Type I pullulanases are enzymes that specifically hydrolyse α-1,6 linkages in polysaccharides. This study reports the analyses of a novel type I pullulanase (PulASK) from Anoxybacillus sp. SK3-4. Purified PulASK (molecular mass of 80 kDa) was stable at pH 5.0-6.0 and was most active at pH 6.0. The optimum temperature for PulASK was 60 °C, and the enzyme was reasonably stable at this temperature. Pullulan was the preferred substrate for PulASK, with 89.90 % adsorbance efficiency (various other starches, 56.26-72.93 % efficiency). Similar to other type I pullulanases, maltotriose was formed on digestion of pullulan by PulASK. PulASK also reacted with β-limit dextrin, a sugar rich in short branches, and formed maltotriose, maltotetraose and maltopentaose. Nevertheless, PulASK was found to preferably debranch long branches at α-1,6 glycosidic bonds of starch, producing amylose, linear or branched oligosaccharides, but was nonreactive against short branches; thus, no reducing sugars were detected. This is surprising as all currently known type I pullulanases produce reducing sugars (predominantly maltotriose) on digesting starch. The closest homologue of PulASK (95 % identity) is a type I pullulanase from Anoxybacillus sp. LM14-2 (Pul-LM14-2), which is capable of forming reducing sugars from starch. With rational design, amino acids 362-370 of PulASK were replaced with the corresponding sequence of Pul-LM14-2. The mutant enzyme formed reducing sugars on digesting starch. Thus, we identified a novel motif involved in substrate specificity in type I pullulanases. Our characterization may pave the way for the industrial application of this unique enzyme.
Type I pullulanase from Anoxybacillus sp. SK3-4 (PulASK) is an unusual debranching enzyme that specifically hydrolyzes starch α-1,6 linkages at long branches producing oligosaccharides (≥G8), but is nonreactive against short branches; thus, incapable of producing reducing sugars (G1-G7). We report on the effects of both single and co-immobilization of PulASK on product specificity. PulASK was purified and immobilized through covalent attachment to three epoxides (ReliZyme EP403/M, Immobead IB-150P, and Immobead IB-150A) and an amino-epoxide (ReliZyme HFA403/M) activated supports. Following immobilization, all PulASK derivatives were active on both short and long branches in starch producing reducing sugars (predominantly maltotriose) and oligosaccharides (≥G8), respectively, a feature that is absent in the free enzyme. This study also demonstrated that co-immobilization of PulASK and α-amylase from Anoxybacillus sp. SK3-4 (TASKA) on ReliZyme HFA403/M significantly changed the product specificity compared to the free enzymes alone or individually immobilized enzymes. In conclusion, individual or co-immobilization caused changes in the product specificity, presumably due to changes in the enzyme binding pocket caused by the influence of carrier surface properties (hydrophobic or hydrophilic) and the lengths of the spacer arms.
α-Amylase from Anoxybacillus sp. SK3-4 (ASKA) is a thermostable enzyme that produces a high level of maltose from starches. A truncated ASKA (TASKA) variant with improved expression and purification efficiency was characterized in an earlier study. In this work, TASKA was purified and immobilized through covalent attachment on three epoxide (ReliZyme EP403/M, Immobead IB-150P, and Immobead IB-150A) and an amino-epoxide (ReliZyme HFA403/M) activated supports. Several parameters affecting immobilization were analyzed, including the pH, temperature, and quantity (mg) of enzyme added per gram of support. The influence of the carrier surface properties, pore sizes, and lengths of spacer arms (functional groups) on biocatalyst performances were studied. Free and immobilized TASKAs were stable at pH 6.0-9.0 and active at pH 8.0. The enzyme showed optimal activity and considerable stability at 60 °C. Immobilized TASKA retained 50% of its initial activity after 5-12 cycles of reuse. Upon degradation of starches and amylose, only immobilized TASKA on ReliZyme HFA403/M has comparable hydrolytic ability with the free enzyme. To the best of our knowledge, this is the first report of an immobilization study of an α-amylase from Anoxybacillus spp. and the first report of α-amylase immobilization using ReliZyme and Immobeads as supports.
