A screening program searching for indigenous microbial control agents of mosquitos in Malaysia is initiated since 1987 and to date at least 20 isolates of mosquitocidal Bacillus thuringiensis serotypes have been obtained. Preliminary field evaluation of several isolates indicated that they are highly effective in the control of medically important mosquito species. For operational purposes, there is an urgent need to produce this agent utilizing cheap and locally available wastes through fermentation biotechnology. Fermentation studies in shake-flasks containing standard nutrient broth and soya bean waste, respectively, indicate that it takes about 37 hours for a Malaysian isolate of B. thuringiensis serotype H-14 to mature. In the grated coconut waste, fishmeal and rice bran, the bacteria took 28 hours, 26 hours and 126 hours respectively to mature. The endotoxin was harvested from the standard nutrient broth at 55 hours and at 50 hours from soybean, grated coconut waste and fishmeal. The endotoxin could only be harvested 150 hours after inoculation from rice bran medium. However, no bacterial growth was detected in palm oil effluent. In terms of endotoxin and biomass production, fishmeal appears to be a suitable medium. Variations in the pH of the fermenting media were also noted.
In this study, we characterized, identified, and determined the effect of salt-tolerant PGPR isolated from coastal saline areas on rice growth and yield. A total of 44 bacterial strains were isolated, and 5 were found to be tolerant at high salt concentration. These isolates were further characterized for salinity tolerance and beneficial traits through a series of quantitative tests. Biochemical characterization showed that bacterial survivability decreases gradually with the increase of salt concentration. One of the strains, UPMRB9, produced the highest amount of exopolysaccharides when exposed to 1.5M of NaCl. Moreover, UPMRB9 absorbed the highest amount of sodium from the 1.5M of NaCl-amended media. The highest floc yield and biofilm were produced by UPMRE6 and UPMRB9 respectively, at 1M of NaCl concentration. The SEM observation confirmed the EPS production of UPMRB9 and UPMRE6 at 1.5M of NaCl concentration. These two isolates were identified as Bacillus tequilensis and Bacillus aryabhattai based on the 16S rRNA gene sequence. The functional group characterization of EPS showed the presence of hydroxyl, carboxyl, and amino groups. This corresponded to the presence of carbohydrates and proteins in the EPS and glucose was identified as the major type of carbohydrate. The functional groups of EPS can help to bind and chelate Na+ in the soil and thereby reduces the plant's exposure to the ion under saline conditions. The plant inoculation study revealed significant beneficial effects of bacterial inoculation on photosynthesis, transpiration, and stomatal conductance of the plant which leads to a higher yield. The Bacillus tequilensis and Bacillus aryabhattai strains showed good potential as PGPR for salinity mitigation practice for coastal rice cultivation.
Palm kernel cake (PKC) has been largely produced in Malaysia as one of the cheap and abundant agro-waste by-products from the palm oil industry and it contains high fiber (mannan) content. The present study aimed to produce β-mannanase by Bacillus subtilis ATCC11774 via optimization of the medium composition using palm kernel cake as substrate in semi-solid fermentation. The fermentation nutrients such as PKC, peptone, yeast extract, sodium chloride, magnesium sulphate (MgSO2), initial culture pH and temperature were screened using a Plackett-Burman design. The three most significant factors identified, PKC, peptone and NaCl, were further optimized using central composite design (CCD), a response surface methodology (RSM) approach, where yeast extract and MgSO2 were fixed as a constant factor. The maximum β-mannanase activity predicted by CCD under the optimum medium composition of 16.50 g/L PKC, 19.59 g/L peptone, 3.00 g/L yeast extract, 2.72 g/L NaCl and 0.2 g/L MgSO2 was 799 U/mL. The validated β-mannanase activity was 805.12 U/mL, which was close to the predicted β-mannanas activity. As a comparison, commercial media such as nutrient broth, M9 and Luria bertani were used for the production of β-mannanase with activities achieved at 204.16 ± 9.21 U/mL, 50.32 U/mL and 88.90 U/mL, respectively. The optimized PKC fermentation medium was four times higher than nutrient broth. Hence, it could be a potential fermentation substrate for the production of β-mannanase activity by Bacillus subtilis ATCC11774.
