Peroxidases have conquered a prominent position in biotechnology and associated research areas (enzymology, biochemistry, medicine, genetics, physiology, histo- and cytochemistry). They are one of ...the most extensively studied groups of enzymes and the literature is rich in research papers dating back from the 19th century. Nevertheless, peroxidases continue to be widely studied, with more than 2000 articles already published in 2002 (according to the Institute for Scientific Information). The importance of peroxidases is emphasised by their wide distribution among living organisms and by their multiple physiological roles. They have been divided into three superfamilies according to their source and mode of action: plant peroxidases, animal peroxidases and catalases. Among all peroxidases, horseradish peroxidase (HRP) has received a special attention and will be the focus of this review. A brief description of the three super-families is included in the first section of this review. In the second section, a comprehensive description of the present state of knowledge of the structure and catalytic action of HRP is presented. The physiological role of peroxidases in higher plants is described in the third section. And finally, the fourth section addresses the applications of peroxidases, especially HRP, in the environmental and health care sectors, and in the pharmaceutical, chemical and biotechnological industries.
The
Saccharomyces cerevisiae W303-1A strain transformed with a centromeric plasmid containing
CYPRO11, which codifies the aspartic protease cyprosin B, was grown in a 3
l bioreactor under aerobic ...conditions. Expression of cyprosin B is directly dependent on the concentration of galactose used as the inducer and carbon source in 1% yeast extract, 2% bactopeptone, and 4% galactose in culture medium. For 4% of galactose, 209 mg·
l
−1 total protein, and 1036 U·ml
−1 recombinant cyprosin B activity were obtained from 6.1 g dcw·
l
−1 biomass. The recombinant cyprosin B, purified by two consecutive anion-exchange chromatographies (diethyl amino-ethyl DEAE-Sepharose and Q-Sepharose XK-16 columns), shows a specific activity of 62×10
3 U·mg
−1, corresponding to a purification degree of 12.5-fold and a recovery yield of 25.6% relative to that in fermentation broth. The proteolytic activity of recombinant cyprosin B is optimal at 42°C and pH 4.5. The recombinant cyprosin B activity is 95% inhibited by pepstatin A, which confirms its aspartic protease nature. The pure recombinant cyprosin B is composed of two subunits, one with 14 and the other with 32 kDa. It exhibits clotting activity, similar to that of the natural enzyme from
Cynara cardunculus flowers. The results reported here show that recombinant cyprosin B, the first clotting protease of plant origin produced in a bioreactor, can now be produced in large scale and may constitute a new and efficient alternative to enzymes of animal or fungal origin that are widely used in cheese making.
Biocatalysis presents a sound alternative to chemical synthesis in the field of drug production, given the highly selective nature of biological catalysts. Penicillin G Acylase (PGA) from
E. coli
is ...currently used to hydrolyze penicillin G (PG) and catalyzes the synthesis of β-lactam antibiotics. In this work, particular emphasis is given to recent developments in penicillin G acylase immobilization, by entrapment simultaneously with nano-magnetic particles in a silica matrix. The sol–gel biocatalytic particles were prepared either by a conventional method (crushed powder) or by a more recent approach, based in an emulsion system using 150 mM AOT/isooctane, which allowed for the formation of spherical micro- and nanobeads. The effects on PGA activity of different sol–gel precursors, additives, enzyme concentration, aging, drying conditions and mechanical stability were evaluated. After these optimization studies, a mechanically stable carrier based on porous xerogels silica matrixes, starting from tetramethoxysilane (TMOS) with 65–67% PGA activity yield in these carriers allowed an immobilization yield of 74 mg protein g
dry sol–gel
−1
and 930 Ug
dry sol–gel
−1
for specific activity were obtained.
The magnetoresistive (MR) biochip concept has emerged a decade ago and since then considerable achievements were made in the field. At the moment there is a strong effort in building up a fully ...integrated, portable and accessible spintronic device for bioanalytical assays. Some of the major challenges and working solutions are addressed here. In a MR-biochip platform five main components can be identified as key points for its success: the MR sensing elements, the magnetic labels, the surface chemistry, the microfluidic system and the read-out electronic set-up. Linear spin valve sensors were fabricated with good sensitivity and proper field range. Magnetic particles were carefully characterized and selected seeking for the best biomolecular labels. The surface chemistry was extensively optimized in order to get it more efficient, specific and reproducible. A microfluidic structure was designed and fabricated in polydimethilsiloxane (PDMS) to work as sample transportation and simultaneously control the wash out steps. Finally, a portable and autonomous electronic microsystem provides the electronic circuitry to control, address and read-out up to 256 sensors. From the assembling of all these components emerges a versatile portable platform. The first results from the platform in a real-time detection of 20mer single stranded DNA sequences labeled with 130
nm magnetic labels are presented.
