Expression of an extremely acidic β-1,4-glucanase from thermoacidophilic Alicyclobacillus sp. A4 in Pichia pastoris is improved by truncating the gene sequence

<p>Abstract</p> <p>Background</p> <p><it>Alicyclobacillus </it>sp. A4 is thermoacidophilic and produces many glycoside hydrolases. An extremely acidic β-1,4-glucanase (CelA4) has been isolated from <it>Alicyclobacillus </it>sp. A4 and purified. This glucanase with a molecular mass of 48.6 kDa decreases the viscosity of barley-soybean feed under simulated gastric conditions. Therefore, it has the potential to improve the nutrient bioavailability of pig feed. For the study reported herein, the full-length gene, <it>CelA4</it>, of this glucanase (CelA4) was identified using the sequences of six peptides and cloned from strain A4. The gene fragment (<it>CelA4</it>_<it>F</it>) encoding the mature protein was expressed in <it>Pichia pastoris</it>. Sequence truncation and glycosylation were found for recombinant CelA4_F, both of which affected the expression efficiency. The physical properties of various forms of CelA4 as they affected enzymatic activity were characterized.</p> <p>Results</p> <p>We located the full-length 2,148-bp gene for CelA4 (<it>CelA4</it>) in the genome of <it>Alicyclobacillus </it>sp. A4. <it>CelA4 </it>encodes a 715-residue polypeptide with a calculated molecular mass of 71.64 kDa, including an N-terminal signal peptide (residues 1-39), a catalytic domain (residues 39-497), and a C-terminal threonine-rich region (residues 498-715). Its deduced amino acid sequence and that of an <it>Alicyclobacillus acidocaldarius </it>endo-β-1,4-glucanase were identical at 44% of the residue positions. When the experimental molecular mass of CelA4_F--a recombinant protein designed to mimic the CelA4 sequence lacking the N-terminal signal peptide that had been expressed in <it>Pichia pastoris</it>--was compared with its hypothetical molecular mass, it was apparent that CelA4_Fwas truncated, possibly at residue 497. An artificially truncated gene fragment (<it>CelA4</it>_<it>T</it>) without C-terminal threonine-rich region was expressed in <it>P. pastoris</it>, and the expression efficiency of CelA4_Twas substantially greater than that of CelA4_F. Purified CelA4_Fand CelA4_Thad similar molecular masses (~60 kDa) and enzymatic properties (optimum pH, 3.4; optimum temperature, 60°C); they were relatively stable between pH 1.2 and 8.2 at 70°C and resistant to acidic and neutral proteases. However, their molecular masses and thermostabilities differed from those of CelA4 isolated from <it>Alicyclobacillus </it>sp. A4. A deglycosylated form of CelA4 (CelA4_D) had properties similar to that of CelA4 except that it was thermoliable at 60°C.</p> <p>Conclusions</p> <p>Truncation during expression of CelA4_For artificial truncation of its gene--both of which produced a form of CelA4 lacking a threonine-rich region that includes a putative linker--increased the level of enzyme produced in comparison with that produced by cultivation of <it>Alicyclobacillus </it>sp. A4. Glycosylation increased the thermostability of CelA4. Of the four forms of CelA4 studied, CelA4_Twas produced in highest yield and had the most favorable physical properties; therefore, it has potential for use in the feed industry.</p

Deep learning-based image segmentation model using an MRI-based convolutional neural network for physiological evaluation of the heart

Background and Objective: Cardiovascular disease is a high-fatality health issue. Accurate measurement of cardiovascular function depends on precise segmentation of physiological structure and accurate evaluation of functional parameters. Structural segmentation of heart images and calculation of the volume of different ventricular activity cycles form the basis for quantitative analysis of physiological function and can provide the necessary support for clinical physiological diagnosis, as well as the analysis of various cardiac diseases. Therefore, it is important to develop an efficient heart segmentation algorithm.Methods: A total of 275 nuclear magnetic resonance imaging (MRI) heart scans were collected, analyzed, and preprocessed from Huaqiao University Affiliated Strait Hospital, and the data were used in our improved deep learning model, which was designed based on the U-net network. The training set included 80% of the images, and the remaining 20% was the test set. Based on five time phases from end-diastole (ED) to end-systole (ES), the segmentation findings showed that it is possible to achieve improved segmentation accuracy and computational complexity by segmenting the left ventricle (LV), right ventricle (RV), and myocardium (myo).Results: We improved the Dice index of the LV to 0.965 and 0.921, and the Hausdorff index decreased to 5.4 and 6.9 in the ED and ES phases, respectively; RV Dice increased to 0.938 and 0.860, and the Hausdorff index decreased to 11.7 and 12.6 in the ED and ES, respectively; myo Dice increased to 0.889 and 0.901, and the Hausdorff index decreased to 8.3 and 9.2 in the ED and ES, respectively.Conclusion: The model obtained in the final experiment provided more accurate segmentation of the left and right ventricles, as well as the myocardium, from cardiac MRI. The data from this model facilitate the prediction of cardiovascular disease in real-time, thereby providing potential clinical utility

