We report on the cloning of the lipase gene from Bacillus licheniformis IBRLCHS2
and the expression of the recombinant lipase. DNA sequencing analysis of the
cloned lipase gene showed that it shares 99% identity with the lipase gene from
B. licheniformis ATCC 14580 and belongs to subfamily 1.4 of true lipases based on amino
acid sequence alignment of various Bacillus lipases. The 612 bp lipase gene was then
cloned into the pET-15b(+) expression vector and the construct was transformed into
E. coli BL21 (DE3) for bulk expression of the lipase. Expression was analysed by SDSPAGE
where the lipase was found to have a molecular weight of about 23 kDa.
Three new open reading frames were found downstream from cbm71, a toxin gene from Clostridium bifermentans malaysia (Cbm) strain CH18. The first one (91bp downstream) called cbm72, is 1857bp long and encodes a 71727-Da protein (Cbm72) with a sequence similar to that of Bacillus thuringiensis delta-endotoxins. This protein shows no significant toxicity to mosquito larvae. The two others, cbm17.1 (462bp) and cbm17.2 (459bp), are copies of the same gene encoding Cbm P18 and P16 polypeptides and located 426bp and 1022bp downstream from cbm72, respectively. They encode 17189-Da and 17451-Da proteins with sequences 44.6% similar to that of Aspergillus fumigatus hemolysin; however, they were not hemolytic in the conditions tested.
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.
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.
Two optimization strategies, codon usage modification and glycine supplementation, were adopted to improve the extracellular production of Bacillus sp. NR5 UPM β-cyclodextrin glycosyltransferase (CGT-BS) in recombinant Escherichia coli. Several rare codons were eliminated and replaced with the ones favored by E. coli cells, resulting in an increased codon adaptation index (CAI) from 0.67 to 0.78. The cultivation of the codon modified recombinant E. coli following optimization of glycine supplementation enhanced the secretion of β-CGTase activity up to 2.2-fold at 12 h of cultivation as compared to the control. β-CGTase secreted into the culture medium by the transformant reached 65.524 U/mL at post-induction temperature of 37 °C with addition of 1.2 mM glycine and induced at 2 h of cultivation. A 20.1-fold purity of the recombinant β-CGTase was obtained when purified through a combination of diafiltration and nickel-nitrilotriacetic acid (Ni-NTA) affinity chromatography. This combined strategy doubled the extracellular β-CGTase production when compared to the single approach, hence offering the potential of enhancing the expression of extracellular enzymes, particularly β-CGTase by the recombinant E. coli.
Protease is an enzyme that catalysed the hydrolysis of protein into peptide. Application of protease in industry has been linked with cost effective substrates and complex of enzyme-substrate stability. Molecular docking approach has identified casein as a preference substrates. However, lack of data on casein mode of binding to protease and enzyme stability represents a limitation for its production and structural optimization. In this study, we have used a molecular dynamic (MD) to examine the stability of complex enzyme-substrate of protease from Bacillus lehensis G1. The 3D structure of protease (BleG1_1979) was docked with substrate casein using AutoDock Vina. Structural analysis of the substrate-binding cleft revealed a binding site of casein was predominantly at the hydrophobic region of BleG1_1979. The MD of complex BleG1_1979-casein was tested with two temperatures; 298 K and 310 K using GROMACS v5.1.4. MD simulation showed a stable behaviour of BleG1_1979 over the 20 ns simulation period. The molecular docking and MD simulation suggested that the production of protease from B. lehensis G1 by utilization of casein and the stability of complex protease-casein could be a potential application to generate a cost effective enzyme to be develop for industrial use.
