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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.125, #3)
Applications of polymer nanofibers in biomedicine and biotechnology by J. Venugopal; S. Ramakrishna (pp. 147-157).
Recent advancements in the electrospinning method enable the production of ultrafine solid and continuous fibers with diameters ranging from a few nanometers to a few hundred nanometers with controlled surface and internal molecular structures. A wide range of biodegradable biopolymers can be electrospun into mats with specific fiber arrangement and structural integrity. Through secondary processing, the nanofiber surface can be functionalized to display specific biochemical characteristics. It is hypothesized that the large surface area of nanofibers with specific surface chemistry facilitates attachment of cells and control of their cellular functions. These features of nanofiber mats are morphologically and chemically similar to the extracellular matrix of natural tissue, which is characterized by a wide range of pore diameter distribution, high porosity, effective mechanical properties, and specific biochemical properties. The current emphasis of research is on exploiting such properties and focusing on determining appropriate conditions for electrospinning various polymers and biopolymers for eventual applications including multifunctional membranes, biomedical structural elements (scaffolds used in tissue engineering, wound dressing, drug delivery, artificial organs, vascular grafts), protective shields in specialty fabrics, and filter media for submicron particles in the separation industry. This has resulted in the recent applications for polymer nanofibers in the field of biomedicine and biotechnology.
Keywords: Electrospinning; nanofibers; tissue engineering; biotechnology; scaffolds
Development of a method to quantify gene expression levels for glycosylation pathway genes in Chinese hamster ovary cell cultures by Kevin J. -R. Clark; Sarah W. Harcum; Jennifer Griffiths; Kevin M. Bailey (pp. 159-173).
Changes in protein glycosylation owing to changes in environmental conditions are not well understood. To better understand these relationships, methods to quantify controlling factors are needed. Because enzymes are translated from genes, the ability to quantify gene expression levels for glycosylation-related enzymes would be advantageous. We developed quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) assays to monitor gene expression in Chinese hamster ovary (CHO) cells for five terminal glycosylation genes. The five enzymes were sialidase, a putative α2,3-sialyltransferase, β1,4-galactosyltransferase, cytosine monophosphate-sialic acid transporter, and uracil diphosphate-galactosyl transporter. Four of these CHO cell genes were publicly available from GenBank; however, the α2,3-sialyltransferase gene for Cricetulus griseus (CHO cell species) was not available and, therefore, was sequenced as a part of this work. The qRT-PCR primers and probes (based on the TaqManTM chemistry) were designed and validated for these five genes. The gene expression profiles were obtained for CHO cells producing the recombinant interleukin-4/13 cytokine trap molecule in batch reactors.
Keywords: Quantitative real-time polymerase chain reaction; mRNA; glycosylation; interleukin-4/13 cytokine trap; α2,3-sialyltransferase; sequencing
Carbohydrate reactions during high-temperature steam treatment of aspen wood by Jiebing Li; Gunnar Henriksson; Göran Gellerstedt (pp. 175-188).
Aspen wood was treated with steam at different time-temperature severity factors. Analysis of the amounts of acids released revealed a relationship between the acidity and the formation of furfural and hydroxymethyl furfural as degradation products from carbohydrates. It is suggested that two concurrent or consecutive mechanisms are responsible for the observed results: a homolytic cleavage and an acid hydrolysis of glucosidic linkages in the polysaccharides. By preimpregnating the wood with alkali, hydrolysis can be eliminated, resulting in a much cleaner depolymerization of the polysaccharides without any further acid-catalyzed degradation. The enzymatic digestibility of the steam-treated wood material for the formation of glucose was compared with that of steam-exploded wood. A more efficient route for glucose production from steam-exploded wood was found as long as the biomass-pretreated material was homogeneous and without shives.
Keywords: Aspen wood; steam treatment; steam explosion; depolymerization; acid-catalyzed degradation; furfural; hydroxymethyl furfural; enzymatic hydrolysis; acid hydrolysis
Eco1524I, a type II restriction endonuclease by Hadeer Lazim; Jytte Josephsen; Assia Ben Hassen; Omrane Belhadj; Férid Limam (pp. 189-199).
Various strains of Escherichia coli, isolated from different patients, were screened for type II restriction endonuclease activity. In 1 out of 23 patients, a type II restriction endonuclease activity was found. The restriction endonuclease designated Eco1524I was purified to near homogeneity, based on hydroxyapatite and heparin sepharose chromatography. Eco1524I exhibited endonuclease restriction activity in the pH range from 6.0 to 10.0 (maximum level at pH 8.0) and required Mg2+ as divalent cation. The enzyme was stable till temperature 55°C and pH range from 6.0 to 10.0. Eco1524I recognized the sequence 6-bp palindromic 5′AGG↑CCT 3′, producing blunt end and is found to be an isoschizomer of Stu I.
Keywords: E. coli ; restriction/modification; type II endonuclease; restriction site; Stu I isoschizomer
Purification and partial characterization of an extracellular ribonuclease from a mutant of Aspergillus niger by Ya-Hong Xiong; Jian-Zhong Liu; Hai-Yan Song (pp. 201-210).
A new extracellular ribonuclease (RNase) from a mutant of Aspergillus niger, named A. niger SA-13-20 RNase, was purified to homogeneity by (NH4)2SO4 fractionation (50–85%), DEAE-cellulose anion-exchange chromatography, ultrafiltration and Sephacryl HR-200 chromatography. The enzyme was purified up to 54.4-fold with a final yield of 24.5%. There were differences in the molecular weight, pI value and some physico-chemical properties between A. niger SA-13-20 RNase and that from the parent strain. The enzyme is monomeric and its molecular weight and isoelectric point were 40.1 kDa and 5.3, respectively. The N-terminal amino acid sequence of A. niger SA-13-20 RNase was TIDTYSSDSP. The optimum pH, temperature and buffer concentration for the enzymatic reaction were 3.5, 65°C, and 0.175 M, respectively. Metal ions, such as K+, NH4 +, Mg2+, and Ca2+ at the concentration of 1.0 mM had a slight activation effect on the enzyme activity and (NH4)2SO4 activated the enzyme significantly. The enzyme was stable at pH lower than 8.5 and was easy to inactivate in strong alkali solution.
Keywords: Ribonuclease; purification; Aspergillus niger ; properties