With the prevalence of edible diacylglycerol (DAG) oil, which is beneficial to human, the generation of 3-monochloropropanediol esters (3-MCPDE) and glycidyl esters (GE) as well as the stability of physical properties during heat-induced processing still need to be explored. In this study, the experiment used olive-based edible oil with different contents of DAG (40, 60, and 80%) to make crackers and fry chicken. They were heated at 160 and 180 °C to determine the changes in 3-MCPDE and GE, the crackers’ hardness and gumminess, and the physical properties of the oil. During baking and frying, 3-MCPDE decreased, while the content of GE slightly increased with the prolonged heating duration. Finally, 3-MCPDE and GE were lower than 1.25 mg/kg and 1.00 mg/kg, respectively. The AV increased proportionally as duration increased and POV was below 0.30 g/100 g. In general, the changes in 3-MCPDE and GE were related to the heating temperature and duration, and not significantly (p > 0.05) related to the content of DAG.
Several recent studies have shown the presence of genes for the key enzyme associated with archaeal methane/alkane metabolism, methyl-coenzyme M reductase (Mcr), in metagenome-assembled genomes (MAGs) divergent to existing archaeal lineages. Here, we study the mcr-containing archaeal MAGs from several hot springs, which reveal further expansion in the diversity of archaeal organisms performing methane/alkane metabolism. Significantly, an MAG basal to organisms from the phylum Thaumarchaeota that contains mcr genes, but not those for ammonia oxidation or aerobic metabolism, is identified. Together, our phylogenetic analyses and ancestral state reconstructions suggest a mostly vertical evolution of mcrABG genes among methanogens and methanotrophs, along with frequent horizontal gene transfer of mcr genes between alkanotrophs. Analysis of all mcr-containing archaeal MAGs/genomes suggests a hydrothermal origin for these microorganisms based on optimal growth temperature predictions. These results also suggest methane/alkane oxidation or methanogenesis at high temperature likely existed in a common archaeal ancestor.
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.
Species of Anoxybacillus are widespread in geothermal springs, manure, and milk-processing plants. The genus is composed of 22 species and two subspecies, but the relationship between its lifestyle and genome is little understood. In this study, two high-quality draft genomes were generated from Anoxybacillus spp. SK3-4 and DT3-1, isolated from Malaysian hot springs. De novo assembly and annotation were performed, followed by comparative genome analysis with the complete genome of Anoxybacillus flavithermus WK1 and two additional draft genomes, of A. flavithermus TNO-09.006 and A. kamchatkensis G10. The genomes of Anoxybacillus spp. are among the smaller of the family Bacillaceae. Despite having smaller genomes, their essential genes related to lifestyle adaptations at elevated temperature, extreme pH, and protection against ultraviolet are complete. Due to the presence of various competence proteins, Anoxybacillus spp. SK3-4 and DT3-1 are able to take up foreign DNA fragments, and some of these transferred genes are important for the survival of the cells. The analysis of intact putative prophage genomes shows that they are highly diversified. Based on the genome analysis using SEED, many of the annotated sequences are involved in carbohydrate metabolism. The presence of glycosyl hydrolases among the Anoxybacillus spp. was compared, and the potential applications of these unexplored enzymes are suggested here. This is the first study that compares Anoxybacillus genomes from the aspect of lifestyle adaptations, the capacity for horizontal gene transfer, and carbohydrate metabolism.
Jeotgalibacillus soli, a bacterium capable of degrading N-acyl homoserine lactone, was isolated from a soil sample in Portugal. J. soli constitutes the only Jeotgalibacillus species isolated from a non-marine source. Here, the draft genome, several interesting glycosyl hydrolases, and its putative N-acyl homoserine lactonases are presented.
The Bacillaceae family members are a good source of bacteria for bioprocessing and biotransformation involving whole cells or enzymes. In contrast to Bacillus and Geobacillus, Anoxybacillus is a relatively new genus that was proposed in the year 2000. Because these bacteria are alkali-tolerant thermophiles, they are suitable for many industrial applications. More than a decade after the first report of Anoxybacillus, knowledge accumulated from fundamental and applied studies suggests that this genus can serve as a good alternative in many applications related to starch and lignocellulosic biomasses, environmental waste treatment, enzyme technology, and possibly bioenergy production. This current review provides the first summary of past and recent discoveries regarding the isolation of Anoxybacillus, its medium requirements, its proteins that have been characterized and cloned, bioremediation applications, metabolic studies, and genomic analysis. Comparisons to some other members of Bacillaceae and possible future applications of Anoxybacillus are also discussed.
Jeotgalibacillus spp. are halophilic bacteria within the family Planococcaceae. No genomes of Jeotgalibacillus spp. have been reported to date, and their metabolic pathways are unknown. How the bacteria survive in hypertonic conditions such as seawater is yet to be discovered. As only few studies have been conducted on Jeotgalibacillus spp., potential applications of these bacteria are unknown. Here, we present the complete genome of J. malaysiensis D5(T) (=DSM 28777(T) =KCTC 33350(T)), which is invaluable in identifying interesting applications for this genus.