The low rate of discovery and rapid spread of resistant pathogens have made antibiotic discovery a worldwide priority. In cell-based screening, the mechanism of action (MOA) is identified after ...antimicrobial activity. This increases rediscovery, impairs low potency candidate detection, and does not guide lead optimization. In this study, high-throughput Fourier-transform infrared (FTIR) spectroscopy was used to discriminate the MOA of 14 antibiotics at pathway, class, and individual antibiotic level. For that, the optimal combinations and parametrizations of spectral preprocessing were selected with cross-validated partial least squares discriminant analysis, to which various machine learning algorithms were applied. This coherently resulted in very good accuracies, independently of the algorithms, and at all levels of MOA. Particularly, an ensemble of subspace discriminants predicted the known pathway (98.6%), antibiotic classes (100%), and individual antibiotics (97.8%) with exceptional accuracy, and similar results were obtained for simulated novel MOA. Even at very low concentrations (1 mug/mL) and growth inhibition (15%), over 70% pathway and class accuracy was achieved, suggesting FTIR spectroscopy can probe the grey chemical matter. Prediction of inhibitory effect was also examined, for which a squared exponential Gaussian process regression yielded a root mean square error of 0.33 and a R.sup.2 of 0.92, indicating that metabolic alterations leading to growth inhibition are intrinsically reflected on FTIR spectra beyond cell density.
The main objective of this work was to study the enzymatic synthesis of short chain ethyl esters, a group of relevant aroma molecules, by
Fusarium solani pisi cutinase in an organic solvent media ...(iso-octane), and to assess the influence of different parameters on the reaction yield.
Cutinase displayed high initial esterification rates in iso-octane, which amounted to 1.15
μmol
min
−1
mg
−1 for ethyl butyrate (C
4 acid chain) and 1.06
μmol
min
−1
mg
−1 for ethyl valerate (C
5 acid chain). High product yields, 84% for ethyl butyrate and 96% for ethyl valerate, were observed after 6
h of reaction, for an initial equimolar concentration of substrates (0.1
M).
The highest product yield (97%) was observed for ethyl caproate (C
6) synthesis, a compound which is a part of natural apple and pineapple flavour, for an alcohol:acid molar ratio of 2 (0.2
M ethanol concentration).
Cutinase affinity for short chain length carboxylic acids (C
4–C
6) in ester synthesis in iso-octane confirmed previous observations in reversed micellar system.
L-Asparaginase (ASNase) is used in the treatment of acute lymphoblastic leukemia, being produced and commercialized only from bacterial sources. Alternative
Saccharomyces cerevisiae
ASNase II coded ...by the
ASP3
gene was biosynthesized by recombinant
Pichia pastoris MUT
s
under the control of the
AOX1
promoter, using different cultivation strategies. In particular, we applied multistage fed-batch cultivation divided in four distinct phases to produce ASNase II and determine the fermentation parameters, namely specific growth rate, biomass yield, and enzyme activity. Cultivation of recombinant
P. pastoris
under favorable conditions in a modified defined medium ensured a dry biomass concentration of 31 g
dcw
.L
−1
during glycerol batch phase, corresponding to a biomass yield of 0.77 g
dcw.
g
glycerol
-
1
and a specific growth rate of 0.21 h
−1
. After 12 h of glycerol feeding under limiting conditions, cell concentration achieved 65 g
dcw.
L
−1
while ethanol concentration was very low. During the phase of methanol induction, biomass concentration achieved 91 g
dcw.
L
−1
, periplasmic specific enzyme activity 37.1 U.g
dcw
-
1
, volumetric enzyme activity 3,315 U.L
−1
, overall enzyme volumetric productivity 31 U.L
−1
.h
−1
, while the specific growth rate fell to 0.039 h
−1
. Our results showed that the best strategy employed for the ASNase II production was using glycerol fed-batch phase with pseudo exponential feeding plus induction with continuous methanol feeding.
