Purification and characterisation of endo-β-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 Vmax of 42.0 &micro;mol reducing sugars produced min&ndash;1 mg protein&ndash;1, a Km of 0.126% (w/v) and an optimum pH range of 5.5&ndash;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 Vmax of 19.6 &micro;mol reducing sugars produced min&ndash;1 mg protein&ndash;1, a Km 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&ndash;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&plusmn;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. <br /

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This is the published version: Allardyce, Benjamin J., Linton, Stuart M. and Saborowski, Reinhard 2010, The last piece in the cellulase puzzle : the characterisation of β-glucosidase from the herbivorous gecarcinid land crab Gecarcoidea natalis, Journal of experimental biology, vol. 213, no. 17, pp. 2950-2957. Available from Deakin Research Online: http://hdl.handle.net/10536/DRO/DU:30031463 Reproduced with the kind permissions of the copyright owner. Copyright : 2010, Company of Biologists 2950 INTRODUCTION Herbivorous terrestrial crustaceans, such as gecarcinid land crabs, consume a wide range of plant material, which is rich in cellulose and hemicellulose. Land crabs possess multiple endogenous endo--1,4-glucanases, one of the key enzyme classes involved in cellulose hydrolysis The traditional model for cellulose digestion in invertebrates reflects what is known about fungal cellulase systems. This model suggests that cellulose hydrolysis involves the synergistic activity of endo--1,4-glucanases (1,4--D-glucan-4-glucanohydrolases, EC 3.2.1.4), exoglucanases, including both cellobiohydrolases (1,4--D-glucan cellobiohydrolases, EC 3.2.1.91) and sometimes glucohydrolases (1,4--D-glucan glucanohydrolases, EC 3.2.1.74) and -glucosidases (-glucoside glucohydrolases, EC 3.2.1.21) Like fungal systems, endo--1,4-glucanases and -glucosidases appear to be key components of the cellulase systems of many marine and terrestrial invertebrate

Large expert-curated database for benchmarking document similarity detection in biomedical literature search

Document recommendation systems for locating relevant literature have mostly relied on methods developed a decade ago. This is largely due to the lack of a large offline gold-standard benchmark of relevant documents that cover a variety of research fields such that newly developed literature search techniques can be compared, improved and translated into practice. To overcome this bottleneck, we have established the RElevant LIterature SearcH consortium consisting of more than 1500 scientists from 84 countries, who have collectively annotated the relevance of over 180 000 PubMed-listed articles with regard to their respective seed (input) article/s. The majority of annotations were contributed by highly experienced, original authors of the seed articles. The collected data cover 76% of all unique PubMed Medical Subject Headings descriptors. No systematic biases were observed across different experience levels, research fields or time spent on annotations. More importantly, annotations of the same document pairs contributed by different scientists were highly concordant. We further show that the three representative baseline methods used to generate recommended articles for evaluation (Okapi Best Matching 25, Term Frequency-Inverse Document Frequency and PubMed Related Articles) had similar overall performances. Additionally, we found that these methods each tend to produce distinct collections of recommended articles, suggesting that a hybrid method may be required to completely capture all relevant articles. The established database server located at https://relishdb.ict.griffith.edu.au is freely available for the downloading of annotation data and the blind testing of new methods. We expect that this benchmark will be useful for stimulating the development of new powerful techniques for title and title/abstract-based search engines for relevant articles in biomedical research.Peer reviewe

Functionality of nano and 3D-microhierarchical TiO₂ particles as coagulants for sericin extraction from the silk degumming wastewater

Amine-functionalised TiO2 particles were employed as coagulants to remove sericin from the silk degumming wastewater. Two types of TiO2 particles including 3D-microhierarchical TiO2 (Micron-size) and TiO2 nanoparticles (Degussa P-25) were used in this study. The surfaces of both types of TiO2 particles were functionalised using 3-aminopropyl trimethoxysilane (APTMS). The impacts of TiO2 type, pH, TiO2 concentration, and settling time on sericin removal efficiency were investigated. The efficiency of TiO2 particles in sericin removal was evaluated by measuring the residual turbidity and UV-vis spectra of the solutions before and after the treatment. Moreover, the COD, SDS-PAGE and protein assay tests were conducted to further analyse the treated solutions. The results demonstrated that the sericin removal efficiency of around 67% and turbidity reduction of 95% were achieved at the optimum conditions of 0.04 g TiO2, pH = 5, and 60 min settling time. Nano and micron-size TiO2 particles showed similar efficiency for sericin removal, but micron-size particles outweighed due to their higher efficiency in inducing greater turbidity reduction after 60 min. The obtained sericin/TiO2 composites were stable after several cycles of wash and could be useful in different fields including cosmetic, and textile finishing, among others

Design and implementation of an organic powder printer

Organic powders are polymers with organic origin in a powdered form. They are biocompatible, biodegradable, and possess positive biological attributes, and can form constructs with high mechanical properties due to their powder form. However, there are various constraints that limit organic powders to be solely used with current 3D printers. Many organic powders cannot be fused by heat and light exposure and not easy to dissolve by the printing ink. Moreover, binding solutions for silk and other organic powders are mostly acidic in nature which cause damage to current 3D powder printers as they use neutral or slight low pH inks. This work aims to address this problem by developing a 3D printer that enables the printing of an organic powder, silk. This paper presents the design and implementation of the first prototype of a novel organic powder printer. The printer incorporates a novel compression mechanism that enables the compression of the powder during the printing process. It also includes a non-corrosive binder supply system consisting of silicone peroxide tubing, a custom-built peristaltic pump, and a nozzle built from a 33-gauge needle. The powder management system consists of a powder canister apparatus, depositing mechanism, and a powder bed designed to be compatible with low flowability powders, such as silk powder. The implementation of each component and the whole printer is presented. Evaluating the functionality of the organic powder printer found that several silk powder scaffolds were able to be printed with a varying magnitude of control over architecture. Future work is required to further advance the printable constructs up to a standard found in commercial 3D printers. This work demonstrated that a 3D printer system can be developed to fabricate constructs solely out of an organic powder. The paper highlights the limitations of the current design and suggests future improvements

Understanding Morphology, Bulk Properties, and Binding of Silk Particles for 3D Printing

Silk fibroin has emerged as a leading biomaterial for biomedical applications. 3D printing has been successfully used for printing with silk fibroin, albeit in the form of a bioink, in direct-write 3D printers. However, in the form of bioinks, stability and mechanical attributes of silk are lost. An innovative alternative to producing 3D printed solid silk constructs is silk milled into powder for printing in a binder jetting printer. In this work, we focus on characteristics of silk powder to determine suitability for use in 3D printing. Two different silk powders are compared with hydroxyapatite powder, a known biomaterial for biomedical constructs. We have investigated powder size and shape by Camsizer X2 and Scanning Electron Microscope and bulk behaviour, dynamic flow behaviour, and shear behaviour by FT4 powder rheometer. Preliminary printing tests were conducted in an in-house custom-built printer designed for silk powder. It was found that silk powder has low flowability and stability. Therefore, to print solely out of silk powder, a 3D printer design will need sophisticated techniques to produce flow to ensure even distribution and consistent thickness of powder layers during the printing process. It was also found that high concentrations of formic acid (&gt;75 to 99 wt.%) can fuse particles and therefore be used as a binder ink for 3D printing. The printer design challenges for silk powder are discussed

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 β-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 β-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 β-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.PostprintPeer reviewe