Heterologous expression of nattokinase from B. subtilis natto using Pichia pastoris GS115 and assessment of its thrombolytic activity.

BACKGROUND: Nattokinase is a fibrinolytic enzyme that has huge market value as a nutritional supplement for health promotion. In order to increase nattokinase yields, fermentation conditions, strains, cultivation media, and feeding strategies have been optimized. Nattokinase has been expressed using several heterologous expression systems. Pichia pastoris heterologous expression system was the alternative. RESULTS: This report aimed to express high levels of nattokinase from B. subtilis natto (NK-Bs) using a Pichia pastoris heterologous expression system and assess its fibrinolytic activity in vivo. Multicopy expression strains bearing 1-7 copies of the aprN gene were constructed. The expression level of the target protein reached a maximum at five copies of the target gene. However, multicopy expression strains were not stable in shake-flask or high-density fermentation, causing significant differences in the yield of the target protein among batches. Therefore, P. pastoris bearing a single copy of aprN was used in shake-flask and high-density fermentation. Target protein yield was 320 mg/L in shake-flask fermentation and approximately 9.5 g/L in high-density fermentation. The recombinant nattokinase showed high thermo- and pH-stability. The present study also demonstrated that recombinant NK-Bs had obvious thrombolytic activity. CONCLUSIONS: This study suggests that the P. pastoris expression system is an ideal platform for the large-scale, low-cost preparation of nattokinase.

QSSR Modeling of Bacillus Subtilis Lipase A Peptide Collision Cross-Sections in Ion Mobility Spectrometry: Local Descriptor Versus Global Descriptor.

To investigate the structure-dependent peptide mobility behavior in ion mobility spectrometry (IMS), quantitative structure-spectrum relationship (QSSR) is systematically modeled and predicted for the collision cross section Ω values of totally 162 single-protonated tripeptide fragments extracted from the Bacillus subtilis lipase A. Two different types of structure characterization methods, namely, local and global descriptor as well as three machine learning methods, namely, partial least squares (PLS), support vector machine (SVM) and Gaussian process (GP), are employed to parameterize and correlate the structures and Ω values of these peptide samples. In this procedure, the local descriptor is derived from the principal component analysis (PCA) of 516 physicochemical properties for 20 standard amino acids, which can be used to sequentially characterize the three amino acid residues composing a tripeptide. The global descriptor is calculated using CODESSA method, which can generate > 200 statistically significant variables to characterize the whole molecular structure of a tripeptide. The obtained QSSR models are evaluated rigorously via tenfold cross-validation and Monte Carlo cross-validation (MCCV). A comprehensive comparison is performed on the resulting statistics arising from the systematic combination of different descriptor types and machine learning methods. It is revealed that the local descriptor-based QSSR models have a better fitting ability and predictive power, but worse interpretability, than those based on the global descriptor. In addition, since the QSSR modeling using local descriptor does not consider the three-dimensional conformation of tripeptide samples, the method would be largely efficient as compared to the global descriptor.

Hierarchical natural move Monte Carlo refines flexible RNA structures into cryo-EM densities.

Ribonucleic acids (RNAs) play essential roles in living cells. Many of them fold into defined three-dimensional (3D) structures to perform functions. Recent advances in single-particle cryo-electron microscopy (cryo-EM) have enabled structure determinations of RNA to atomic resolutions. However, most RNA molecules are structurally flexible, limiting the resolution of their structures solved by cryo-EM. In modeling these molecules, several computational methods are limited by the requirement of massive computational resources and/or the low efficiency in exploring large-scale structural variations. Here we use hierarchical natural move Monte Carlo (HNMMC), which takes advantage of collective motions for groups of nucleic acid residues, to refine RNA structures into their cryo-EM maps, preserving atomic details in the models. After validating the method on a simulated density map of tRNA, we applied it to objectively obtain the model of the folding intermediate for the specificity domain of ribonuclease P from Bacillus subtilis and refine a flexible ribosomal RNA (rRNA) expansion segment from the Mycobacterium tuberculosis (Mtb) ribosome in different conformational states. Finally, we used HNMMC to model atomic details and flexibility for two distinct conformations of the complete genomic RNA (gRNA) inside MS2, a single-stranded RNA virus, revealing multiple pathways for its capsid assembly.

