activity, first isolated from Pedobacter heparinus, has since been reported from several other microorganisms. Significant progress has been made in the production, characterisation and improvement of microbial heparinases in response to application demands in terms of heparinase yield and purity, which is likely to extend their usefulness in various applications. This review focuses on recent developments in the identification, characterisation and improvement of microbial heparinases and their established and emerging industrial, clinical and therapeutic applications.

Recombinant production and characterisation of two chitinases from Rasamsonia emersonii, and assessment of their potential industrial applicability.

Rasamsonia emersonii (previously Talaromyces emersonii) is a thermophilic filamentous fungus displaying optimum growth at 45 °C. It has a history of use in commercial food enzyme production. Its unfractionated chitinolytic secretome was partially characterised in the early 1990s; however, no individual chitinase from this source has been described in literature previously. This study describes two GH18 chitinases originating from the R. emersonii genome, expressed in the methylotrophic yeast P. pastoris. Chit1 comprises of a GH18 catalytic domain and Chit2 comprises of a GH18 catalytic domain and a chitin-binding motif at the C-terminal. The chitinases were expressed as glycoproteins. The apparent molecular weight of Chit1 was 35.8-42.1 kDa with a smearing tail associated with glyco-sidechains visible up to 72.2 kDa. This became two bands of 30.8 and 29.0 kDa upon de-glycosylation. The apparent molecular weight of Chit2 was 50.4 kDa, reducing to 48.2 kDa upon de-glycosylation. Both chitinases displayed endo-chitinase and chitobiosidase activity, temperature optima of 50-55 °C and low pH optima (pH 4.5 or lower); Chit1 displayed a pH optimum of 3.5, retaining > 60% maximum activity at pH 2.2, a pH range lower than most enzymes of fungal origin. Chit2 displayed the highest chitin-degrading ability at 3456 µmol/mg on 4-NP-triacetylchitotriose, but lost activity faster than Chit1, which displayed 403 µmol/mg on the same substrate. The predicted D values (time required to reduce the enzyme activity to 10% of its original value at 50 °C) were 19.2 and 2.3 days for Chit1 and Chit2, respectively. Thus, Chit1 can be considered one of few hyperthermostable chitinase enzymes described in literature to date. Their physicochemical properties render these chitinases likely suitable for shrimp chitin processing including one-step chitin hydrolysis and alternative sustainable protein processing and the attractive emerging application of mushroom food waste valorisation.Key points• Two GH18 chitinases originating from the industrially relevant thermophilic fungus R. emersonii were cloned and expressed in P. pastoris.• The purified recombinant chitinases showed low pH and high temperature optima and appreciable thermostability at industrially relevant temperatures.• The chitinases displayed characteristics that indicate their likely suitability to several industrial applications including sustainable alternative protein processing, food waste valorisation of commercial mushroom production and one-step shrimp chitin processing.

Flexible Chemiresistive Cyclohexanone Sensors Based on Single-Walled Carbon Nanotube-Polymer Composites.

We report a chemiresistive cyclohexanone sensor on a flexible substrate based on single-walled carbon nanotubes (SWCNTs) functionalized with thiourea (TU) derivatives. A wrapper polymer containing both 4-vinylpyridine (4VP) groups and azide groups (P(4VP-VBAz)) was employed to obtain a homogeneous SWCNT dispersion via noncovalent functionalization of SWCNTs. The P(4VP-VBAz)-SWCNT composite dispersion was then spray-coated onto an organosilanized flexible poly(ethylene terephthalate) (PET) film to achieve immobilizing quaternization between the pyridyl groups from the polymer and the functional PET substrate, thereby surface anchoring SWCNTs. Subsequent surface functionalization was performed to incorporate a TU selector into the composites, resulting in P(Q4VP-VBTU)-SWCNT, for the detection of cyclohexanone via hydrogen bonding interactions. An increase in conductance was observed as a result of the hydrogen-bonded complex with cyclohexanone resulting in a higher hole density and/or mobility in SWCNTs. As a result, a sensor device fabricated with P(Q4VP-VBTU)-SWCNT composites exhibited chemiresistive responses (ΔG/G0) of 7.9 ± 0.6% in N2 (RH 0.1%) and 4.7 ± 0.4% in air (RH 5%), respectively, upon exposure to 200 ppm cyclohexanone. Selective cyclohexanone detection was achieved with minor responses (ΔG/G0 < 1.4% at 500 ppm) toward interfering volatile organic compounds (VOC). analytes. We demonstrate a robust sensing platform using the polymer-SWCNT composites on a flexible PET substrate for potential application in wearable sensors.

