Keratinase is an important enzyme that can degrade recalcitrant keratinous wastes to form beneficial recyclable keratin hydrolysates. Keratinase is not only important as an alternative to reduce environmental pollution caused by chemical treatments of keratinous wastes, but it also has industrial significance. Currently, the bioproduction of keratinase from native keratinolytic host is considered low, and this hampers large-scale usage of the enzyme. Straightforward approaches of cloning and expression of recombinant keratinases from native keratinolytic host are employed to elevate the amount of keratinase produced. However, this is still insufficient to compensate for the lack of its large-scale production to meet the industrial demands. Hence, this review aimed to highlight the various sources of keratinase and the strategies to increase its production in native keratinolytic hosts. Molecular strategies to increase the production of recombinant keratinase such as plasmid selection, promoter engineering, chromosomal integration, signal peptide and propeptide engineering, codon optimization, and glycoengineering were also described. These mentioned strategies have been utilized in heterologous expression hosts, namely, Escherichia coli, Bacillus sp., and Pichia pastoris, as they are most widely used for the heterologous propagations of keratinases to further intensify the production of recombinant keratinases adapted to better suit the large-scale demand for them. KEY POINTS: • Molecular strategies to enhance keratinase production in heterologous hosts. • Construction of a prominent keratinolytic host from a native strain. • Patent analysis of keratinase production shows rapid high interest in molecular field.
Bacillus velezensis FS26 is a bacterium from the genus Bacillus that has been proven as a potential probiotic in aquaculture with a good antagonistic effect on Aeromonas spp. and Vibrio spp. Whole-genome sequencing (WGS) allows a comprehensive and in-depth analysis at the molecular level, and it is becoming an increasingly significant technique in aquaculture research. Although numerous probiotic genomes have been sequenced and investigated recently, there are minimal data on in silico analysis of B. velezensis as a probiotic bacterium isolated from aquaculture sources. Thus, this study aims to analyse the general genome characteristics and probiotic markers from the B. velezensis FS26 genome with secondary metabolites predicted against aquaculture pathogens. The B. velezensis FS26 genome (GenBank Accession: JAOPEO000000000) assembly proved to be of high quality, with eight contigs containing 3,926,371 bp and an average G + C content of 46.5%. According to antiSMASH analysis, five clusters of secondary metabolites from the B. velezensis FS26 genome showed 100% similarity. These clusters include Cluster 2 (bacilysin), Cluster 6 (bacillibactin), Cluster 7 (fengycin), Cluster 8 (bacillaene), and Cluster 9 (macrolactin H), which signify promising antibacterial, antifungal, and anticyanobacterial agents against pathogens in aquaculture. The probiotic markers of B. velezensis FS26 genome for adhesion capability in the hosts' intestine, as well as the acid and bile salt-tolerant genes, were also detected through the Prokaryotic Genome Annotation System (Prokka) annotation pipeline. These results are in agreement with our previous in vitro data, suggesting that the in silico investigation facilitates establishing B. velezensis FS26 as a beneficial probiotic for use in aquaculture.
In nature, bacteria are ubiquitous and can be categorized as beneficial or harmless to humans, but most bacteria have one thing in common which is their ability to produce biofilm. Biofilm is encased within an extracellular polymeric substance (EPS) which provides resistance against antimicrobial agents. Protease enzymes have the potential to degrade or promote the growth of bacterial biofilms. In this study, the effects of a recombinant intracellular serine protease from Bacillus sp. (SPB) on biofilms from Staphylococcus aureus, Acinetobacter baumannii, and Pseudomonas aeruginosa were analyzed. SPB was purified using HisTrap HP column and concentrated using Amicon 30 ultra-centrifugal filter. SPB was added with varying enzyme activity and assay incubation period after biofilms were formed in 96-well plates. SPB was observed to have contrasting effects on different bacterial biofilms, where biofilm degradations were observed for both 7-day-old A. baumannii (37.26%) and S. aureus (71.51%) biofilms. Meanwhile, SPB promoted growth of P. aeruginosa biofilm up to 176.32%. Compatibility between protein components in S. aureus biofilm with SPB as well as a simpler membrane structure morphology led to higher biofilm degradation for S. aureus compared to A. baumannii. However, SPB promoted growth of P. aeruginosa biofilm due likely to its degrading protein factors that are responsible for biofilm detachment and dispersion, thus resulting in more multi-layered biofilm formation. Commercial protease Savinase which was used as a comparison showed degradation for all three bacterial biofilms. The results obtained are unique and will expand our understanding on the effects that bacterial proteases have toward biofilms.