Economical production of isomaltulose from agricultural residues in a system with sucrose isomerase displayed on Bacillus subtilis spores.

A safe, efficient, environmentally friendly process for producing isomaltulose is needed. Here, the biocatalyst, sucrose isomerase (SIase) from Erwinia rhapontici NX-5, displayed on the surface of Bacillus subtilis 168 spores (food-grade strain) was applied for isomaltulose production. The anchored SIase showed relatively high bioactivity, suggesting that the surface display system using CotX as the anchoring protein was successful. The stability of the anchored SIase was also significantly better. Thermal stability analysis showed that 80% of relative activity was retained after incubation at 40 °C and 45 °C for 60 min. To develop an economical industrial fermentation medium, untreated beet molasses (30 g/L) and cold-pressed soybean powder (50 g/L) were utilised as the main broth components for SIase pilot-scale production. Under the optimal conditions, the productive spores converted 92% of sucrose after 6 h and the conversion rate was 45% after six cycles. Isomaltulose production with this system using the agricultural residues, untreated beet molasses and soybean powder, as substrates is cost-effective and environmentally friendly and can help to overcome issues due to the genetic background.

Cost-effective fibrinolytic enzyme production by Bacillus subtilis WR350 using medium supplemented with corn steep powder and sucrose.

The goal of this study was to develop a cheap and simple medium and to optimize fermentation parameters for fibrinolytic enzyme production by Bacillus subtilis WR350. A low-cost medium containing 35 g/L sucrose, 20 g/L corn steep powder and 2 g/L MgSO4·7H2O was developed via single-factor and orthogonal experiments. A cheap nitrogen source, corn steep powder, was used to replace the soy peptone present in the initial medium. The highest fibrinolytic activity of 5865 U/mL was achieved using the optimized medium in a 100-L fermenter with an aeration rate of 1.0 vvm and an agitation speed of 200 rpm. The resulting enzyme yield was among the highest described in the literature with respect to fibrinolytic activity, as determined by the fibrin plate method. Techno-economic evaluation indicated that the cost of the optimized medium was only 8.5% of the cost of the initial medium, and the total fermentation cost of fibrinolytic enzyme production using the optimized medium was 23.35% of the cost of using the initial medium.

Biosynthesis and production of quercitols and their application in the production of pharmaceuticals: current status and prospects.

(-)-vibo-Quercitol is a deoxyinositol (1L-1,2,4/3,5-cyclohexanepentol) that occurs naturally in low concentrations in oak species, honeydew honey, and Gymnema sylvestre. The author's research group recently reported that (-)-vibo-quercitol and scyllo-quercitol (2-deoxy-myo-inositol, 1,3,5/2,4-cyclohexanepentol), a stereoisomer of (-)-vibo-quercitol, are stereoselectively synthesized from 2-deoxy-scyllo-inosose by the reductive reaction of a novel (-)-vibo-quercitol 1-dehydrogenase in Burkholderia terrae and of a known scyllo-inositol dehydrogenase in Bacillus subtilis, respectively. The author's research group therefore identified two enzymes capable of producing both stereoisomers of deoxyinositols, which are rare in nature. (-)-vibo-Quercitol and scyllo-quercitol are potential intermediates for pharmaceuticals. In this review, the author describes the biosynthesis and enzymatic production of quercitols and myo-inositol stereoisomers and their application in the production of potential pharmaceuticals.

Engineering NOG-pathway in Escherichia coli for poly-(3-hydroxybutyrate) production from low cost carbon sources.

