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Introduction
Scientist | Protein Engineering | Cell & Molecular Biology | Synthetic Biology | Team Player
Additional affiliations
February 2020 - present
Innate Immunity LLC
Position
- Engineer
March 2014 - present
August 2007 - February 2014
Publications
Publications (34)
All biosensing platforms rest on two pillars: specific biochemical recognition of a particular analyte and transduction of that recognition into a readily detectable signal. Most existing biosensing technologies utilize proteins that passively bind to their analytes and therefore require wasteful washing steps, specialized reagents, and expensive i...
The development of precise and modulated methods for customized manipulation of DNA is an important objective for the study and engineering of biological processes and is essential for the optimization of gene therapy, metabolic flux, and synthetic gene networks. The clustered regularly interspaced short palindromic repeat- (CRISPR-) associated pro...
Background:
Product inhibition can reduce catalytic performance of enzymes used for biofuel production. Different mechanisms can cause this inhibition and, in most cases, the use of classical enzymology approach is not sufficient to overcome this problem. Here we have used a semi-rational protein fusion strategy to create a product-stimulated enzy...
Background
Saccharification of lignocellulosic material by xylanases and other glycoside hydrolases is generally conducted at high concentrations of the final reaction products, which frequently inhibit the enzymes used in the saccharification process. Using a random nonhomologous recombination strategy, we have fused the GH11 xylanase from Bacillu...
Protein switches have a variety of potential applications in biotechnology and medicine that motivate efforts to accelerate their development. Switches can be built by the proper fusion of two proteins with the prerequisite input and output functions. However, the exact fusion geometry for switch creation, which typically involves insertion of one...
A novel antifungal peptide, PcAFP (6.48 kDa, pI 8.83), was obtained from the culture supernatant of the fungus Penicillium crustosum. The gene encoding the PcAFP peptide was isolated based on its homologue in Penicillium chrysogenum, PgAFP. PcAFP is a small, cystine-rich peptide, and the mature peptide consists of 58 amino acid residues. The immatu...
Hospital-associated infections (HAIs) are a leading cause of morbidity and mortality in intensive care units (ICUs) and neonatal intensive care units (NICUs). Organisms causing these infections are often present on surfaces around the patient. Given that microbiota may vary across different ICUs, the HAI-related microbial signatures within these un...
Protein engineering emerged as a powerful approach to generate more robust and efficient biocatalysts for bio-based economy applications, an alternative to ecologically toxic chemistries that rely on petroleum. On the quest for environmentally friendly technologies, sustainable and low-cost resources such as lignocellulosic plant-derived biomass ar...
Hospital-associated infections (HAIs) are the leading cause of morbidity and mortality in intensive care units (ICUs) and neonatal intensive care units (NICUs). Organisms causing these infections are often present on surfaces around the patient. Given that microbiotas may vary across different ICUs, the HAI-related microbial signatures within these...
The cyclic AMP (cAMP)-dependent protein kinase A (PKA) signaling pathway is well conserved across eukaryotes, and previous work has shown that it plays an important role in regulating development, growth, and virulence in a number of fungi. PKA is activated in response to extracellular nutrients and acts to regulate metabolism and growth. While a n...
Carbohydrate binding modules (CBMs) are non-catalytic domains within larger multidomain polypeptides. The CelH from Ruminoclostridium (Clostridium) thermocellum contains a family 11 CBM (RtCBM11) with high binding affinity for the linear polysaccharide β-glucan, and low affinity for the branched xyloglucan. Screening a random RtCBM11 mutant phage l...
The present study describes the one-step purification and biochemical characterization of an endo-1,4-β-xylanase from Aspergillus tamarii Kita. Extracellular xylanase was purified to homogeneity 7.43-fold through CM-cellulose. Enzyme molecular weight and pI were estimated to be 19.5kDa and 8.5, respectively. The highest activity of the xylanase was...
A protein switch is a protein that changes between inactive (“off”) and active (“on”) states in response to a biomolecule or physical signal. These switches can be created by fusing two domains in such a way that the activity of the output domain is regulated by the input domain’s recognition of an input signal (such as the binding of a molecule, r...
Filamentous fungi are attractive hosts for heterologous protein expression due to their capacity to secrete large amounts of enzymes into the extracellular medium. Xyloglucanases, which specifically hydrolyze xyloglucan, have been recently applied in lignocellulosic biomass degradation and conversion in many other industrial processes. In this cont...
Bioproducts and biobased technologies are increasingly taking
on outstanding position in the global market. Although
there is debate on the longevity of the oil reserves, the global
warming and its devastating consequences cannot be denied
and probably will put an end to the fossil fuels era and
start a new one with dependence on renewable energy....
: Protein engineering has become the most important tool for enabling industrial application of biocatalysts. Advances in structure-guided methods and novel techniques for directed evolution and high-throughput screening have facilitated the design of enzymes with improved properties such as functional expression, stability, and catalytic activity....
Xyloglucan-specific endo-β-1,4-glucanases (Xegs, EC 3.2.1.151) exhibit high catalytic specificity for β-1,4 linkages of xyloglucan, a branched hemicellulosic polysaccharide abundant in dicot primary cell walls and present in many monocot species. In nature, GH12 Xegs are not associated with carbohydrate-binding modules (CBMs), and here, we have inv...
The search for novel thermostable xylanases for industrial use has intensified in recent years, and thermophilic fungi are a promising source of useful enzymes. The present work reports the heterologous expression and biochemical characterization of a novel thermostable xylanase (GH10) from the thermophilic fungus Malbranchea pulchella, the influen...