Azoxystrobin (AZ) is a broad-spectrum synthetic fungicide widely used in agriculture globally. However, there are concerns about its fate and effects in the environment. It is reportedly transformed into azoxystrobin acid as a major metabolite by environmental microorganisms. Bacillus licheniformis strain TAB7 is used as a compost deodorant in commercial compost and has been found to degrade some phenolic and agrochemicals compounds. In this article, we report its ability to degrade azoxystrobin by novel degradation pathway. Biotransformation analysis followed by identification by electrospray ionization-mass spectrometry (MS), high-resolution MS, and nuclear magnetic resonance spectroscopy identified methyl (E)-3-amino-2-(2-((6-(2-cyanophenoxy)pyrimidin-4-yl)oxy)phenyl)acrylate, or (E)-azoxystrobin amine in short, and (Z) isomers of AZ and azoxystrobin amine as the metabolites of (E)-AZ by TAB7. Bioassay testing using Magnaporthe oryzae showed that although 40 μg/mL of (E)-AZ inhibited 59.5 ± 3.5% of the electron transfer activity between mitochondrial Complexes I and III in M. oryzae, the same concentration of (E)-azoxystrobin amine inhibited only 36.7 ± 15.1% of the activity, and a concentration of 80 μg/mL was needed for an inhibition rate of 56.8 ± 7.4%, suggesting that (E)-azoxystrobin amine is less toxic than the parent compound. To our knowledge, this is the first study identifying azoxystrobin amine as a less-toxic metabolite from bacterial AZ degradation and reporting on the enzymatic isomerization of (E)-AZ to (Z)-AZ, to some extent, by TAB7. Although the fate of AZ in the soil microcosm supplemented with TAB7 will be needed, our findings broaden our knowledge of possible AZ biotransformation products.
Quinoa (Chenopodium quinoa Willd.) is a highly nutritious food product with a comprehensive development prospect. Here, we discussed the effect of Bacillus amyloliquefaciens 11B91 on the growth, development and salt tolerance (salt concentrations: 0, 150, 300 mmol·L-1) of quinoa and highlighted a positive role for the application of plant growth-promoting rhizobacteria bacteria in quinoa. In this artical, the growth-promoting effect of Bacillus amyloliquefaciens 11B91 on quinoa (Longli No.1) and the changes in biomass, chlorophyll content, root activity and total phosphorus content under salt stress were measured. The results revealed that plants inoculated with 11B91 exhibited increased maximum shoot fresh weight (73.95%), root fresh weight (75.36%), root dry weight (136%), chlorophyll a (65.32%) contents and chlorophyll b (58.5%) contents, root activity (54.44%) and total phosphorus content (16.66%). Additionally, plants inoculated with 11B91 under salt stress plants showed significantly improved, fresh weight (107%), dry weight (133%), chlorophyll a (162%) contents and chlorophyll b (76.37%) contents, root activity (33.07%), and total phosphorus content (42.73%).
Bacterial biofilms are a preferred mode of growth for many types of microorganisms in their natural environments. The ability of pathogens to integrate within a biofilm is pivotal to their survival. The possibility of biofilm formation in Lactobacillus communities is also important in various industrial and medical settings. Lactobacilli can eliminate the colonization of different pathogenic microorganisms. Alternatively, new opportunities are now arising with the rapidly expanding potential of lactic acid bacteria biofilms as bio-control agents against food-borne pathogens.