Four marine-derived fungi strains—Penicillium citrinum CBMAI 1186, Aspergillus sydowii CBMAI 934, Aspergillus sydowii CBMAI 935 and Mucor racemosus CBMAI 847—were studied as potential microbial ...producers of cellulases under solid-state fermentation. The cultivation parameters (time, pH and temperature) and the performance of the cellulases in the hydrolysis of cellulose from sugarcane bagasse were evaluated. The best cultivation time in solid-state fermentation for producing cellulases was 3 days for all of the strains except for A. sydowii CBMAI 934, which exhibited its highest filter paper activity (FPU) after 7 days. After optimizing pH and temperature, activities of 0.73, 1.9, 2.4 and 1.3 FPU g−1 (of substrate) were obtained for A. sydowii CBMAI 935, A. sydowii CBMAI 934, M. racemosus CBMAI 847 and P. citrinum CBMAI 1186, respectively. The hydrolysis of cellulose was studied using rind and pith fractions of sugarcane bagasse, with and without alkali pretreatment. Independent of the fungi strains, untreated bagasse was resistant to saccharification. After the treatment, the degree of saccharification was 78% for A. sydowii CBMAI 934 and 24% for M. racemosus CBMAI 847. The different anatomical and morphological characteristics of the rind and pith cells influenced the saccharification by cellulases for the different marine fungi strains. The hydrolysis by cellulases from A. sydowii CBMAI 934 produced 56% and 81% saccharification in the rind and pith, respectively. This fungus strain exhibits significant potential for the production of fermentable sugars resulting from the hydrolysis of cellulose from sugarcane bagasse, mainly after pretreatment with NaOH solution.
•Four marine-derived fungi were achieved for the first time as cellulase producers.•Different optimum pH and temperature for FPU activity were obtained for each strain.•Aspergillus sydowii CBMAI 934 performed the saccharification of sugarcane bagasse.•This is the first report of sugarcane bagasse saccharification by marine-derived fungi.
The main objective of this work was studying and testing the nature and influence of reaction media (organic solvent vs. miniemulsion system) on the synthesis of alkyl esters catalyzed by Fusarium ...solani pisi cutinase. Ester synthesis and cutinase selectivity for different chain length of acids and alcohols (ethyl and hexyl) were evaluated. In iso‐octane, after 1 h of reaction, cutinase exhibits rates of esterification between 0.24 μmol x mg–1 x min–1 for ethyl oleate and 1.15 μmol x mg–1 x min–1 for ethyl butyrate, while in a miniemulsion system the rates were from 0.05 for ethyl heptanoate to 0.76 μmol x mg–1 x min–1 for ethyl decanoate. The reaction rate for the synthesis of hexyl esters in a miniemulsion system was from 0.19 for hexyl heptanoate to 1.07 μmol x mg–1 x min–1 for hexyl decanoate. High conversion yields of 95% at equilibrium after 8 h of reaction in iso‐octane for pentanoic acid (C5) with ethanol at equimolar concentration (0.1 M) was achieved. Additionally, this work showed that a significant and unexpected shift in cutinase selectivity occurred towards longer chain length carboxylic acids (C8–C10) in miniemulsion system as compared to organic solvent (iso‐octane) and previous studies in reverse micellar systems. The possibility of working with higher concentration of substrates, without inhibitory effect on the enzyme, was another advantage of the miniemulsion system.
The aim of this work is to devise an efficient enzymatic process for the production of linear alkyl esters in aqueous miniemulsion systems. The esterification reactions of linear alcohols and ...carboxylic acids were performed with three different enzymes, commercial Amano lipase PS from Pseudomonas cepacia, Lipase type VII from Candida rugosa, and lyophilized Fusarium solani pisi cutinase expressed in Saccharomyces cerevisiae SU50. The miniemulsion system shows a high potential for the synthesis of linear alkyl esters, for example, hexyl octanoate, which could be synthesized with an ester yield of 94% using Amano lipase PS. Even with hydrophilic alcohols as ethanol, ethyl decanoate could be obtained with a concentration of 0.45 M and a yield of 62% using F. s. pisi cutinase as catalyst. High esterification rates for ethyl- and hexyloleate in miniemulsion showed a significant shift in cutinase selectivity towards longer chain length carboxylic acids. The stepwise addition of the alcohol led to an increase of the esterification yield. Moreover, increasing the amount of dispersed organic phase, mainly consisting of the substrates, led to a significant increase of the final ester concentration (e.g., concentration of 1.4 M for ethyl decanoate for the esterification with Amano Lipase PS). Biotechnol. Bioeng. 2010;106: 507-515.
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