Bacillus Halodurans Research Articles

Pesticide application inhibit the microbial carbonic anhydrase-mediated carbon sequestration in a soil microcosm.

Heterotrophic system for carbon sequestration is gaining importance in the recent decades. Carbonic anhydrase (CA) is a major enzyme involved in carbon sequestration and biomineralization process. In this paper, we evaluate the effect of pesticide on CA activity using inhibitory assay. 2,4-D, being one of the most extensively used pesticide, being deleterious to soil health, its usage should be minimized to regain the soil health. Maximum inhibitory constant (Ki) was observed for 5% 2,4-D (49.53 mM) followed by 5% glyphosate (43.92 mM). The maximum Km increase with increase in pesticide concentration by 3.05-fold was in case of glyphosate which was higher than that of 2,4-D (2.08-fold) and dichlorvos (2.38-fold). Moreover, we evaluated the carbon sequestration using CA enzyme in the soil microcosm. In the present study, we identified the negative impact of 2,4-D on carbonic anhydrase produced by Bacillus halodurans PO15. The inhibition was a mixed type and had significantly lowered the carbon reduction to about 2.38 ± 0.17% in a soil microcosm study. Based on the molecular docking, the inhibition was contributed due to weak H-bonding interaction with amino acid residues (Gly65, Gly95, Val147, Ser150 and Gly65, Ser146, and Ser150).

Structural analysis of the manganese transport regulator MntR from Bacillus halodurans in apo and manganese bound forms.

The manganese transport regulator MntR is a metal-ion activated transcriptional repressor of manganese transporter genes to maintain manganese ion homeostasis. MntR, a member of the diphtheria toxin repressor (DtxR) family of metalloregulators, selectively responds to Mn2+ and Cd2+ over Fe2+, Co2+ and Zn2+. The DtxR/MntR family members are well conserved transcriptional repressors that regulate the expression of metal ion uptake genes by sensing the metal ion concentration. MntR functions as a homo-dimer with one metal ion binding site per subunit. Each MntR subunit contains two domains: an N-terminal DNA binding domain, and a C-terminal dimerization domain. However, it lacks the C-terminal SH3-like domain of DtxR/IdeR. The metal ion binding site of MntR is located at the interface of the two domains, whereas the DtxR/IdeR subunit contains two metal ion binding sites, the primary and ancillary sites, separated by 9 Å. In this paper, we reported the crystal structures of the apo and Mn2+-bound forms of MntR from Bacillus halodurans, and analyze the structural basis of the metal ion binding site. The crystal structure of the Mn2+-bound form is almost identical to the apo form of MntR. In the Mn2+-bound structure, one subunit contains a binuclear cluster of manganese ions, the A and C sites, but the other subunit forms a mononuclear complex. Structural data about MntR from B. halodurans supports the previous hypothesizes about manganese-specific activation mechanism of MntR homologues.

Genome-based reclassification of Bacillus okuhidensis as a later heterotypic synonym of Bacillus halodurans.

The present study was carried out to clarify the taxonomic relationship between two closely related Bacillus species, Bacillus okuhidensis Li et al. 2002 and Bacillus halodurans (ex Boyer 1973) Nielsen et al. 1995. The maximum-likelihood tree based on the 16S rRNA gene sequence and the phylogenomic tree based on concatenation of 16 protein-marker genes showed that these species were similar. Average nucleotide identity (ANIm 99.25 %, ANIb 98.2 %) and digital DNA-DNA hybridization values (93.5 %) between B. okuhidensis DSM 13666T and B. halodurans DSM 497T were greater than the threshold values for bacterial species delineation, indicating that they belong to the same species. Therefore, B. okuhidensis Li et al. 2002 should be reclassified as a later heterotypic synonym of B. halodurans (ex Boyer 1973) Nielsen et al. 1995.

Disruption of the OLE ribonucleoprotein complex causes magnesium toxicity in Bacillus halodurans.