This study aimed to isolate, identify, and evaluate the probiotic properties of Bacillus species from honey of the stingless bee Heterotrigona itama. Bacillus spp. were isolated from five different H. itama meliponicultures, and the isolates were characterized through Gram-staining and a catalase test. Tolerance to acidic conditions and bile salt (0.3%), hydrophobicity, and autoaggregation tests were performed to assess the probiotic properties of the selected isolates, B. amyloliquefaciens HTI-19 and B. subtilis HTI-23. Both Bacillus isolates exhibited excellent antimicrobial activity against both Gram-positive and Gram-negative bacteria and possessed significantly high survival rates in 0.3% bile solution for 3 h. Their survival rates in acidic conditions were also comparable to a commercial probiotic strain, Lactobacillus rhamnosus GG. Interestingly, the hydrophobicity and autoaggregation percentage showed no significant difference from L. rhamnosus GG, a commercial probiotic strain. The results from this study suggest that B. amyloliquefaciens HTI-19 and B. subtilis HTI-23 isolated from stingless bee honey have considerably good probiotic properties. Therefore, more studies should be done to investigate the effects of these bacteria cultures on gastrointestinal health.
There are increasing applications of diazotrophic rhizobacteria in the sustainable agriculture system. A field experiment on young immature oil palm was conducted to quantify the uptake of N derived from N₂ fixation by the diazotroph Bacillus sphaericus strain UPMB-10, using the ¹⁵N isotope dilution method. Eight months after ¹⁵N application, young immature oil palms that received 67% of standard N fertilizer application together with B. sphaericus inoculation had significantly lower ¹⁵N enrichment than uninoculated palms that received similar N fertilizers. The dilution of labeled N served as a marker for the occurrence of biological N₂ fixation. The proportion of N uptake that was derived from the atmosphere was estimated as 63% on the whole plant basis. The inoculation process increased the N and dry matter yields of the palm leaflets and rachis significantly. Field planting of young, immature oil palm in soil inoculated with B. sphaericus UPMB-10 might mitigate inorganic fertilizer-N application through supplementation by biological nitrogen fixation. This could be a new and important source of nitrogen biofertilizer in the early phase of oil palm cultivation in the field.
This study aimed to improve lipid and gamma-linolenic acid (GLA) production of an oleaginous fungus, Mucor plumbeus, through coculturing with Bacillus subtilis bacteria, optimising the environmental and nutritional culture conditions, and scaling them for batch fermentation. The maximum levels of biomass, lipid, fatty acid, and GLA in a 5 L bioreactor containing cellobiose and ammonium sulfate as the optimal carbon and nitrogen sources, respectively, achieved during the coculturing processes were 14.5 ± 0.4 g/L, 41.5 ± 1.3, 24 ± 0.8, and 20 ± 0.5%, respectively. This strategy uses cellobiose in place of glucose, decreasing production costs. The nutritional and abiotic factor results suggest that the highest production efficiency is achieved at 6.5 pH, 30 °C temperature, 10% (v/v) inoculum composition, 200 rpm agitation speed, and a 5-day incubation period. Interestingly, the GLA concentration of cocultures (20.0 ± 0.5%) was twofold higher than that of monocultures (8.27 ± 0.11%). More importantly, the GC chromatograms of cocultures indicated the presence of one additional peak corresponding to decanoic acid (5.32 ± 0.20%) that is absent in monocultures, indicating activation of silent gene clusters via cocultivation with bacteria. This study is the first to show that coculturing of Mucor plumbeus with Bacillus subtilis is a promising strategy with industrialisation potential for the production of GLA-rich microbial lipids and prospective biosynthesis of new products.
Thirteen strains among 3 species of entomopathogenic bacteria were tested against 3 medically important mosquito species in French Polynesia. Two strains of Bacillus thuringiensis were highly toxic to Aedes polynesiensis, Aedes aegypti, and Culex quinquefasciatus. Six of 7 strains of Bacillus sphaericus tested were highly toxic to Cx. quinquefasciatus but not to the Aedes spp. Clostridium bifermentans serovar. malaysia was more toxic to Ae. polynesiensis than to the other 2 species. Entomopathogenic bacteria merit field testing for larval mosquito control in French Polynesia.
