Silk Industry Waste Protein-Derived Sericin Hybrid Nanoflowers for Antibiotics Remediation via Circular Economy.

Hybrid protein-copper nanoflowers have emerged as promising materials with diverse applications in biocatalysis, biosensing, and bioremediation. Sericin, a waste biopolymer from the textile industry, has shown potential for fabricating such nanoflowers. However, the influence of the molecular weight of sericin on nanoflower morphology and peroxidase-like activity remains unexplored. This work focused on the self-assembly of nanoflowers using high- and low-molecular-weight (HMW and LMW) silk sericin combined with copper(II) as an inorganic moiety. The peroxidase-like activity of the resulting nanoflowers was evaluated using 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and hydrogen peroxide (H2O2). The findings revealed that high-molecular-weight sericin hybrid nanoflowers (HMW-ShNFs) exhibited significantly higher peroxidase-like activity than low-molecular-weight sericin hybrid nanoflowers (LMW-ShNFs). Furthermore, HMW-ShNFs demonstrated superior reusability and storage stability, thereby enhancing their potential for practical use. This study also explored the application of HMW-ShNF for ciprofloxacin degradation to address the environmental and health hazards posed by this antibiotic in water. The results indicated that HMW-ShNFs facilitated the degradation of ciprofloxacin, achieving a maximum degradation of 33.2 ± 1% at pH 8 and 35 °C after 72 h. Overall, the enhanced peroxidase-like activity and successful application in ciprofloxacin degradation underscore the potential of HMW-ShNFs for a sustainable and ecofriendly remediation process. These findings open avenues for the further exploration and utilization of hybrid nanoflowers in various environmental applications.

Biomedical Applications of Electro-Initiated Polymerisation on Ti6Al4†V Titanium Alloy using Silk Fibroin Coatings for Antibiotic Delivery and Improved Cell Metabolism.

Silk fibroin interactions with metallic surfaces can provide utility for medical materials and devices. Toward this goal, titanium alloy (Ti6Al4 V) was covalently grafted with polyacrylamide via electrochemically reducing 4-nitrobenzene diazonium salt in the presence of acrylamide. Analysis of the modified surfaces with FT-IR spectra, SEM and AFM were consistent with surface grafting. Functionalised titanium samples with a silk fibroin membrane, with and without impregnated therapeutics, were used to assess cytocompatibility and drug delivery. Initial cytocompatibility experiments using fibroblasts showed that the functionalised samples, both with and without silk fibroin coatings, supported significant increases between 72-136 % in cell metabolism, compared to the controls after 7 days. A 7-days release profiling showed consistent bacterial inhibition through gentamicin release with average inhibition zones of 239 mm2 . Over a 5-week period, silk fibroin coated samples, both with and without growth factors, supported better human mesenchymal stem cell metabolism with increases reaching 1031 % and 388 %, respectively, compared to samples without the silk fibroin coating with.

Methods for Silk Property Analyses across Structural Hierarchies and Scales.

Silk from silkworms and spiders is an exceptionally important natural material, inspiring a range of new products and applications due to its high strength, elasticity, and toughness at low density, as well as its unique conductive and optical properties. Transgenic and recombinant technologies offer great promise for the scaled-up production of new silkworm- and spider-silk-inspired fibres. However, despite considerable effort, producing an artificial silk that recaptures the physico-chemical properties of naturally spun silk has thus far proven elusive. The mechanical, biochemical, and other properties of pre-and post-development fibres accordingly should be determined across scales and structural hierarchies whenever feasible. We have herein reviewed and made recommendations on some of those practices for measuring the bulk fibre properties; skin-core structures; and the primary, secondary, and tertiary structures of silk proteins and the properties of dopes and their proteins. We thereupon examine emerging methodologies and make assessments on how they might be utilized to realize the goal of developing high quality bio-inspired fibres.

Promoting Silk Fibroin Adhesion to Stainless Steel Surfaces by Interface Tailoring.

