Oriana Salazar

Enzymatic lysis of microbial cells.

Cell wall lytic enzymes are valuable tools for the biotechnologist, with many applications in medicine, the food industry, and agriculture, and for recovering of intracellular products from yeast or bacteria. The diversity of potential applications has conducted to the development of lytic enzyme systems with specific characteristics, suitable for satisfying the requirements of each particular application. Since the first time the lytic enzyme of excellence, lysozyme, was discovered, many investigations have contributed to the understanding of the action mechanisms and other basic aspects of these interesting enzymes. Today, recombinant production and protein engineering have improved and expanded the area of potential applications. In this review, some of the recent advances in specific enzyme systems for bacteria and yeast cells rupture and other applications are examined. Emphasis is focused in biotechnological aspects of these enzymes.

Toward Tailor-Made Biocide Materials Based on Poly(propylene)/Copper Nanoparticles

A set of poly(propylene) composites containing different amounts of copper nanoparticles (CNP) were prepared by the melt mixed method and their antimicrobial behavior was quantitatively studied. The time needed to reduce the bacteria to 50% dropped to half with only 1 v/v % of CNP, compared to the polymer without CNP. After 4 h, this composite killed more than 99.9% of the bacteria. The biocide kinetics can be controlled by the nanofiller content; composites with CNP concentrations higher than 10 v/v % eliminated 99% of the bacteria in less than 2 h. X-ray photoelectron spectroscopy did not detect CNP at the surface, therefore the biocide behavior was attributed to copper in the bulk of the composite.

Characterization of the two rRNA gene operons present in Thiobacillus ferrooxidans

The organization of rRNA genes from the autotrophic, acidophilic bacterium Thiobacillus ferrooxidans has been examined. Two rRNA operons were found in this microorganism by means of genomic hybridization studies. Recombinant plasmids, pTR‐3 and pTR‐1 that carry a portion of 16/23 S rDNA from one operon and the 5′‐flanking region of the second operon, respectively, were identified and characterized.

Cloning of novel cellulases from cellulolytic fungi: heterologous expression of a family 5 glycoside hydrolase from Trametes versicolor in Pichia pastoris.

Total cDNA isolated from cellulolytic fungi cultured in cellulose was examined for the presence of sequences encoding for endoglucanases. Novel sequences encoding for glycoside hydrolases (GHs) were identified in Fusarium oxysporum, Ganoderma applanatum and Trametes versicolor. The cDNA encoding for partial sequences of GH family 61 cellulases from F. oxysporum and G. applanatum shares 58 and 68% identity with endoglucanases from Glomerella graminicola and Laccaria bicolor, respectively. A new GH family 5 endoglucanase from T. versicolor was also identified. The cDNA encoding for the mature protein was completely sequenced. This enzyme shares 96% identity with Trametes hirsuta endoglucanase and 22% with Trichoderma reesei endoglucanase II (EGII). The enzyme, named TvEG, has N-terminal family 1 carbohydrate binding module (CBM1). The full length cDNA was cloned into the pPICZαB vector and expressed as an active, extracellular enzyme in the methylotrophic yeast Pichia pastoris. Preliminary studies suggest that T. versicolor could be useful for lignocellulose degradation.

Bacteria and yeast cell disruption using lytic enzymes.

Enzymatic methods provide a convenient alternative for overcoming technical disadvantages of mechanical disruption. Protocols for protein extraction from bacteria and Saccharomyces cerevisiae using lytic enzymes are presented in this chapter. Adaptation of the yeast protocol to a microtiter plate format makes this protocol amenable for proteomic applications and high-throughput screening of libraries expressing genetic variants in yeast. This methodology can also be applied to bacteria.

Cloning, expression and decoding of the cold adaptation of a new widely represented thermolabile subtilisin-like protease.

Cloning, expression and characterization of a new cold‐adapted protease with potential biotechnological application, isolated from Antarctic bacteria.

Bioenergy II: Biological Pretreatment with Fungi as a Tool for Improvement of the Enzymatic Saccharification of Eucalyptus globulus Labill to Obtain Bioethanol