This paper describes a new bacterial white spot syndrome (BWSS) in cultured tiger shrimp Penaeus monodon. The affected shrimp showed white spots similar to those caused by white spot syndrome virus (WSSV), but the shrimp remained active and grew normally without significant mortalities. The study revealed no evidence of WSSV infection using electron microscopy, histopathology and nested polymerase chain reaction. Electron microscopy indicated bacteria associated with white spot formation, and with degeneration and discoloration of the cuticle as a result of erosion of the epicuticle and underlying cuticular layers. Grossly the white spots in BWSS and WSS look similar but showed different profiles under wet mount microscopy. The bacterial white spots were lichen-like, having perforated centers unlike the melanized dots in WSSV-induced white spots. Bacteriological examination showed that the dominant isolate in the lesions was Bacillus subtilis. The occurrence of BWSS may be associated with the regular use of probiotics containing B. subtilis in shrimp ponds. The externally induced white spot lesions were localized at the integumental tissues, i.e., cuticle and epidermis, and connective tissues. Damage to the deeper tissues was limited. The BWS lesions are non-fatal in the absence of other complications and are usually shed through molting.
The production, optimization, and characterization of the bioflocculant QZ-7 synthesized by a novel Bacillus salmalaya strain 139SI isolated from a private farm soil in Selangor, Malaysia, are reported. The flocculating activity of bioflocculant QZ-7 present in the selected strain was found to be 83.3%. The optimal culture for flocculant production was achieved after cultivation at 35.5 °C for 72 h at pH 7 ± 0.2, with an inoculum size of 5% (v/v) and sucrose and yeast extract as carbon and nitrogen sources. The maximum flocculating activity was found to be 92.6%. Chemical analysis revealed that the pure bioflocculant consisted of 79.08% carbohydrates and 15.4% proteins. The average molecular weight of the bioflocculant was calculated to be 5.13 × 10⁵ Da. Infrared spectrometric analysis showed the presence of carboxyl (COO-), hydroxyl (-OH), and amino (-NH₂) groups, polysaccharides and proteins. The bioflocculant QZ-7 exhibited a wide pH stability range from 4 to 7, with a flocculation activity of 85% at pH 7 ± 0.2. In addition, QZ-7 was thermally stable and retained more than 80% of its flocculating activity after being heated at 80 °C for 30 min. SEM analysis revealed that QZ-7 exhibited a clear crystalline brick-shaped structure. After treating wastewater, the bioflocculant QZ-7 showed significant flocculation performance with a COD removal efficiency of 93%, whereas a BOD removal efficiency of 92.4% was observed in the B. salmalaya strain 139SI. These values indicate the promising applications of the bioflocculant QZ-7 in wastewater treatment.
Maltooligosaccharides (MOSs) are emerging oligosaccharides in food-based applications and can be synthesized through the enzymatic synthesis of maltogenic amylase from Bacillus lehensis G1 (Mag1). However, the lack of enzyme stability makes this approach unrealistic for industrial applications. The formation of cross-linked enzyme aggregates (CLEAs) is a promising tool for improving enzyme stability, and the substrate accessibility problem of CLEA formation was overcome by the addition of porous agents to generate porous CLEAs (p-CLEAs). However, p-CLEAs exhibited high enzyme leaching and low solvent tolerance. To address these problems, p-CLEAs of Mag1 (Mag1-p-CLEAs) were entrapped in calcium alginate beads (CA). Mag1-p-CLEAs-CA prepared with 2.5% (w/v) sodium alginate and 0.6% (w/v) calcium chloride yielded 53.16% (17.0 U/mg) activity and showed a lower deactivation rate and longer half-life than those of entrapped free Mag1 (Mag1-CA) and entrapped non-porous Mag1-CLEAs (Mag1-CLEAs-CA). Moreover, Mag1-p-CLEAs-CA exhibited low enzyme leaching and high tolerance in various solvents compared to Mag1-p-CLEAs. A kinetic study revealed that Mag1-p-CLEAs-CA exhibited relatively high affinity towards beta-cyclodextrin (β-CD) (Km = 0.62 mM). MOSs (300 mg/g) were synthesized by Mag1-p-CLEAs-CA at 50 °C. Finally, the reusability of Mag1-p-CLEAs-CA makes them as a potential biocatalyst for the continuous synthesis of MOSs.