Bonding dissimilar materials has been a persistent challenge for decades. This paper presents a method to modify a stainless steel surface (316 L), routinely used in medical applications to enable the significant adhesion of a biopolymer (silk fibroin). The metallic surface was first covalently grafting with polyacrylamide, to enable a hydrogen bonding compatible surface. The polymerisation was initiated via the irreversible electrochemical reduction of a 4-nitrobenzene diazonium salt (20 mM), in the presence of an acrylamide monomer (1 M) at progressively faster scan rates (0.01 V/s to 1 V/s). Examination of the modified samples by FT-IR was consistent with successful surface modification, via observations of the acrylamide carbonyl (1600-1650 cm-1 ) was observed, with more intense peaks correlating to slower scan rates. Similar observations were made with respect to increasing surface polarity, assessed by water contact angle. Reductions of >60° were observed for the grafted surfaces, relative to the unmodified control materials, indicating a surface able to undergo significant hydrogen bonding. The adhesion of silk to the metallic surface was quantified using a lap shear test, effectively using silk fibroin as an adhesive. Adhesion improvements of 5-7-fold, from 4.1 MPa to 29.3 MPa per gram of silk fibroin, were observed for the treated samples, highlighting the beneficial effect of this surface treatment. The methods developed in this work can be transferred to any metallic (or conductive) surface and can be tailored to complement any desired interface.

Toughening Wet-Spun Silk Fibers by Silk Nanofiber Templating.

Regenerated silk fibers typically fall short of silkworm cocoon fibers in mechanical properties due to reduced fiber crystal structure and alignment. One approach to address this has been to employ inorganic materials as reinforcing agents. The present study avoids the need for synthetic additives, demonstrating the first use of exfoliated silk nanofibers to control silk solution crystallization, resulting in all-silk pseudocomposite fibers with remarkable mechanical properties. Incorporating only 0.06 wt% silk nanofibers led to a ≈44% increase in tensile strength (over 600 MPa) and ≈33% increase in toughness (over 200 kJ kg-1 ) compared with fibers without silk nanofibers. These remarkable properties can be attributed to nanofiber crystal seeding in conjunction with fiber draw. The crystallinity nearly doubled from ≈17% for fiber spun from pure silk solution to ≈30% for the silk nanofiber reinforced sample. The latter fiber also shows a high degree of crystal orientation with a Herman's orientation factor of 0.93, a value which approaches that of natural degummed B. mori silk cocoon fiber (0.96). This study provides a strong foundation to guide the development of simple, eco-friendly methods to spin regenerated silk with excellent properties and a hierarchical structure that mimics natural silk.

Using In Situ Polymerization to Increase Puncture Resistance and Induce Reversible Formability in Silk Membranes.

Silk fibroin is an excellent biopolymer for application in a variety of areas, such as textiles, medicine, composites and as a novel material for additive manufacturing. In this work, silk membranes were surface modified by in situ polymerization of aqueous acrylic acid, initiated by the reduction of various aryldiazonium salts with vitamin C. Treatment times of 20 min gave membranes which possessed increased tensile strength, tensile modulus, and showed significant increased resistance to needle puncture (+131%), relative to 'untreated' standards. Most interestingly, the treated silk membranes were able to be reversibly formed into various shapes via the hydration and plasticizing of the surface bound poly(acrylic acid), by simply steaming the modified membranes. These membranes and their unique properties have potential applications in advanced textiles, and as medical materials.

Tunable Biodegradable Silk-Based Memory Foams with Controlled Release of Antibiotics.

Sustained, local delivery of the antibiotic ciprofloxacin under different formats from porous silk protein-based memory foam systems was studied. Similarly, protease XIV was incorporated during processing to provide control of the degradation kinetics of the silk materials. In vitro antibiotic release studies combined with degradation assessments were utilized to assess the mechanisms and kinetics of release from the silk materials. The sequestered protease XIV affected the degradation profiles of the silk foams yet did not impact the release kinetics of the ciprofloxacin, which was controlled by solubility and diffusion of the drug. The ability to tune the release of ciprofloxacin between 1 and 200 days, combined with the option to modulate the degradation rate up to 80% in 2 weeks via incorporation of a protease, suggests utility for drug release devices. Further, we anticipate that this approach could also be extended to other medical implant needs and other drugs.

