scholarly journals Recombinant Production and One-Pot Purification for Enhancing Activity of Haloacid Dehalogenase from Bacillus cereus IndB1

REAKTOR ◽  
2021 ◽  
Vol 21 (2) ◽  
pp. 59-64
Author(s):  
Enny Ratnaningsih ◽  
Sulistiya Nirta Sunaryo ◽  
Idris Idris ◽  
Rindia Maharani Putri
Keyword(s):  
Bacillus Cereus ◽  
Recombinant Expression ◽  
Host Cells ◽  
Monochloroacetic Acid ◽  
Affinity Column ◽  
One Pot ◽  
Crude Enzyme Extract ◽  
Local Strains ◽  
Haloacid Dehalogenase ◽  
Purification System

In recent years we have witnessed the emergence of organohalogen utilization in various chemical-based industries, particularly polymer-based, agricultural, and pharmaceutical sectors. Despite this, organohalogen compounds are actually very dangerous to the environment, as they are difficult to be naturally degraded and generally toxic to organisms. A green and biocompatible method to overcome this issue is by employing enzymes that could convert organohalogens into non-toxic compounds, such as the class of enzymes known as haloacid dehalogenases. To enhance the activity of haloacid dehalogenase isolated from local strains of Bacillus cereus IndB1, we have developed a recombinant expression system using pET-bcfd1 plasmid in E. coli BL21 (DE3) host cells. Following enzyme production, we also demonstrated a one-pot purification system for the expressed dehalogenase, harnessing the presence of His-tag in the recombinant clones. Purification was carried out using Ni-NTA affinity column chromatography, using imidazole eluent with a concentration gradient of 10 mM to 500 mM. The enzyme activity was tested against the monochloroacetic acid (MCA) substrate according to the Bergmann and Sanik method, and the protein content in the solution was measured using the Bradford method. The purity of the enzyme after one-pot purification was confirmed by SDS-PAGE analyses, showing a single band of 40 kDa in size. Remarkably, the purified haloacid dehalogenase specific activity was increased by 12-fold compared to its crude enzyme extract. Therefore, the expression and purification system developed in this study allow further exploration of dehalogenases from local strains as an efficient catalyst for MCA biodegradation.Keywords: recombinant expression, haloacid dehalogenase, monochloroacetic acid, enzyme purification

Optimization of Monochloroacetic Acid Biodegradation by Recombinant E. coli BL21 (DE3)/pET-bcfd1 Carrying Haloacid Dehalogenase Gene from Bacillus cereus IndB1

2021 ◽  
Vol 10 (4) ◽  
pp. 857-863
Author(s):  
Enny Ratnaningsih ◽  
Rachmad Ade ◽  
Rindia Maharani Putri ◽  
Idris Idris
Keyword(s):  
Bacillus Cereus ◽  
Chloride Ion ◽  
Local Strain ◽  
Luria Bertani ◽  
Chloride Ions ◽  
Monochloroacetic Acid ◽  
Iptg Induction ◽  
Haloacid Dehalogenase ◽  
E Coli ◽  
Dehalogenase Gene

In recent years, attention to microbial dehalogenase has continually increased due to its potential application, both in bioremediation and in the biosynthesis of fine chemicals. Many microbial recombinant strains carrying dehalogenase gene have been developed, particularly to increase the dehalogenase production and its quality. In this study, we aimed to find the optimum condition for the production of active haloacid dehalogenase by E. coli BL21 (DE3) harboring recombinant plasmid pET-bcfd1 that carried haloacid dehalogenase gene from Bacillus cereus IndB1 local strain. This would be examined by assessing the ability of whole cell life culture to degrade monochloroacetic acid (MCA) and quantifying the chloride ion released into the medium. Several variables were evaluated to find this optimal condition. We found that the best condition for MCA biodegradation using this recombinant clone was at 0.2 mM MCA, 10 μM of isopropyl β-D-1-thiogalactopyranoside (IPTG), 6 hours of pre-induction incubation at 37ºC with shaking, 2 hours IPTG induction at 30ºC with shaking, at pH 7 in Luria Bertani (LB) liquid medium without NaCl, which produced about 0.056 mM chloride ions. Inducer concentration, pre-induction incubation time and temperature, as well as induction time and temperature were apparent to be associated with the expression of the protein, while the MCA concentration and the pH of the medium influenced the ability of the recombinant E. coli BL21 (DE3)/pET-bcfd1 to grow in toxic environment. Our findings laid the foundation for exploration of dehalogenases from local Bacillus strains through genetic engineering for MCA biodegradation

scholarly journals Cloning and expression of haloacid dehalogenase gene from Bacillus cereus IndB1