OLE RNAs represent an unusual class of bacterial noncoding RNAs common in Gram-positive anaerobes. The OLE RNA of the alkaliphile Bacillus halodurans is highly expressed and naturally interacts with at least two RNA-binding proteins called OapA and OapB. The phenotypes of the corresponding knockouts include growth inhibition when exposed to ethanol or other short-chain alcohols or when incubated at modestly reduced temperatures (e.g. 20°C). Intriguingly, the OapA 'PM1' mutant, which carries two amino acid changes to a highly conserved region, yields a dominant-negative phenotype that causes more severe growth defects under these same stress conditions. Herein, we report that the PM1 strain also exhibits extreme sensitivity to elevated Mg2+ concentrations, beginning as low as 2mM. Suppressor mutants predominantly map to genes for aconitate hydratase and isocitrate dehydrogenase, which are expected to alter cellular citrate concentrations. Citrate reduces the severity of the Mg2+ toxicity phenotype, but neither the genomic mutations nor the addition of citrate to the medium overcomes ethanol toxicity or temperature sensitivity. These findings reveal that OLE RNA and its protein partners are involved in biochemical responses under several stress conditions, wherein the unusual sensitivity to Mg2+ can be independently suppressed by specific genomic mutations.

Improving Catalytic Efficiency and Changing Substrate Spectrum for Asymmetric Biocatalytic Reductive Amination.

With the advantages of biocatalytic method, enzymes have been excavated for the synthesis of chiral amino acids by the reductive amination of ketones, offering a promising way of producing pharmaceutical intermediates. In this work, a robust phenylalanine dehydrogenase (PheDH) with wide substrate spectrum and high catalytic efficiency was constructed through rational design and active-site-targeted, site-specific mutagenesis by using the parent enzyme from Bacillus halodurans. Active sites with bonding substrate and amino acid residues surrounding the substrate binding pocket, 49L-50G-51G, 74M,77K, 122G-123T-124D-125M, 275N, 305L and 308V of the PheDH, were identified. Noticeably, the new mutant PheDH (E113D-N276L) showed approximately 6.06-fold increment of kcat/Km in the oxidative deamination and more than 1.58-fold in the reductive amination compared to that of the wide type. Meanwhile, the PheDHs exhibit high capacity of accepting benzylic and aliphatic ketone substrates. The broad specificity, high catalytic efficiency and selectivity, along with excellent thermal stability, render these broad-spectrum enzymes ideal targets for further development with potential diagnostic reagent and pharmaceutical compounds applications.

Biochemical characterization of a novel exo-oligoxylanase from Paenibacillus barengoltzii suitable for monosaccharification from corncobs

BackgroundXylan is the major component of hemicelluloses, which are the second most abundant polysaccharides in nature, accounting for approximately one-third of all renewable organic carbon resources on earth. Efficient degradation of xylan is the prerequisite for biofuel production. Enzymatic degradation has been demonstrated to be more attractive due to low energy consumption and environmental friendliness, when compared with chemical degradation. Exo-xylanases, as a rate-limiting factor, play an important role in the xylose production. It is of great value to identify novel exo-xylanases for efficient bioconversion of xylan in biorefinery industry.ResultsA novel glycoside hydrolase (GH) family 8 reducing-end xylose-releasing exo-oligoxylanase (Rex)-encoding gene (PbRex8) was cloned from Paenibacillus barengoltzii and heterogeneously expressed in Escherichia coli. The deduced amino acid sequence of PbRex8 shared the highest identity of 74% with a Rex from Bacillus halodurans. The recombinant enzyme (PbRex8) was purified and biochemically characterized. The optimal pH and temperature of PbRex8 were 5.5 and 55 °C, respectively. PbRex8 showed prominent activity on xylooligosaccharides (XOSs), and trace activity on xylan. It also exhibited β-1,3-1,4-glucanase and xylobiase activities. The enzyme efficiently converted corncob xylan to xylose coupled with a GH family 10 endo-xylanase, with a xylose yield of 83%. The crystal structure of PbRex8 was resolved at 1.88 Å. Structural comparison suggests that Arg67 can hydrogen-bond to xylose moieties in the -1 subsite, and Asn122 and Arg253 are close to xylose moieties in the -3 subsite, the hypotheses of which were further verified by mutation analysis. In addition, Trp205, Trp132, Tyr372, Tyr277 and Tyr369 in the grove of PbRex8 were found to involve in glucooligosaccharides interactions. This is the first report on a GH family 8 Rex from P. barengoltzii.ConclusionsA novel reducing-end xylose-releasing exo-oligoxylanase suitable for xylose production from corncobs was identified, biochemically characterized and structurally elucidated. The properties of PbRex8 may make it an excellent candidate in biorefinery industries.

