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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.141, #1)
Purification and properties of a β-galactosidase with potential application as a digestive supplement by S. O'Connell; G. Walsh (pp. 1-13).
Functional-based screening of crude β-galactosidase activities from 42 yeast strains resulted in the selection of a single enzyme of potential interest as a digestive supplement. β-Galactosidase produced by Kluyveromyces marxianus DSM5418 was purified to homogeneity by a combination of gel filtration, ion-exchange, and hydroxylapatite chromatographies. The denatured (123 kDa) and native molecular masses (251 kDa) suggest that the enzyme is a homodimer. The optimum pH and temperature of the purified enzyme were 6.8 and 37°C, respectively. The unpurified β-galactosidase in particular displayed a high level of stability when exposed to simulated intestinal conditions in vitro for 4 h. Matrix-assisted laser desorption ionization mass sectrometry analysis revealed that the enzyme's trypsin-generated peptide mass fingerprint shares several peptide fragment hits with β-galactosidases from Kluyveromyces lactis. This confirms the enzyme's identity and indicates that significant sequence homology exists between these enzymes.
Keywords: β-Galactosidase; Kluyveromyces marxianus ; lactose intolerance; small intestinal conditions; peptide mass fingerprinting
A parametric study on biphasic medium conditions for the enantioselective production of naproxen by Candida rugosa lipase by Serpil Takaç; Deniz Mutlu (pp. 15-26).
A parametric study to increase the enantioselectivity of Candida rugosa lipase (CRL) toward S-Naproxen production by the hydrolysis of racemic Naproxen methyl ester in an aqueous-organic biphasic batch system was carried out. Effects of organic solvent type, aqueous phase/organic solvent volume ratio, agitation rate, concentrations of the substrate and the enzyme, pH of the aqueous phase, and temperature on the en antiomeric excess for the product (eep), on the enantiomeric ratio (E) and on the conversion (x) were evaluated. Employing isooctane as the solvent resulted in higher eep, E, and x than those obtained in hexane, cyclohexane, and toluene. The higher the volume ratio of aqueous phase/organic solvent employed, the higher the conversion and enantioselectivity achieved. The increase in agitation rate increased the hydrolysis rate. Higher concentration of racemic Naproxen methyl ester than 10 mg/mL decreased both the conversion and enantioselectivity. The increase in crude CRL concentration resulted in enhancement of x, but the decrease of feep and E. Acidic pH led to higher conversion and enantioselectivity than the medium and alkaline pH values. A further increase in temperature to over 45°C decreased the conversion and enantioselectivity. The highest enantiomeric ratio achieved in the S-Naproxen production was E=171.1, with x=49.8% and eep=95.7%.
Keywords: Biphasic systems; Candida rugosa lipase; enantioselectivity; Naproxen methyl ester; S-Naproxen
Evaluation of mesophilic biodegraded grape marc as soil fertilizer by Ana B. Moldes; Manuel Vázquez; José M. Domínguez; Francisco Díaz-Fierros; María T. Barral (pp. 27-36).
The wine industry generates a large amount of wastes, including grape marc and vinification lees. These substances can be used to produce enzymes or other food additives. Composting could be a successful strategy for the sustainable and complete recycling of grape marc. In this work, the mesophilic biodegradation of grape marc during 60 d under microaerobic conditions using several ratios of seeds, skin, and stem were studied. The presence of Penicillium spp. was detected at the beginning of the composting. Changes in chemical and biological parameters were evaluated. Biodegraded grape marc with stem showed the best organic matter properties (C/N ratio of 14 and N content of 37 g/kg) and a germination index of 155% for the growth of ray grass seeds. The results suggest that the biodegraded of grape marc could be used as fertilizer, especially for ray grass crops.
Keywords: Biodegradation; compost; winery; grape marc; seeds; germination index
Degradation of eucalypt waste components by Lentinula edodes strains detected by chemical and near-infrared spectroscopy methods by M. Brienzo; E. M. Silva; A. M. F. Milagres (pp. 37-49).
There are many changes, both qualitative and quantitative, in eucalypt waste during growth and fructification of Lentinula edodes. Wet chemical analysis and near-infrared (NIR) spectroscopy were used in conjunction with multivariate regression and principal components analysis to monitor biodegradation of eucalyptus waste during growth of several L. edodes strains. Weight and component losses of eucalypt residue after biodegradation by L. edodes strains were compared for periods of 1 to 5 mo. Decrease in cellulose, hemicellulose, and lignin contents occurred, however it was not concomitant. Measurement of lignin degradation by NIR and wet chemical analysis indicated its attack in the early stages of biodegradation. Selective lignin degradation by L. edodes was observed up to 2 mo of biodegradation for strains DEBIQ and FEB-14. One group of degraded substrate was identified based on the principal component analysis (PCA) of the data on their biodegradation time. Samples treated for 5 months by L. edodes strains (DEBIQ, UFV or FEB-14) differed from other, but no discrimination was observed among them. By the end of 5 mo, NIR analyses showed decrease of about 18–47% cellulose, 35–47% polyose and 39–60% lignin. These data were used for comparison with those obtained by wet chemical method for the degradation of the substrate by other five L. edodes strains cultivated at the same conditons. NIR calibration developed in this study was proven to be perfectly suitable as an analytical method to predict the changes in lignocellulose composition during biodegradation.