A Joint Inspection-Based Preventive Maintenance and Spare Ordering Optimization Policy Using a Three-Stage Failure Process

Separated decision-making for maintenance and spare ordering is unrealistic in the industry, so this paper aims to optimize them together. A joint policy of inspection-based preventive maintenance (PM) and spare ordering considering two modes of spare ordering, namely, a regular order and an emergency order, is proposed for single-unit systems using a three-stage failure process. If the system is recognized to be in the minor defective stage, the original inspection interval is shortened and a regular order is placed. However, replacement is undertaken preventively or correctively if the severe defective stage is identified or a failure occurs. Depending on the system state and the state of the regular ordered spare when replacement is needed, all possible scenarios are considered to construct optimization model I. The decision variables are the optimal inspection interval and the times of shortening the original inspection interval. Additionally, model II on the basis of an assumption that the spare is always ordered at time 0 is also developed. The results from a numerical example illustrated the applicability and the effectiveness of model I compared to model II, and the irregular inspection policy is validated to be cost-saving compared to the regular inspection policy

Straightforward Synthesis of Bifunctional Phosphorus Phenols via Phosphination of In Situ Generated o-Quinone Methides

An efficient and practical approach towards bifunctional phosphorus phenols has been developed through a reaction of diphenylphosphine oxide and the o-quinone methides in situ generated from 2-tosylalkyl phenols under basic conditions. This protocol features simple experimental procedures under mild conditions and is easily scaled up. With this method, a variety of diarylmethyl phosphine oxides can be produced with up to 92% yield

Transformation of CuO Nanoparticles in the Aquatic Environment: Influence of pH, Electrolytes and Natural Organic Matter

Many studies have shown the effect of solution chemistry on the environmental behavior of metal-based nanoparticles (NPs), except CuO NPs. Here, we investigated the agglomeration, sedimentation, dissolution, and speciation of CuO NPs by varying pH, ionic strength, ionic valence, and natural organic matter (NOM). The results showed that as the pH moved away from 6, the size of CuO agglomerates decreased, along with the enhanced NP suspension stabilization, due to the increase of electrostatic repulsive force. Increasing ionic strength and valence intensified the agglomeration and sedimentation of CuO NPs because of the compression of electrical double layers. The presence of humic acid and citric acid enhanced the dispersion and stabilization of CuO NP suspension, but l-cysteine showed a different impact. Decreasing pH, increasing ionic strength and all NOM improved the dissolution of CuO NPs, but the divalent electrolyte (CaCl2) inhibited the Cu2+ release from CuO NPs compared to the monovalent electrolyte (NaCl). In addition, X-ray absorption near edge structure (XANES) analysis demonstrated that the presence of l-cysteine transformed more than 30% of CuO NPs to Cu(I)-cysteine by coordinating with thiol group. This study can give us an in-depth understanding on the environmental behavior and fate of CuO NPs in the aquatic environment

Electrocatalytic activity and stability of carbon nanotubes-supported Pt-on-Au, Pd-on-Au, Pt-on-Pd-on-Au, Pt-on-Pd, and Pd-on-Pt catalysts for methanol oxidation reaction

For optimizing both the activity and stability of Pt-based catalysts for methanol oxidation reaction (MOR), several carbon nanotubes(CNTs)-supported catalysts such as Pt-on-Au/CNTs, Pd-on-Au/CNTs, Pt-on-Pdon- Au/CNTs, Pt-on-Pd/CNTs, and Pd-on-Pt/CNTs catalysts are synthesized mainly through electrodeposition method. The activity and stability comparisons show that Pt-on-Au/CNTs has a higher MOR activity but a lower stability than Pd-on-Au/CNTs. To utilize the merits of the Pt and Pd components, Pt-on-Pd-on- Au/CNTs and Pt-on-Pd/CNTs catalysts are synthesized. The Pt-on-Pd-on-Au/CNTs and Pt-on-Pd/CNTs catalysts shows higher MOR activity than Pd-on-Au/CNTs and Pd/CNTs catalysts and higher stability than Pt-on-Au/CNTs, suggesting a synergistic interaction between Pt and Pd in catalyzing methanol oxidation reaction. Calculation shows that the total mass activity of Pt-on-Pd/CNTs with quite low Pt amount is on a similar level as that of Pt/CNTs for MOR oxidation, indicating the Pt-on-Pd catalyst could have promising potential as a low-Pt catalyst for MOR in alkaline media.Peer reviewed: YesNRC publication: Ye