Comparative laboratory bioassays of three formulations of Bacillus thuringiensis H-14 (IPS-78, San 402-I and Bactimos) were conducted against late 3rd/early 4th instar larvae of four species of mosquito, viz., Aedes aegypti, Culex quinquefasciatus, Anopheles balabacensis and Mansonia (Mansonioides) indiana, in Malaysia. From the average response of the mosquito larvae to the three formulations of B. thuringiensis H-14, Ae. aegypti was found to be most susceptible, followed by Cx. quinquefasciatus, An. balabacensis and M. (M.) indiana in decreasing order. The LC50 values for Ae. aegypti, Cx. quinquefasciatus, An. balabacensis and M. (M.) indiana after a 48-hour exposure to IPS-78 formulation were 50.9, 129.3, 117.8 and 169.6 International Toxic Unit (ITU) Ae. ae./l; to San 402-I formulation were 54.6, 223.1, 405.1 and 177.6 ITU Ae. ae/l and to Bactimos formulation were 57.2, 175.7, 35.6 and 514.5 ITU Ae. ae./l respectively. The efficacy of the bacterial product was also found to be determined by its formulation in relation to the feeding and resting habits of the mosquito larvae. No delayed pupation or emergence was observed on the larvae exposed to B. thuringiensis H-14 at sub-lethal concentrations.
The current experiment was conducted to evaluate and compare the efficacy of two different probiotics Bacillus subtilis WB60 and Lactobacillus plantarum KCTC3928 in diet of Japanese eel, Anguilla japonica. Seven experimental diets were formulated to contain no probiotics (CON), three graded levels of B. subtilis at 106 (BS1), 107 (BS2), 108 (BS3) and L. plantarum at 106 (LP1), 107 (LP2), 108 (LP3) CFU/g diet. Twenty fish averaging 8.29 ± 0.06 g were distributed in to 21 aquaria and were randomly assigned to one of the experimental diets in triplicate groups. Average weight gain (WG), feed efficiency (FE), and protein efficiency ratio (PER) of fish fed B. subtilis at 107 (BS2) and 108 (BS3) CFU/g diet were significantly higher than those of fish fed other experimental diets (P
This study presents the initial structural model of L-haloacid dehalogenase (DehLBHS1) from Bacillus megaterium BHS1, an alkalotolerant bacterium known for its ability to degrade halogenated environmental pollutants. The model provides insights into the structural features of DehLBHS1 and expands our understanding of the enzymatic mechanisms involved in the degradation of these hazardous pollutants. Key amino acid residues (Arg40, Phe59, Asn118, Asn176, and Trp178) in DehLBHS1 were identified to play critical roles in catalysis and molecular recognition of haloalkanoic acid, essential for efficient binding and transformation of haloalkanoic acid molecules. DehLBHS1 was modeled using I-TASSER, yielding a best TM-score of 0.986 and an RMSD of 0.53 Å. Validation of the model using PROCHECK revealed that 89.2% of the residues were located in the most favored region, providing confidence in its structural accuracy. Molecular docking simulations showed that the non-simulated DehLBHS1 preferred 2,2DCP over other substrates, forming one hydrogen bond with Arg40 and exhibiting a minimum energy of -2.5 kJ/mol. The simulated DehLBHS1 exhibited a minimum energy of -4.3 kJ/mol and formed four hydrogen bonds with Arg40, Asn176, Asp9, and Tyr11, further confirming the preference for 2,2DCP. Molecular dynamics simulations supported this preference, based on various metrics, including RMSD, RMSF, gyration, hydrogen bonding, and molecular distance. MM-PBSA calculations showed that the DehLBHS1-2,2-DCP complex had a markedly lower binding energy (-21.363 ± 1.26 kcal/mol) than the DehLBHS1-3CP complex (-14.327 ± 1.738 kcal/mol). This finding has important implications for the substrate specificity and catalytic function of DehLBHS1, particularly in the bioremediation of 2,2-DCP in contaminated alkaline environments. These results provide a detailed view of the molecular interactions between the enzyme and its substrate and may aid in the development of more efficient biocatalytic strategies for the degradation of halogenated compounds.Communicated by Ramaswamy H. Sarma.
The protozoan parasites such as Cryptosporidiumparvum and Giardialamblia have been recognized as a frequent cause of recent waterborne disease outbreaks because of their strong resistance against chlorine disinfection. In this study, ozone and Fe(VI) (i.e., FeO(4)(2-)) were compared in terms of inactivation efficiency for Bacillus subtilis spores which are commonly utilized as an indicator of protozoan pathogens. Both oxidants highly depended on water pH and temperature in the spore inactivation. Since redox potential of Fe(VI) is almost the same as that of ozone, spore inactivation efficiency of Fe(VI) was expected to be similar with that of ozone. However, it was found that ozone was definitely superior over Fe(VI): at pH 7 and 20°C, ozone with the product of concentration×contact time (C¯T) of 10mgL(-1)min inactivate the spores more than 99.9% within 10min, while Fe(VI) with C¯T of 30mgL(-1) min could inactivate 90% spores. The large difference between ozone and Fe(VI) in spore inactivation was attributed mainly to Fe(III) produced from Fe(VI) decomposition at the spore coat layer which might coagulate spores and make it difficult for free Fe(VI) to attack live spores.