2018 ◽  
Vol 22 (2) ◽  
pp. 55
Author(s):  
Enny Ratnaningsih ◽  
Idris Idris
Keyword(s):  
Recombinant Protein ◽  
Bacillus Cereus ◽  
Tertiary Structure ◽  
Specific Activity ◽  
Halogen Bond ◽  
Expression System ◽  
Industrial Sector ◽  
Cloning And Expression ◽  
Haloacid Dehalogenase ◽  
Organohalogen Compounds

Organohalogen compounds, widely used as pesticides in agriculture and solvents in the industrial sector, cause environmental pollution and health problems due to their toxicity and persistence. Numerous studies have been conducted on the biodegradation of organohalogen compounds, with many focusing on the use of dehalogenase from bacteria. Haloacid dehalogenase is a group of enzymes that cleaves the carbon-halogen bond in halogenated aliphatic acids. In a previous study, the bcfd1 gene encoded haloacid dehalogenase from Bacillus cereus IndB1 was successfully isolated and characterized. This research aimed to create an expression system of the bcfd1 gene by subcloning this gene into pET expression vector and to overexpress the gene in Escherichia coli BL21 (DE3). In addition, the recombinant protein was characterized to gain a better understanding of the catalytic action of this enzyme. A high expression of bcfd1 was obtained by inducing the culture at OD550 0.8–1.0  using 0.01 mM IPTG as determined by SDS-PAGE. Zymogram analysis proved that the recombinant protein possessed dehalogenase activity. Bcfd1 activity toward monochloroacetic acid (MCA) showed specific activity of 37 U/mg at 30°C, pH 9. The predicted tertiary structure of Bcfd1 was estimated has conserved α/ß hydrolase folding motif for haloacid dehalogenase superfamily.

scholarly journals Bacterial Quorum-Quenching Lactonase Hydrolyzes Fungal Mycotoxin and Reduces Pathogenicity of Penicillium expansum—Suggesting a Mechanism of Bacterial Antagonism

2021 ◽  
Vol 7 (10) ◽  
pp. 826
Author(s):  
Shlomit Dor ◽  
Dov Prusky ◽  
Livnat Afriat-Jurnou
Keyword(s):  
Cell Wall ◽  
Quorum Sensing ◽  
Virulence Factors ◽  
Molecular Mechanisms ◽  
Recombinant Expression ◽  
Bacterial Species ◽  
Quorum Quenching ◽  
Host Cells ◽  
Penicillium Expansum ◽  
Fungal Colonization

Penicillium expansum is a necrotrophic wound fungal pathogen that secrets virulence factors to kill host cells including cell wall degrading enzymes (CWDEs), proteases, and mycotoxins such as patulin. During the interaction between P. expansum and its fruit host, these virulence factors are strictly modulated by intrinsic regulators and extrinsic environmental factors. In recent years, there has been a rapid increase in research on the molecular mechanisms of pathogenicity in P. expansum; however, less is known regarding the bacteria–fungal communication in the fruit environment that may affect pathogenicity. Many bacterial species use quorum-sensing (QS), a population density-dependent regulatory mechanism, to modulate the secretion of quorum-sensing signaling molecules (QSMs) as a method to control pathogenicity. N-acyl homoserine lactones (AHLs) are Gram-negative QSMs. Therefore, QS is considered an antivirulence target, and enzymes degrading these QSMs, named quorum-quenching enzymes, have potential antimicrobial properties. Here, we demonstrate that a bacterial AHL lactonase can also efficiently degrade a fungal mycotoxin. The mycotoxin is a lactone, patulin secreted by fungi such as P. expansum. The bacterial lactonase hydrolyzed patulin at high catalytic efficiency, with a kcat value of 0.724 ± 0.077 s−1 and KM value of 116 ± 33.98 μM. The calculated specific activity (kcat/KM) showed a value of 6.21 × 103 s−1M−1. While the incubation of P. expansum spores with the purified lactonase did not inhibit spore germination, it inhibited colonization by the pathogen in apples. Furthermore, adding the purified enzyme to P. expansum culture before infecting apples resulted in reduced expression of genes involved in patulin biosynthesis and fungal cell wall biosynthesis. Some AHL-secreting bacteria also express AHL lactonase. Here, phylogenetic and structural analysis was used to identify putative lactonase in P. expansum. Furthermore, following recombinant expression and purification of the newly identified fungal enzyme, its activity with patulin was verified. These results indicate a possible role for patulin and lactonases in inter-kingdom communication between fungi and bacteria involved in fungal colonization and antagonism and suggest that QQ lactonases can be used as potential antifungal post-harvest treatment.

scholarly journals One-pot simultaneous production and sustainable purification of fibrinolytic protease from Bacillus cereus using natural deep eutectic solvents