Promiscuity of host cell proteins in the purification of histidine tagged recombinant xylanase A by IMAC procedures: A case study with a Ni2+-tacn-based IMAC system

Determination of the extent of host cell protein (HCP) contamination is an essential pre-requisite to validate the chromatographic purification of recombinant proteins. This study explores how different experimental conditions affect the HCP profiles generated during the immobilised metal ion affinity chromatographic (IMAC) purification with a Ni2+-1,4,7-triaza-cyclononane (tacn) Sepharose FF™ sorbent of the Bacillus halodurans N- and C-terminal His6-tagged xylanase A, expressed by Escherichia coli BL21(DE3) cells, and captured directly from cell lysates. Comparative studies were also carried out under identical loading, wash and elution conditions using nitrilotriacetic acid (NTA), also immobilised onto an agarose support and complexed with Ni2+ ions. High-resolution tandem mass spectrometry of the tryptic peptides derived from the proteins present in the IMAC flow-through, wash and elution fractions confirmed that the E. coli BL21(DE3) HCP profiles were dependent on the choice of adsorbent. With feedstocks containing the N- or C-terminal His6-tagged xylanase A, in several instances the same E. coli BL21(DE3) HCPs were found to co-elute with the tagged protein from either adsorbent, indicating a preferential ability of some HCPs to bind to both the IMAC resin and to the recombinant protein. This promiscuous behaviour has been found to be due to factors other than just the presence of histidine-rich motifs within the amino acid sequences of these HCPs. This case study demonstrates that the choice of protein expression and separation conditions impact on the levels of HCP contamination when different IMAC systems are employed.

Characterization of thermostable alkaline protease from Bacillus halodurans SE5 and its application in degumming coupled with sericin hydrolysate production from yellow cocoon

Abstract Bacillus halodurans SE5 was newly isolated and grew well on a medium containing crude sericin extract from cocoon. Thermostable alkaline serine protease (protease_SE5) capable of decomposing sericin extract was purified to homogeneity from culture supernatant with a final yield of 25% and 2.01 × 104 U/mg. Among the six natural proteins tested, protease_SE5 showed the highest activity toward sericin. The sericin degumming, bio-bleaching coupled with sericin hydrolysate production from yellow cocoon by protease_SE5 and commercial Alcalase were demonstrated in the presence or absence of dithiothreitol (DTT). The addition of DTT enhanced the efficacy of both proteases. However, without DTT, the protease_SE5 had higher degumming ability and produced sericin hydrolysate 4-times higher than commercial enzyme based on the soluble protein concentration. SDS-PAGE and size exclusion chromatography analysis revealed that the maximal concentration of oligopeptides was observed with the hydrolysate prepared by protease_SE5 and showed higher antioxidant activity than those from Alcalase. The appreciable radical scavenging activities of the crude peptide (1.36 ± 0.07 mM) on ABTS, DPPH, and FRAP assay were 1568 ± 78, 3.6 ± 1.6, and 13.6 ± 0.4 μmol TE/L, respectively. Protease_SE5 has potential application for one step degumming and preparation of bioactive peptides from yellow cocoon.