The compatibility of the commercial aqueous Bacillus thuringiensis serovar israelensis (B.t.i.) formulation, Vectobac 12AS, with the chemical insecticides Actellic 50EC, Aqua Resigen, Resigen, and Fendona SC, for the simultaneous control of Aedes larvae and adults was studied by dispersing nine different formulations using a portable mist blower, in single story half-brick houses. The effectiveness of the treatment was evaluated by measuring the larval mortality, adult mortality, and droplet analysis at varying distances from the sprayer. Persistence of the larvicidal activity of the chemical insecticides and B.t.i was also determined by measuring the larval mortality in the test samples 7 days posttreatment. The sprayed particles in all the trials were 50-60 microns in size, indicating that the particles were those of mist spray. Test samples placed within 3 m from the sprayer gave the maximum larval and adult mortality. Chemical insecticides exhibited maximum larval mortality in the 1 h posttreatment test samples and it was comparable to the larvicidal activity of B.t.i. The larvicidal toxins of B.t.i were more stable and were able to affect sufficient larval mortality for 7 days posttreatment. The larvicidal activity of the mixtures, i.e., chemical insecticides with B.t.i, in the 1 h posttreatment test samples was not significantly different from the larvicidal activity of the chemical insecticides and it was comparable to the larvicidal activity of B.t.i alone. However, the larvicidal activity of the mixtures was significantly more than the chemical insecticides alone in the 7 days posttreatment test samples except for the Actellic 50EC and Vectobac 12AS mixture. In all the trials, with or without B.t.i, there was no significant difference in adult mortality, indicating that this B.t.i formulation, Vectobac 12AS, was not antagonistic to the adulticidal activity of the chemical insecticides. From this study, it can be concluded that chemical insecticides can be used effectively for both adult and larval control, but the chemical insecticides do not exhibit residual larvicidal activity. Hence, for an effective control of both Aedes larvae and adults, it is advisable to add B.t.i. to the chemical insecticides, as B.t.i is specifically larvicidal and is also able to effect extended residual larvicidal activity.
Marilyn Charlene Montini Maluda, Michelle May D. Goroh, Tan, Eric Chee How, Syed Sharizman Syed Abdul Rahim, Richard Avoi, Mohammad Saffree Jeffree, et al.
Introduction: Melioidosis, also known as Whitmore disease, is caused by the gram-negative bacillus, Burkholderia pseudomallei and remains a public health concern in Southeast Asia and northern parts of Australia. This study attempts to identify all possible complications of melioidosis and its outcomes.
Methods: Literature search was conducted from databases such as PubMed, Science Direct and Scopus from 1st January 2000 to 31st August 2019. Medical Subject Headings (MeSH) search strategy was used with the terms ‘Melioidosis’ or ‘Burkholderia pseudomallei’ and ‘Complications’.
Results: A total of 162 titles were identified and 22 articles were included in the review. Findings showed that among the 22 articles, the ratio of male to female melioidosis incidence was 2.3 to 1, with most cases (86.4%) aged older than 14 years old and showed a mean age of 46 years old. A third (7/22) of the papers reported the involvement of the nervous system as a complication of melioidosis followed by cardiovascular complications. Among the 23 cases reported, 13 had underlying medical conditions with most of them (84.6%) having diabetes mellitus or newly diagnosed with diabetes mellitus. Overall, only one case (4.3%) had resulted in mortality, while 17.4% developed complications and 78.3% managed a full recovery after undergoing treatment for melioidosis.
Conclusion: The most commonly found complication of melioidosis involved the nervous system but patient outcomes were favourable. Rare complications included mycotic aneurysm that can be fatal. Melioidosis can affect almost any organ leading to various complications.
