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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.110, #2)
Biological responses of suspension cultures of Taxus chinensis var. mairei to shear stresses in the short term by Zhong-Dong Shi; Ying-Jin Yuan; Jin-Chuan Wu; Gui-Min Shang (pp. 61-74).
The biological responses of Taxus chinensis var. mairei suspension cultures to a range of shear rates, were investigated in a Couette-type shear reactor. It was found that the shear rate below 458 s−1 enhanced the primary metabolism, increasing mitochondrial activity and protein expression and inhibiting the activity of phenylalanine ammonia lyase (PAL), an enzyme relevant to the secondary metabolism. However, the shear rate over 719 s−1 damaged taxus cells to some extent, decreasing the mitochondrial activity, increasing the membrane permeability, and even causing cell hypersensitive responses. As a result, PAL and intracellular peroxidase, an enzyme in cells relevant to reactive oxygen species scavenging, were activated and extracellular phenolics were accumulated. Additionally, shear-induced accumulation of free radicals and alkalization of the culture medium were observed. The results might shed light on a better understanding of the mechanism of plant cells responses to shearstress through the signal transduction pathway.
Keywords: Suspension culture; Taxus chinensis var. mairei ; shear; biological activity; secondary metabolism; hypersensitive response
1H-NMR study of Na alginates extracted from Sargassum spp. in relation to metal biosorption by Thomas A. Davis; Francisco Llanes; Bohumil Volesky; Guillermo Diaz-Pulido; Laurence McCook; Alfonso Mucci (pp. 75-90).
The use of a number of species of marine brown algae in the implementation of bioremediation strategies for toxic heavy metals is being considered and evaluated. The biosorption capacity of these algae for heavy metals resides mainly in a group of linear polysaccharides known as alginates that occur as a gel in the algal thallus. The potential for selective metal binding by the biomass of two species of Sargassum was evaluated by 1H-NMR (nuclear magnetic resonance) following a high temperature, alkaline extraction and purification of their alginate polysaccharide. The alkaline extraction protocol applied to Sargassum fluitans and Sargassum siliquosum yielded alginate samples of low viscosity, suitable for direct acquisition of well-resolved spectra. Estimates of both the ratio of β-d-mannopyranuronosyl (M) and α-l-gulopyranuronosyl (G) residues along the polymer chain and the frequencies of occurrence of diad uronic acid residue pairs were obtained. Guluronic acid (G) was the major component in all, extracts and the GG diads accounted for more than 49% of the polymer diads. Whereas the performance of Sargassum spp. in the metal biosorption process is a function of both its alginate content and composition, the occurrence of “G-blocks” in both purified alginates and in the raw brown seaweed is critical because it results in a well-established selectivity for divalentions, potentially increasing the commercial effectiveness of targeted biosorption as a means of remediation.
Keywords: Alginate; brown seaweed; biosorption; extraction; heavy metals; nuclear magnetic resonance; uronic acid; Sargassum
Characterization of polysaccharide production of Haemophilus influenzae type b and its relationship to bacterial cell growth by Mickie Takagi; Joaquin Cabrera-Crespo; Júlia Baruque-Ramos; Teresa Cristina Zangirolami; Isaias Raw; Martha Massako Tanizaki (pp. 91-100).
Haemophilus influenzae type b (Hib) causes invasive infections in infants and young children. Vaccines consisting of Hib capsular polysaccharide (polymer of ribosylribitol phosphate [PRP]) conjugated to a protein are effective in the prevention of such infections. The production of capsular polysaccharide type b was studied in three cultivation conditions: single, glucose pulse, and repeated batch. Specific polysaccharide production (Y p/x ) was calculated for all experiments, showing the following values: 67 (single-batch cultivation), 71 (glucose pulse), 75 (repeated-batch cultivation, first batch), and 87 mg of PRP/g of dry cell weight (DCW) (repeated-batch cultivation, second batch). Biomass concentration reached ∼1.8 g of DCW/L, while polysaccharide concentration was about ∼132 mg/L in the three fermentation runs. Polysaccharide synthesis is associated with cell growth in all studied conditions as established by Kono's analysis and Luedeking-Piret's model.
Keywords: Haemophilus influenzae ; capsular polysaccharide; polymer of ribosylribitol phosphate; batch cultivation
Improvement in enzyme activity and stability by addition of low molecular weight polyethylene glycol to sodium bis(2-ethyl-L-hexyl)sulfosuccinate/isooctane reverse micellar system by M. M. R. Talukder; T. Takeyama; Y. Hayashi; J. C. Wu; T. Kawanishi; N. Shimizu; C. Ogino (pp. 101-112).
The activity and stability of Chromobacterium viscosum lipase (glycerolester hydrolase, EC 3.1.1.3)-catalyzed olive oil hydrolysis in sodium bis (2-ethyl-1-hexyl)sulfosuccinate (AOT)/isooctane reverse micelles is increased appreciably when low molecular weight polyethylene glycol (PEG 400) is added to the reverse micelles. To understand the effect of PEG 400 on the phase behavior of the reverse micellar system, the phase diagram of AOT/PEG 400/water/isooctane system was studied. The influences of relevant parameters on the catalytic activity in AOT/PEG 400 reverse micelles were investigated and compared with the results in the simple AOT reverse micelles. In the presence of PEG 400, the linear decreasing trend of the lipase activity with AOT concentration, which is observed in the simple AOT reverse micelles, disappeared. Enzyme entrapped in AOT/PEG reverse micelles was very stable, retaining>75% of its initial activity after 60 d, whereas the half-life in simple AOT reverse micelles was 38 d. The kinetics parameter maximum velocity (V max)exhibiting the temperature dependence and the activation energy obtained by Arrhenius plot was suppressed significantly by the addition of PEG 400.
Keywords: Enzyme stability; reverse micelles; polyethylene glycol; phase diagram; activation energy
Production of C-terminal amidated recombinant salmon calcitonin in Streptomyces lividans by Bin Hong; Bingyuan Wu; Yuan Li (pp. 113-123).
Salmon calcitonin (sCT) is one of the many bioactive peptides that require C-terminal amidation for full biologic activity. To produce fully bioactive sCT in large scale, we constructed Streptomyces lividans [pMSA], an engineering Streptomyces strain. In the expression vector, glycine-extended sCT, the substrate for amidation, and rat α-amidating enzyme cDNA were cloned under the control of the strong constitutive promoter from the Streptomyces fradiae aph gene in pIJ680. Both were expressed in a secretory manner by the recombinant strain using the expression and secretion signals of melC1. Extracellularly expressed recombinant sCT was purified to near homogeneity and characterized by enzyme immunoassay, followed by direct amino-terminal sequencing. High-performance liquid chromatography, matrixassisted laser desorption ionization-time-of-flight mass spectrometry, and bioassay in vivo demonstrated purified product to be equivalent to synthetic standard. Thus, the engineered Streptomyces strain can produce bioactive, C-terminal amidated recombinant sCT in the culture supernatant directly. The ease of the recombinant process, as well as its potential for scale-up, makes it adaptable to production demands for sCT, and it may be applied to other bioactive peptides that need C-terminal amidation.
Keywords: Salmon calcitonin; α-amidating enzyme; Streptomyces lividans