The partitioning of β-mannanase derived from Bacillus subtilis ATCC 11774 in aqueous two-phase system (ATPS) was studied. The ATPS containing different molecular weight of polyethylene glycol (PEG) and types of salt were employed in this study. The PEG/salt composition for the partitioning of β-mannanase was optimized using response surface methodology. The study demonstrated that ATPS consists of 25% (w/w) of PEG 6000 and 12.52% (w/w) of potassium citrate is the optimum composition for the purification of β-mannanase with a purification fold (PF) of 2.28 and partition coefficient (K) of 1.14. The study on influences of pH and crude loading showed that ATPS with pH 8.0 and 1.5% (w/w) of crude loading gave highest PF of 3.1. To enhance the partitioning of β-mannanase, four ionic liquids namely 1-butyl-3-methylimidazolium tetrafluoroborate ([Bmim]BF4), 1-ethyl-3-methylimidazolium tetrafluoroborate ([Emim]BF4), 1-butyl-3-methylimidazolium bromide ([Bmim]Br), 1-ethyl-3-methylimidazolium bromide ([Emim]Br) was added into the system as an adjuvant. The highest recovery yield (89.65%) was obtained with addition of 3% (w/w) of [Bmim]BF4. The SDS-PAGE analysis revealed that the β-mannanase was successfully recovered in the top phase of ATPS with the molecular size of 36.7kDa. Therefore, ATPS demonstrated a simple and efficient approach for recovery and purification of β-mannanase from fermentation broth in one single-step strategy.
A new mosquitocidal Bacillus thuringiensis subsp., jegathesan, has recently been isolated from Malaysia. Parasporal crystal inclusions were purified from this strain and bioassayed against fourth-instar larvae of Culex quinquefasciatus, Aedes aegypti, Aedes togoi, Aedes albopictus, Anopheles maculatus, and Mansonia uniformis. The 50% lethal concentration of crystal inclusions for each species was 0.34, 8.08, 0.34, 17.59, 3.91, and 120 ng/ml, respectively. These values show that parasporal inclusions from this new subspecies have mosquitocidal toxicity comparable to that of inclusions isolated from B. thuringiensis subsp. israelensis. Solubilized and chymotrypsin-activated parasporal inclusions possessed low-level hemolytic activity. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that the crystals were composed of polypeptides of 77, 74, 72, 68, 55, 38, 35, 27, and 23 kDa. Analysis by Western blotting (immunoblotting) with polyclonal antisera raised against toxins purified from B. thuringiensis subsp. israelensis reveals that proteins in parasporal inclusions of subsp. jegathesan are distinct, because little cross-reactivity was shown. Analysis of the plasmid content of B. thuringiensis subsp. jegathesan indicates that the genes for toxin production may be located on 105- to 120-kb plasmids. Cry- clones that have been cured of these plasmids are nontoxic. Southern blot analysis of plasmid and chromosomal DNA from subsp. jegathesan showed little or low homology to the genes coding for CryIVA, CryIVB, and CryIVD from B. thuringiensis subsp. israelensis.
Bacillus strain NS 8, a lipase-producing bacterium isolated from a Malaysian hot spring, is able to tolerate a broad range of temperature and pH, which makes it beneficial for this study. It generated PCR products with molecular weight of 1,532 bp, and the 16S rRNA sequence analysis identified it as Bacillus subtilis with accession number AB110598. It showed a 71% similarity index with B. subtilis using Biolog Microstation System. Its lipase production was optimized using a shake flask system by changing the physical (agitation speed, pH and temperature) and nutritional (nitrogen, carbon and minerals) factors. The most suitable combination of the basal medium for lipase production was 2.5% olive oil (carbon), 1.5% peptone (nitrogen), 0.1% MgSO(4) (mineral) at an optimum temperature of 50°C, pH 7.5 and 150 rpm agitation, giving an enzyme yield of 4.23 U/ml. Statistical optimization using response surface methodology was carried out. An optimum lipase production of 5.67 U/ml was achieved when olive oil concentration of 3%, peptone 2%, MgSO(4)·7H(2)O 0.2% and an agitation rate of 200 rpm were combined. Lipase production was further carried out inside a 2-liter bioreactor, which yielded an enzyme activity of 14.5 U/ml after 15 h of incubation.
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.