Maltogenic amylase (MAG1) from Bacillus lehensis G1 displayed the highest hydrolysis activity on β-cyclodextrin (β-CD) to produce maltose as a main product and exhibited high transglycosylation activity on malto-oligosaccharides with polymerization degree of three and above. These substrate and product specificities of MAG1 were elucidated from structural point of view in this study. A three-dimensional structure of MAG1 was constructed using homology modeling. Docking of β-CD and malto-oligosaccharides was then performed in the MAG1 active site. An aromatic platform in the active site was identified which is responsible in substrate recognition especially in determining the enzyme's preference toward β-CD. Molecular dynamics (MD) simulation showed MAG1 structure is most stable when docked with β-CD and least stable when docked with maltose. The docking analysis and MD simulation showed that the main subsites for substrate stabilization in the active site are -2, -1, +1 and +2. A bulky residue, Trp359 at the +2 subsite was identified to cause steric interference to the bound linear malto-oligosaccharides thus prevented it to occupy subsite +3, which can only be reached by a highly bent glucose molecule such as β-CD. The resulted modes of binding from docking simulation show a good correlation with the experimentally determined hydrolysis pattern. The subsite structure generated from this study led to a possible mode of action that revealed how maltose was mainly produced during hydrolysis. Furthermore, maltose only occupies subsite +1 and +2, therefore could not be hydrolyzed or transglycosylated by the enzyme. This important knowledge has paved the way for a novel structure-based molecular design for modulation of its catalytic activities.
Nowadays consumer is more demand on natural foodstuff instead of synthetic product due to their concern on health. The objective of this study is to investigate the effect of C. caudatus extract on the number of microflora in oyster mushroom at different concentration of C. caudatus extract and exposure time using dilution method. The results showed that the number of microorganisms (Log10 CFU/g) in oyster mushroom in term of Total Plate Count (TPC), Bacillus cereus, Escherichia coli and Staphylococcus aureus were 6.13 ± 0.04, 6.15 ± 0.09, 5.97 ± 0.04, and 6.46 ± 0.00, respectively. The effect of C. caudatus extract on microflora in oyster mushroom at concentrations of 0.00%, 0.05%, 0.5%, and 5.0% with exposure time of 0, 5, 10, and 15 min demonstrated that the reduction number of microflora in oyster mushroom was dependent on the concentration of C. caudatus extract and exposure times. The number of TPC (Log10 CFU/g) in oyster mushroom was significantly reduced after treated with C. caudatus extract at concentration of 0.05% for 15 min; 6.13 ± 0.04 reduced to 2.62 ± 0.07. Moreover, B. cereus (Log10 CFU/g) in oyster mushroom was significantly reduced by treatment of C. caudatus extract at concentration of 0.05% for 5 min; 6.15 ± 0.09 reduced to 3.77 ± 0.15. Meanwhile, the number of E. coli (Log10 CFU/g) in oyster mushroom was significantly reduced at concentration of 0.05% for 10 min; 5.97 ± 0.04 reduced to 3.21 ± 0.13. Lastly, the survival number of S. aureus in oyster mushroom was significantly reduced after treated with C. caudatus extract at concentration of 0.05% for 15 min; 6.46 ± 0.00 reduced to 4.83 ± 0.07. In conclusion, C. caudatus extract has potentiality to be developed as natural sanitizer for rinsing raw food materials such as oyster mushroom.
A field population of Plutella xylostella from Malaysia (SERD4) was divided into five sub-populations and four were selected (G2-G5) with the Bacillus thuringiensis insecticidal crystal (Cry) toxins Cry1Ac, Cry1Ab, Cry1Ca and Cry1Da. Bioassay at G6 gave resistance ratios of 88, 5, 2 and 3 for Cry1Ac, Cry1Ab, Cry1Ca and Cry1Da respectively compared with the unselected sub-population (UNSEL-SERD4). The Cry1Ac-selected population showed little cross-resistance to Cry1Ab, Cry1Ca and Cry1Da, (3-, 2- and 3-fold compared with UNSEL-SERD4), whereas the Cry1Ab-SEL sub-population showed marked cross-resistance to Cry1Ac (40-fold), much greater than Cry1Ab itself. In contrast, the Cry1Ca- and Cry1Da-SEL sub-population showed little if any cross-resistance to Cry1Ac and Cry1Ab. The mode of inheritance of resistance to Cry1Ac was examined in Cry1Ac-selected SERD4 by standard reciprocal crosses and back-crosses using a laboratory insecticide-susceptible population (ROTH). Logit regression analysis of F1 reciprocal crosses indicated that resistance to Cry1Ac was inherited as an incompletely dominant trait. At the highest dose of Cry1Ac tested, resistance was recessive, while at the lowest dose it was almost completely dominant. The F2 progeny from a back-cross of F1 progeny with ROTH were tested with a concentration of Cry1Ac that would kill 100% of ROTH. The mortality ranged between 50 and 95% in seven families of back-cross progeny, which indicated that more than one allele on separate loci were responsible for resistance to Cry1Ac.