Purification and Characterization of a Novel ß-Galactosidase From the Thermoacidophile Alicyclobacillus vulcanalis.

Thermoacidophiles are microorganisms capable of optimum growth under a combination of high temperature and low pH. These microorganisms are a rich source of thermo- and acid- active/stable glycosyl hydrolases. Such enzymes could find use as novel biocatalysts in industrial processes, as operation at elevated temperature can increase substrate solubility, decrease viscosity, and reduce the risk of microbial contamination. We report the purification and characterization of an intracellular ß-galactosidase from the thermoacidophile Alicyclobacillus vulcanalis DSM 16176. The enzyme was purified 110-fold, with a 5.89% yield. Denatured (83.7 kDa) and native (179 kDa) molecular masses were determined by SDS-PAGE and gel filtration, respectively, and suggest the enzyme functions as a homodimer. LC-MS/MS analysis confirmed identity, and bioinformatic analysis indicates the enzyme to be a member of the glycosyl hydrolase family 42 (GH42). Highest activity was measured at 70 °C and pH 6.0. The Km on the substrates ONPG and lactose were 5 and 258 mM, respectively. This enzyme is thermostable, retaining 76, 50, and 42% relative activity after 30, 60, and 120 min, respectively, at 70 °C. This property could lend its use to high-temperature industrial processes requiring a thermo-active ß-galactosidase.

Purification and characterization of a novel thermophilic ß-galactosidase from Picrophilus torridus of potential industrial application.

Intracellular ß-galactosidase (E.C 3.2.1.23) produced by the thermoacidophilic archeon Picrophilus torridus DSM 9790 was purified to homogeneity using a combination of DEAE Sepharose, gel filtration, hydroxyapatite and chromatofocusing chromatographies. LC-MS/MS analysis was used to confirm the identity of the purified protein. The enzyme was found to be a homotrimer, with a molecular mass of 157.0 kDa and an isoelectric point of 5.7. To our knowledge, this enzyme has the lowest pH optimum of any intracellular ß-galactosidase characterized to date. Maximal activity was exhibited at acidic pH values of 5.0-5.5 and at 70 °C. The enzyme retained > 95% activity after heating to 70 °C for 1 h, or after incubation at pH 5.5 for 1 h. The enzyme may be of interest for high-temperature bioprocessing, such as in the production of lactulose. This investigation suggests that the ß-galactosidase activity produced by P. torridus is potentially more useful than several enzymes already characterized for such an application.

Expression and characterisation of a thermophilic endo-1,4-ß-glucanase from Sulfolobus shibatae of potential industrial application.

An endo-1,4-ß-D-glucanase gene was cloned from the thermophilic archaea Sulfolobus shibatae and expressed in E. coli. The recombinant enzyme was purified by heat denaturation and affinity chromatography prior to characterisation. The purified recombinant enzyme exhibited maximum activity at 95-100 °C and displayed a broad pH profile with over 91% of its maximum activity observed at pH 3-5. Upon assessment of enzyme thermal stability, full activity was observed after 1 h incubation at 75, 80 and 85 °C while 98%, 90% and 84% of original activity was detected after 2 h at 75, 80 and 85 °C, respectively. Maximum activity was observed on barley ß-glucan and lichenan and the purified enzyme also hydrolysed CMC and xylan. Endoglucanase activity was confirmed by viscometric assay with a rapid decrease in substrate viscosity observed immediately upon incubation with barley ß-glucan or CMC. The crude enzyme released reducing sugars from acid-pretreated straw at 75-85 °C. The thermophilic nature and biochemical properties of the enzyme indicate its potential suitability in industrial applications undertaken at high temperature, such as the production of second-generation bioethanol from lignocellulosic feedstocks and in the brewing industry. This is the first known report of an endoglucanase from S. shibatae.

Switchable Single-Walled Carbon Nanotube-Polymer Composites for CO2 Sensing.

We report a chemiresistive CO2 sensor based on single-walled carbon nanotubes (SWCNTs) noncovalently functionalized with a CO2 switchable copolymer containing amidine pendant groups that transform into amidinium bicarbonates in response to CO2. To fabricate a robust surface-anchored polymer-SWCNT dispersion via spray coating, we first designed and synthesized a precursor copolymer, P(4VP-VBAz), bearing both 4-vinylpyridine (4VP) groups and azide groups. The SWCNT dispersant group, 4VP, is capable of debundling and stabilizing nanotubes to improve their solubility in organic solvents for solution processing. Well-dispersed P(4VP-VBAz)-SWCNT composites are covalently immobilized onto a glass substrate functionalized with alkyl bromides, and then the amidine moieties are subsequently attached to form the resulting CO2-switchable P(Q4VP-VBAm)-SWCNT composites via a copper(I)-catalyzed azide-alkyne cycloaddition click reaction at the film surface. The amidine groups are strong donors that compensate or pin carriers in the SWCNTs. In the presence of CO2 under humid conditions, the generated amidinium bicarbonates from the polymer wrapping increase the concentration and/or liberate the hole carriers in the nanotubes, thereby increasing the net conductance of the composites. The amidinium moieties revert back to the amidines when purged with a CO2-free carrier gas with a reversible decrease in conductance. We also demonstrate high selectivity to CO2 over the other atmospheric gases such as O2 and Ar.