Isolation of a Functional Gene Encoding Homologous Lysophospholipase from Indonesian Indigenous Bacillus halodurans CM1

Lipase is a biocatalyst widely used in industry, for example detergent, pharmaceutical, food, or oil purification. One of the most widely lipase used for oil purification is lysophospholipase. As much as 50% of industrial enzyme needs are supplied from microorganisms. However, enzyme productivity from wild type microbial strain is usually limited and not applicable in industry, so that genetic engineering is necessary. Cloning gene encoding for lysophospholipase from Aspergillus niger and Cryptococcus neoformans have been conducted, but has never been conducted from alkalothermophilic bacteria, such as Bacillus halodurans. Bacillus halodurans CM1 is an alkalothermophilic bacterial strain isolated previously that has many industrially potential enzymes. This study aimed to isolate one of the gene encoding lipase from Bacillus halodurans CM1 and cloned into Escherichia coli DH5α using the pGEM-T easy vector. The gene fragment encoding lysophospholipase obtained with size 783 base pairs and had 100% similarity with gene encoding lysophospholipase from Bacillus halodurans C-125 (No access GenBank: BA000004.3). E. coli harbouring the recombinant plasmid with the gene also showed activity on trybutiryn medium compared to negative control.

Gene Cloning of Xylanase Glycoside Hydrolase Family 11 from Bacillus halodurans CM1 in Escherichia coli DH5α

Xylanase is an enzyme that can break down xylan into xylose and xylooligosaccharide that is widely used in industry. Seeing the many applications of this enzyme, researchers conducted many studies on how to increase the productivity and effectiveness of the xylanase enzyme. One of the method that can be used to increase the xylanase enzyme production process is by using recombinant DNA technology such as cloning. Bacillus halodurans CM1 is a local alkalothermophilic bacterium that potential producer for xylanase and other industrial enzymes. This research was conducted to clone the GH11 xylanase coding gene from B. halodurans CM1 using pJET 1.2 / blunt plasmid as vector into Escherichia coli DH5α as cell host and determine the nucleotide base sequence of the GH11 xylanase coding gene from B. halodurans CM1. The results showed the GH11 xylanase gene from B. halodurans CM1 was successfully cloned in E. coli DH5α and based on the results of BLAST nucleotides had 99% similarities with that of endo-1,4-beta -xylanhydrolase (xyn11A) from B. halodurans C-125. Key words: Bacillus halodurans CM1, cloning, xylanase glycoside hydrolase family 11

The effect of an oligosaccharide reducing-end xylanase, BhRex8A, on the synergistic degradation of xylan backbones by an optimised xylanolytic enzyme cocktail

Xylan, the most abundant hemicellulose in lignocellulosic biomass, requires a consortium of xylanolytic enzymes to achieve its complete de-polymerisation. As global interest in using xylan-containing lignocellulosic feedstocks for biofuel production increases, an accompanying knowledge on how to efficiently depolymerise these feedstocks into fermentable sugars is required. Since it has been observed that the same enzyme [i.e. an enzyme with the same EC (Enzyme Commission) classification] from different GH families can display different substrate specificities and properties, we evaluated GH10 (XT6) and 11 (Xyn2A) xylanase performance alone, and in combination, during xylan depolymerisation. Synergistic enhancement with respect to reducing sugar release was observed when Xyn2A at 75% loading was supplemented with 25% loading of XT6 for both beechwood glucuronoxylan (1.14-fold improvement) and wheat arabinoxylan (1.1-fold improvement) degradation. Following this, the optimised xylanase mixture was dosed with an oligosaccharide reducing-end xylanase (Rex8A) from either Bifidobacterium adolescentis or Bacillus halodurans for further synergistic enhancement. Dosing 75% of the xylanase mixture (Xyn2A:XT6 at 75:25%) with 25% loading of Rex8A led to an enhancement of reducing sugar (up to an 1.1-fold improvement) and xylose release (up to an 1.5-fold improvement); however, this effect was both xylan and Rex8A specific. Using thin layer chromatography, synergism appeared to be a result of the GH10 and 11 xylanases liberating xylo-oligomers that are preferred substrates of the processive Rex8As. Rex8As then hydrolysed xylo-oligomers to xylose - and xylobiose which was the preferred substrate for xylosidase, SXA. This likely explains why there was a significant improvement in xylose release in the presence of Rex8As. Here, it was shown that Rex8As are key enzymes in the efficient saccharification of hetero-xylan into xylose, a major component of lignocellulosic substrates.