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Senthil Kumar Rathnasamy ◽  
Aadhavan Durai ◽  
A. A. Vigneshkumar ◽  
C. Purushothaman ◽  
Devi Sri Rajendran ◽  
...  
Keyword(s):  
Bacillus Cereus ◽  
Deep Eutectic Solvents ◽  
One Pot ◽  
Simultaneous Production ◽  
Natural Deep Eutectic Solvents

scholarly journals Menaquinone-Specific Prenyl Reductase from the Hyperthermophilic Archaeon Archaeoglobus fulgidus

2005 ◽  
Vol 187 (6) ◽  
pp. 1937-1944 ◽  
Author(s):  
Hisashi Hemmi ◽  
Yoshihiro Takahashi ◽  
Kyohei Shibuya ◽  
Toru Nakayama ◽  
Tokuzo Nishino
Keyword(s):  
High Performance ◽  
Recombinant Expression ◽  
Reversed Phase ◽  
Host Cells ◽  
Archaeoglobus Fulgidus ◽  
In Vitro Assays ◽  
Hyperthermophilic Archaeon ◽  
Quinone Profile ◽  
New Compounds

ABSTRACT Four genes that encode the homologues of plant geranylgeranyl reductase were isolated from a hyperthermophilic archaeon Archaeoglobus fulgidus, which produces menaquinone with a fully saturated heptaprenyl side chain, menaquinone-7(14H). The recombinant expression of one of the homologues in Escherichia coli led to a distinct change in the quinone profile of the host cells, although the homologue is the most distantly related to the geranylgeranyl reductase. The new compounds found in the profile had successively longer elution times than those of ordinary quinones from E. coli, i.e., menaquinone-8 and ubiquinone-8, in high-performance liquid chromatography on a reversed-phase column. Structural analyses of the new compounds by electron impact-mass spectrometry indicated that their molecular masses progressively increase relative to the ordinary quinones at a rate of 2 U but that they still contain quinone head structures, strongly suggesting that the compounds are quinones with partially saturated prenyl side chains. In vitro assays with dithionite as the reducing agent showed that the prenyl reductase is highly specific for menaquinone-7, rather than ubiquinone-8 and prenyl diphosphates. This novel enzyme noncovalently binds flavin adenine dinucleotide, similar to geranylgeranyl reductase, but was not able to utilize NAD(P)H as the electron donor, unlike the plant homologue.

scholarly journals Hosts for Hostile Protein Production: The Challenge of Recombinant Immunotoxin Expression

Biomedicines ◽  
2019 ◽  
Vol 7 (2) ◽  
pp. 38 ◽  
Author(s):  
Stefania Zuppone ◽  
Maria Serena Fabbrini ◽  
Riccardo Vago
Keyword(s):  
Protein Production ◽  
Recombinant Expression ◽  
Chimeric Protein ◽  
Host Cells ◽  
Mammalian Host ◽  
Crucial Step ◽  
Protein Toxin ◽  
Recombinant Immunotoxin ◽  
Accurate Design ◽  
Production Host

For the recombinant expression of toxin-based drugs, a crucial step lies not only in the choice of the production host(s) but also in the accurate design of the protein chimera. These issues are particularly important since such products may be toxic to the expressing host itself. To avoid or limit the toxicity to productive cells while obtaining a consistent yield in chimeric protein, several systems from bacterial to mammalian host cells have been employed. In this review, we will discuss the development of immunotoxin (IT) expression, placing special emphasis on advantages and on potential drawbacks, as one single perfect host for every chimeric protein toxin or ligand does not exist.

scholarly journals The interaction between flagellin and the glycosphingolipid Gb3 on host cells contributes to Bacillus cereus acute infection

Virulence ◽  
2020 ◽  
Vol 11 (1) ◽  
pp. 769-780 ◽  
Author(s):  
Song Gao ◽  
Chengpei Ni ◽  
Wenhua Huang ◽  
Huaijie Hao ◽  
Hua Jiang ◽  
...  
Keyword(s):  
Bacillus Cereus ◽  
Acute Infection ◽  
Host Cells

scholarly journals Biosynthesis of l-Sorbose and l-Psicose Based on C—C Bond Formation Catalyzed by Aldolases in an Engineered Corynebacterium glutamicum Strain

2015 ◽  
Vol 81 (13) ◽  
pp. 4284-4294 ◽  
Author(s):  
Jiangang Yang ◽  
Jitao Li ◽  
Yan Men ◽  
Yueming Zhu ◽  
Ying Zhang ◽  
...  
Keyword(s):  
Corynebacterium Glutamicum ◽  
Host Cells ◽  
Dihydroxyacetone Phosphate ◽  
Microbial Fermentation ◽  
One Pot ◽  
Content Type ◽  
Stereochemical Control ◽  
Fed Batch Culture ◽  
Fermentation Strategy ◽  
First Time