Poly-(3-hydroxybutyrate) (P3HB) is a polyester with biodegradable and biocompatible characteristics suitable for bio-plastics and bio-medical use. In order to reduce the raw material cost, cheaper carbon sources such as xylose and glycerol were evaluated for P3HB production. We first conducted genome-scale metabolic network analysis to find the optimal pathways for P3HB production using xylose or glycerol respectively as the sole carbon sources. The results indicated that the non-oxidative glycolysis (NOG) pathway is important to improve the product yields. We then engineered this pathway into E. coli by introducing foreign phophoketolase enzymes. The results showed that the carbon yield improved from 0.19 to 0.24 for xylose and from 0.30 to 0.43 for glycerol. This further proved that the introduction of NOG pathway can be used as a general strategy to improve P3HB production.

Bacillus Probiotic Enzymes: External Auxiliary Apparatus to Avoid Digestive Deficiencies, Water Pollution, Diseases, and Economic Problems in Marine Cultivated Animals.

Exploitation of marine fishes is the main source of several life-supporting feed compounds such as proteins, lipids, and carbohydrates that maintain the production of most trading marine organisms by aquaculture. However, at this rate the marine inventory will go to the end soon, since fishery resources are finite. In this sense, the availability of the principal ingredients obtained from marine fishes is going to decrease considerably, increasing the diet prices and affecting the economy of this activity. Therefore, aquaculture industry needs to find nonexpensive land unconventional resources of protein, carbohydrates, and lipids and use bacterial probiotics to improve digestion-assimilation of these unfamiliar compounds. Bacillus subtilis is a cosmopolitan probiotic bacterium with a great enzymatic profile that could improve nutrient digestion-assimilation, induce healthy growth, and avoid water pollution, decreasing economic problems and increasing yields in the aquaculture industry. In this chapter, we present how Bacillus enzymes can help marine animals to assimilate nutrients from unconventional and economic plant resources.

Assessment of hemolytic activity, enzyme production and bacteriocin characterization of Bacillus subtilis LR1 isolated from the gastrointestinal tract of fish.

In the present investigation, probiotic potential (antagonistic activity, enzyme production, hemolytic activity, biosafety, antibiotic sensitivity and bile tolerance level) of Bacillus subtilis LR1 was evaluated. Bacteriocin produced by the bacterial strain B. subtilis LR1 isolated from the gastrointestinal tract of Labeo rohita was purified and characterized. The molecular weight of the purified bacteriocin was ~50 kDa in 12 % Native PAGE and showed inhibitory activity against four fish pathogens such as Bacillus mycoides, Aeromonas salmonicida, Pseudomonas fluorescens and Aeromonas hydrophila. The purified bacteriocin was maximally active at temperature 40 °C and pH 7.0, while none of the tested surfactants affect the bacteriocin activity. Extracellular enzyme activity of the selected bacterial strain was also evaluated. Amylase activity was estimated to be highest (38.23 ± 1.15 µg of maltose liberated mg-1 protein ml-1 of culture filtrate) followed by cellulase and protease activity. The selected bacterium was sensitive to most of the antibiotics used in this experiment, can tolerate 0.25 % bile salt and non-hemolytic in nature. Finally, the efficiency of the proposed probiotic candidate was evaluated in in vivo condition. It was detected that the bacterial strain can effectively reduce bacterial pathogenicity in Indian major carps.

Phylogenetic distribution and evolutionary history of bacterial DEAD-Box proteins.