Cloning and expression of alkalophilic xylanase gene from an Indonesia local Bacillus halodurans CM1 in Escherichia coli and its application on deinking process of waste paper

Cloning and expression of alkalophilic xylanase gene from an Indonesia local Bacillus halodurans CM1 in Escherichia coli and its application on deinking process of waste paper

Cloning of a Gene Encoding Protease from Bacillus halodurans CM1 into Escherichia coli DH5 and Expression Analyses of the Gene Product

Bacillus halodurans strain CM1 is an Indonesia alkalothermophilic bacterium isolated from Cimanggu Hot Spring, Bandung, West Java. This bacterial strain produces high levels of thermoalkalophilic xylanase. It has also been predicted to produce other potential industrial enzymes, including protease. For production and application of protease in the future, the protease gene from B. halodurans CM1 was cloned into Escherichia coli . The protease gene was isolated from B. halodurans CM1 by the PCR approach using primers designed based on the GenBank . The PCR product was then ligated into pGEM-T Easy vector, transformed into E. coli DH5α, verified, and analyzed based on DNA sequencing data using the BLAST search tool. A 1086-bp protease gene was obtained that exhibited a very high sequence similarity (99%) with that of alkaline protease gene from B. halodurans C-125. When the culture of this positive recombinant E. coli DH5α containing the protease gene was spotted onto calcium caseinate agar, a clear zone appeared after incubation at 50°C. This result demonstrated that the protease gene was expressed in this recombinant E.coli DH5α.

A second RNA-binding protein is essential for ethanol tolerance provided by the bacterial OLE ribonucleoprotein complex

OLE (ornate, large, extremophilic) RNAs comprise a class of structured noncoding RNAs (ncRNAs) found in many extremophilic bacteria species. OLE RNAs constitute one of the longest and most widespread bacterial ncRNA classes whose major biochemical function remains unknown. In the Gram-positive alkaliphile Bacillus halodurans, OLE RNA is abundant, and localizes to the cell membrane by association with the transmembrane OLE-associated protein called OapA (formerly OAP). These characteristics, along with the well-conserved sequence and structural features of OLE RNAs, suggest that the OLE ribonucleoprotein (RNP) complex performs important biological functions. B. halodurans strains lacking OLE RNA (∆ole) or OapA (∆oapA) are less tolerant of cold (20 °C) and short-chain alcohols (e.g., ethanol). Here, we describe the effects of a mutant OapA (called PM1) that more strongly inhibits growth under cold or ethanol stress compared with strains lacking the oapA gene, even when wild-type OapA is present. This dominant-negative effect of PM1 is reversed by mutations that render OLE RNA nonfunctional. This finding demonstrates that the deleterious PM1 phenotype requires an intact RNP complex, and suggests that the complex has one or more additional undiscovered components. A genetic screen uncovered PM1 phenotype suppressor mutations in the ybzG gene, which codes for a putative RNA-binding protein of unknown biological function. We observe that YbzG protein (also called OapB) selectively binds OLE RNA in vitro, whereas a mutant version of the protein is not observed to bind OLE RNA. Thus, YbzG/OapB is an important component of the functional OLE RNP complex in B. halodurans.