Keywords: Lentinula edodes ; biodegradation; Near infrared; lignin selectivity
Structure-function relationships of a catalytically efficient β-D-xylosidase by Douglas B. Jordan; Xin-Liang Li; Christopher A. Dunlap; Terence R. Whitehead; Michael A. Cotta (pp. 51-76).
β-d-Xylosidase from Selenomonas ruminantium is revealed as the best catalyst known (k cat, k cat/K m) for promoting hydrolysis of 1,4-β-d-xylooligosaccharides. 1H nuclear magnetic resonance experiments indicate the family 43 glycoside hydrolase acts through an inversion mechanism on substrates 4-nitrophenyl-β-d-xylopyranoside (4NPX) and 1,4-β-d-xylobiose (X2). Progress curves of 4-nitrophenyl-β-d-xylobioside, xylotetraose and xylohexaose reactions indicate that one residue from the nonreducing end of substrate is cleaved per catalytic cycle without processivity. Values of k cat and k cat/K m decrease for xylooligosaccharides longer than X2, illustrating the importance to catalysis of subsites −1 and +1 and the lack there of subsite +2. Homology models of the enzyme active site with docked substrates show that subsites bey ond−1 are blocked by protein and subsites bey ond +1 are not formed; they suggest that D14 and E186 serve catalysis as general base and general acid, respectively. Individual mutations, D14A and E186A, erode k cat and k cat/K m by <103 and to asimilar extent for substrates 4NPX and 4-nitrophenyl-α-l-arabinofuranoside (4NPA), indicating that the two substrates share the same active site. With 4NPX and 4NPA, pH governs k cat/K m with pK a values of 5.0 and 7.0 assigned to D14 and E186, respectively. k cat (4NPX) has a pK a value of 7.0 and k cat (4NPA) is pH independent above pH 4.0, suggesting that the catalytically inactive, “dianionic” enzyme form (D14-E187-) binds 4NPX but not 4NPA.
Keywords: Glycoside hydrolase; stereochemistry; substrate specificity; xylan; arabinofuranosidase
Thermostability of Cromobacterium viscosum lipase in AOT/isooctane reverse micelle by M. M. R. Talukder; M. M. Zaman; Y. Hayashi; J. C. Wu; T. Kawanishi (pp. 77-83).
The thermostability of Cromobacterium viscosum lip ase (EC 3.1.1.3) entrapped in AOT (sodium bis-[2-ethylhexyl] sulfosuccinate) reverse micelles was in creased by the addition of short-chain polyethylene glycol (PEG 400). Two different approaches were considered: (1) the determination of half-life time and (2) the mechanistic analysis of deactivation kinetics. The half-life of lipase entrapped in AOT/isooctane reverse micelles with PEG 400 at 60°C was 28h, ninefold higher than that in reverse micelles without PEG 400. The lip ase entrapped in both reverse micellar systems followed a series-type deactivation mechanism involving two first-order steps. The deactivation constant for the first step at 60°C in PEG containing reverse micelles was 0.055 h11, 11-fold lower than that in reverse micelles without PEG, whereas it remained almost constant for the second step. The inactivation energy of the lip ase entrapped in reverse micelles with and without PEG 400 was 88.12 and 21.97 kJ/mol, respectively.
Keywords: Cromobacterium viscosum ; lip ase; polyethylene glycol; reverse micelles; thermostability
A review of biodegradation of synthetic plastic and foams by R. Gautam; A. S. Bassi; E. K. Yanful (pp. 85-108).
Synthetic polymeric foams have pervaded every aspect of modern life. Although foams provide numerous benefits, they also cause a significant environmental litter problem because of their recalcitrant and xenobiotic nature. Biodegradation may provide solution to the problem, but not enough is known about the biodegradation process of synthetic plastic and plastic-based foams. This review has been written to provide an overview of the current state of plastic foam biodegradation. Several biodegradation pathways of a few select synthetic polymers are also presented along with a discussion on some of the physico-chemical factors that can influence the biodegradation of plastic foams.
Keywords: Plastic foam; synthetic polymer; biodegradation
Computational prediction of the coupling specificity of G protein-coupled receptors by Zhenran Jiang; Cuiping Guan; Yanhong Zhou (pp. 109-118).
Gprotein-coupled receptors (GPCRs) represent one of the most important categories of membrane proteins that play important roles in signaling pathways. GPCRs transduce the extracellular stimuli into intracellular second messengers via their coupling to specific class of heterotrimeric GTP-binding proteins (G proteins) and the subsequent regulation of a diverse variety of effectors. Understanding the coupling specificity of GPCRs is critical for further comprehending their function, and is of tremendous clinical significance because GPCRs are the most successful drug targets. This minireview addresses the computational approaches that have been created for the prediction of coupling specificity of GPCRs and high lights the perspective of bioinformatics strategies that may be used to tackle this important task. In addition, some of the important resources of this field are also provided.
