High yield expression of an AHL-lactonase from Bacillus sp. B546 in Pichia pastoris and its application to reduce Aeromonas hydrophila mortality in aquaculture

<p>Abstract</p> <p>Background</p> <p><it>Aeromonas hydrophila </it>is a serious pathogen and can cause hemorrhagic septicemia in fish. To control this disease, antibiotics and chemicals are widely used which can consequently result in "superbugs" and chemical accumulation in the food chain. Though vaccine against <it>A. hydrophila </it>is available, its use is limited due to multiple serotypes of this pathogen and problems of safety and efficacy. Another problem with vaccination is the ability to apply it to small fish especially in high numbers. In this study, we tried a new way to attenuate the <it>A. hydrophila </it>infection by using a quorum quenching strategy with a recombinant AHL-lactonase expressed in <it>Pichia pastoris</it>.</p> <p>Results</p> <p>The AHL-lactonase (AiiA_B546) from <it>Bacillus </it>sp. B546 was produced extracellularly in <it>P. pastoris </it>with a yield of 3,558.4 ± 81.3 U/mL in a 3.7-L fermenter when using 3-oxo-C8-HSL as the substrate. After purification with a HiTrap Q Sepharose column, the recombinant homogenous protein showed a band of 33.6 kDa on SDS-PAGE, higher than the calculated molecular mass (28.14 kDa). Deglycosylation of AiiA_B546with Endo H confirmed the occurrence of <it>N</it>-glycosylation. The purified recombinant AiiA_B546showed optimal activity at pH 8.0 and 20°C, exhibited excellent stability at pH 8.0-12.0 and thermal stability at 70°C, was firstly confirmed to be significantly protease-resistant, and had wide substrate specificity. In application test, when co-injected with A. <it>hydrophila </it>in common carp, recombinant AiiA_B546decreased the mortality rate and delayed the mortality time of fish.</p> <p>Conclusions</p> <p>Our results not only indicate the possibility of mass-production of AHL-lactonase at low cost, but also open up a promising foreground of application of AHL-lactonase in fish to control <it>A. hydrophila </it>disease by regulating its virulence. To our knowledge, this is the first report on heterologous expression of AHL-lactonase in <it>P. pastoris </it>and attenuating <it>A. hydrophila </it>virulence by co-injection with AHL-lactonase.</p

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

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

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

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

An alkaline and surfactant-tolerant lipase from Trichoderma lentiforme ACCC30425 with high application potential in the detergent industry

Abstract Alkaline lipases with adaptability to low temperatures and strong surfactant tolerance are favorable for application in the detergent industry. In the present study, a lipase-encoding gene, TllipA, was cloned from Trichoderma lentiforme ACCC30425 and expressed in Pichia pastoris GS115. The purified recombinant TlLipA was found to have optimal activities at 50 °C and pH 9.5 and retain stable over the pH range of 6.0–10.0 and 40 °C and below. When using esters of different lengths as substrates, TlLipA showed preference for the medium length p-nitrophenyl octanoate. In comparison to commercial lipases, TlLipA demonstrated higher tolerance to various surfactants (SDS, Tween 20, and Triton X100) and retained more activities after incubation with Triton X100 for up to 24 h. These favorable characteristics make TlLipA prospective as an additive in the detergent industry

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

Synergistic effect of acetyl xylan esterase from Talaromyces leycettanus JCM12802 and xylanase from Neocallimastix patriciarum achieved by introducing carbohydrate-binding module-1

Abstract Wheat bran is an effective raw material for preparation xylooligosaccharides; however, current research mainly focuses on alkali extraction and enzymatic hydrolysis methods. Since ester bonds are destroyed during the alkali extraction process, xylanase and arabinofuranosidase are mainly used to hydrolyze xylooligosaccharides. However, alkali extraction costs are very high, and the method also causes pollution. Therefore, this study focuses on elucidating a method to efficiently and directly degrade destarched wheat bran. First, an acidic acetyl xylan esterase (AXE) containing a carbohydrate-binding module-1 (CBM1) domain was cloned from Talaromyces leycettanus JCM12802 and successfully expressed in Pichia pastoris. Characterization showed that the full-length acetyl xylan esterase AXE + CBM1 was similar toe uncovered AXE with an optimum temperature and pH of 55 °C and 6.5, respectively. Testing the acetyl xylan esterase and xylanase derived from Neocallimastix patriciarum in a starch-free wheat bran cooperative experiment revealed that AXE + CBM1 and AXE produced 29% and 16% reducing sugars respectively, compared to when only NPXYN11 was used. In addition, introduced the CBM1 domain into NPXYN11, and the results indicated that the CBM1 domain showed little effect on NPXYN11 properties. Finally, the systematically synergistic effects between acetyl xylan esterase and xylanase with/without the CBM1 domain demonstrated that the combined ratio of AXE + CBM1 coming in first and NPXYN11 + CBM1 s increased reducing sugars by almost 35% with AXE and NPXYN11. Furthermore, each component’s proportion remained the same with respect to xylooligosaccharides, with the largest proportion (86%) containing of 49% xylobiose and 37% xylotriose