Parallel sorting of orbital and spin angular momenta of light in a record large number of channels.

Parallel sorting of orbital and spin angular momentum components of structured optical beams is demonstrated. Both spin channels are multiplexed within the novel orbital angular momentum (OAM) sorter, reducing the size, weight, and number of elements. The sorted states are linearly spaced over 70 topological charge values. We experimentally and theoretically evaluate the operational range and crosstalk between neighboring channels and find that 30 orbital angular momentum states are available per spin channel for quantum communication or cryptography. This is achieved using an angular momentum sorter that we developed based on geometric phase optical elements. We present two devices consisting of liquid crystal polymer films photoaligned with complex two-dimensional patterns.

Purification and Characterisation of a Thermostable ß-Xylosidase from Aspergillus niger van Tieghem of Potential Application in Lignocellulosic Bioethanol Production.

A locally isolated strain of Aspergillus niger van Tieghem was found to produce thermostable ß-xylosidase activity. The enzyme was purified by cation and anion exchange and hydrophobic interaction chromatography. Maximum activity was observed at 70-75 °C and pH 4.5. The enzyme was found to be thermostable retaining 91 and 87% of its original activity after incubation for 72 h at 60 and 65 °C, respectively, with 52% residual activity detected after 18 h at 70 °C. Available data indicates that the purified ß-xylosidase is more thermostable over industrially relevant prolonged periods at high temperature than those reported from other A. niger strains. Maximum activity was observed on p-nitrophenyl-ß-D-xylopyranoside and the enzyme also hydrolysed p-nitrophenyl ß-D-glucopyranoside and p-nitrophenyl α-L-arabinofuranoside. The purified enzyme acted synergistically with A. niger endo-1,4-ß-xylanase in the hydrolysis of beechwood xylan at 65 °C. During hydrolysis of pretreated straw lignocellulose at 70 °C using a commercial lignocellulosic enzyme cocktail, inclusion of the purified enzyme resulted in a 19-fold increase in the amount of xylose produced after 6 h. The results observed indicate potential suitability for industrial application in the production of lignocellulosic bioethanol where thermostable ß-xylosidase activity is of growing interest to maximise the enzymatic hydrolysis of lignocellulose.

Pancharatnam-Berry optical element sorter of full angular momentum eigenstate.

We propose and numerically demonstrate a Pancharatnam-Berry optical element (PBOE) device that simultaneously sorts spin (SAM) and orbital (OAM) angular momentum. This device exploits the circular polarization selective properties of PBOEs to modulate independently the orthogonal SAM eigenstates within a geometric optical transformation that sorts OAM, enabling single measurement characterization of the full angular momentum eigenstate. This expands the available state space for OAM communication and enables characterization of the eigenmode composition of structured polarization beams. We define the two-dimensional orientation patterns of the transversely varying half-waveplate PBOEs that implement the angular momentum sorter. We show that the device discriminates the OAM and SAM eigenstates of optical beams including laser cavity modes such as Laguerre-Gaussian OAM eigenmodes, Hermite-Gaussian modes, and hybrid modes with complex structured polarization. We also demonstrate that it can determine the m parameter of higher order LGml Laguerre-Gaussian modes. The ability of this device to decode information from spatially structured optical phase has potential for applications in communication, encryption, modal characterization, and scientific measurements.

Characterisation of a novel thermostable endoglucanase from Alicyclobacillus vulcanalis of potential application in bioethanol production.

A novel endoglucanase encoding gene was cloned from Alicyclobacillus vulcanalis and expressed in E. coli. The deduced amino acid sequence showed highest identity with α-L-arabinofuranosidase-like proteins from glycoside hydrolase family 51. The recombinant enzyme was purified by affinity chromatography and characterised in terms of its potential suitability for lignocellulose hydrolysis at high temperature in the production of bioethanol. The purified enzyme displayed maximum activity at 80 °C and pH 3.6-4.5. Tween 20 was found to have a beneficial effect on enzyme activity and thermal stability. When incubated in the presence of 0.1% Tween 20, the enzyme retained full activity after 72 h at 70 °C and 78% of original activity after 72 h at 75 °C. Maximum activity was observed on carboxymethyl cellulose, and the purified enzyme also hydrolysed lichenan, barley ß-glucan and xylan. The purified enzyme decreased the viscosity of carboxymethyl cellulose when assessed at 70-85 °C and was capable of releasing reducing sugars from acid-pretreated straw at 70 and 75 °C. The results indicate the potential suitability of the enzyme for industrial application in the production of cellulosic bioethanol.

