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Biotechnology Progress

Kavleen Kaur, Gursharan Singh, Vijaya Gupta, Neena Capalash, Prince Sharma
Laccases are multicopper oxidases known to catalyze the transformation of a wide range of phenolic and non-phenolic substrates using oxygen as electron acceptor and forming water as the only by product. Their potential relevance in several industries requires the constant search for novel laccases. Positive outcome of the isolation of laccase producing bacteria depends on the nature and concentration of media constituents. Several attempts to isolate laccase producing bacteria failed when the phosphate-containing M9 minimal medium was used...
November 14, 2016: Biotechnology Progress
João H P M Santos, Iris M Costa, João V D Molino, Mariana S M Leite, Marcela V Pimenta, João A P Coutinho, Adalberto Pessoa, Sónia P M Ventura, André M Lopes, Gisele Monteiro
L-asparaginase (ASNase) is a biopharmaceutical widely used to treat child leukemia. However, it presents some side effects, and in order to provide an alternative biopharmaceutical, in this work, the genes encoding ASNase from Saccharomyces cerevisiae (Sc_ASNaseI and Sc_ASNaseII) were cloned in the prokaryotic expression system Escherichia coli. In the 93 different expression conditions tested, the Sc_ASNaseII protein was always obtained as an insoluble and inactive form. However, the Sc_ASNaseI (His)6 -tagged recombinant protein was produced in large amounts in the soluble fraction of the protein extract...
November 14, 2016: Biotechnology Progress
Amin Bornadel, Mohamed Ismail, Mahmoud Sayed, Rajni Hatti-Kaul, Sang-Hyun Pyo
Six-membered cyclic carbonates with hydroxyl and methoxycarbonyloxy functional groups were prepared by transesterification of trimethylolpropane (TMP) with dimethylcarbonate (DMC) by solvent-free lipase-mediated flow reaction followed by thermal cyclization. The flow reaction efficiency was evaluated using different configurations of reactor consisting of packed beds of Novozym®435 (immobilized Candida antarctica lipase B-CalB-a.k.a. N435) and molecular sieves, flowrate, and biocatalyst loads. The mixed column of the biocatalyst and molecular sieves, allowing rapid and efficient removal of the by-product-methanol-was the most efficient setup...
November 14, 2016: Biotechnology Progress
Fernie G Mitchelson, Jessica P Mondia, Erik H Hughes
The ability to control charge heterogeneity in monoclonal antibodies is important to demonstrate product quality comparability and consistency. This paper addresses the control of C-terminal lysine processing through copper supplementation to yeast hydrolysate powder, a raw material used in the cell culture process. Large-scale production of a murine cell line exhibited variation in the C-terminal lysine levels of the monoclonal antibody. Analysis of process data showed that this variation correlated well with shifts in cell lactate metabolism and pH levels of the production culture...
November 14, 2016: Biotechnology Progress
Nora Luniak, Peter Meiser, Sonja Burkart, Rolf Müller
Expression of proteases in heterologous hosts remains an ambitious challenge due to severe problems associated with digestion of host proteins. On the other hand, proteases are broadly used in industrial applications and resemble promising drug candidates. Bromelain is an herbal drug that is medicinally used for treatment of oedematous swellings and inflammatory conditions and consists in large part of proteolytic enzymes. Even though various experiments underline the requirement of active cysteine proteases for biological activity, so far no investigation succeeded to clearly clarify the pharmacological mode of action of bromelain...
November 14, 2016: Biotechnology Progress
Jamie Sharp, Patrick Vermette
Perfusion bioreactors, unlike traditional in vitro cell culture systems, offer stringent control of physiological parameters such as pH, flow, temperature, and dissolved oxygen concentration which have been shown to have an impact on cellular behaviour and viability. Due to the relative infancy and the growing interest in these in vitro culture systems, detection methods to monitor cell function under dynamic perfusion bioreactor conditions remains one of the main challenges. In this study, INS-1 cells, a cell line which exhibit glucose-stimulated insulin secretion, were encapsulated in fibrin and cultured under perfusion bioreactor conditions for 48h and then exposed to either a high-, or low-glucose concentration for 24h...
November 14, 2016: Biotechnology Progress
Chare Li Kueh, Chean Yeah Yong, Seyedehsara Masoomi Dezfooli, Subha Bhassu, Soon Guan Tan, Wen Siang Tan
Macrobrachium rosenbergii nodavirus (MrNV) is a virus native to giant freshwater prawn. Recombinant MrNV capsid protein has been produced in Escherichia coli, which self-assembled into virus-like particles (VLPs). However, this recombinant protein is unstable, degrading and forming heterogenous VLPs. In the current study, MrNV capsid protein was produced in insect Spodoptera frugiperda (Sf9) cells through a baculovirus system. Dynamic light scattering (DLS) and transmission electron microscopy (TEM) revealed that the recombinant protein produced by the insect cells self-assembled into highly stable, homogenous VLPs each of approximately 40 nm in diameter...