DEAD-box proteins are found in all domains of life and participate in almost all cellular processes that involve RNA. The presence of DEAD and Helicase_C conserved domains distinguish these proteins. DEAD-box proteins exhibit RNA-dependent ATPase activity in vitro, and several also show RNA helicase activity. In this study, we analyzed the distribution and architecture of DEAD-box proteins among bacterial genomes to gain insight into the evolutionary pathways that have shaped their history. We identified 1,848 unique DEAD-box proteins from 563 bacterial genomes. Bacterial genomes can possess a single copy DEAD-box gene, or up to 12 copies of the gene, such as in Shewanella. The alignment of 1,208 sequences allowed us to perform a robust analysis of the hallmark motifs of DEAD-box proteins and determine the residues that occur at high frequency, some of which were previously overlooked. Bacterial DEAD-box proteins do not generally contain a conserved C-terminal domain, with the exception of some members that possess a DbpA RNA-binding domain (RBD). Phylogenetic analysis showed a separation of DbpA-RBD-containing and DbpA-RBD-lacking sequences and revealed a group of DEAD-box protein genes that expanded mainly in the Proteobacteria. Analysis of DEAD-box proteins from Firmicutes and γ-Proteobacteria, was used to deduce orthologous relationships of the well-studied DEAD-box proteins from Escherichia coli and Bacillus subtilis. These analyses suggest that DbpA-RBD is an ancestral domain that most likely emerged as a specialized domain of the RNA-dependent ATPases. Moreover, these data revealed numerous events of gene family expansion and reduction following speciation.

A simple and cost-saving approach to optimize the production of subtilisin NAT by submerged cultivation of Bacillus subtilis natto.

Subtilisin NAT, formerly designated nattokinase or subtilisin BSP, is a potent cardiovascular drug because of its strong fibrinolytic activity and safety. In this study, one Bacillus subtilis natto strain with high fibrinolytic activity was isolated. We further studied the optimal conditions for subtilisin NAT production by submerged cultivation and three variables/three levels of response surface methodology (RSM) using various inoculum densities, glucose concentrations, and defatted soybean concentrations as the three variables. According to the RSM analysis, while culturing by 2.93% defatted soybean, 1.75% glucose, and 4.00% inoculum density, we obtained an activity of 13.78 SU/mL. Processing the batch fermentation with this optimal condition, the activity reached 13.69 SU/mL, which is equal to 99.3% of the predicted value.

Gene cloning and characterization of a thermostable phytase from Bacillus subtilis US417 and assessment of its potential as a feed additive in comparison with a commercial enzyme.

An extracellular phytase from Bacillus subtilis US417 (PHY US417) was purified and characterized. The purified enzyme of 41 kDa was calcium-dependent and optimally active at pH 7.5 and 55 degrees C. The thermal stability of PHY US417 was drastically improved by calcium. Indeed, it recovered 77% of its original activity after denaturation for 10 min at 75 degrees C in the presence of 5 mM CaCl2, while it retained only 22% of activity when incubated for 10 min at 60 degrees C without calcium. In addition, PHY US417 was found to be highly specific for phytate and exhibited pH stability similar to Phyzyme, a commercial phytase with optimal activity at pH 5.5 and 60 degrees C. The phytase gene was cloned by PCR from Bacillus subtilis US417. Sequence analysis of the encoded polypeptide revealed one residue difference from PhyC of Bacillus subtilis VTTE-68013 (substitution of arginine in position 257 by proline in PHY US417) which was reported to exhibit lower thermostability especially in the absence of calcium. With its neutral pH optimum as well as its great pH and thermal stability, the PHY US417 enzyme presumed to be predominantly active in the intestine has a high potential for use as feed additive.

Metastable liquid clusters in super- and undersaturated protein solutions.

Dense liquid phases, metastable with respect to a solid phase, but stable with respect to the solution, have been known to form in solutions of proteins and small-molecule substances. Here, with the protein lumazine synthase as a test system, using dynamic and static light scattering and atomic force microscopy, we demonstrate submicron size clusters of dense liquid. In contrast to the macroscopic dense liquid, these clusters are metastable not only with respect to the crystals, but also with respect to the low-concentration solution: the characteristic cluster lifetime is limited to approximately 10 s, after which they decay. The cluster population is detectable only if they occupy >10(-6) of the solution volume and have a number density >105 cm-3 for 3 to 11% of the monitored time. The cluster volume fraction varies within wide limits and reaches up to 10(-3). Increasing protein concentration increases the frequency of cluster detection but does not affect the ranges of the cluster sizes, suggesting that a preferred cluster size exists. A simple Monte Carlo model with protein-like potentials reproduces the metastable clusters of dense liquid with limited lifetimes and variable sizes and suggests that the mean cluster size is determined by the kinetics of growth and decay and not by thermodynamics.

