The bioconversion of vitamin D3 catalyzed by cytochrome P450 (CYP) requires 25-hydroxylation and subsequent 1α-hydroxylation to produce the hormonal activated 1α,25-dihydroxyvitamin D3. Vitamin D3 25-hydroxylase catalyses the first step in the vitamin D3 biosynthetic pathway, essential in the de novo activation of vitamin D3. A CYP known as CYP107CB2 has been identified as a novel vitamin D hydroxylase in Bacillus lehensis G1. In order to deepen the understanding of this bacterial origin CYP107CB2, its detailed biological functions as well as biochemical characteristics were defined. CYP107CB2 was characterized through the absorption spectral analysis and accordingly, the enzyme was assayed for vitamin D3 hydroxylation activity. CYP-ligand characterization and catalysis optimization were conducted to increase the turnover of hydroxylated products in an NADPH-regenerating system. Results revealed that the over-expressed CYP107CB2 protein was dominantly cytosolic and the purified fraction showed a protein band at approximately 62 kDa on SDS-PAGE, indicative of CYP107CB2. Spectral analysis indicated that CYP107CB2 protein was properly folded and it was in the active form to catalyze vitamin D3 reaction at C25. HPLC and MS analysis from a reconstituted enzymatic reaction confirmed the hydroxylated products were 25-hydroxyitamin D3 and 1α,25-dihydroxyvitamin D3 when the substrates vitamin D3 and 1α-hydroxyvitamin D3 were used. Biochemical characterization shows that CYP107CB2 performed hydroxylation activity at 25 °C in pH 8 and successfully increased the production of 1α,25-dihydroxyvitamin D3 up to four fold. These findings show that CYP107CB2 has a biologically relevant vitamin D3 25-hydroxylase activity and further suggest the contribution of CYP family to the metabolism of vitamin D3.
Infections caused by methicillin-resistant Staphylococcus aureus (MRSA) have reached epidemic proportions globally. Therefore, there is an urgent need for a continuous supply of antibiotics to combat the problem. In this study, bacteria initially identified as species belonging to the Bacillus amyloliquefaciens operational group were re-identified based on the housekeeping gene, gyrB. Cell-free supernatants (CFS) from the strains were used for antimicrobial tests using the agar well diffusion assay against MRSA and various types of pathogenic bacteria. The minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC) and physicochemical characteristics of the CFS were determined. Based on gyrB sequence analysis, five strains (PD9, B7, PU1, BP1 and L9) were identified as Bacillus velezensis. The CFS of all B. velezensis strains showed broad inhibitory activities against Gram-negative and -positive as well as MRSA strains. Strain PD9 against MRSA ATCC 33742 was chosen for further analysis as it showed the biggest zone of inhibition (21.0 ± 0.4 mm). The MIC and MBC values obtained were 125 μl/ml. The crude antimicrobial extract showed bactericidal activity and was stable at various temperatures (40-80°C), pH (4-12), surfactants (Tween 20, Tween 80, SDS and Triton X-100) and metal ions (MgCI2, NaCI2, ZnNO3 and CuSO4) when tested. However, the crude extract was not stable when treated with proteinase K. All these properties resembled the characteristics of peptides. The antimicrobial compound from the selected strain was purified by using solvent extraction method and silica gel column chromatography. The purified compound was subjected to High Performance Liquid Chromatography which resulted in a single peak of the anti-MRSA compound being detected. The molecular weight of the anti-MRSA compound was determined by using SDS-PAGE and zymogram. The size of the purified antimicrobial peptide was approximately ~ 5 kDa. The antimicrobial peptide produced from B. velezensis strain PD9 is a promising alternative to combat the spread of MRSA infections in the future.
This study investigated the capability of a biosurfactant produced by a novel strain of Bacillus salmalaya to enhance the biodegradation rates and bioavailability of organic contaminants. The biosurfactant produced by cultured strain 139SI showed high physicochemical properties and surface activity in the selected medium. The biosurfactant exhibited a high emulsification index and a positive result in the drop collapse test, with the results demonstrating the wetting activity of the biosurfactant and its potential to produce surface-active molecules. Strain 139SI can significantly reduce the surface tension (ST) from 70.5 to 27 mN/m, with a critical micelle concentration of 0.4%. Moreover, lubricating oil at 2% (v/v) was degraded on Day 20 (71.5). Furthermore, the biosurfactant demonstrated high stability at different ranges of salinity, pH, and temperature. Overall, the results indicated the potential use of B. salmalaya 139SI in environmental remediation processes.