November 14, 2016: Biotechnology Progress
María F Baieli, Nicolás Urtasun, María J Martinez, Daniela B Hirsch, Ana M R Pilosof, María V Miranda, Osvaldo Cascone, Federico J Wolman
Casein glycomacropeptide (CMP) is a 64- amino acid peptide found in cheese whey, which is released after κ-casein specific cleavage by chymosin. CMP lacks aromatic amino acids, a characteristic that makes it usable as a nutritional supplement for people with phenylketonuria. CMP consists of two nonglycosylated isoforms (aCMP A and aCMP B) and its different glycosylated forms (gCMP A and gCMP B). The most predominant carbohydrate of gCMP is N-acetylneuraminic acid (sialic acid). Here, we developed a CMP purification process based on the affinity of sialic acid for wheat germ agglutinin (WGA)...
November 7, 2016: Biotechnology Progress
Jiayi Sun, Li Ma, Ka-Yiu San, Christie A M Peebles
Hairy root cultures generated using Agrobacterium rhizogenes are an extensively investigated system for the overproduction of various secondary metabolite based pharmaceuticals and chemicals. This study demonstrated a transgenic Catharanthus roseus hairy root line carrying a feedback-insensitive anthranilate synthase (AS) maintained chemical and genetic stability for 11 years. The AS gene was originally inserted in the hairy root genome under the control of a glucocorticoid inducible promoter. After 11 years continuous maintenance of this hairy root line, genomic PCR of the ASA gene showed the presence of ASA gene in the genome...
November 4, 2016: Biotechnology Progress
Jan-Hendrik Grosch, David Wagner, Vasilios Nistelkas, Antje C Spieß
The reaction medium has major impact on biocatalytic reaction systems and on their economic significance. To allow for tailored medium engineering, thermodynamic phenomena, intrinsic enzyme kinetics and enzyme-solvent interactions have to be discriminated. To this end, enzyme reaction kinetic modeling was coupled with thermodynamic calculations based on investigations of the alcohol dehydrogenase from Lactobacillus brevis (LbADH) in monophasic water/methyl tert-butyl ether (MTBE) mixtures as a model solvent...
November 4, 2016: Biotechnology Progress
Cyrus D Agarabi, Brittany K Chavez, Scott C Lute, Erik K Read, Sarah Rogstad, David Awotwe-Otoo, Matthew R Brown, Michael T Boyne, Kurt A Brorson
Linkage of upstream cell culture with downstream processing and purification is an aspect of Quality by Design crucial for efficient and consistent production of high quality biopharmaceutical proteins. In a previous Plackett-Burman screening study of parallel bioreactor cultures we evaluated main effects of 11 process variables, such as agitation, sparge rate, feeding regimens, dissolved oxygen set point, inoculation density, supplement addition, temperature, and pH shifts. In this follow-up study, we observed linkages between cell culture process parameters and downstream capture chromatography performance and subsequent antibody attributes...
November 4, 2016: Biotechnology Progress
John McReynolds, Yu Wen, Xiaofei Li, Jianjun Guan, Sha Jin
Three-dimensional (3D) scaffold culture of pancreatic β-cell has been proven to be able to better mimic physiological conditions in the body. However, one critical issue with culturing pancreatic β-cells is that β-cells consume large amounts of oxygen, and hence insufficient oxygen supply in the culture leads to loss of β-cell mass and functions. This becomes more significant when cells are cultured in a 3D scaffold. In this study, in order to understand the effect of oxygen tension inside a cell-laden collagen culture on β-cell proliferation, a culture model with encapsulation of an oxygen-generator was established...
November 1, 2016: Biotechnology Progress
Pui-Fong Jee, Fez-Shin Chen, Meng-Hooi Shu, Won Fen Wong, Raha Abdul Rahim, Sazaly AbuBakar, Li-Yen Chang
Heterologous protein displayed on the surface of Lactococcus lactis using the binding domain of N-acetylmuramidase (AcmA) has a potential application in vaccine delivery. In this study, we developed a non-recombinant L. lactis surface displaying the influenza A (H1N1) 2009 hemagglutinin (HA1). Three recombinant proteins, HA1/L/AcmA, HA1/AcmA and HA1 were overexpressed in Escherichia coli, and purified. In the binding study using flow cytometry, the HA1/L/AcmA, which contained the single-chain variable fragment (scFv) peptide linker showed significantly higher percentage of binding counts and mean fluorescence binding intensity (MFI) (51...