Enhancing Resistance of Silk Fibroin Material to Enzymatic Degradation by Cross-Linking Both Crystalline and Amorphous Domains.

Silk fibroin (SF) membranes are finding widespread use as biomaterial scaffolds in a range of tissue engineering applications. The control over SF scaffold degradation kinetics is usually driven by the proportion of SF crystalline domains in the formulation, but membranes with a high ß-sheet content are brittle and still contain amorphous domains, which are highly susceptible to enzymatic degradation. In this work, photo-cross-linking of SF using a ruthenium-based method, and with the addition of glycerol, was used to generate robust and flexible SF membranes for long-term tissue engineering applications requiring slow degradation of the scaffolds. The resulting mechanical properties, protein secondary structure, and degradation rate were investigated. In addition, the cytocompatibility and versatility of porous micropatterning of SF films were assessed. The photo-cross-linking reduced the enzymatic degradation of SF in vitro without interfering with the ß-sheet content of the SF material, while adding glycerol to the composition grants flexibility to the membranes. By combining these methods, the membrane resistance to protease degradation was significantly enhanced compared to either method alone, and the SF mechanical properties were not impaired. We hypothesize that photo-cross-linking protects the SF amorphous regions from enzymatic degradation and complements the natural protection offered by ß-sheets in the crystalline region. Overall, this approach presents broad utility in tissue engineering applications that require a long-term degradation profile and mechanical support.

Silk particles, microfibres and nanofibres: A comparative study of their functions in 3D printing hydrogel scaffolds.

Silk, with highly crystalline structure and well-documented biocompatibility, is promising to be used as reinforcing material and build functionalized composite scaffolds. In the present study, we developed chitosan/silk composite scaffolds using silk particles, silk microfibres and nanofibres via 3D printing method. The three forms of silk fillers with varied shapes and dimensions were obtained via different processing methods and evaluated of their morphology, crystalline structure and thermal property. All silk fillers showed different degrees of improvement on printability in terms of ink rheology and printing shape fidelity. Different silk fillers led to different scaffold surface morphology and different roughness, while all reduced the contact angle compared to pure chitosan. Similar reinforcements were observed on compressive modulus, while oscillatory gel strength reinforcement was found to be positively correlated to the filler aspect ratio. Addition of silk introduced no cytotoxicity for that all scaffolds supported a steady cell growth using human fibroblasts. Meanwhile different cellular behaviours were observed on different scaffold surfaces, which can possibly intriguer specific application on soft tissue engineering.

Facile and versatile solid state surface modification of silk fibroin membranes using click chemistry.

Reported is a fast and versatile protocol to surface modify pre-cast silk membranes targeting tyrosine residues. Enriched alkyne silk membranes were prepared using this method and azides possessing a range of functional groups were tethered to the membrane surface using click chemistry to give a range of water contact angles from 85 ± 3° to 34 ± 6°.

3D Printing of Silk Particle-Reinforced Chitosan Hydrogel Structures and Their Properties.

Hydrogel bioprinting is a major area of focus in the field of tissue engineering. However, 3D printed hydrogel scaffolds often suffer from low printing accuracy and poor mechanical properties because of their soft nature and tendency to shrink. This makes it challenging to process them into structural materials. In this study, natural chitosan hydrogel scaffolds were, for the first time, reinforced with milled silk particles and fabricated by 3D printing. Compared with pure chitosan scaffolds, the addition of silk particles resulted in up to a 5-fold increase in compressive modulus as well as significantly better printing accuracy and improved scaffold stability. The chitosan/silk inks flowed well during printing; loading of up to 300% silk (w/w) resulted in only minor changes in the rheological properties of the ink. Particle loading also enabled tuning of the surface roughness of the scaffolds and improved scaffolds' biodegradability. The printed composite hydrogel scaffolds showed no cytotoxicity and supported adherence and growth of human fibroblast cells.

cDNA sequences of GHF9 endo-ß-1,4-glucanases in terrestrial Crustacea.