Heterologous expression and enhanced production of β-1,4-glucanase of Bacillus halodurans C-125 in Escherichia coli

Abstract Background Recombinant DNA technology enables us to produce proteins with desired properties and insubstantial amount for industrial applications. Endo-1, 4-β-glucanases (Egl) is one of the major enzyme involved in degradation of cellulose, an important component of plant cell wall. The present study was aimed at enhancing the production of endo-1, 4-β-glucanases (Egl) of Bacillus halodurans in Escherichia coli. Results A putative Egl gene of Bacillus Halodurans was expressed in E. coli by cloning in pET 22b (+). On induction with isopropyl-b- d -1-thiogalactopyranoside, the enzyme expression reached upto ~ 20% of the cell protein producing 29.2 mg/liter culture. An increase in cell density to 12 in auto-inducing LB medium (absorbance at 600 nm) enhanced β-glucanase production up to 5.4 fold. The molecular mass of the enzyme was determined to be 39 KDa, which is nearly the same as the calculated value. Protein sequence was analyzed by CDD, Pfam, I TASSER, COACH, PROCHECK Servers and putative amino acids involved in the formation of catalytic, substrate and metal binding domains were identified. Phylogenetic analysis of the β-glucanases of B. halodurans was performed and position of Egl among other members of the genus Bacillus producing endo-glucanases was determined. Temperature and pH optima of the enzyme were found to be 60°C and 8.0, respectively, under the assay conditions. Conclusion Production of endo-1, 4 β-glucanase enzymes from B. halodurans increased several folds when cloned in pET vector and expressed in E. coli. To our knowledge, this is the first report of high-level expression and characterization of an endo-1, 4 β-glucanases from B. halodurans. How to cite: Zeeshan N, Naz S, Naz S et al. Heterologous expression and enhanced production of β-1,4-glucanase of Bacillus halodurans C-125 in E. coli. Electron J Biotechnol 2018;34. https://doi.org/10.1016/j.ejbt.2018.05.001 .

Resolution Mechanism and Characterization of an Ammonium Chloride-Tolerant, High-Thermostable, and Salt-Tolerant Phenylalanine Dehydrogenase from Bacillus halodurans.

As phenylalanine dehydrogenase (PheDH) plays an important role in the synthesis of chiral drug intermediates and detection of phenylketonuria, it is significant to obtain a PheDH with specific and high activity. Here, a PheDH gene, pdh, encoding a novel BhPheDH with 61.0% similarity to the known PheDH from Microbacterium sp., was obtained. The BhPheDH showed optimal activity at 60°C and pH 7.0, and it showed better stability in hot environment (40-70°C) than the PheDH from Nocardia sp. And its activity and thermostability could be significantly increased by sodium salt. After incubation for 2h in 3M NaCl at 60°C, the residual activity of the BhPheDH was found to be 1.8-fold higher than that of the control group (without NaCl). The BhPheDH could tolerate high concentration of ammonium chloride and its activity could be also enhanced by the high concentration of ammonium salts. These characteristics indicate that the BhPheDH possesses better thermostability, ammonium chloride tolerance, halophilic mechanism, and high salt activation. The mechanism of thermostability and high salt tolerance of the BhPheDH was analyzed by molecular dynamics simulation. These results provide useful information about the enzyme with high-temperature activity, thermostability, halophilic mechanism, tolerance to high concentration of ammonium chloride, higher salt activation and enantio-selectivity, and the application of molecular dynamics simulation in analyzing the mechanism of these distinctive characteristics.

Characterization of the Dual Pathways for B. halodurans Asparaginyl‐tRNA Formation

Protein synthesis requires the attachment of an amino acid to its cognate transfer RNA (tRNA). Two distinct pathways for attaching asparagine (Asn) to tRNAAsn are known: the direct pathway, in which Asn is directly attached to tRNAAsn by the enzyme AsnRS; and the indirect pathway, in which Asn is attached to tRNA by a non‐discriminating AspRS and GatCAB. Bacillus halodurans uses both routes for Asn‐tRNAAsn formation. To better understand why B. halodurans possesses both pathways, we have purified its AsnRS and AspRS in order to test and compare their activities under different physiologically relevant conditions such as in the presence of reactive oxygen species and changes in pH. This research will provide insight into how B. halodurans has adapted to survive in different environmental conditions.Support or Funding InformationNational Science Foundation (MCB‐1615770)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Investigation of the Structure and Function of Phosphoserine Phosphatases