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.
Soy sauce fermentation was simulated in a laboratory and subjected to 10min of sonication. A full factorial design, including different cycles, probe size, and amplitude was used. The composition of 17 free-amino acids (FAAs) was determined by the AccQ-Tag method with fluorescent detection. Main effect plots showed total FAAs extraction was favoured under continuous sonication at 100% amplitude using a 14mm diameter transducer probe, reaching 1214.2±64.3mg/100ml of total FAAs. Moreover, after 7days of fermentation, sonication treatment caused significantly higher levels (p<0.05) of glutamic acids (343.0±22.09mg/100g), total FAAs (1720.0±70.6mg/100g), and essential FAAs (776.3±7.0mg/100g) 3days sooner than the control. Meanwhile, enzymatic and microbial behaviours remained undisturbed. Collectively, the sonication to moromi resulted in maturation 57% faster than the untreated control.
The detection of 3- and 2-MCPD ester and glycidyl ester was transformed from selected ion monitoring (SIM) mode to multiple reaction monitoring (MRM) mode by gas chromatography triple quadrupole spectrometry. The derivatization process was adapted from AOCS method Cd 29a-13. The results showed that the coefficient of determination (R2) of all detected compounds obtained from both detection mode was comparable, which falls between 0.997 and 0.999. The limit of detection and quantification (LOD and LOQ) were improved in MRM mode as compared to SIM mode. In MRM mode, the LOD of 3- and 2-MCPD ester was achieved 0.01 mg/kg while the LOQ was 0.05 mg/kg. Besides, LOD and LOQ of glycidyl ester were 0.024 and 0.06 mg/kg respectively. A blank spiked with MCPD esters (0.03, 0.10 and 0.50 mg/kg) and GE (0.06, 0.24 and 1.20 mg/kg) were chosen for repeatability and recovery tests. MRM mode showed better repeatability in area ratio and recovery with relative standard deviation (RSD %)
The technique of Fourier transform infrared spectroscopy is widely used to generate spectral data for use in the detection of food contaminants. Monochloropropanediol (MCPD) is a refining process-induced contaminant that is found in palm-based fats and oils. In this study, a chemometric approach was used to evaluate the relationship between the FTIR spectra and the total MCPD content of a palm-based cooking oil. A total of 156 samples were used to develop partial least squares regression (PLSR), artificial neural network (nnet), average artificial neural network (avNNET), random forest (RF) and cubist models. In addition, a consensus approach was used to generate fusion result consisted from all the model mentioned above. All the models were evaluated based on validation performed using training and testing datasets. In addition, the box plot of coefficient of determination (R2), root mean square error (RMSE), slopes and intercepts by 100 times randomization was also compared. Evaluation of performance based on the testing R2 and RMSE suggested that the cubist model predicted total MCPD content with the highest accuracy, followed by the RF, avNNET, nnet and PLSR models. The overfitting tendency was assessed based on differences in R2 and RMSE in the training and testing calibrations. The observations showed that the cubist and avNNET models possessed a certain degree of overfitting. However, the accuracy of these models in predicting the total MCPD content was high. Results of the consensus model showed that it slightly improved the accuracy of prediction as well as significantly reduced its uncertainty. The important variables derived from the cubist and RF models suggested that the wavenumbers corresponding to the MCPDs originated from the -CH=CH2 or CH=CH (990-900 cm-1) and C-Cl stretch (800-700 cm-1) regions of the FTIR spectrum data. In short, chemometrics in combination with FTIR analysis especially for the consensus model represent a potential and flexible technique for estimating the total MCPD content of refined vegetable oils.
Thermovorax subterraneus 70BT is a thermophile found in a geothermically active underground mine. The strain 70BT belongs to the class of Clostridia, order of Thermosediminibacterales, and family of Thermosediminibacteraceae. Strain 70BT was the only type strain since the genus was discovered >10 years ago. Strain 70BT was compared to strains from other genera in terms of its phenotypics, chemotaxonomics, and phylogenetics (16S rRNA gene) in previous studies. However, the genome sequence of this strain has not been described. We herein described the genome sequence of strain 70BT. In total, the assembled genome of strain 70BT has a size of 2,451,552 bp, contributed by 44 contigs, with a coverage of 445X, a N50 of 86,294 bp, and a GC% of 43.8. A total of 2,540 genes were encoded in the genome, including 2,431 protein-coding sequences, 46 pseudogenes, and 63 RNA genes. Through the Cluster of Orthologous Groups (COGs) analysis, a total of 2,404 protein-coding genes were functionally assigned to COGs in the genome of strain 70BT. Among the members of Thermosediminibacteraceae family, strain 70BT has the closest relationship to Caldanaerovirga acetigignens JW/SA-NV4T based on the genome-to-genome comparison indexes (i.e., ANI, dDDH, AAI, and POCP). An earlier study reported that strain 70BT could produce hydrogen. We discovered genes encoding [FeFe] hydrogenase through gene mining analysis. For future research, this genome data will be used as a reference for all matters pertaining to the genus Thermovorax and family Thermosediminibacteraceae.