This study aimed to sequence and identify a glycosyl hydrolase family 9 (GHF9) endo-ß-1,4-glucanase expressed in the midgut gland of the herbivorous gecarcinid land crab, Gecarcoidea natalis. Hence this would explain the gene responsible for the production of previously purified and characterised endo-ß-1,4-glucanases. Three different transcripts, two complete and one partial were sequenced from cDNA and an open reading frame of 1383bp was produced. Translated, this would produce a putative protein of 460 amino acid residues, including a 16 amino acid residue signal peptide. The mature protein (without signal peptide) is predicted to have a molecular mass of 47.6-47.7kDa; this closely matches the molecular mass (47.4kDa) of one of the three endo-ß-1,4-glucanase/lichenase enzymes purified previously from G. natalis. It is therefore proposed that the gene described here encodes one of the previously characterised enzymes. The presence of multiple transcripts suggests gene duplication. To confirm that the gene is widely expressed within the Crustacea, cDNA encoding a GHF9 endo-ß-1,4-glucanase was also sequenced in diverse crustaceans, the deposit feeding soldier crab, Mictyris platycheles and the terrestrial hermit crabs, Coenobita purlatus and C. brevimanus. An open reading frame of 1356bp was sequence from M. platycheles, while an incomplete open reading frames of 1384 and 1523bp were respectively sequenced from Coenobita brevimanus and C. perlatus. The midgut gland of M. platycheles contained activity (0.704±0.218µmol reducing sugars produced. min-1·mg-1 tissue wet weight) of a 26.3±0.3(5) endo-ß-1,4-glucanase isozyme (determined from activity staining). These species, particularly M. platycheles does not consume and digest significant amounts of plant cellulose. This implies that the ancestral enzyme is not a cellulase, but rather it may be involved in hydrolysing cellulose like polysaccharides within other organisms such as algae.

Glycerol-plasticised silk membranes made using formic acid are ductile, transparent and degradation-resistant.

Regenerated silk fibroin membranes tend to be brittle when dry. The use of plasticisers such as glycerol improve membrane ductility, but, when combined with aqueous processing, can lead to a higher degradation rate than solvent-annealed membranes. This study investigated the use of formic acid as the solvent with glycerol to make deformable yet degradation-resistant silk membranes. Here we show that membranes cast using formic acid had low light scattering, with a diffuse transmittance of less than 5% over the visible wavelengths, significantly lower than the 20% transmittance of aqueous derived silk/glycerol membranes. They had 64% ß-sheet content and lost just 30% of the initial silk weight over 6h when tested with an accelerated enzymatic degradation assay, in comparison the aqueous membranes completely degraded within this timeframe. The addition of glycerol also improved the maximum elongation of formic acid derived membranes from under 3% to over 100%. They also showed good cytocompatibility and supported the adhesion and migration of human tympanic membrane keratinocytes. Formic acid based, silk/glycerol membranes may be of great use in medical applications such as repair of tympanic membrane perforation or ocular applications where transparency and resistance to enzymatic degradation are important.

Comparative acoustic performance and mechanical properties of silk membranes for the repair of chronic tympanic membrane perforations.

The acoustic and mechanical properties of silk membranes of different thicknesses were tested to determine their suitability as a repair material for tympanic membrane perforations. Membranes of different thickness (10-100µm) were tested to determine their frequency response and their resistance to pressure loads in a simulated ear canal model. Their mechanical rigidity to pressure loads was confirmed by tensile testing. These membranes were tested alongside animal cartilage, currently the strongest available myringoplasty graft as well as paper, which is commonly used for simpler procedures. Silk membranes showed resonant frequencies within the human hearing range and a higher vibrational amplitude than cartilage, suggesting that silk may offer good acoustic energy transfer characteristics. Silk membranes were also highly resistant to simulated pressure changes in the middle ear, suggesting they can resist retraction, a common cause of graft failure resulting from chronic negative pressures in the middle ear. Part of this strength can be explained by the substantially higher modulus of silk films compared with cartilage. This allows for the production of films that are much thinner than cartilage, with superior acoustic properties, but that still provide the same level of mechanical support as thicker cartilage. Together, these in vitro results suggest that silk membranes may provide good hearing outcomes while offering similar levels of mechanical support to the reconstructed middle ear.