With the ever increasing availability of new genome sequences every year, there is a growing need for functional annotation both on a structural and cellular level. In the case of large superfamilies of proteins with a great deal of sequence divergence, assignment of function based on sequence alone remains difficult. For instance, although Bacillus subtilis is the model gram positive bacterium, only 30% of the genome has assigned activity. YsaA (BSU28940), a B. subtilis protein discovered in a knockout screen for metabolic mutants, has been confirmed to have phosphoserine phosphatase activity by determining the steady‐state kinetic constants. Despite this, YsaA shares only 12% sequence identity to the known E. coli phosphoserine phosphatase SerB. We are currently working to determine the molecular structure of YsaA, along with a Bacillus halodurans protein BH2155 (60% similarity with YsaA) which will provide better understanding of the structure‐function relationship between these new proteins and SerB. It is very possible that although YsaA and SerB are within the same super family, they may have independently evolved the same function.BH2155 has been over‐expressed in E.coli, purified, and subjected to sparse‐matrix high throughput screens yielding initial crystallization conditions. Additionally, experiments have been performed using a phosphate assay to obtain the steady state kinetic constants for BH2155 and compare them to YsaA and known phosphoserine phosphatase SerB. By determining the structures of these proteins as well as profiling their activity we hope to gain insight into the structure‐function relationship and the possibility of pseudo‐convergent evolution in phosphoserine phosphatases.Support or Funding InformationWe thank the Undergraduate Research Opportunities Program at Boston University for funding supporting this project (to MCB).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Microbial-Enhanced Heavy Oil Recovery under Laboratory Conditions by Bacillus firmus BG4 and Bacillus halodurans BG5 Isolated from Heavy Oil Fields

Microbial Enhanced Oil Recovery (MEOR) is one of the tertiary recovery methods. The high viscosity and low flow characteristics of heavy oil makes it difficult for the extraction from oil reservoirs. Many spore-forming bacteria were isolated from Oman oil fields, which can biotransform heavy crude oil by changing its viscosity by converting heavier components into lighter ones. Two of the isolates, Bacillus firmus BG4 and Bacillus halodurans BG5, which showed maximum growth in higher concentrations of heavy crude oil were selected for the study. Gas chromatography analysis of the heavy crude oil treated with the isolates for nine days showed 81.4% biotransformation for B. firmus and 81.9% for B. halodurans. In both cases, it was found that the aromatic components in the heavy crude oil were utilized by the isolates, converting them to aliphatic species. Core flooding experiments conducted at 50 °C, mimicking reservoir conditions to prove the efficiency of the isolates in MEOR, resulted in 10.4% and 7.7% for B. firmus and B. halodurans, respectively, after the nine-day shut-in period. These investigations demonstrated the potential of B. firmus BG4 and B. halodurans BG5 as an environmentally attractive approach for heavy oil recovery.

First report on molecular characterization of novel betaine aldehyde dehydrogenase from the halotolerant eubacteria, Bacillus halodurans

Betaine aldehyde dehydrogenase (BADH) is an effective compatible solute, which maintains fluidity of membranes and protects the biological structure of the organisms under stress. Betaine aldehyde dehydrogenase catalyses the last step in the biosynthetic pathway that leads to glycine betaine. In this study, betaine aldehyde dehydrogenase (BADH1) from the halotolerant Bacillus halodurans SMBPL06 was heterologously expressed in Escherichia coli M15 (pREP4). The recombinant enzyme was purified by column chromatography using DEAE sepharose. The purified enzyme revealed a five-fold increase in the activity with choline as substrate and phenazine methosulfate as electron acceptor, compared to the control strain. The betaine aldehyde dehydrogenase gene sequences reported in this study contains several base substitutions with that of reported sequences in GenBank, resulting in the altered amino acid sequences of the translated proteins.