An investigation into transition metal ion binding properties of silk fibers and particles using radioisotopes

Silk is a structural protein fiber that is stable over a wide pH range making it attractive for use in medical and environmental applications. Variation in amino acid composition has the potential for selective binding for ions under varying conditions. Here we report on the metal ion separation potential of Mulberry and Eri silk fibers and powders over a range of pH. Highly sensitive radiotracer probes, ⁶⁴Cu²⁺, ¹⁰⁹Cd²⁺, and ⁵⁷Co²⁺ were used to study the absorption of their respective stable metal ions Cu²⁺, Cd²⁺, and Co²⁺ into and from the silk sorbents. The total amount of each metal ion absorbed and time taken to reach equilibrium occurred in the following order: Cu²⁺ > Cd²⁺ > Co²⁺. In all cases the silk powders absorbed metal ions faster than their respective silk fibers. Intensive degumming of the fibers and powders significantly reduced the time to absorb respective metal ions and the time to reach equilibrium was reduced from hours to 5–15 min at pH 8. Once bound, 45–100% of the metal ions were released from the sorbents after exposure to pH 3 buffer for 30 min. The transition metal ion loading capacity for the silk sorbents was considerably higher than that found for commercial ion exchange resins (AG MP‐50 and AG 50W‐X2) under similar conditions. Interestingly, total Cu²⁺ bound was found to be higher than theoretically predicted values based on known specific Cu²⁺ binding sites (AHGGYSGY), suggesting that additional (new) sites for transition metal ion binding sites are present in silk fibers. © 2010 Wiley Periodicals, Inc. J Appl Polym Sci, 2011     

Mechanical properties and structure of silkworm cocoons: A comparative study of Bombyx mori,Antheraea assamensis,Antheraea pernyi and Antheraea mylitta silkworm cocoons

As a protective shell against environmental damage and attack by natural predators, the silkworm cocoon has outstanding mechanical properties. In particular, this multilayer non-woven composite structure can be exceptionally tough to enhance the chance of survival for silkworms while supporting their metabolic activity. Peel, out-of-plane compression and nano-indentation tests and micro-structure analysis were performed on four types of silkworm cocoon walls (domesticated Bombyx mori, semi-domesticated Antheraea assamensis and wild Antheraea pernyi and Antheraea mylitta silkworm cocoons) to understand the structure and mechanical property relationships. The wild silkworm cocoons were shown to be uniquely tough composite structures. The maximum work-of-fracture for the wild cocoons (A. pernyi and A. mylitta) was approximately 1000 J/m², which was almost 10 times the value for the domesticated cocoon (Bombyx mori) and 3 ~ 4 times the value for the semi-domesticated cocoon (A. assamensis). Calcium oxalate crystals were found to deposit on the outer surfaces of the semi-domesticated and wild cocoons. They did not show influence in enhancing the interlaminar adhesion between cocoon layers but exhibited much higher hardness than the cocoon pelades.     

Ultra-fine silk powder preparation through rotary and ball milling

Three commercial silk varieties, namely mulberry, muga and eri, were used to prepare ultra-fine silk particles. Degummed silk fibres were chopped into short snippets and then pulverised using rotary and planetary ball milling. The effects of degree of degumming, size of milling media, water and lubricant on particle refinement were studied. Before milling, single fibre strength tests were conducted on silk fibres degummed under different conditions. The results indicate that while reducing fibre strength via harsh degumming could cut milling time drastically, too severe a reduction in fibre strength is actually detrimental to achievable minimum particle size due to increased particle aggregation. Water played an important role in affecting the performance of ball milling. With the milling processes used in this study, a volume based median particle size (d(0.5)) of around 200 nm was achieved, which is much smaller than previously reported results. To achieve a similar particle size, mulberry silk required more milling time, even though the overall milling behaviour was quite similar for the three silk varieties examined. SEM observations revealed axial spitting and fragmentation of micro and nanofibrillar architecture of silk fibres due to milling. Unlike ball milling, which produced particles with variable shapes, rotary milled particles remained fibrous through the size reduction process.     

Effect of solvent on ionic liquid dissolved regenerated antheraea assamensis silk fibroin

We report on the dissolution of semi‐domestic silk type Antheraea assamensis using ionic liquids. We investigated the impact of different coagulating solvents, including isopropanol and water on the structure and the morphology of the regenerated silk. We found that the water regenerated silk film showed a high β‐sheet content and a native silk‐like XRD pattern. © 2012 Wiley Periodicals, Inc. J. Appl. Polym. Sci., 2013     

Enzyme‐catalyzed copolymerization of oxiranes with dicarboxylic acid anhydrides

Ring‐opening copolymerizations of the oxiranes glycidyl phenyl ether (GPE) and diglycidyl ether of bisphenol A (DGEBA) with a dicarboxylic acid anhydride [methyl hexahydrophthalic anhydride, nadic anhydride, maleic anhydride (MA), or itaconic anhydride (IA)] were carried out with the lipases Candida cylindracea (CCL), Lipozyme TL‐IM (LIM), and Novozyme 435 (N435) as catalysts. The CCL‐catalyzed reaction of DGEBA with MA or IA (at a 1:2 molar ratio) at 80°C resulted in only partial curing. We monitored the reactions by Fourier transform infrared spectroscopy and by following the changes in the intensities of carbonyl stretching frequencies of the anhydride and ester groups. The reactivity of the oxirane group in GPE was higher than that in DGEBA; this may have been due to the higher viscosity of DGEBA. The reactivities of the enzymes for the copolymerization of the oxiranes and dicarboxylic acid anhydride were in the order LIM > CCL > N435. © 2005 Wiley Periodicals, Inc. J Appl Polym Sci 97: 697–704, 2005     

Steric Constraints Dependent on Nucleobase Pair Orientation Vary in Different DNA Polymerase Active Sites

Finding the right fit : Herein, we report on the development of novel steric probes and present initial insights into their interplay with DNA polymerases. Our findings provide experimental evidence for varied enzyme–substrate interactions that might account for the varied selectivity previously observed.