A glycosyl hydrolase family 16 gene is responsible for the endogenous production of ß-1,3-glucanases within decapod crustaceans.

To identify the gene responsible for the production of a ß-1,3-glucanase (laminarinase) within crustacea, a glycosyl hydrolase family 16 (GHF16) gene was sequenced from the midgut glands of the gecarcinid land crab, Gecarcoidea natalis and the freshwater crayfish, Cherax destructor. An open reading frame of 1098 bp for G. natalis and 1095 bp for C. destructor was sequenced from cDNA. For G. natalis and C. destructor respectively, this encoded putative proteins of 365 and 364 amino acids with molecular masses of 41.4 and 41.5 kDa. mRNA for an identical GHF16 protein was also expressed in the haemolymph of C. destructor. These putative proteins contained binding and catalytic domains that are characteristic of a ß-1,3-glucanase from glycosyl hydrolase family 16. The amino acid sequences of two short 8-9 amino acid residue peptides from a previously purified ß-1,3-glucanase from G. natalis matched exactly that of the putative protein sequence. This plus the molecular masses of the putative proteins matching that of the purified proteins strongly suggests that the sequences obtained encode for a catalytically active ß-1,3-glucanase. A glycosyl hydrolase family 16 cDNA was also partially sequenced from the midgut glands of other amphibious (Mictyris platycheles and Paragrapsus laevis) and terrestrial decapod species (Coenobita rugosus, Coenobita perlatus, Coenobita brevimanus and Birgus latro) to confirm that the gene is widely expressed within this group. There are three possible hypothesised functions and thus evolutionary routes for the ß-1,3-glucanase: 1) a digestive enzyme which hydrolyses ß-1,3-glucans, 2) an enzyme which cleaves ß-1,3-glycosidic bonds within cell walls to release cell contents or 3) an immune protein which can hydrolyse the cell walls of potentially pathogenic micro-organisms.

The last piece in the cellulase puzzle: the characterisation of beta-glucosidase from the herbivorous gecarcinid land crab Gecarcoidea natalis.

A 160 kDa enzyme with beta-glucosidase activity was purified from the midgut gland of the land crab Gecarcoidea natalis. The enzyme was capable of releasing glucose progressively from cellobiose, cellotriose or cellotetraose. Although beta-glucosidases (EC 3.2.1.21) have some activity towards substrates longer than cellobiose, the enzyme was classified as a glucohydrolase (EC 3.2.1.74) as it had a preference for larger substrates (cellobiose

Functional morphology of the gastric mills of carnivorous, omnivorous, and herbivorous land crabs.

Terrestrial decapods consume a wide variety of plant and animal material. The potential adaptations of carnivorous, omnivorous, and herbivorous terrestrial crustaceans were studied by examining the functional morphology of the gastric mill. Two closely related species from each feeding preference group were examined to identify which features of the mill were due to phylogeny and which were due to adaptation. The morphology of the gastric mill matched the diet well; the gastric mills of the carnivorous species (Geograpsus grayi and Geograpsus crinipes) possessed a blunt, rounded medial tooth and flattened lateral teeth with a longitudinal grinding groove. These features make them well suited to a carnivorous diet of soft animal tissue as well as hard material, such as arthropod exoskeleton. In contrast, the mill of the herbivorous gecarcinids (Gecarcoidea natalis and Discoplax hirtipes) consisted of a medial tooth with sharp transverse ridges and lateral teeth with sharp interlocking cusps and ridges and no grinding surface. These features would efficiently shred fibrous plant material. The morphology of the mill of the omnivorous coenobitids (Coenobita perlatus and Birgus latro) was more generalized toward a mixed diet. However, the mill of B. latro was more adapted to deal with highly nutritious food items, such as nuts and heavily calcified decapods. Its mill possessed lateral teeth with extended ridges, which sat close to the calcified cardiopyloric valve to form a flattened floor. Hard items trapped in the mill would be crushed against this surface by the medial tooth.

Food utilisation and digestive ability of aquatic and semi-terrestrial crayfishes, Cherax destructor and Engaeus sericatus (Astacidae, Parastacidae).