This study is focused on the effect of the application of biological pretreatment of Eucalyptus globulus Labill wood pieces on the cellulose digestibility by depolymerizing enzymes. Wood chips were incubated with five different white-rot fungi (WRF) for 30, 45 and 60 days at 25°C. The effect of the fungal action was determined as weight losses, changes in chemical composition and released sugars in the wood chips. Enzymatic hydrolysis was conducted in a discontinuous reactor at 37°C, using a commercial cellulase preparation from Trichoderma reesei. The enzyme/substrate ratio was 0.04 g/g. The highest weight losses were obtained using Stereum hirsutum and Lentinus edodes for all the incubation times, reaching 27.2% and 25.8% at 60 days, respectively. The lowest weight losses were produced by Coriolus versicolor and Pleurotus ostreatus, reaching 6.0 and 9.1%, respectively after 60 days. For all the tested fungi, the yield of reducing sugars and glucose increased significantly over the untreated controls, with Stereum hirsutum producing after 30 days pretreatment the best values (140 mM reducing sugars and 53 mM glucose); after this time no additional increments were observed. Pre-treated wood pieces showed changes in chemical composition in comparison to control samples. Total extractable substances contents are higher in wood pieces subjected to fungal action. However, lignin and alpha-cellulose values are lower in comparison to control samples. Some net holocellulose consumption could be observed, mainly during pretreatment with S. hirsutum by 45 and 60 days. Altogether these results support the potential of Eucalyptus globulus pre-treatment with S. hirsutum and L. edodes by periods not longer than 30 days, as a tool to increase the wood accessibility to depolymerizing action of hydrolytic enzymes. This preliminary study contributes to the identification of fungal pretreatment conditions for more effective cellulose degradation, a vital step in the utilization of monomer sugars process from cellulose to produce ethanol. Additional analysis of the solid and liquid fractions after saccharification is necessary to complement this information.

Improvement of the lytic properties of a β-1,3-glucanase by directed evolution

BGLII is a bacterial endoglucanase that hydrolyzes the β-1,3-glucan present in yeast cell walls, resulting in lysis of Saccharomyces cerevisiae. As a result of this property, BGLII is considered a potential tool for downstream processing and recovery of biotechnological products produced in yeast. Here we describe the improvement of the yeast lytic activity of BGLII, achieved by a directed evolution approach involving random mutagenesis and screening for variants with improved catalytic activity, combined with site-directed mutagenesis. A BGLII variant having three times the wild-type hydrolytic activity on laminarin was identified. The purified enzyme also exhibited higher lytic activity on yeast cells. Mutations causing the improvements are located very close to each other in the amino acid sequence, suggesting that the region should be considered as a target for further improvements of the glucanase activity. These results demonstrate the feasibility of molecular evolution methods for the improvement of the BGLII hydrolytic activity, and open a window for further improvement of this or other properties in glycosyl hydrolases in general.

The effect of a lytic polysaccharide monooxygenase and a xylanase from Gloeophyllum trabeum on the enzymatic hydrolysis of lignocellulosic residues using a commercial cellulase

Hydrolysis of lignocellulosic biomass depends on the concerted actions of cellulases and accessory proteins. In this work we examined the combined action of two auxiliary proteins from the brown rot fungus Gloeophyllum trabeum: a family AA9 lytic polysaccharide monooxygenase (GtLPMO) and a GH10 xylanase (GtXyn10A). The enzymes were produced in the heterologous host Pichia pastoris. In the presence of an electron source, GtLPMO increased the activity of a commercial cellulase on filter paper, and the xylanase activity of GtXyn10A on beechwood xylan. Mixtures of GtLPMO, GtXyn10A and Celluclast 1.5L were used for hydrolysis of pretreated wheat straw. Results showed that a mixture of 60% Celluclast 1.5L, 20% GtXyn10A and 20% GtLPMO increased total reducing sugar production by 54%, while the conversions of glucan to glucose and xylan to xylose were increased by 40 and 57%, respectively. This suggests that GtLPMO can contribute to lignocellulose hydrolysis, not only by oxidative activity on glycosidic bonds, but also to hemicellulose through the oxidation of xylosyl bonds in xylan. The concerted action of these auxiliary enzymes may significantly improve large-scale recovery of sugars from lignocellulose.

A Tyrosine-Dependent Riboswitch Controls the Expression of a Tyrosyl-tRNA Synthetase from Acidithiobacillus ferrooxidans

Expression of aminoacyl-tRNA synthetases is regulated by a variety of mechanisms at the level of transcription or translation. A T-box dependent transcription termination / antitermination riboswitch system that responds to charged / uncharged tRNA regulates expression of aminoacyl tRNA synthetase genes in Gram-positive bacteria. TyrZ, the gene encoding tyrosyl-tRNA synthetase from Acidithiobacillus ferrooxidans, a Gram-negative acidophilic bacterium that participates in bioleaching of minerals, resembles the gene from Bacillus subtilis including the 5 ́-untranslated region encoding the riboswitch. Transcription of A. ferrooxidans tyrZ is induced by the presence of tyrosine by a mechanism involving antitermination of transcription. This mechanism is probably adapted to the low supply of amino acids of acidic environments of autotrophic bioleaching microorganisms.