Bacillus thuringiensis Berliner (Bt) crystal (Cry) toxins are expressed in various transgenic crops and are also used as sprays in integrated pest management and organic agricultural systems. The diamondback moth (Plutella xylostella L.) is a major worldwide pest of crucifer crops and one that has readily acquired field resistance to a broad range of insecticides.
An organic solvent-tolerant lipase from Bacillus sp. strain 42 was crystallized using the capillary-tube method. The purpose of studying this enzyme was in order to better understand its folding and to characterize its properties in organic solvents. By initially solving its structure in the native state, further studies on protein-solvent interactions could be performed. X-ray data were collected at 2.0 Å resolution using an in-house diffractometer. The estimated crystal dimensions were 0.09×0.19×0.08 mm. The crystal belonged to the monoclinic space group C2, with unit-cell parameters a=117.41, b=80.85, c=99.44 Å, β=96.40°.
Purified thermostable recombinant L2 lipase from Bacillus sp. L2 was crystallized by the counter-diffusion method using 20% PEG 6000, 50 mM MES pH 6.5 and 50 mM NaCl as precipitant. X-ray diffraction data were collected to 2.7 A resolution using an in-house Bruker X8 PROTEUM single-crystal diffractometer system. The crystal belonged to the primitive orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 87.44, b = 94.90, c = 126.46 A. The asymmetric unit contained one single molecule of protein, with a Matthews coefficient (V(M)) of 2.85 A(3) Da(-1) and a solvent content of 57%.
Ticks are vectors of bacteria, protozoa and viruses capable of causing serious and life threatening diseases in humans and animals. Disease transmission occurs through the transfer of pathogen from tick bites to susceptible humans or animals. Most commonly known tick-borne pathogens are obligate intracellular microorganisms but little is known on the prevalence of culturable pathogenic bacteria from ticks capable of growth on artificial nutrient media. One hundred and forty seven ticks originating from dairy cattle, goats and rodents were collected from nine selected sites in Peninsular Malaysia. The culture of surfacesterilized tick homogenates revealed the isolation of various pathogenic bacteria including, Staphylococcus sp., Corynebacterium sp., Rothia sp., Enterococcus faecalis, Klebsiella pneumoniae, Escherichia coli and Bacillus sp. and its derived genera. These pathogens are among those that affect humans and animals. Findings from this study suggest that in addition to the regular intracellular pathogens, ticks could also harbor extracellular pathogenic bacteria. Further studies, hence, would be needed to determine if these extracellular pathogens could contribute to human or animal infection.
Teratomas of anterior mediastinum are rare. They are often slow growing, asymptomatic, and detected incidentally on chest imaging. Mycobacterium abscessus (M. abscessus) is an acid-fast bacillus that is classified as a pathogenic "rapid growing" non-tuberculous mycobacteria. It is an uncommon cause of human pathology, which may cause skin and soft tissue infection after skin injury following inoculation, minor trauma, and surgery. Here, we present an unusual case of benign cystic teratoma mimicking recurrent pleural effusion, which was subsequently complicated by M. abscessus infection following thoracotomy. Cystic teratoma is rare, but it needs to be considered whenever clinical and investigative work-up fails to provide a convincing diagnosis. A combined clinical, radiological, surgical, and histopathological assessment is important to arrive at the correct diagnosis. Rapidly growing mycobacteria needs to be included in the differential diagnosis of patients with non-resolving infected post-thoracotomy wound and who do not respond to broad-spectrum antibiotics.