Lignification of the plant cell wall could serve as the first line of defense against pathogen attack, but the molecular mechanisms of virulence and disease between oil palm and Ganoderma boninense are poorly understood. This study presents the biochemical, histochemical, enzymology and gene expression evidences of enhanced lignin biosynthesis in young oil palm as a response to G. boninense (GBLS strain). Comparative studies with control (T1), wounded (T2) and infected (T3) oil palm plantlets showed significant accumulation of total lignin content and monolignol derivatives (syringaldehyde and vanillin). These derivatives were deposited on the epidermal cell wall of infected plants. Moreover, substantial differences were detected in the activities of enzyme and relative expressions of genes encoding phenylalanine ammonia lyase (EC 4.3.1.24), cinnamate 4-hydroxylase (EC 1.14.13.11), caffeic acid O-methyltransferase (EC 2.1.1.68) and cinnamyl alcohol dehydrogenase (CAD, EC 1.1.1.195). These enzymes are key intermediates dedicated to the biosynthesis of lignin monomers, the guaicyl (G), syringyl (S) and ρ-hydroxyphenyl (H) subunits. Results confirmed an early, biphasic and transient positive induction of all gene intermediates, except for CAD enzyme activities. These differences were visualized by anatomical and metabolic changes in the profile of lignin in the oil palm plantlets such as low G lignin, indicating a potential mechanism for enhanced susceptibility toward G. boninense infection.
Thousands of prokaryotic genera have been published, but methodological bias in the study of prokaryotes is noted. Prokaryotes that are relatively easy to isolate have been well-studied from multiple aspects. Massive quantities of experimental findings and knowledge generated from the well-known prokaryotic strains are inundating scientific publications. However, researchers may neglect or pay little attention to the uncommon prokaryotes and hard-to-cultivate microorganisms. In this review, we provide a systematic update on the discovery of underexplored culturable and unculturable prokaryotes and discuss the insights accumulated from various research efforts. Examining these neglected prokaryotes may elucidate their novelties and functions and pave the way for their industrial applications. In addition, we hope that this review will prompt the scientific community to reconsider these untapped pragmatic resources.
Life on Earth has displayed remarkable adaptability to the harshest environments, spanning polar regions, scorching deserts, abyssal oceans, lightless caves, noxious lakes, boiling hot springs, and nuclear waste sites. These resilient organisms, known as extremophiles or polyextremophiles, owe their survival due to their unique genetic adaptations. This collection, titled ‘Genomics of Extreme Environments’, comprises several articles published in the esteemed journal Scientific Reports. Each article within this collection investigated genetic signature and adaptation in different extreme environments, including the cold polar region, arid desert, oxygen-deprived Tibetan mountains and others. These studies provide invaluable understanding of how life thrives and evolves under extreme conditions, shedding light on genetic mechanisms and adaptation strategies.
Shortening derived from palm oil is widely used in baking applications. However, palm oil and the related products are reported to contain high levels of monochloropropandiol (MCPD) ester and glycidyl ester (GE). MCPD and glycidol are known as process contaminants, which are carcinogenic and genotoxic compounds, respectively. The objective was to evaluate the effects of antioxidant addition in palm olein and stearin to the content of MCPD esters and GE in baked cake. Butylated hydroxyanisole (BHA), rosemary extract and tocopherol were used to fortify the samples at 200 mg/kg and in combinations (400, 600 and 800 mg/kg rosemary or tocopherol combined with 200 mg/kg BHA). The MCPD esters and GE content, radical formation and the quality of the fats portion were analyzed. The results showed that palm olein fortified with rosemary extract yielded less 2-MCPD ester. The GE content was lower when soft stearin was fortified with rosemary. ESR spectrometry measurements showed that the antioxidants were effective to reduce radical formation. The synergistic effects of combining antioxidants controlled the contaminants formation. In conclusion, oxidation stability was comparable either in the single or combined antioxidants. Tocopherol in combination with BHA was more effective in controlling the MCPD esters and GE formation.