Protein engineering is a very useful tool for probing structure-function relationships in proteins. Specifically, site-directed mutagenized proteins can provide useful insights into structural, binding and catalytic mechanisms of a protein, particularly when coupled with crystallization. In this chapter, we describe two protocols for performing site-directed mutagenesis of any protein-coding sequence, namely, megaprimer PCR and overlapping extension PCR (OE-PCR). We use as an example how these two SDM methods enhanced the function of a cyclodextrin glucosyltransferase (CGTase) from Bacillus lehensis strain G1.
Studies related to the engineering of calcium binding sites of CGTase are limited. The calcium binding regions that are known for thermostability function were subjected to site-directed mutagenesis in this study. The starting gene-protein is a variant of CGTase Bacillus sp. G1, reported earlier and denoted as "parent CGTase" herein. Four CGTase variants (S182G, S182E, N132R and N28R) were constructed. The two variants with a mutation at residue 182, located adjacent to the Ca-I site and the active site cleft, possessed an enhanced thermostability characteristic. The activity half-life of variant S182G at 60 °C was increased to 94 min, while the parent CGTase was only 22 min. This improvement may be attributed to the formation of a shorter α-helix and the alleviation of unfavorable steric strains by glycine at the corresponding region. For the variant S182E, an extra ionic interaction at the A/B domain interface increased the half-life to 31 min, yet it reduced CGTase activity. The introduction of an ionic interaction at the Ca-I site via the mutation N132R disrupted CGTase catalytic activity. Conversely, the variant N28R, which has an additional ionic interaction at the Ca-II site, displayed increased cyclization activity. However, thermostability was not affected.
The xylanase gene from Bacillus pumilus PJ19 amplified by polymerase chain reaction (PCR) was cloned into pCRII vector and transformed into Escherichia coli strain INValphaF'. Starting from an ATG as an initiator codon, an open reading frame coding for 202 amino acids was obtained. The recombinant xylanase sequence showed a 96% homology with the xylanase sequence from B. pumilus IPO strain and had an estimated molecular weight of 22,474. Xylanase activity expressed by E. coli INValphaF' harboring the cloned gene was located primarily in the cytoplasmic fraction.
Environmental contamination by petroleum hydrocarbons, mainly crude oil waste from refineries, is becoming prevalent worldwide. This study investigates the bioremediation of water contaminated with crude oil waste. Bacillus salamalaya 139SI, a bacterium isolated from a private farm soil in the Kuala Selangor in Malaysia, was found to be a potential degrader of crude oil waste. When a microbial population of 108 CFU ml-1 was used, the 139SI strain degraded 79% and 88% of the total petroleum hydrocarbons after 42 days of incubation in mineral salt media containing 2% and 1% of crude oil waste, respectively, under optimum conditions. In the uninoculated medium containing 1% crude oil waste, 6% was degraded. Relative to the control, the degradation was significantly greater when a bacteria count of 99 × 108 CFU ml-1 was added to the treatments polluted with 1% oil. Thus, this isolated strain is useful for enhancing the biotreatment of oil in wastewater.
A heterologous signal peptide (SP) from Bacillus sp. G1 was optimized for secretion of recombinant cyclodextrin glucanotransferase (CGTase) to the periplasmic and, eventually, extracellular space of Escherichia coli. Eight mutant SPs were constructed using site-directed mutagenesis to improve the secretion of recombinant CGTase. M5 is a mutated SP in which replacement of an isoleucine residue in the h-region to glycine created a helix-breaking or G-turn motif with decreased hydrophobicity. The mutant SP resulted in 110 and 94% increases in periplasmic and extracellular recombinant CGTase, respectively, compared to the wild-type SP at a similar level of cell lysis. The formation of intracellular inclusion bodies was also reduced, as determined by sodium dodecyl sulfate-polyacrylamyde gel electrophoresis, when this mutated SP was used. The addition of as low as 0.08% glycine at the beginning of cell growth improved cell viability of the E. coli host. Secretory production of other proteins, such as mannosidase, also showed similar improvement, as demonstrated by CGTase production, suggesting that the combination of an optimized SP and a suitable chemical additive leads to significant improvements of extracellular recombinant protein production and cell viability. These findings will be valuable for the extracellular production of recombinant proteins in E. coli.
