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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.119, #2)
Identification of microbial inhibitory functional groups in corn stover hydrolysate by carbon-13 nuclear magnetic resonance spectroscopy by F. A. Agblevor; J. Fu; B. Hames; J. D. McMillan (pp. 97-120).
Dilute-acid biomass hydrolysates contain biomass degradation products that are inhibitory to cell growth and fermentation. Overliming with Ca(OH)2 has been found to be one of the most effective methods for detoxifying the dilute-acid hydrolysate for ethanol production. However, the mechanism of overliming is not well understood. Carbon-13 nuclear magnetic resonance (13C-NMR) spectroscopy was used to elucidate the functional groups involved in the overliming reaction. The 13C-NMR spectra showed that the major functional groups removed during the overliming process were aliphatic and aromatic acids or esters, and other aromatic and aliphatic compounds. Ketone and aldehyde functionalities were not detected in the spectra. This is the first time that 13C-NMR has been used to elucidate the overliming reaction.
Keywords: Overliming; inhibition; corn stover hydrolysate; carbon-13 nuclear magnetic resonance; acetone
Immobilization of glucoamylase onto novel porous polymer supports of vinylene carbonate and 2-hydroxyethyl methacrylate by Yanli Huo; Yue Li; Zhi Yuan; Jiaxian Huang (pp. 121-131).
Glucoamylase was immobilized onto novel porous polymer supports. The properties of immobilized glucoamylase and the relationship between the activity of immobilized enzyme and the properties of porous polymer supports were investigated. Compared with the native enzyme, the temperature profile of immobilized glucoamylase was widened, and the optimum pH was also changed. The optimum substrate concentration of immobilized glucoamylase was higher than that of native enzyme. After storage for 23 d, the immobilized glucoamylase still maintained about 84% of its initial activity, whereas the native enzyme only maintained about 58% of the initial activity. Moreover, after using repeatedly seven times, the immobilized enzyme maintained about 85% of its initial activity. Furthermore, the properties of porous polymer supports had an effect on the activity of the immobilized glucoamylase.
Keywords: Glucoamylase; immobilization; porous supports; vinylene carbonate; 2-hydroxyethyl methacrylate
Capacity of Bacillus thuringiensis S-layer protein displaying polyhistidine peptides on the cell surface by Li Wang; Ming Sun; Ziniu Yu (pp. 133-143).
S-layer protein of Bacillus thuringiensis strain CTC was used as the carrier protein to display polyhistidine (poly[6His]) peptides on the cell surface. Poly(6His) n was fused with S-layer protein at two different sites, inserting just downstream of the S-layer protein homologous domain (slh) and replacing the non-slh region of S-layer protein, respectively. The two series chimeric proteins were both expressed by crystal negative B. thuringiensis strain 4Q7 and strain 171, respectively, as shown by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The recombinant B. thuringiensis cells gained Ni2+- and Cd2+-binding ability and had a capacity to display up to nine copies of poly(6His). The Cd2+ adsorption quantity of the recombinant strain with the strongest adsorption ability was twice that of the host strain.
Keywords: Bacillus thuringiensis ; cell-surface display; S-layer protein; polyhistidine; csaAB operon; ctc gene
Xylanase production by the thermophilic mold Humicola lanuginosa in solid-state fermentation by Pankaj Kamra; T. Satyanarayana (pp. 145-157).
Among the lignocellulosic substrates tested, wheat bran supported a high xylanase (EC 3.2.1.8) secretion by Humicola lanuginosa in solid-state fermentation (SSF). Enzyme production reached a peak in 72 h followed by a decline thereafter. Enzyme production was very high (7832 U/g of dry moldy bran) when wheat bran was moistened with tap water at a substrate-to-moistening agent ratio of 1:2.5 (w/v) and an inoculum level of 3 × 106 spores/10 g of wheat bran at a water activity (a w ) of 0.95. Cultivation of the mold in large enamel trays yielded a xylanase titer comparable with that in flasks. Parametric optimization resulted in a 31% increase in enzyme production in SSF. Xylanase production was approx 23-fold higher in SSF than in submerged fermentation (SmF). A threshold constitutive level of xylanase was secreted by H. lanuginosa in a medium containing glucose as the sole carbon source. The enzyme was induced by xylose and xylan. Enzyme synthesis was repressed beyond 1.0% (w/v) xylose in SmF, whereas it was unaffected up to 3.0% (w/w) in SSF, suggesting a minimization of catabolite repression in SSF.
Keywords: Humicola lanuginosa ; solid-state fermentation; submerged fermentation; wheat bran; xylanase
Optimization of medium by orthogonal matrix method for submerged mycelial culture and exopolysaccharide production in Collybia maculata by Jong Min Lim; Sang Woo Kim; Hye Jin Hwang; Ji Hoon Joo; Hyun Oh Kim; Jang Won Choi; Jong Won Yun (pp. 159-170).