Both Engaeus sericatus and Cherax destructor are omnivorous crayfishes consuming a variety of food items. Materials identified in the faeces of both E. sericatus and C. destructor consisted of mainly plant material with minor amounts of arthropod animals, algae and fungi. The morphology of the gastric mill of C. destructor suggests that it is mainly involved in crushing of food material while the gastric mill of E. sericatus appears to be better suited to cutting of food material. Given this, the gastric mill of E. sericatus may be better able to cut the cellulose and hemicellulose fibres associated with fibrous plant material. In contrast, the gastric mill of C. destructor appears to be more efficient in grinding soft materials such as animal protein and algae. Both species accumulated high amounts of lipids in their midgut glands (about 60% of the dry mass) which were dominated by triacylglycerols (81-82% of total lipids). The dominating fatty acids were 16:0, 16:1(n-7), 18:1(n-9), 18:2(n-6), and 18:3(n-3). The two latter fatty acids can only be synthesised by plants, and are thus indicative of the consumption of terrestrial plants by the crayfishes. The similarity analysis of the fatty acid patterns showed three distinct clusters of plants and each of the crayfish species. The complement of digestive enzymes, proteinases, total cellulase, endo-beta-1,4-glucanase, beta-glucosidase, laminarinase and xylanase within midgut gland suggests that both C. destructor and E. sericatus are capable of hydrolysing a variety of substrates associated with an omnivorous diet. Higher activities of total cellulase, endo-beta-1,4-glucanase and beta-glucosidase indicate that E. sericatus is better able to hydrolyse cellulose within plant material than C. destructor. In contrast to E. sericatus, higher total protease and N-acetyl-beta-D-glucosaminidase activity in the midgut gland of C. destructor suggests that this species is better able to digest animal materials in the form of arthropods. Differences in total cellulase and gastric mill morphology suggest that E. sericatus is more efficient at digesting plant material than C. destructor. However, the contents of faecal pellets and the fatty acid compositions seem to indicate that both species opportunistically feed on the most abundant and easily accessible food items.

Purification and characterisation of endo-beta-1,4-glucanase and laminarinase enzymes from the gecarcinid land crab Gecarcoidea natalis and the aquatic crayfish Cherax destructor.

Laminarinase and endo-beta-1,4-glucanase were purified and characterised from the midgut gland of the herbivorous land crab Gecarcoidea natalis and the crayfish Cherax destructor. The laminarinase isolated from G. natalis was estimated to have a molecular mass of 41 kDa by SDS-PAGE and 71 kDa by gel filtration chromatography. A similar discrepancy was noted for C. destructor. Possible reasons for this are discussed. Laminarinase (EC 3.2.1.6) from G. natalis had a V(max) of 42.0 micromol reducing sugars produced min(-1) mg protein(-1), a K(m) of 0.126% (w/v) and an optimum pH range of 5.5-7, and hydrolysed mainly beta-1,3-glycosidic bonds. In addition to the hydrolysis of beta-1,3-glycosidic bonds, laminarinase (EC 3.2.1.39) from C. destructor was capable of significant hydrolysis of beta-1,4-glycosidic bonds. It had a V(max) of 19.6 mumol reducing sugars produced min(-1) mg protein(-1), a K(m) of 0.059% (w/v) and an optimum pH of 5.5. Laminarinase from both species produced glucose and other short oligomers from the hydrolysis of laminarin. Endo-beta-1,4-glucanase (EC 3.2.1.4) from G. natalis had a molecular mass of 52 kDa and an optimum pH of 4-7. It mainly hydrolysed beta-1,4-glycosidic bonds, but was also capable of significant hydrolysis of beta-1,3-glycosidic bonds. Two endo-beta-1,4-glucanases, termed 1 and 2, with respective molecular masses of 53+/-3 and 52 kDa, were purified from C. destructor. Endo-beta-1,4-glucanase 1 was only capable of hydrolysing beta-1,4-glycosidic bonds and had an optimum pH of 5.5. Endo-beta-1,4-glucanases from both species produced some glucose, cellobiose and other short oligomers from the hydrolysis of carboxymethyl cellulose.