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 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.
Molybdenum is very toxic to agricultural animals. Mo-reducing bacterium can be used to immobilize soluble molybdenum to insoluble forms, reducing its toxicity in the process. In this work the isolation of a novel molybdate-reducing Gram positive bacterium tentatively identified as Bacillus sp. strain A.rzi from a metal-contaminated soil is reported. The cellular reduction of molybdate to molybdenum blue occurred optimally at 4 mM phosphate, using 1% (w/v) glucose, 50 mM molybdate, between 28 and 30 °C and at pH 7.3. The spectrum of the Mo-blue product showed a maximum peak at 865 nm and a shoulder at 700 nm. Inhibitors of bacterial electron transport system (ETS) such as rotenone, sodium azide, antimycin A, and potassium cyanide could not inhibit the molybdenum-reducing activity. At 0.1 mM, mercury, copper, cadmium, arsenic, lead, chromium, cobalt, and zinc showed strong inhibition on molybdate reduction by crude enzyme. The best model that fitted the experimental data well was Luong followed by Haldane and Monod. The calculated value for Luong's constants p max, K(s), S(m), and n was 5.88 μmole Mo-blue hr(-1), 70.36 mM, 108.22 mM, and 0.74, respectively. The characteristics of this bacterium make it an ideal tool for bioremediation of molybdenum pollution.
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.
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.
An organic solvent tolerant (OST) lipase gene from Bacillus sphaericus 205y was successfully expressed extracellularly. The expressed lipase was purified using two steps purification; ultrafiltration and hydrophobic interaction chromatography (HIC) to 8-fold purity and 32% recovery. The purified 205y lipase revealed homogeneity on denaturing gel electrophoresis and the molecular mass was at approximately 30 kDa. The optimum pH for the purified 205y lipase was 7.0-8.0 and its stability showed a broad range of pH value between pH 5.0 to 13.0 at 37 degrees C. The purified 205y lipase exhibited an optimum temperature of 55 degrees C. The activity of the purified lipase was stimulated in the presence of Ca2+ and Mg2+. Ethylenediaminetetraacetic acid (EDTA) has no effect on its activity; however inhibition was observed with phenylmethane sulfonoyl fluoride (PMSF) a serine hydrolase inhibitor. Organic solvents such as dimethylsulfoxide (DMSO), methanol, p-xylene and n-decane enhanced the activity. Studies on the effect of oil showed that the lipase was most active in the presence of tricaprin (C10). The lipase exhibited 1,3 positional specificity.
The α-amylases from Anoxybacillus species (ASKA and ADTA), Bacillus aquimaris (BaqA) and Geobacillus thermoleovorans (GTA, Pizzo and GtamyII) were proposed as a novel group of the α-amylase family GH13. An ASKA yielding a high percentage of maltose upon its reaction on starch was chosen as a model to study the residues responsible for the biochemical properties. Four residues from conserved sequence regions (CSRs) were thus selected, and the mutants F113V (CSR-I), Y187F and L189I (CSR-II) and A161D (CSR-V) were characterised. Few changes in the optimum reaction temperature and pH were observed for all mutants. Whereas the Y187F (t1/2 43 h) and L189I (t1/2 36 h) mutants had a lower thermostability at 65°C than the native ASKA (t1/2 48 h), the mutants F113V and A161D exhibited an improved t1/2 of 51 h and 53 h, respectively. Among the mutants, only the A161D had a specific activity, k(cat) and k(cat)/K(m) higher (1.23-, 1.17- and 2.88-times, respectively) than the values determined for the ASKA. The replacement of the Ala-161 in the CSR-V with an aspartic acid also caused a significant reduction in the ratio of maltose formed. This finding suggests the Ala-161 may contribute to the high maltose production of the ASKA.
A bioflocculant-producing bacterial strain with highly mucoid and ropy colony morphological characteristics identified as Bacillus spp. UPMB13 was found to be a potential bioflocculant-producing bacterium. The effect of cation dependency, pH tolerance and dosage requirement on flocculating ability of the strain was determined by flocculation assay with kaolin as the suspended particle. The flocculating activity was measured as optical density and by flocs formation. A synergistic effect was observed with the addition of monovalent and divalent cations, namely, Na⁺, Ca²⁺, and Mg²⁺, while Fe²⁺ and Al³⁺ produced inhibiting effects on flocculating activity. Divalent cations were conclusively demonstrated as the best cation source to enhance flocculation. The bioflocculant works in a wide pH range, from 4.0 to 8.0 with significantly different performances (P < 0.05), respectively. It best performs at pH 5.0 and pH 6.0 with flocculating performance of above 90%. A much lower or higher pH would inhibit flocculation. Low dosage requirements were needed for both the cation and bioflocculant, with only an input of 50 mL/L for 0.1% (w/v) CaCl₂ and 5 mL/L for culture broth, respectively. These results are comparable to other bioflocculants produced by various microorganisms with higher dosage requirements.