Optimization of submerged culture conditions for the production of mycelial growth and exopolysaccharides (EPSs) by Collybia maculata was investigated. The optimum temperature and the initial pH for EPS production in a shake-flask culture of C. maculata were found to be 20°C and 5.5, respectively. Among the various medium’s constituents examined, glucose, Martone A-1, K2HPO4, and CaCl2 were the most suitable carbon, nitrogen, and mineral sources for EPS production, respectively. The optimum concentration of the medium’s ingredients determined using the orthogonal matrix method was as follows: 30 g/L of glucose, 20 g/L of Martone A-1, 1g/L of K2HPO4, and 1g/L of CaCl2. Under the optimized culture conditions, the maximum concentration of EPSs in a 5-L stirred-tank reactor was 2.4 g/L, which was approximately five times higher than that in the basal medium. A comparative fermentation result showed that the EPS productivity in an airlift reactor was higher than that in the stirred-tank reactor despite the lower mycelial growth rate. The specific productivities and the yield coefficients in the airlift reactor were higher than those in the stirred-tank reactor even though the volumetric productivities were higher in the stirred-tank reactor than in the airlift reactor.
Keywords: Collybia maculata ; exopolysaccharide; optimization; orthogonal matrix method; submerged culture
Coupled hydroperoxide lyase and alcohol dehydrogenase for selective synthesis of aldehyde or alcohol by Mohamed Gargouri; Najla Ben Akacha; Marie-Dominique Legoy (pp. 171-180).
The main objective of this work was to improve the selective synthesis of a volatile compound: aldehyde or alcohol using a coupled-enzyme system. A novel method of synthesis of C6-aldehyde or alcohol was carried out in the presence of hydroperoxide lyase (HPLS) activity coupled to alcohol dehydrogenase (ADH) activity. After cleavage of the initial substrate, hydroperoxy fatty acid catalyzed by HPLS, the second enzyme, ADH, can catalyze the reduction of the aldehyde to the corresponding alcohol, or the oxidation of contaminating alcohol into aldehyde, depending on the cofactor present in the medium (oxidized or reduced form). We succeeded in improving the synthesis of one of the products. When coupling HPLS to NADP, the selectivity of hexanal production from 13-hydroperoxy linoleic acid was improved, and hexanol production was reduced 5 to 10 times after 15 min of reaction at 15 °C and pH 7.0. In another experiment, HPLS was coupled to ADH in the presence of NADH. The production of alcohol (hexenols) was then favored especially when using 13-hydroperoxy linolenic acid as substrate at concentrations >15 mM, reaching 95% of the products. Coupling of the enzymatic reactions (cleavage reduction) not only reduced the number of steps but also allowed us to increase the conversion rate of the initial substrate (hydroperoxy fatty acid). Structures of the compounds produced in this work were confirmed using gas chromatography-mass spectroscopy analysis. Each of these products has its own delicately different fresh odor that can be used in various applications.
Keywords: Alcohol dehydrogenase; coupled enzymes; specific synthesis; hexanal; hexanol; hexenal; hexenol; hydroperoxide lyase; hydroperoxy fatty acid
Effect of expression of manganese superoxide dismutase in baculovirus-infected insect cells by Ying Wang; Larry W. Oberley; Dale Howe; Donald L. Jarvis; Gaurav Chauhan; David W. Murhammer (pp. 181-193).
It has previously been demonstrated that baculovirus infection of the Spodoptera frugiperda Sf-9 (Sf-9) and Trichoplusia ni BTI-Tn-5B1-4 (Tn-5B1-4) insect cell lines leads to oxidative stress as measured by protein and membrane lipid oxidation and that this oxidative damage contributes to cell death. As a result of these findings, it was hypothesized that baculovirus infection stimulates superoxide radical (O 2 ·— synthesis in the mitochondria and that the resulting O 2 ·— accumulation overwhelms the cells’ antioxidant defenses. We investigated the ability of manganese superoxide dismutase (MnSOD) expression (which reduces O 2 ·— to H2O2) to overcome the oxidative damage caused by baculovirus infection. It was found that MnSOD expression significantly reduced oxidative damage in baculovirus-infected Tn-5B1-4 cells but had no significant effect on oxidative damage in baculovirus-infected Sf-9 cells. The results are consistent with the hypothesis that O 2 ·— accumulation in the mitochondria is at least partially responsible for the oxidative damage resulting from the baculovirus infection of insect cells.
Keywords: Spodoptera frugiperda Sf-9; Trichoplusia ni BTI-Tn-5B1-4; baculovirus expression vector system; manganese superoxide dismutase; catalase; protein oxidation; lipid oxidation