Immobilisation of cyclodextrin glucanotransferase (CGTase) on nanofibres was demonstrated. CGTase solution (1% v/v) and PVA (8 wt%) solution were mixed followed by electrospinning (-9 kV, 3 h). CGTase/PVA nanofibres with an average diameter of 176 ± 46 nm were successfully produced. The nanofibres that consist of immobilised CGTase were crosslinked with glutaraldehyde vapour. A CGTase/PVA film made up from the same mixture and treated the same way was used as a control experiment. The immobilised CGTase on nanofibres showed superior performance with nearly a 2.5 fold higher enzyme loading and 31% higher enzyme activity in comparison with the film.
Penghasilan CGTase daripada Bacillus sp. G1 berjaya ditingkatkan dengan menggunakan sistem kultur selanjar mengatasi penghasilan daripada kultur kelompok. Aktiviti CGTase tertinggi yang didapati dalam kultur kelompok ialah 28.1 U/ml. Kajian kultur selanjar difokuskan kepada masa kemasukan medium segar yang berbeza (12, 24 dan 48 jam ), kadar pencairan ditetapkan pada 0.03 per jam. Hasil menunjukkan masa memulakan pam medium segar tidak memberi perubahan yang signifikan terhadap aktiviti CGTase (25.7, 26.3 dan 26.1 U/ml masing-masing) dan produktiviti CGTase (0.77, 0.79 dan 0.78 U/ml/j masing-masing) pada keadaan mantap tetapi produktiviti CGTase (0.77 U/ml/j) akan lebih tinggi berbanding produktiviti kultur kelompok apabila masa di antara larian kultur kelompok diambil kira. Malah peningkatan berpotensi ditingkatkan lagi dengan memulakan pam medium segar lebih awal daripada 12 jam dan juga dengan meningkatkan kadar pencairan.
An N-acylhomoserine lactone (AHL)-degrading bacterial strain, L62, was isolated from a sample of fermentation brine of Chinese soya sauce by using rich medium agar supplemented with soya sauce (10% v/v). L62, a rod-shaped Gram positive bacterium with amylolytic activity, was phylogentically related to Bacillus sonorensis by 16S ribosomal DNA and rpoB sequence analyses. B. sonorensis L62 efficiently degraded N-3-oxohexanoyl homoserine lactone and N-octanoylhomoserine lactone. However, the aiiA homologue, encoding an autoinducer inactivation enzyme catalyzing the degradation of AHLs, was not detected in L62, suggesting the presence of a different AHL-degrading gene in L62. To the best of our knowledge, this is the first report of AHL-degrading B. sonorensis from soya sauce liquid state fermentation.
Lead is among the most common toxic heavy metals and its contamination is of great public concern. Bacillus coagulans is the probiotic which can be considered as the lead absorption sorbent to apply in the lead contaminant water directly or indirectly. A better understanding of the lead resistance and tolerance mechanisms of B. coagulans would help further its development and utilization. Wild-type Bacillus coagulans strain R11 isolated from a lead mine, was acclimated to lead-containing culture media over 85 passages, producing two lead-adapted strains, and the two strains shown higher lead intracellular accumulation ability (38.56-fold and 19.36-fold) and reducing ability (6.94-fold and 7.44-fold) than that of wild type. Whole genome sequencing, genome resequencing, and comparative transcriptomics identified lead resistance and tolerance process significantly involved in these genes which regulated glutathione and sulfur metabolism, flagellar formation and metal ion transport pathways in the lead-adapted strains, elucidating the relationships among the mechanisms regulating lead deposition, deoxidation, and motility and the evolved tolerance to lead. In addition, the B. coagulans mutants tended to form flagellar and chemotaxis systems to avoid lead ions rather than export it, suggesting a new resistance strategy. Based on the present results, the optimum lead concentration in environment should be considered when employed B. coagulans as the lead sorbent, due to the bacteria growth ability decreased in high lead concentration and physiology morphology changed could reduce the lead removal effectiveness. The identified deoxidization and compound secretion genes and pathways in B. coagulans R11 also are potential genetic engineering candidates for synthesizing glutathione, cysteine, methionine, and selenocompounds.