Ticks are vectors in the transmission of many important infectious diseases in human and animals. Ticks can be readily found in the semi-forested areas such as the settlements of the indigenous people in Malaysia, the Orang Asli. There is still minimal information available on the bacterial agents associated with ticks found in Malaysia. We performed a survey of the bacterial communities associated with ticks collected from domestic animals found in two Orang Asli villages in Malaysia. We collected 62 ticks, microscopically and molecularly identified as related to Haemaphysalis wellingtoni, Haemaphysalis hystricis and Haemaphysalis bispinosa. Bacterial 16s rRNA hypervariable region (V6) amplicon libraries prepared from the tick samples were sequenced on the Ion Torrent PGM platform. We detected a total of 392 possible bacterial genera after pooling and sequencing 20 samples, indicating a diverse bacterial community profile. Dominant taxa include the potential tick endosymbiont, Coxiella. Other dominant taxa include the tick-associated pathogen, Rickettsia, and environmental bacteria such as Bacillus, Mycobacterium, Sphingomonas and Pseudomonas. Other known tick-associated bacteria were also detected, including Anaplasma, Ehrlichia, Rickettsiella and Wolbachia, albeit at very low abundance. Specific PCR was performed on selected samples to identify Rickettsia and Coxiella. Sequence of Rickettsia felis, which causes spotted fever in human and cats, was identified in one sample. Coxiella endosymbionts were detected in three samples. This study provides the baseline knowledge of the microbiome of ticks in Malaysia, focusing on tick-associated bacteria affecting the Orang Asli communities. The role of the herein found Coxiella and Rickettsia in tick physiology or disease transmission merits further investigation.
A Na(+)/H(+) antiporter gene was isolated from alkaliphilic Bacillus sp. G1. The full-length sequence of the Na(+)/H(+) antiporter gene was obtained using a genome walking method, and designated as g1-nhaC. An ORF preceded by a promoter-like sequence and a Shine-Dalgarno sequence, and followed by a terminator-like sequence was identified. The deduced amino acid sequence consists of 535 amino acids, and a calculated molecular mass of 57 776 Da. g1-nhaC was subsequently cloned into pET22b(+) and expressed in Escherichia coli BL21 (DE3). Recombinant E. coli harboring the g1-nhaC gene was able to grow in modified L medium at various concentrations of NaCl (0.2-2.0 M) at different pH values. The recombinant bacteria grew well in the medium with concentrations of NaCl as high as 1.75 M at pH 8.0-9.0. Minimal growth was observed at 2.0 M NaCl, pH 8.0-9.0. At pH 10, the recombinant bacteria grew well in a medium with a low concentration of NaCl (0.2 M). These results suggested that the g1-NhaC antiporter from Bacillus sp. G1 plays a role in Na(+) extrusion at lower pH values and in pH homeostasis at pH 10 under Na(+)-limiting conditions.
Thermostable and organic solvent-tolerant enzymes have significant potential in a wide range of synthetic reactions in industry due to their inherent stability at high temperatures and their ability to endure harsh organic solvents. In this study, a novel gene encoding a true lipase was isolated by construction of a genomic DNA library of thermophilic Aneurinibacillus thermoaerophilus strain HZ into Escherichia coli plasmid vector. Sequence analysis revealed that HZ lipase had 62% identity to putative lipase from Bacillus pseudomycoides. The closely characterized lipases to the HZ lipase gene are from thermostable Bacillus and Geobacillus lipases belonging to the subfamily I.5 with ≤ 57% identity. The amino acid sequence analysis of HZ lipase determined a conserved pentapeptide containing the active serine, GHSMG and a Ca(2+)-binding motif, GCYGSD in the enzyme. Protein structure modeling showed that HZ lipase consisted of an α/β hydrolase fold and a lid domain. Protein sequence alignment, conserved regions analysis, clustal distance matrix and amino acid composition illustrated differences between HZ lipase and other thermostable lipases. Phylogenetic analysis revealed that this lipase represented a new subfamily of family I of bacterial true lipases, classified as family I.9. The HZ lipase was expressed under promoter Plac using IPTG and was characterized. The recombinant enzyme showed optimal activity at 65 °C and retained ≥ 97% activity after incubation at 50 °C for 1h. The HZ lipase was stable in various polar and non-polar organic solvents.