     

Tensile stress–strain and recovery behavior of Indian silk fibers and their structural dependence

The tensile stress–strain and recovery behavior of all the four commercial varieties of Indian silk fibers, namely Mulberry, Tasar, Eri, and Muga, have been studied along with their structures. Compared to the non‐Mulberry silk fibers, Mulberry silk fiber is much finer and has crystallites of smaller size, higher molecular orientation, and a more compact overall packing of molecules. These structural differences have been shown to result in (1) the presence of a distinct yield and a yield plateau in non‐Mulberry silk and their absence in Mulberry silk, and (2) relatively higher initial modulus and tenacity along with lower elongation‐to‐break and toughness and superior elstic recovery behavior of mulberry silk compared to non‐Mulberry silk. It is also observed that fine silk fibers have a relatively more ordered and compact structure with higher orientation compared to their coarse counterparts, and this gives rise to higher initial modulus and higher strength in the finer fibers. © 2000 John Wiley & Sons, Inc. J Appl Polym Sci 77: 2418–2429, 2000     

Stress relaxation and inverse stress relaxation in silk fibers

Stress‐relaxation experiments on four varieties of Indian silk fiber show that stress relaxation is significantly greater in non‐Mulberry silks than in the Mulberry silk and that the differences among non‐Mulberry silk fibers are relatively small. All the fibers studied also exhibit inverse stress relaxation. It has been shown that the Maxwell–Wiechert model, with two Maxwell elements in parallel, can be used to analyze and explain both the stress‐relaxation and inverse stress‐relaxation behaviors. © 2001 John Wiley & Sons, Inc. J Appl Polym Sci 82: 1147–1154, 2001     

Molecular weight and secondary structure change in eri silk during alkali degumming and powdering

Changes in molecular weight and secondary structure of eri silk during alkali degumming and silk powdering were studied. An increase in silk degumming intensity, through increased alkali concentration, treatment temperature, and time, reduced the fibroin molecular weight and, therefore, the fiber tenacity, but at the same time, increased the β‐sheet fraction. These changes reduced the time required to mill the degummed silk fibers into fine powders. Mechanical forces used in wet attritor and air jet milling disturbed intermolecular packing along the direction of side chains, but the conformation remained essentially β‐sheet even in the sub‐micron silk particles. Dry milling drastically reduced molecular weight and changed the conformation of the fibroin chains. The rate of the spontaneous conformation transition in regenerated fibroin solution prepared from fibers and powders increased with a decrease in fibroin molecular weight, affecting the time fibroin solutions could be stored before gelling. Overall, the study showed that molecular weight and secondary structure of silk powders could be manipulated by suitably changing the degumming and milling conditions. It also suggests that wet media milling and air jet milling are better than dry media milling to prepare less degraded and more crystalline ultrafine silk particles. © 2010 Wiley Periodicals, Inc. J Appl Polym Sci, 2011     

Varied active-site constraints in the klenow fragment of E. coli DNA polymerase I and the lesion-bypass Dbh DNA polymerase

We report on comparative pre-steady-state kinetic analyses of exonuclease-deficient Escherichia coli DNA polymerase I (Klenow fragment, KF-) and the archaeal Y-family DinB homologue (Dbh) of Sulfolobus solfataricus. We used size-augmented sugar-modified thymidine-5'-triphosphate (T(R)TP) analogues to test the effects of steric constraints in the active sites of the polymerases. These nucleotides serve as models for study of DNA polymerases exhibiting both relatively high and low intrinsic selectivity. Substitution of a hydrogen atom at the 4'-position in the nucleotide analogue by a methyl group reduces the maximum rate of nucleotide incorporation by about 40-fold for KF- and about twelve fold for Dbh. Increasing the size to an ethyl group leads to a further twofold reduction in the rates of incorporation for both enzymes. Interestingly, the affinity of KF- for the modified nucleotides is only marginally affected, which would indicate no discrimination during the binding step. Dbh even has a higher affinity for the modified analogues than it does for the natural substrate. Misincorporation of either TTP or T(Me)TP opposite a G template causes a drastic decline in incorporation rates for both enzymes. At the same time, the binding affinities of KF- for these nucleotides drop by about 16- and fourfold, respectively, whereas Dbh shows only a twofold reduction. Available structural data for ternary complexes of relevant DNA polymerases indicate that both enzymes make close contacts with the sugar moiety of the dNTP. Thus, the varied proficiencies of the two enzymes in processing the size-augmented probes indicate varied flexibility of the enzymes' active sites and support the notion of active site tightness being a criterion for DNA polymerase selectivity.