Cytochrome P450s are a superfamily of heme monooxygenases which catalyze a wide range of biochemical reactions. The reactions involve the introduction of an oxygen atom into an inactivated carbon of a compound which is essential to produce an intermediate of a hydroxylated product. The diversity of chemical reactions catalyzed by cytochrome P450s has led to their increased demand in numerous industrial and biotechnology applications. A recent study showed that a gene sequence encoding a CYP was found in the genome of Bacillus lehensis G1, and this gene shared structural similarity with the bacterial vitamin D hydroxylase (Vdh) from Pseudonocardia autotrophica. The objectives of present study was to mine, for a novel CYP from a new isolate B. lehensis G1 alkaliphile and determine the biological properties and functionalities of CYP in this bacterium. Our study employed the usage of computational methods to search for the novel CYP from CYP structural databases to identify the conserved pattern, functional domain and sequence properties of the uncharacterized CYP from B. lehensis G1. A computational homology model of the protein's structure was generated and a docking analysis was performed to provide useful structural knowledge on the enzyme's possible substrate and their interaction. Sequence analysis indicated that the newly identified CYP, termed CYP107CB2, contained the fingerprint heme binding sequence motif FxxGxxxCxG at position 336-345 as well as other highly conserved motifs characteristic of cytochrome P450 proteins. Using docking studies, we identified Ser-79, Leu-81, Val-231, Val-279, Val-383, Ala-232, Thr-236 and Thr-283 as important active site residues capable of stabilizing interactions with several potential substrates, including vitamin D3, 25-hydroxyvitamin D3 and 1α-hydroxyvitamin D3, in which all substrates docked proximally to the enzyme's heme center. Biochemical analysis indicated that CYP107CB2 is a biologically active protein to produce 1α,25-dihydroxyvitamin D3 from 1α-hydroxyvitamin D3. Based on these results, we conclude that the novel CYP107CB2 identified from B. lehensis G1 is a putative vitamin D hydroxylase which is possibly capable of catalyzing the bioconversion of parental vitamin D3 to calcitriol, or related metabolic products.
Antibacterial activity of honey is mainly dependent on a combination of its peroxide activity and non-peroxide components. This study aims to investigate antibacterial activity of five varieties of Malaysian honey (three monofloral; acacia, gelam and pineapple, and two polyfloral; kelulut and tualang) against Staphylococcus aureus, Bacillus cereus, Escherichia coli, and Pseudomonas aeruginosa.
Matched MeSH terms: Bacillus cereus/drug effects; Bacillus cereus/growth & development
Recombinant protein fused to an N-terminal signal peptide can be translocated to the periplasm and, eventually, to the extracellular medium of Escherichia coli under specific conditions. In this communication, we described the use and optimization of a heterologous signal peptide (G1 signal peptide) from a Bacillus sp for improved recombinant protein secretion and cell viability in E. coli. Significant advantages in maintaining high cell viability and high specificity of target protein secretion were achieved by using G1 signal peptide compared to the well-known PelB signal peptide. Signal peptide sequence analysis and site-directed mutagenesis of G1 signal peptide demonstrated that an 'MKK' sequence in n-region and the presence of a helix-breaking residue at the centre of h-region are important elements for the design of an optimal signal peptide.
Bacillus thuringiensis (Bt), an ubiquitous gram-positive spore-forming bacterium forms parasporal proteins during the stationary phase of its growth. Recent findings of selective human cancer cell-killing activity in non-insecticidal Bt isolates resulted in a new category of Bt parasporal protein called parasporin. However, little is known about the receptor molecules that bind parasporins and the mechanism of anti-cancer activity. A Malaysian Bt isolate, designated Bt18 produces parasporal protein that exhibit preferential cytotoxic activity for human leukaemic T cells (CEM-SS) but is non-cytotoxic to normal T cells or other cancer cell lines such as human cervical cancer (HeLa), human breast cancer (MCF-7) and colon cancer (HT-29) suggesting properties similar to parasporin. In this study we aim to identify the binding protein for Bt18 in human leukaemic T cells.
Bacteria with amine oxidase activity have become a particular interest to reduce biogenic amines concentration in food products such as meat and fish sausages. However, little information is available regarding the application of these bacteria in fish sauce. Hence, our study was aimed to investigate the effect of such starter cultures in reducing biogenic amines accumulation during fish sauce fermentation. Staphylococcus carnosus FS19 and Bacillus amyloliquefaciens FS05 isolated from fish sauce which possess amine oxidase activity were used as starter cultures in this study. Fermentation was held for 120 days at 35 °C. The pH value increased in all samples, while salt concentration remained constant throughout fermentation. Aerobic bacteria count was significantly lower (p < 0.05) in the control than in inoculated samples as a result of starter cultures addition. However, it decreased during fermentation due to the growth inhibition by high salt concentration. Proteolytic bacterial count decreased during fermentation with no significant difference (p > 0.05) among samples. These bacteria hydrolyzed protein in anchovy to produce free amino acid precursors for amines formation by decarboxylase bacteria. The presence of biogenic amines producing bacteria in this study was considered to be indigenous from raw material or contamination during fermentation, since our cultures were negative histamine producers. Amino acid histidine, arginine, lysine and tyrosine concentration decreased at different rates during fermentation as they were converted into their respective amines. In general, biogenic amines concentration namely histamine, putrescine, cadaverine and tyramine increased throughout fermentation. However, their concentrations were markedly higher (p < 0.05) in the control (without starter cultures) as compared to the samples treated with starter cultures. Histamine concentration was reduced by 27.7% and 15.4% by Staphylococcus carnosus FS19 and Bacillus amyloliquefaciens FS05, respectively. Both cultures could also reduce other amines during fermentation. After 120 days of fermentation, the overall biogenic amines concentration was 15.9% and 12.5% less in samples inoculated with Staphylococcus carnosus FS19 and Bacillus amyloliquefaciens FS05, respectively, as compared to control samples. These findings emphasized that application of starter cultures with amines oxidase activity in fish sauce fermentation was found to be effective in reducing biogenic amines accumulation.
