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

Redistribution of the astrocyte phenotypes in the medial vestibular nuclei after unilateral labyrinthectomy

Astrocytes are highly heterogeneous and involved in different aspects of fundamental functions in the central nervous system (CNS). However, whether and how this heterogeneous population of cells reacts to the pathophysiological challenge is not well understood. To investigate the response status of astrocytes in the medial vestibular nucleus (MVN) after vestibular loss, we examined the subtypes of astrocytes in MVN using single-cell sequencing technology in a unilateral labyrinthectomy mouse model. We discovered four subtypes of astrocytes in the MVN with each displaying unique gene expression profiles. After unilateral labyrinthectomy, the proportion of the astrocytic subtypes and their transcriptional features on the ipsilateral side of the MVN differ significantly from those on the contralateral side. With new markers to detect and classify the subtypes of astrocytes in the MVN, our findings implicate potential roles of the adaptive changes of astrocyte subtypes in the early vestibular compensation following peripheral vestibular damage to reverse behavioral deficits

Improving the Substrate Affinity and Catalytic Efficiency of β-Glucosidase Bgl3A from <i>Talaromyces leycettanus</i> JCM12802 by Rational Design

Improving the substrate affinity and catalytic efficiency of β-glucosidase is necessary for better performance in the enzymatic saccharification of cellulosic biomass because of its ability to prevent cellobiose inhibition on cellulases. Bgl3A from Talaromyces leycettanus JCM12802, identified in our previous work, was considered a suitable candidate enzyme for efficient cellulose saccharification with higher catalytic efficiency on the natural substrate cellobiose compared with other β-glucosidase but showed insufficient substrate affinity. In this work, hydrophobic stacking interaction and hydrogen-bonding networks in the active center of Bgl3A were analyzed and rationally designed to strengthen substrate binding. Three vital residues, Met36, Phe66, and Glu168, which were supposed to influence substrate binding by stabilizing adjacent binding site, were chosen for mutagenesis. The results indicated that strengthening the hydrophobic interaction between stacking aromatic residue and the substrate, and stabilizing the hydrogen-bonding networks in the binding pocket could contribute to the stabilized substrate combination. Four dominant mutants, M36E, M36N, F66Y, and E168Q with significantly lower Km values and 1.4–2.3-fold catalytic efficiencies, were obtained. These findings may provide a valuable reference for the design of other β-glucosidases and even glycoside hydrolases

Kimura disease

Kimura disease is a rare, benign, chronic, immune-mediated inflammatory disorder. We report a 46-year-old man who presented with a cutaneous nodule behind his left ear. Surgical removal of the growth confirmed the histological diagnosis of KD. There was no recurrence found after 3 years follow-up

A Novel Thermostable GH3 β-Glucosidase from Talaromyce leycettanus with Broad Substrate Specificity and Significant Soybean Isoflavone Glycosides-Hydrolyzing Capability

A novel β-glucosidase gene (Bgl3B) of glycoside hydrolase (GH) family 3 was cloned from the thermophilic fungus Talaromyce leycettanus JM12802 and successfully expressed in Pichia pastoris. The deduced Bgl3B contains 860 amino acid residues with a calculated molecular mass of 91.2 kDa. The purified recombinant Bgl3B exhibited maximum activities at pH 4.5 and 65°C and remained stable at temperatures up to 60°C and pH 3.0−9.0, respectively. The enzyme exhibited broad substrate specificities, showing β-glucosidase, glucanase, cellobiase, xylanase, and isoflavone glycoside hydrolase activities, and its activities were stimulated by short-chain alcohols. The catalytic efficiencies of Bgl3B were 693 and 104/mM/s towards pNPG and cellobiose, respectively. Moreover, Bgl3B was highly effective in converting isoflavone glycosides to aglycones at 37°C within 10 min, with the hydrolysis rates of 95.1%, 76.0%, and 75.3% for daidzin, genistin, and glycitin, respectively. These superior properties make Bgl3B potential for applications in the food, animal feed, and biofuel industries

Molecular Characterization of a New Alkaline-Tolerant Xylanase from Humicola insolens Y1

An endo-1,4-β-xylanase-encoding gene, xyn11B, was cloned from the thermophilic fungus Humicola insolens Y1. The gene encodes a multimodular xylanase that consists of a typical hydrophobic signal sequence, a catalytic domain of glycoside hydrolase (GH) family 11, a glycine-rich linker, and a family 1 carbohydrate binding module (CBM1). Deduced Xyn11B shares the highest identity of 74% with a putative xylanase from Podospora anserina S mat+. Recombinant Xyn11B was successfully expressed in Pichia pastoris and purified to electrophoretic homogeneity. Xyn11B had a high specific activity of 382.0 U mg−1 towards beechwood xylan and showed optimal activity at pH 6.0 and 50°C. Distinct from most reported acidic fungal xylanases, Xyn11B was alkaline-tolerant, retaining 30.7% of the maximal activity at pH 9.0. The Km and Vmax values for beechwood xylan were 2.2 mg mL−1 and 462.8 μmol min−1 mg−1, respectively. The enzyme exhibited a wider substrate specificity and produced a mixture of xylooligosaccharides. All these favorable enzymatic properties make Xyn11B attractive for potential applications in various industries

Role of N-linked glycosylation in the enzymatic properties of a thermophilic GH 10 xylanase from Aspergillus fumigatus expressed in Pichia pastoris.

N-Glycosylation is a posttranslational modification commonly occurred in fungi and plays roles in a variety of enzyme functions. In this study, a xylanase (Af-XYNA) of glycoside hydrolase (GH) family 10 from Aspergillus fumigatus harboring three potential N-glycosylation sites (N87, N124 and N335) was heterologously produced in Pichia pastoris. The N-glycosylated Af-XYNA (WT) exhibited favorable temperature and pH optima (75°C and pH 5.0) and good thermostability (maintaining stable at 60°C). To reveal the role of N-glycosylation on Af-XYNA, the enzyme was deglycosylated by endo-β-N-acetylglucosaminidase H (DE) or modified by site-directed mutagenesis at N124 (N124T). The deglycosylated DE and mutant N124T showed narrower pH adaptation range, lower specific activity, and worse pH and thermal stability. Further thermodynamic analysis revealed that the enzyme with higher N-glycosylation degree was more thermostable. This study demonstrated that the effects of glycosylation at different degrees and sites were diverse, in which the glycan linked to N124 played a key role in pH and thermal stability of Af-XYNA