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

Crystalline structures and crystallization behaviors of poly(L-lactide) in poly(L-lactide)/graphene nanosheet composites

GNS existence in PLLA favors α′ crystal formation more than α crystal formation resulting in a shift of α′–α crystal formation transition toward high Tcs.</p

Distinct hyperuricemia trajectories are associated with different risks of incident diabetes: A prospective cohort study

Background and aim: Conflicting results suggest a link between serum uric acid and diabetes and previous studies ignored the effect of continuous exposure of serum uric acid on diabetes risk. This study aims to characterize hyperuricemia trajectories in middle-aged adults and to examine its potential impact on diabetes risk, considering the role of obesity, dyslipidemia, and hypertension. Methods and results: The cohort included 9192 participants who were free of diabetes before 2013. The hyperuricemia trajectories during 2009–2013 were identified by latent class growth models. Incident diabetes during 2014–2018 was used as the outcome. Modified Poisson regression models were used to assess the association of trajectories with diabetes. Furthermore, marginal structural models were used to estimate the mediating effects of the relationship between hyperuricemia trajectories and diabetes. We identified three discrete hyperuricemia trajectories: high-increasing (n = 5794), moderate-stable (n = 2049), and low-stable (n = 1349). During 5 years of follow-up, we documented 379 incident diabetes cases. Compared with the low-stable pattern, the high-increasing pattern had a higher risk of developing diabetes (RR, 1.42; 95% CI: 1.09–1.84). In addition, the percentages of total effect between the high-increasing hyperuricemia pattern and diabetes mediated by obesity, dyslipidemia, and hypertension were 24.41%, 18.26%, and 6.29%. However, the moderate-stable pattern was not associated with an increased risk of diabetes. Conclusions: These results indicate that the high-increasing hyperuricemia trajectory is significantly associated with an increased risk of diabetes. Furthermore, obesity, dyslipidemia, and hypertension play mediating roles in the relationship between the high-increasing hyperuricemia pattern and increased diabetes risk

Based on Network Pharmacology and Molecular Docking to Discuss the Mechanism of Antitussive and Expectorant Action of Ruanerli

The antitussive and expectorant effects of Ruanerli and its mechanism were investigated by methods of network pharmacology. The outcomes predicted were verified by molecular docking and animal experiments. The components and targets of Ruanerli were obtained by literature investigation and TCMSP database screen. Mapping with two groups of genes related to "cough" and "sputum" from GeneCards database, the target genes of antitussive and expectorant effects of Ruanerli were obtained. GO and KEGG enrichment analysis of the target genes was performed by Metascape platform. The PPI network among the target genes was constructed through STRING data platform. Cytoscape plugin CytoHubba was used to screen the Top10 genes related to antitussive and expectorant effects of Ruanerli, and KEGG pathway enrichment was performed on the Top10 genes through Metascape data platform to predict the possible signal pathways involved in antitussive and expectorant effects of Ruanerli. Autodock Vina was used for molecular docking between the predicted Top10 gene proteins and the Top 3 active ingredients of Ruanerli. Finally, the predicted results were verified by ammonia induced cough test and phenol red excretion test. According to the analysis of multiple databases, 51 chemical components and 282 corresponding targets have been reported, eighty of them were related to the antitussive and expectorant effects of Ruanerli. The Top10 genes selected by Degree value were mainly concentrated in infection and immune-related pathways. Molecular docking test showed that the Top10 genes had strong binding activity with the Top3 chemical components (Caffeic acid, Rutin and Valeraldehyde) in PPI network. Animal experiments showed that the cough induced by ammonia was significantly inhibited when treated with Ruanerli in mice. The levels of IL-6 and IL-13 in serum were reduced and the excretion of phenol red in mice trachea was increased. PCR and WB detection showed that the mRNA levels and protein expressions of inflammatory genes IL6, IL1B, VEGFA, PTGS2 and MAPK3 were decreased, suggesting that the antitussive and expectorant effects of Ruanerli might be related to decreasing the expression of inflammatory genes and the release of inflammatory factors

Intracavity generated visible self-reconstructing Bessel-like laser beams by thermal effect

Abstract(#br)In this paper we report the intracavity realization of visible reconstructing Bessel-like beams when a thin Nd:YVO 4 crystal is inserted in a diode-pumped Pr:YLF laser at 639.5 nm. The position of the thin crystal and the intracavity power determine the Bessel-like mode generated. The self-reconstructing characteristic of the obtained Bessel-like beam is also demonstrated

Switchable and tunable multiple-channel erbium-doped fiber laser using graphene-polymer nanocomposite and asymmetric two-stage fiber Sagnac loop filter

A high-performance multiple-channel erbium-doped fiber laser (EDFL) is proposed and experimentally demonstrated, using graphene-polymer nanocomposite as a multiwavelength equalizer and an asymmetric two-stage polarization-maintaining fiber (PMF) Sagnac loop as a flexible comb filter. At first, the filtering characteristics of the PMF Sagnac loop filter (SLF) are investigated. Both theoretical and experimental results show that it can provide a flexibly switchable and tunable comblike filtering. Then, the two-stage PMF SLF is inserted into a graphene-assisted EDFL cavity for generating multiwavelength oscillation. The extreme-high third-order optical nonlinearity of graphene is exploited to suppress the mode competition of the EDFL, and a stable multiple-channel lasing is observed. By carefully adjusting the polarization controllers in the two-stage PMF SLF, not only can the lasing-line number per channel be switchable between single and multiple wavelengths, but also the wavelength spacing in the triple-wavelength condition can be tunable. In the case of triple wavelengths per channel, up to 12 wavelengths with four channels stable oscillations can be achieved. The multiple-channel EDFL can keep a high extinction ratio of >40 dB and a narrow linewidth of <0.01 nm. (C) 2011 Optical Society of Americ