The fermentation of Malaysian fish sauce (budu) varies from one to twelve months depending on the producer, resulting in inconsistent quality. The microbiota, their predicted metabolic pathways and volatile metabolites profiles were determined at different stages of budu fermentation. Budu fermented for 1 and 3 months were characterized by the presence of Gram negative Enterobacterales, Gammaproteobacteria, and Fusobacteriaceae, which continuously decrease in abundance over fermentation time. The metabolic pathways prediction grouped 1- and 3- month budu in a cluster enriched with degradation reactions. 6-month budu were dominated by Halanaerobium and Staphylococcus, while the 12-month were dominated by Lentibacillus, Bacilli, and Halomonas. Biosynthesis-type predicted pathways involving protein and lipid derivatives were enriched in 6- and 12-month fermented budu, accumulating 2,6-dimethylpyrazine, methyl 2-ethyldecanoate, 2-phenylacetaldehyde, 3-methylbutanal, and 3-methylbutanoic acid. These compounds may indicate budu maturity and quality. This result may assist as a reference for quality control and fermentation monitoring.
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.
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%.
Agricultural plantations in Indonesia and Malaysia yield substantial waste, necessitating proper disposal to address environmental concerns. Yet, these wastes, rich in starch and lignocellulosic content, offer an opportunity for value-added product development, particularly amino acid production. Traditional methods often rely on costly commercial enzymes to convert biomass into fermentable sugars for amino acid production. An alternative, consolidated bioprocessing, enables the direct conversion of agricultural biomass into amino acids using selected microorganisms. This review provides a comprehensive assessment of the potential of agricultural biomass in Indonesia and Malaysia for amino acid production through consolidated bioprocessing. It explores suitable microorganisms and presents a case study on using Bacillus subtilis ATCC 6051 to produce 9.56 mg/mL of amino acids directly from pineapple plant stems. These findings contribute to the advancement of sustainable amino acid production methods using agricultural biomass especially in Indonesia and Malaysia through consolidated bioprocessing, reducing waste and enhancing environmental sustainability.
In an effort to develop a more effective technique in dispersing a microbial control agent, Bacillus thuringiensis (Bt), a truck-mounted ultra low volume (ULV) generator (Scorpion) was used to disperse B. thuringiensis israelensis (Bti) and Bti with malathion. Complete larval and adult mortalities for all tested mosquito species within the first 70-80 feet from the ULV generator were achieved. Beyond that distance less than 50% mortality was achieved as insufficient sprayed particles reached the area. A minimum of 10(3) Bti colony forming units per ml is required to cause 100% larval mortality. The sprayed Bti larvicidal toxins were persistent in the test water 7 days post ULV. The effectiveness of B. thuringiensis jegathesan (Btj), a new mosquitocidal Bt serotype was also evaluated. Similar mortality results as Bti were achieved except that the Btj toxins underwent degradation in the test water, since less than 50% less in larval mortality was observed in 7 days post ULV samples. This ULV method has the potential to disperse Bt and malathion effectively for a simultaneous control of mosquito adults and larvae.
Microbial strain optimization focuses on improving technological properties of the strain of microorganisms. However, the complexities of the metabolic networks, which lead to data ambiguity, often cause genetic modification on the desirable phenotypes difficult to predict. Furthermore, vast number of reactions in cellular metabolism lead to the combinatorial problem in obtaining optimal gene deletion strategy. Consequently, the computation time increases exponentially with the increase in the size of the problem. Hence, we propose an extension of a hybrid of Bees Algorithm and Flux Balance Analysis (BAFBA) by integrating OptKnock into BAFBA to validate the result. This paper presents a number of computational experiments to test on the performance and capability of BAFBA. Escherichia coli, Bacillus subtilis and Clostridium thermocellum are the model organisms in this paper. Also included is the identification of potential reactions to improve the production of succinic acid, lactic acid and ethanol, plus the discussion on the changes in the flux distribution of the predicted mutants. BAFBA shows potential in suggesting the non-intuitive gene knockout strategies and a low variability among the several runs. The results show that BAFBA is suitable, reliable and applicable in predicting optimal gene knockout strategy.