Keywords: G protein-coupled receptor; coupling specificity; orphan receptor; bioinformatics
Reusability of entrapped cells of Pseudomonas diminuta for production of 7-aminocephalosporanic acid by V. K. Nigam; S. Kundu; P. Ghosh (pp. 119-125).
Entrapped cells of P. diminuta were used for the production of 7-aminocephalosporanic acid (7-ACA), a key intermediate required for the production of most of the clinically used cephalosporin derivatives, i.e., semisynthetic cephalosporins. The repeated batch production of 7-ACA with entrapped cells of P. diminuta in different carriers were carried out for six cycles at optimal conditions. It was found that 33%, 38%, and 47% of activity was lost with chitosan, gelatin, and agar, respectively as immobilizing supports after the sixth cycle of operation.
Keywords: 7-ACA; CPC acylase; GL-7-ACA; GL-7-ACA acylase; immobilization; repeated batch production
Molecular cloning and biochemical characterization of a family-9 endoglucanase with an unusual structure from the gliding bacteria Cytophaga hut chinsonii by Clifford Louime; Michael Abazinge; Elijah Johnson; Lekan Latinwo; Christopher Ikediobi; Ann-Marie Clark (pp. 127-138).
Cytophaga hutchinsonii was originally isolated from sugarcane piles. This microorganism therefore probably produces an array of enzymes allowing it to digest cellulosic substrates. C. hutchinsonii thus represents a rich source of potentially effective cellulase enzymes that can be harnessed for conversion of biomass to simple sugars. These sugars can then be used as feedstock for ethanol production or other chemical syntheses. In this study, we report the PCR cloning of an endoglucanase gene (Cel9A) from C. hutchinsonii using degenerated primers directed at the catalytic domain. Alignment of the amino acids sequence revealed that Cel9A has a gene structure totally different from the other known cellulose degraders. The most striking feature of this cloned protein is the absence of a cellulose-binding domain (CBD), which to date was believed to be imperative in cellulose hydrolysis. Consequently, the Cel9A gene, encoding β-1,4 endoglucanase from C. hutchinsonii was over-expressed in Escherichia coli with a His-Tag based expression vector. The resulting polypeptide, with a molecular mass of 105 KDa, was purified from cell extracts by affinity chromatography on cellulose. Mature Cel9A was optimally active at pH 5.0 and 45°C. The enzyme efficiently hydrolyzes carboxymethyl-cellulose (CMC). Analysis of CMC and filter paper hydrolysis suggests that Cel9A is a nonprocessive enzyme with endo-cellulase activities.
Keywords: Cytophaga; cellulase; cellulose-binding domain
Synthesis of isoamyl laurate and isoamyl stearate in supercritical carbon dioxide by Mahesh N. Varma; Giridhar Madras (pp. 139-147).
The synthesis of isoamyl laurate and isoamyl stearate was studied in supercritical carbon dioxide with three lipases, Novozym 435, Lipolase 100T, and Candida rugosa. The maximum conversion of 37% and 53%, respectively for isoamyl laurate and isoamyl stearate was obtained when Novozym 435 was used. The effect of various parameters such as molar ratio of alcohol to acid, presence of water, time and temperature was investigated. An optimum temperature of 40–45°C was observed for all reactions. The kinetics of reactions was fast and equilibrium was achieved in 2–3 h. Although the presence of excess alcohol did not reduce conversion, excess water reduced conversion significantly.
Keywords: Isoamyl laurate; Isoamyl stearate; esterification; supercritical carbon dioxide; lipase
Large-scale purification of human granulocyte-macrophage colony-stimulating factor expressed in Bombyx mori pupae by Jian Chen; Zuo-Ming Nie; Zheng-Bing Lü; Cheng-Gang Zhu; Cheng-Zhi Xu; Yong-Feng Jin; Xiang-Fu Wu; Yao-Zhou Zhang (pp. 149-159).
Human granulocyte-macrophage colony-stimulating factor (hGM-CSF) acts on many different kinds of cells, including monocytes, macrophages, granulocytes, eosinophils, and multipotential stem cells. To explore further explore pharmaceutical action, we expressed hGM-CSF by the Bombyx mori nucleopolyhedrovirus expression system in silkworm pupae. However, purifying recombinant proteins from silkworm pupae on a large scale has been a big challenge. To establish purification methods suitable for mass production, we tried two crude preparation methods: (NH4)2SO4 fractional precipitation and isoelectric precipitation with a combination of gel filtration andion-exchange chromatography. The isoelectric precipitation method was found to be more efficient. With this method, we eventually obtained approx 11.7 mg of 95% pure product from 1000 g of infected silkworm pupae. The recovery of purified protein was greatly increased, by approx 40%, compared with the other method. The biologic activity of this protein was determined up to 9.0×106 colony-forming units/mg in the final purified product.
Keywords: Bombyx mori nucleopolyhedrovirus; silkworm pupae; human granulocyte-macrophage colony-stimulating factor; purification; isoelectric precipitation; (NH4)2SO4 fractional precipitation; target protein