November 1, 2016: Biotechnology Progress
Noah D Weiss, Lisbeth Garbrecht Thygesen, Christian Roslander, Keith Gourlay, Claus Felby
The underlying mechanisms of the recalcitrance of biomass to enzymatic deconstruction are still not fully understood, and this hampers the development of biomass based fuels and chemicals. With water being necessary for most biological processes, it is suggested that interactions between water and biomass may be key to understanding and controlling biomass recalcitrance. This study investigates the correlation between biomass recalcitrance and the constraint and retention of water by the biomass, using SO2 pretreated spruce, a common feedstock for lignocellulosic biofuel production, as a substrate to evaluate this relationship...
November 1, 2016: Biotechnology Progress
Miao Zhang, Harriëtte Oldenhof, Harald Sieme, Willem F Wolkers
Fibroblasts take up trehalose during freezing and thawing, which facilitates cryosurvival of the cells. The aim of this study was to investigate if trehalose uptake via fluid-phase endocytosis prefreeze increases cryosurvival. To determine endocytic trehalose uptake in attached as well as suspended fibroblasts, intracellular trehalose concentrations were determined during incubation at 37°C using an enzymatically based trehalose assay. In addition, freezing-induced trehalose uptake of extracellularly added trehalose was determined...
November 1, 2016: Biotechnology Progress
Thomas E Murphy, Leslie E Prufert-Bebout, Brad M Bebout
The results of a numerical study on the simulation of pulse amplitude modulated (PAM) fluorometry within dense suspensions of photosynthetic microorganisms are presented. The Monte Carlo method was used to solve the radiative transfer equation in an algae-filled cuvette, taking into account absorption, anisotropic scattering, and fluorescence, as well as Fresnel reflections at interfaces. This method was used to simulate the transport of excitation and fluorescence light in a common laboratory fluorometer. In this fluorometer, detected fluorescence originates from a multitude of locations within the algal suspension, which can be exposed to very different fluence rates...
November 1, 2016: Biotechnology Progress
Stevan C Albers, Christie A M Peebles
Cyanobacteria are enticing microbial factories, but little is understood how their gene control elements respond to the periodic availability to light. This research tested the capability of PpsbAII to control gene expression during light/dark conditions when moved to a neutral location within the Synechocystis sp. PCC 6803 genome. When the eYFP reporter gene was run by PpsbAII in the promoter's native genomic location, mutants exposed to 12-hour light conditions experienced a 15.8X increase in transcript abundance over that observed from the same construct exposed to 12-hour dark conditions...
October 31, 2016: Biotechnology Progress
Jared S Bee, LeeAnn M Machiesky, Li Peng, Kristin C Jusino, Matthew Dickson, Jeffrey Gill, Douglas Johnson, Hung-Yu Lin, Kenneth Miller, Jenny Heidbrink Thompson, Richard L Remmele
Recombinant therapeutic monoclonal antibodies (mAbs) must be purified from host cell proteins (HCPs), DNA, and other impurities present in Chinese hamster ovary (CHO) cell culture media. HCPs can potentially result in adverse clinical responses in patients and, in specific cases, have caused degradation of the final mAb product. As reported previously, residual traces of cathepsin D caused particle formation in the final product of mAb-1. The current work was focused on identification of a primary sequence in mAb-1 responsible for the binding and consequent co-purification of trace levels of CHO cathepsin D...
October 31, 2016: Biotechnology Progress
Christina L Ross
Directed cell migration and adhesion is essential to embryonic development, tissue formation and wound healing. For decades it has been reported that electric field (EF), magnetic field (MF) and electromagnetic field (EMF) can play important roles in determining cell differentiation, migration, adhesion, and evenwound healing. Combinations of these techniques have revealed new and exciting explanations for how cells move and adhere to surfaces; how the migration of multiple cells are coordinated and regulated; how cellsinteract with neighboring cells, and also to changes in their microenvironment...
October 31, 2016: Biotechnology Progress
Tatjana Krivorotova, Ramune Staneviciene, Juliana Luksa, Elena Serviene, Jolanta Sereikaite
The relationship between pectin structure and the antimicrobial activity of nisin-loaded pectin particles was examined. The antimicrobial activity of five different nisin-loaded pectin particles, i.e., nisin-loaded high methoxyl pectin, low methoxyl pectin, pectic acid, dodecyl pectin with 5.4 and 25% degree of substitution were tested in the pH range of 4.0-7.0 by agar-diffusion assay and agar plate count methods. It was found that the degree of esterification of carboxyl group of galacturonic acid in pectin molecule is important for the antimicrobial activity of nisin-loaded pectin particles...
October 28, 2016: Biotechnology Progress
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