A total of 97 amino acids, considered as the signal peptide and transmembrane segments were removed from 205y lipase gene using polymerase chain reaction technique that abolished the low activity of this enzyme. The mature enzyme was expressed in Escherichia coli using pBAD expression vector, which gave up to a 13-fold increase in lipase activity. The mature 205y lipase (without signal peptide and transmembrane; -SP/TM) was purified to homogeneity using the isoelectric focusing technique with 53% recovery. Removing of the signal peptide and transmembrane segments had resulted in the shift of optimal pH, an increase in optimal temperature and tolerance towards more water-miscible organic solvents as compared to the characteristics of open reading frame (ORF) of 205y lipase. Also, in the presence of 1mM inhibitors, less decrease in the activity of mature 205y lipase was observed compared to the ORF of the enzyme. Protein structure modeling showed that 205y lipase consisted of an α/β hydrolase fold without lid domain. However, the transmembrane segment could effect on the enzyme activity by covering the active site or aggregation the protein.
Heavy metals from industrial effluents and sewage contribute to serious water pollution in most developing countries. The constant penetration and contamination of heavy metals into natural water sources may substantially raise the chances of human exposure to these metals through ingestion, inhalation, or skin contact, which could lead to liver damage, cancer, and other severe conditions in the long term. Biosurfactant as an efficient biological surface-active agent may provide an alternative solution for the removal of heavy metals from industrial wastes. Biosurfactants exhibit the properties of reducing surface and interfacial tension, stabilizing emulsions, promoting foaming, high selectivity, and specific activity at extreme temperatures, pH, and salinity, and the ability to be synthesized from renewable resources. This study aimed to produce biosurfactant from renewable feedstock, which is used cooking oil (UCO), by a local isolate, namely Bacillus sp. HIP3 for heavy metals removal. Bacillus sp. HIP3 is a Gram-positive isolate that gave the highest oil displacement area with the lowest surface tension, of 38 mN/m, after 7 days of culturing in mineral salt medium and 2% (v/v) UCO at a temperature of 30 °C and under agitation at 200 rpm. An extraction method, using chloroform:methanol (2:1) as the solvents, gave the highest biosurfactant yield, which was 9.5 g/L. High performance liquid chromatography (HPLC) analysis confirmed that the biosurfactant produced by Bacillus sp. HIP3 consists of a lipopeptide similar to standard surfactin. The biosurfactant was capable of removing 13.57%, 12.71%, 2.91%, 1.68%, and 0.7% of copper, lead, zinc, chromium, and cadmium, respectively, from artificially contaminated water, highlighting its potential for bioremediation.
Bacillus lehensis G1 is a Gram-positive, moderately alkalitolerant bacterium isolated from soil samples. B. lehensis produces cyclodextrin glucanotransferase (CGTase), an enzyme that has enabled the extensive use of cyclodextrin in foodstuffs, chemicals, and pharmaceuticals. The genome sequence of B. lehensis G1 consists of a single circular 3.99 Mb chromosome containing 4017 protein-coding sequences (CDSs), of which 2818 (70.15%) have assigned biological roles, 936 (23.30%) have conserved domains with unknown functions, and 263 (6.55%) have no match with any protein database. Bacillus clausii KSM-K16 was established as the closest relative to B. lehensis G1 based on gene content similarity and 16S rRNA phylogenetic analysis. A total of 2820 proteins from B. lehensis G1 were found to have orthologues in B. clausii, including sodium-proton antiporters, transport proteins, and proteins involved in ATP synthesis. A comparative analysis of these proteins and those in B. clausii and other alkaliphilic Bacillus species was carried out to investigate their contributions towards the alkalitolerance of the microorganism. The similarities and differences in alkalitolerance-related genes among alkalitolerant/alkaliphilic Bacillus species highlight the complex mechanism of pH homeostasis. The B. lehensis G1 genome was also mined for proteins and enzymes with potential viability for industrial and commercial purposes.