Biotoxicity assessment on reusability of municipal solid waste incinerator (MSWI) ash.

This study provides a first attempt of dose-response analysis and margin of safety using Escherichia coli DH5alpha, Bacillus subtilis as indicator microorganisms to put forward, in general terms and explanations, the toxicity rankings of various ashes of municipal solid waste incinerator (MSWI) for feasibility in further applications. Since the MSWI ash often contains cations of Si, Ca, Al and Fe, it is frequently considered to be recycled for construction building-materials. Growth inhibition of E. coli DH5alpha occurred at concentrations over 0.156, 0.625 and 0.0195 g/L for bottom ash (BA), cyclone ash (CA), scrubber ash (SA), respectively, suggesting the toxicity ranking of SA>BA>CA. In contrast, except for SA (ca. 0.313 g/L), almost same inhibitory levels of ashes to cell growth were also observed in Bacillus subtilis. Evidently, biotoxicity responses were strongly dependent upon the characteristics of indicator microorganism. Based on DH5alpha, the margins of safety (MOS) were thus 0.195, 1.56 and 6.25 mg/L for SA, BA and CA, respectively. Nearly identical levels of MOS were also suggested by B. subtilis, except for SA (3.13 mg/L). Although MSWI residual ashes qualified EPA's standard test of Toxicity Characteristic Leaching Procedure (TCLP), they might still contain other toxic residues (e.g., chloride ions and/or anions) to cause existing toxicity as indicated in this toxicity study.

Fed-batch optimization of alpha-amylase and protease-producing Bacillus subtilis using Markov chain methods.

A stoichiometry-based model for the fed-batch culture of the recombinant bacterium Bacillus subtilis ATCC 6051a, producing extracellular alpha-amylase as a desirable product and proteases as undesirable products, was developed and verified. The model was then used for optimizing the feeding schedule in fed-batch culture. To handle higher-order model equations (14 state variables), an optimization methodology for the dual-enzyme system is proposed by integrating Pontryagin's optimum principle with fermentation measurements. Markov chain Monte Carlo (MCMC) procedures were appropriate for model parameter and decision variable estimation by using a priori parameter distributions reflecting the experimental results. Using a simplified Metropolis-Hastings algorithm, the specific productivity of alpha-amylase was maximized and the optimum path was confirmed by experimentation. The optimization process predicted a further 14% improvement of alpha-amylase productivity that could not be realized because of the onset of sporulation. Among the decision variables, the switching time from batch to fed-batch operation (t(s)) was the most sensitive decision variable.

Contributions of conformational compression and preferential transition state stabilization to the rate enhancement by chorismate mutase.

The rate enhancement provided by the chorismate mutase (CM) enzyme for the Claisen rearrangement of chorismate to prephenate has been investigated by application of the concept of near attack conformations (NACs). Using a combined QM/MM Monte Carlo/free-energy perturbation (MC/FEP) method, 82% and 100% of chorismate conformers were found to be NAC structures in water and in the CM active site, respectively. Consequently, the conversion of non-NACs to NACs does not contribute to the free energy of activation from preorganization of the substrate into NACs. The FEP calculations yielded differences in free energies of activation that well reproduce the experimental data. Additional calculations indicate that the rate enhancement by CM over the aqueous phase results primarily from conformational compression of NACs by the enzyme and that this process is enthalpically controlled. This suggests that preferential stabilization of the transition state in the enzyme environment relative to water plays a secondary role in the catalysis by CM.