Thermostable lipase produced by a genotypically identified extremophilic Bacillus subtilis NS 8 was purified 500-fold to homogeneity with a recovery of 16% by ultrafiltration, DEAE-Toyopearl 650M and Sephadex G-75 column. The purified enzyme showed a prominent single band with a molecular weight of 45 kDa. The optimum pH and temperature for activity of lipase were 7.0 and 60°C, respectively. The enzyme was stable in the pH range between 7.0 and 9.0 and temperature range between 40 and 70°C. It showed high stability with half-lives of 273.38 min at 60°C, 51.04 min at 70°C and 41.58 min at 80°C. The D-values at 60, 70 and 80°C were 788.70, 169.59 and 138.15 min, respectively. The enzyme's enthalpy, entropy and Gibb's free energy were in the range of 70.07-70.40 kJ mol(-1), -83.58 to -77.32 kJ mol(-1)K(-1) and 95.60-98.96 kJ mol(-1), respectively. Lipase activity was slightly enhanced when treated with Mg(2+) but there was no significant enhancement or inhibition of the activity with Ca(2+). However, other metal ions markedly inhibited its activity. Of all the natural vegetable oils tested, it had slightly higher hydrolytic activity on soybean oil compared to other oils. On TLC plate, the enzyme showed non-regioselective activity for triolein hydrolysis.
Matched MeSH terms: Bacillus subtilis/enzymology*; Bacillus subtilis/growth & development
Cyclodextrin glucanotransferase (CGTase) is an extracellular enzyme which catalyzes the formation of cyclodextrin from starch. The production of CGTase using lactic acid bacterium is an attractive alternative and safer strategy to produce CGTase. In this study, we report the construction of genetically modified Lactococcus lactis strains harboring plasmids that secrete the Bacillus sp. G1 β-CGTase, with the aid of the signal peptides (SPs) SPK1, USP45 and native SP (NSP). Three constructed vectors, pNZ:NSP:CGT, pNZ:USP:CGT and pNZ:SPK1:CGT, were developed in this study. Each vector harbored a different SP fused to the CGTase. The formation of halo zones on starch plates indicated the production and secretion of β-CGTase by the recombinants. The expression of this enzyme is shown by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and zymogram analysis. A band size of ∼75 kDa corresponding to β-CGTase is identified in the intracellular and the extracellular environments of the host after medium modification. The replacement of glucose by starch in the medium was shown to induce β-CGTase production in L. lactis. Although β-CGTase production is comparatively low in NZ:SPK1:CGT, the SP SPK1 was shown to have higher secretion efficiency compared to the other SPs used in this study.
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.
Treatment of Bacillus licheniformis α-amylase (BLA) with guanidine hydrochloride (GdnHCl) produced both denatured and aggregated forms of the enzyme as studied by circular dichroism, fluorescence, UV difference spectroscopy, size exclusion chromatography (SEC), and enzymatic activity. The presence of CaCl(2) in the incubation mixture produced significant recovery in spectral signals, being complete in presence of 10 mM CaCl(2), as well as in enzymatic activity, which is indicative of protein stabilization. However, the SEC results obtained with GdnHCl-denatured BLA both in the absence and the presence of 10 mM CaCl(2) suggested significant aggregation of the protein in the absence of CaCl(2) and disaggregation in its presence. Although partial structural stabilization with significant retention of enzymatic activity was observed in the presence of calcium, it was far from the native state, as reflected by spectral probes. Hence, spectral results as to BLA stabilization should be treated with caution in the presence of aggregation.