Enhanced second harmonic generation from InAs nano-wing structures on silicon.

We demonstrate morphology-dependent second-harmonic generation (SHG) from InAs V-shaped nanomembranes. We show SHG correlation with the nano-wing shape and size, experimentally quantify the SHG efficiency, and demonstrate a maximum SHG enhancement of about 500 compared to the bulk. Experimental data are supported by rigorous calculations of local electromagnetic field spectra.

Enhanced second harmonic generation from Au nanoparticle arrays by femtosecond laser irradiation.

Ultrafast laser irradiation of planar Au nanoparticle arrays is shown to double the particle plasmon quality factor and to enhance second harmonic generation 40-fold. It is found that the irradiation induces a morphological change of nano-cylinders into asymmetric droplet-like shapes with reduced surface roughness without altering their lithographically defined locations. These findings can be used to improve the performance of linear and nonlinear plasmonic devices.

Enhanced second harmonic generation by photonic-plasmonic Fano-type coupling in nanoplasmonic arrays.

In this communication, we systematically investigate the effects of Fano-type coupling between long-range photonic resonances and localized surface plasmons on the second harmonic generation from periodic arrays of Au nanoparticles arranged in monomer and dimer geometries. Specifically, by scanning the wavelength of an ultrafast tunable pump laser over a large range, we measure the second harmonic excitation spectra of these arrays and demonstrate their tunability with particle size and separation. Moreover, through a comparison with linear optical transmission spectra, which feature asymmetric Fano-type lineshapes, we demonstrate that the second harmonic generation is enhanced when coupled photonic-plasmonic resonances of the arrays are excited at the fundamental pump wavelength, thus boosting the intensity of the electromagnetic near-fields. Our experimental results, which are supported by numerical simulations of linear optical transmission and near-field enhancement spectra based on the Finite Difference Time Domain method, demonstrate a direct correlation between the onset of Fano-type coupling and the enhancement of second harmonic generation in arrays of Au nanoparticles. Our findings enable the engineering of the nonlinear optical response of Fano-type coupled nanoparticle arrays that are relevant to a number of device applications in nonlinear nano-optics and plasmonics, such as on-chip frequency generators, modulators, switchers, and sensors.

Microfluidics integration of aperiodic plasmonic arrays for spatial-spectral optical detection.

We demonstrate successful integration of aperiodic arrays of metal nanoparticles with microfluidics technology for optical sensing using the spectral-colorimetric responses of nanostructured arrays to refractive index variations. Different aperiodic arrays of gold (Au) nanoparticles with varying interparticle separations and Fourier spectral properties are fabricated using Electron Beam Lithography (EBL) and integrated with polydimethylsiloxane (PDMS) microfluidics structures by soft-lithographic micro-imprint techniques. The spectral shifts of scattering spectra and the distinctive modifications of structural color patterns induced by refractive index variations were simultaneously measured inside microfluidic flow cells by dark-field spectroscopy and image correlation analysis in the visible spectral range. The integration of engineered aperiodic arrays of Au nanoparticles with microfluidics devices provides a novel sensing platform with multiplexed spatial-spectral responses for opto-fluidics applications and lab-on-a-chip optical biosensing.

Engineering plasmon-enhanced Au light emission with planar arrays of nanoparticles.

By systematically investigating the light emission and scattering properties of arrays of Au nanoparticles with varying size and separation, we demonstrate tunability and control of metal photoluminescence and unveil the critical role of near-field plasmonic coupling for the engineering of active metal nanostructures. We show that the decay of photoexcited electron-hole pairs into localized surface plasmons (LSPs) dramatically modifies the Au emission wavelength, line shape, and quantum efficiency depending both on particles size and separation. In particular, in arrays with near-field coupled nanoparticles we demonstrate broad light scattering and emission spectra that scale differently with respect to nanoparticle size due to the enhanced LSP nonradiative decay caused by near-field interparticle coupling. Our experimental results are fully supported by semianalytical extinction simulations based on rigorous coupled wave analysis, which demonstrate the importance of tuning plasmonic near-field coupling for the engineering of active devices based on light emitting arrays of metallic nanoparticles.