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

Evaluation of Cage Designs and Feeding Regimes for Honey Bee (Hymenoptera: Apidae) Laboratory Experiments

The aim of this study was to improve cage systems for maintaining adult honey bee (Apis mellifera L.) workers under in vitro laboratory conditions. To achieve this goal, we experimentally evaluated the impact of different cages, developed by scientists of the international research network COLOSS (Prevention of honey bee COlony LOSSes), on the physiology and survival of honey bees. We identified three cages that promoted good survival of honey bees. The bees from cages that exhibited greater survival had relatively lower titers of deformed wing virus, suggesting that deformed wing virus is a significant marker reflecting stress level and health status of the host. We also determined that a leak- and drip-proof feeder was an integral part of a cage system and a feeder modified from a 20-ml plastic syringe displayed the best result in providing steady food supply to bees. Finally, we also demonstrated that the addition of protein to the bees' diet could significantly increase the level of vitellogenin gene expression and improve bees' survival. This international collaborative study represents a critical step toward improvement of cage designs and feeding regimes for honey bee laboratory experiment

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

Engineering of a Bacillus amyloliquefaciens Strain with High Neutral Protease Producing Capacity and Optimization of Its Fermentation Conditions.

The neutral protease has high potential for industrial applications, and attempts to improve enzyme expression level have important application values. In the present study, a neutral protease-encoding gene, Banpr, was cloned from Bacillus amyloliquefaciens strain K11, and a genetic manipulation method specific for this difficult-to-transform strain was developed for the high-level expression of neutral protease. The recombinant plasmid pUB110-Banpr was constructed in Bacillus subtilis strain WB600 and then transformed into strain K11 under optimized conditions. A positive transformant 110N-6 with the highest protease secreting capacity on skim milk plates and great genetic stability for more than 100 generations was selected for further study. Optimization of the fermentation conditions increased the enzyme activity of strain 110N-6 to 8995 ± 250 U/ml in flask culture and 28084 ± 1282 U/ml in 15-l fermentor, which are significantly higher than that of the native strain K11 and industrial strain B. subtilis AS.1398, respectively. The high expression level and extreme genetic stability make B. amyloliquefaciens strain 110N-6 more favorable for mass production of neutral protease for industrial uses

Identification of WxL and S-Layer Proteins from Lactobacillus brevis with the Ability to Bind Cellulose and Xylan

Xylanase releases xylo-oligosaccharides from dietary xylan, which stimulate the growth of the gut bacteria lactobacilli. Many lactobacilli adhere to dietary fibers, which may facilitate the assimilation of xylo-oligosaccharides and help them gain competence in the gut, but the underlying mechanisms remain elusive. Herein we report, from the highly abundant transcripts of Lactobacillus brevis cultured in wheat arabinoxylan supplemented with a xylanase, the identification of genes encoding four putative cell-surface WxL proteins (Lb630, Lb631, Lb632, and Lb635) and one S-layer protein (Lb1325) with either cellulose- or xylan-binding ability. The repetitively occurring WxL proteins were encoded by a gene cluster, among which Lb630 was chosen for further mutational studies. The analysis revealed three aromatic residues (F30, W61, and W156) that might be involved in the interaction of the protein with cellulose. A homology search in the genome of Enterococcus faecium identified three WxL proteins with conserved counterparts of these three aromatic residues, and they were also found to be able to bind cellulose and xylan. The findings suggested a role of the cell-surface WxL and S-layer proteins in assisting the cellular adhesion of L. brevis to plant cell wall polysaccharides

PUL-Mediated Plant Cell Wall Polysaccharide Utilization in the Gut Bacteroidetes

Plant cell wall polysaccharides (PCWP) are abundantly present in the food of humans and feed of livestock. Mammalians by themselves cannot degrade PCWP but rather depend on microbes resident in the gut intestine for deconstruction. The dominant Bacteroidetes in the gut microbial community are such bacteria with PCWP-degrading ability. The polysaccharide utilization systems (PUL) responsible for PCWP degradation and utilization are a prominent feature of Bacteroidetes. In recent years, there have been tremendous efforts in elucidating how PULs assist Bacteroidetes to assimilate carbon and acquire energy from PCWP. Here, we will review the PUL-mediated plant cell wall polysaccharides utilization in the gut Bacteroidetes focusing on cellulose, xylan, mannan, and pectin utilization and discuss how the mechanisms can be exploited to modulate the gut microbiota

Characterization of two thermophilic cellulases from Talaromyces leycettanus JCM12802 and their synergistic action on cellulose hydrolysis.

Talaromyces leycettanus JCM12802 is a great producer of thermophilic glycoside hydrolases (GHs). In this study, two cellulases (TlCel5A and TlCel6A) belonging to GH5 and GH6 respectively were expressed in Pichia pastoris and functionally characterized. The enzymes had acidic and thermophilic properties, showing optimal activities at pH 3.5-4.5 and 75-80°C, and retained stable at temperatures up to 60°C and over a broad pH range of 2.0-8.0. TlCel5A and TlCel6A acted against several cellulose substrates with varied activities (3,101.1 vs. 92.9 U/mg to barley β-glucan, 3,905.6 U/mg vs. 109.0 U/mg to lichenan, and 840.3 and 0.09 U/mg to CMC-Na). When using Avicel, phosphoric acid swollen cellulose (PASC) or steam-exploded corn straw (SECS) as the substrate, combination of TlCel5A and TlCel6A showed significant synergistic action, releasing more reduced sugars (1.08-2.87 mM) than the individual enzymes. These two cellulases may represent potential enzyme additives for the efficient biomass conversion and bioethanol production

Diversity of Beta-Propeller Phytase Genes in the Intestinal Contents of Grass Carp Provides Insight into the Release of Major Phosphorus from Phytate in Nature▿

Phytate is the most abundant organic phosphorus compound in nature, and microbial mineralization of phytate by phytase is a key process for phosphorus recycling in the biosphere. In the present study, beta-propeller phytase (BPP) gene fragments were readily amplified from the intestinal contents of grass carp (Ctenopharyngodon idellus) directly or from phytate-degrading isolates from the same source, confirming the widespread occurrence of BPP in aquatic communities. The amounts of sequences collected using these two methods differed (88 distinct genes versus 10 isolates), but the sequences showed the same general topology based on phylogenetic analysis. All of the sequences fell in five clusters and were distinct from those of Anabaena, Gloeobacter, Streptomyces, Flavobacterium, Prosthecochloris, and Desulfuromonas, which have never been found in the grass carp intestine. Analysis of the microbial diversity by denaturing gradient gel electrophoresis demonstrated that unculturable bacteria were dominant bacteria in the grass carp intestine and thus the predominant phytate-degrading organisms. The predominant cultured species corresponding to the phytate-degrading isolates, Pseudomonas, Bacillus and Shewanella species, might be the main source of known BPPs. A phytase from Brevundimonas was first obtained from cultured species. Combining our results with Lim et al.'s inference that phytate-mineralizing bacteria are widely distributed and highly diverse in nature (B. L. Lim, P. Yeung, C. Cheng, and J. E. Hill, ISME J. 1:321-330, 2007), we concluded that BPP is the major phytate-degrading enzyme in nature, that most of this enzyme might originate from unculturable bacteria, and that the distribution of BPP may be related to the type of niche. To our knowledge, this is the first study to experimentally estimate BPP diversity in situ