The cyclodextrin glucanotransferase (CGTase, EC 2.4.1.19) gene from Bacillus sp. G1 was successfully isolated and cloned into Escherichia coli. Analysis of the nucleotide sequence revealed the presence of an open reading frame of 2,109 bp and encoded a 674 amino acid protein. Purified CGTase exhibited a molecular weight of 75 kDa and had optimum activity at pH 6 and 60 degrees C. Heterologous recombinant protein expression in E. coli is commonly problematic causing intracellular localization and formation of inactive inclusion bodies. This paper shows that the majority of CGTase was secreted into the medium due to the signal peptide of Bacillus sp. G1 that also works well in E. coli, leading to easier purification steps. When reacted with starch, CGTase G1 produced 90% beta-cyclodextrin (CD) and 10% gamma-CD. This enzyme also preferred the economical tapioca starch as a substrate, based on kinetics studies. Therefore, CGTase G1 could potentially serve as an industrial enzyme for the production of beta-CD.
A Bacillus sphaericus strain (205y) that produces an organic solvent-tolerant lipase was isolated in Port Dickson, Malaysia. The gene for the lipase was recovered from a genomic library and sequenced. Phylogenetic analysis was performed based on an alignment of thirteen microbial lipase sequences obtained from the NCBI database. The analysis suggested that the B. sphaericus lipase gene is a novel gene, as it is distinct from other lipase genes in Families I.4 and I.5 reported so far. Expression in Escherichia coli under the control of the lacZ promoter resulted in an eight-fold increase in enzyme activity after a 3-h induction with 1 mM IPTG. The crude enzyme thus obtained showed a slight (10%) enhancement in activity after a 30-min incubation in 25% (v/v) n-hexane at 37 degrees C, and retained 90% of its activity after a similar period in 25% (v/v) p-xylene.
Five out of the nine benzene-toulene-ethylbenzene-xylene (BTEX) tolerant bacteria that demonstrated high protease activity on skim milk agar were isolated. Among them, isolate 115b identified as Bacillus pumilus exhibited the highest protease production. The protease produced was stable in 25% (v/v) benzene and toluene and it was activated 1.7 and 2.5- fold by n-dodecane and n-tetradecane, respectively. The gene encoding the organic solvent tolerant protease was cloned and its nucleotide sequence determined. Sequence analysis revealed an open reading frame (ORF) of 1,149 bp that encoded a polypeptide of 383 amino acid residues. The polypeptide composed of 29 residues of signal peptide, a propeptide of 79 residues and a mature protein of 275 amino acids with a calculated molecular mass of 27,846 Da. This is the only report available to date on organic solvent tolerant protease from B. pumilus.
Regulation of RNA transcription in controlling the expression of genes at promoter and terminator regions is crucial as the interaction of RNA polymerase occurred at both sites. Gene encoding cyclodextrin glycosyltransferase (CGTase) from Bacillus sp. NR5 UPM isolated in the previous study was used for further construction of pTZCGT-SS, pTZCGT-BS and pTZCGT-BT expression systems for enhancement of CGTase production. The putative promoter regions, -35 and -10 sequences were found in the upstream of the mature gene start codon. Whereas, long inverted repeats sequences which can form a stable stem and loop structure was found downstream of the open reading frame (ORF) of Bacillus sp. NR5 UPM CGTase. The construction of E. coli strain harbouring pTZCGT-BS showed increment of 3.2-fold in CGTase activity compared to the wild type producer. However, insertion of terminator downstream of CGTase gene in E. coli strain harbouring pTZCGT-BT only resulted in 4.42 % increment of CGTase production compared to E. coli strain containing pTZCGT-BS, perhaps due to low intrinsic termination efficiency. Thus, it is suggested that the insertion of the putative promoter regions upstream of the coding sequence for the construction of CGTase expression system will further enhance in the recombinant enzyme production.