ABSTRACTThe property of loose stereochemical control at aldol products from aldolases helped to synthesize multiple polyhydroxylated compounds with nonnatural stereoconfiguration. In this study, we discovered for the first time that some fructose 1,6-diphosphate aldolases (FruA) and tagatose 1,6-diphosphate (TagA) aldolases lost their strict stereoselectivity when usingl-glyceraldehyde and synthesized not onlyl-sorbose but also a high proportion ofl-psicose. Among the aldolases tested, TagA fromBacillus licheniformis(BGatY) showed the highest enzyme activity withl-glyceraldehyde. Subsequently, a “one-pot” reaction based on BGatY and fructose-1-phosphatase (YqaB) generated 378 mg/literl-psicose and 199 mg/literl-sorbose from dihydroxyacetone-phosphate (DHAP) andl-glyceraldehyde. Because of the high cost and instability of DHAP, a microbial fermentation strategy was used further to producel-sorbose/l-psicose from glucose andl-glyceraldehyde, in which DHAP was obtained from glucose through the glycolytic pathway, and some recombination pathways based on FruA or TagA and YqaB were constructed inEscherichia coliandCorynebacterium glutamicumstrains. After evaluation of different host cells and combinations of FruA or TagA with YqaB and optimization of gene expression, recombinantC. glutamicumstrain WT(pXFTY) was selected and produced 2.53 g/liter total ketoses, with a yield of 0.50 g/gl-glyceraldehyde. Moreover, deletion of genecgl0331, encoding the Zn-dependent alcohol dehydrogenase inC. glutamicum, was confirmed for the first time to significantly decrease conversion ofl-glyceraldehyde to glycerol and to increase yield of target products. Finally, fed-batch culture of strain SY14(pXFTY) produced 3.5 g/literl-sorbose and 2.3 g/literl-psicose, with a yield of 0.61 g/gl-glyceraldehyde. This microbial fermentation strategy also could be applied to efficiently synthesize otherl-sugars.

scholarly journals The Morphogenetic Protein CotE Positions Exosporium Proteins CotY and ExsY during Sporulation of Bacillus cereus

mSphere ◽  
2021 ◽  
Vol 6 (2) ◽  
Author(s):  
Armand Lablaine ◽  
Mònica Serrano ◽  
Christelle Bressuire-Isoard ◽  
Stéphanie Chamot ◽  
Isabelle Bornard ◽  
...  
Keyword(s):  
Fluorescence Microscopy ◽  
Bacillus Cereus ◽  
Basal Layer ◽  
Super Resolution ◽  
Host Cells ◽  
Surface Layers ◽  
Content Type ◽  
Localization Pattern ◽  
Morphogenetic Protein ◽  
Environmental Interactions

ABSTRACT The exosporium is the outermost spore layer of some Bacillus and Clostridium species and related organisms. It mediates the interactions of spores with their environment, modulates spore adhesion and germination, and has been implicated in pathogenesis. In Bacillus cereus, the exosporium consists of a crystalline basal layer, formed mainly by the two cysteine-rich proteins CotY and ExsY, surrounded by a hairy nap composed of glycoproteins. The morphogenetic protein CotE is necessary for the integrity of the B. cereus exosporium, but how CotE directs exosporium assembly remains unknown. Here, we used super-resolution fluorescence microscopy to follow the localization of SNAP-tagged CotE, CotY, and ExsY during B. cereus sporulation and evidenced the interdependencies among these proteins. Complexes of CotE, CotY, and ExsY are present at all sporulation stages, and the three proteins follow similar localization patterns during endospore formation that are reminiscent of the localization pattern of Bacillus subtilis CotE. We show that B. cereus CotE guides the formation of one cap at both forespore poles by positioning CotY and then guides forespore encasement by ExsY, thereby promoting exosporium elongation. By these two actions, CotE ensures the formation of a complete exosporium. Importantly, we demonstrate that the assembly of the exosporium is not a unidirectional process, as previously proposed, but occurs through the formation of two caps, as observed during B. subtilis coat morphogenesis, suggesting that a general principle governs the assembly of the spore surface layers of Bacillaceae. IMPORTANCE Spores of Bacillaceae are enveloped in an outermost glycoprotein layer. In the B. cereus group, encompassing the Bacillus anthracis and B. cereus pathogens, this layer is easily recognizable by a characteristic balloon-like appearance and separation from the underlying coat by an interspace. In spite of its importance for the environmental interactions of spores, including those with host cells, the mechanism of assembly of the exosporium is poorly understood. We used super-resolution fluorescence microscopy to directly visualize the formation of the exosporium during the sporulation of B. cereus, and we studied the localization and interdependencies of proteins essential for exosporium morphogenesis. We discovered that these proteins form a morphogenetic scaffold before a complete exosporium or coat is detectable. We describe how the different proteins localize to the scaffold and how they subsequently assemble around the spore, and we present a model for the assembly of the exosporium.

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