Screening different filamentous fungi for thermostable xylanases and cellulases that would not be inhibited by xylose and glucose, respectively.
A gene circuit for the controlled expression of a marker gene and for the assay of xylose concentration in Escherichia coli has been designed and tested. The xylF coding sequence for the xylose binding protein (XBP) was cloned in pT7T318U downstream from the promoter for xylanase A from B. subtilis (Pbsu), together with the GFP coding sequence (gfp...
The aim of this chapter is to give the reader an overview of some of the
most recent techniques of protein engineering applied to fungal enzymes
emphasizing the important relationship between basic science and its
application. We will divide the engineering strategies in three main points:
rational, not rational and semi-rational design.
##Assembly-Data-START## Sequencing Technology :: Sanger dideoxy sequencing ##Assembly-Data-END##
A bifunctional enzyme has been created by fusing two Bacillus subtilis enzymes: the β-1,3-1,4-glucanase (BglS, EC 3.2.1.73) that hydrolyzes plant cell wall β-glucans and the copper-dependent oxidase laccase (CotA, EC 1.10.3.2) that catalyzes the oxidation of aromatic compounds with simultaneous reduction of oxygen to water. The chimeric laccase/β-1...
Xyloglucan is a major structural polysaccharide of the primary (growing) cell wall of higher plants. It consists of a cellulosic backbone (beta-1,4-linked glucosyl residues) that is frequently substituted with side chains. This report describes Aspergillus nidulans strain A773 recombinant secretion of a dimeric xyloglucan-specific endo-β-1,4-glucan...
Xyloglucan is a major structural polysaccharide of the primary (growing) cell wall of higher plants. It consists
of a cellulosic backbone (beta-1,4-linked glucosyl residues) that is frequently substituted with side chains.
This report describes Aspergillus nidulans strain A773 recombinant secretion of a dimeric xyloglucanspecific
endo-β-1,4-glucano...
a b s t r a c t The sucrose content of soybean seeds affects the final flavor of soy-derived products. The aim of this work was to develop a simple, low-cost, spectrophotometric method for sucrose quantification in soybean seeds. To achieve this goal, we combined the action of invertase, an enzyme that hydrolyses sucrose into fructose and glucose,...
Two bifunctional enzymes exhibiting combined xylanase and laccase activities were designed, constructed, and characterized
by biochemical and biophysical methods. The Bacillus subtilis cotA and xynA genes were used as templates for gene fusion, and the xynA coding sequence was inserted into a surface loop of the cotA. A second chimera was built rep...
β-1,3–1,4-Glucanases (E.C. 3.2.1.73) hydrolyze linked β-d-glucans, such as lichenan and barley β-glucan. Recombinant β-1,3–1,4-glucanase from Bacillus subtilis expressed in Escherichia coli and purified by Ni-NTA chromatography exhibited optimum activity at 50 °C and pH 6.0. The catalytic half-life at 60 °C decreased from 90 to 5 min when the enzym...
Phaseolus vulgaris
beans
rust diseases
Uromyces appendiculatus
plant pathogenic fungi
pathogenicity
inoculation methods
disease resistance
virulence
leaves
germplasm evaluation
rapid methods
genetic resistance
genes
cultivars
genetic variation
pathotypes
The pyramiding of resistance genes (R) to overcome diseases incited by different
pathogens has been proposed as a strategy to obtain wide spectrum durable resistance. However, this
procedure is difficult to accomplish when conventional inoculation techniques are used to evaluate the
plant-pathogen interaction (Faleiro et al., 2003). Conventional...
Questions
Questions (17)
I have seen on literature some purified glycoside hydrolases, without cysteines, that showed their activity increased by addicition of DTT or β-mercaptoethanol on enzimatic assay. What are the possible explanations?
I'm looking for a good server for creating DNA 3D structure based on small sequences.
I have produced a number of circular permutations for a protein. There are
several structures available for the wild type protein. Is MODELLER
able to process circular permutations? If so, how would I encode this in
my alignment file? Thanks in advance for your help!
Would anyone know what is the best system to get a good amount of protein after in vitro expression? I'm using Rabbit Reticulocyte Lysate System, but I didn't get protein enough to see SDS-PAGE (coomassie).
I have a library with variants of the same gene, and after selection steps (antibiotic resistance) I have observed a large number of variants with deletions. I don't have these deletions in my naive library, they are found only in the selected clones. I use the SNO 301 strain (AMPD1, ampA1, ampC8, pyrB, recA, rpsL) for screenings. Anybody have any suggestions about this issue?
I have tried to purify my protein after the in vitro translation using his tag (or poly dT followed by his tag). In the either case I can detect activity after the purification, but I can not see the band on the gel (I tried: Coomassie, silver and fluorescence, once that fluorescein is attached to the RNA), I didn't make a Western blot yet. Does anyone know some tricks to increase the in vitro expression and obtain greater amount of protein after purification steps? I'm using Rabbit Reticulocyte Lysate System.
Would someone know which of these purification techniques performs better: Anti-Flag M2-agarose affinity gel or Ni-NTA resin?
What are the major differences between protein unfolding and misfolding? And how can I pick up the most thermostable proteins?
I did a inverse PCR and I saw a lot of undesired bands in my PCR. My primers Tms is about 55C and, I used 65C to anneal. My template have some repeated sequences. My desired band has 8kb and I saw many smaller bands. I'm using phusion polymerase and a extension time of 6 min. Does anyone have any tips on what to do?
Computational tools for protein engineering.
