Gene cloning and molecular characterization of a thermostable chitosanase from Bacillus cereus TY24.

BACKGROUND: An important conceptual advance in health and the environment has been recognized that enzymes play a key role in the green processing industries. Of particular interest, chitosanase is beneficial for recycling the chitosan resource and producing chitosan oligosaccharides. Also, chitosan gene expression and molecular characterization will promote understanding of the biological function of bacterial chitosanase as well as explore chitosanase for utilizing chitosan resources. RESULTS: A chitosanase-producing bacterium TY24 was isolated and identified as Bacillus cereus. Moreover, the chitosanase gene was cloned and expressed in Escherichia coli. Sequence analysis reveals that the recombinant chitosanase (CHOE) belongs to the glycoside hydrolases 8 family. The purified CHOE has a molecular weight of about 48 kDa and the specific activity of 1150 U/mg. The optimal pH and temperature of CHOE were 5.5 and 65 °C, respectively. The enzyme was observed stable at the pH range of 4.5-7.5 and the temperature range of 30-65 °C. Especially, the half-life of CHOE at 65 °C was 161 min. Additionally, the activity of CHOE was remarkably enhanced in the presence of Mn2+, Cu2+, Mg2+ and K+, beside Ca2+ at 5 mM. Especially, the activity of CHOE was enhanced to more than 120% in the presence of 1% of various surfactants. CHOE exhibited the highest substrate specificity toward colloid chitosan. CONCLUSION: A bacterial chitosanase was cloned from B. cereus and successfully expressed in E. coli (BL21) DE3. The recombinant enzyme displayed good stability under acid pH and high-temperature conditions.

Production of surfactant-stable keratinase from Bacillus cereus YQ15 and its application as detergent additive.

BACKGROUND: With the growing concern for the environment, there are trends that bio-utilization of keratinous waste by keratinases could ease the heavy burden of keratinous waste from the poultry processing and leather industry. Especially surfactant-stable keratinases are beneficial for the detergent industry. Therefore, the production of keratinase by Bacillus cereus YQ15 was improved; the characterization and use of keratinase in detergent were also studied. RESULTS: A novel alkaline keratinase-producing bacterium YQ15 was isolated from feather keratin-rich soil and was identified as Bacillus cereus. Based on the improvement of medium components and culture conditions, the maximum keratinase activity (925 U/mL) was obtained after 36 h of cultivation under conditions of 35 °C and 160 rpm. Moreover, it was observed that the optimal reacting temperature and pH of the keratinase are 60 °C and 10.0, respectively; the activity was severely inhibited by PMSF and EDTA. On the contrary, the keratinase showed remarkable stability in the existence of the various surfactants, including SDS, Tween 20, Tween 60, Tween 80, and Triton X-100. Especially, 5% of Tween 20 and Tween 60 increased the activity by 100% and 60%, respectively. Furtherly, the keratinase revealed high efficiency in removing blood stains. CONCLUSION: The excellent compatibility with commercial detergents and the high washing efficiency of removing blood stains suggested its suitability for potential application as a bio-detergent additive.

Biochemical properties of a novel glycoside hydrolase family 1 ß-glucosidase (PtBglu1) from Paecilomyces thermophila expressed in Pichia pastoris.

A novel ß-glucosidase gene (PtBglu1) from the thermophilic fungus, Paecilomyces thermophila, was cloned and expressed in Pichia pastoris. PtBglu1 contained an open reading frame of 1440-bp nucleotides and encoded a protein of 479 amino acids which showed significant similarity to other fungal ß-glucosidases from glycoside hydrolase (GH) family 1. The recombinant ß-glucosidase (PtBglu1) was secreted at high level of 190.2 U mL(-1) in high cell density fermentor (5L). PtBglu1 was purified to homogeneity, and was found to be a glycoprotein with molecular mass of 56.7 kDa. The purified PtBglu1 showed optimum catalytic activity at pH 6.0 and 55 °C. The enzyme exhibited broad substrate specificity with highest activity toward pNP-ß-D-glucopyranoside, followed by pNP-ß-D-galactopyranoside and cellobiose. The K(m) values for pNP-ß-D-glucopyranoside, cellobiose, gentiobiose and salicin were 0.55 mM, 1.0 mM, 1.74 mM and 6.85 mM, respectively. These properties make PtBglu1 a potential candidate for various industrial applications.

Expression of DnaJ gene in Alicyclobacillus acidoterrestris under stress conditions by quantitative real-time PCR.

UNLABELLED: This article describes the cloning, sequence analysis and expression of the DnaJ gene from Alicyclobacillus acidoterrestris. The genome walking technique was used to clone the full-length sequence of DnaJ and quantitative real-time PCR was used to analyze DnaJ expression under stress conditions. AadnaJ (GenBank accession nr: HQ893544) containing an open reading frame of 1137 bp encoding 378 amino acid residues was cloned from A. acidoterrestris DSM 3922(T). The nucleotide sequence of AadnaJ shows 77% homology with the DnaJ of A. acidocaldarius LAA1. The DnaJ expression level was upgraded rapidly under heat or acid stress. Its mRNA expression level reached a peak value at 25 min after the onset of heat stress (70 °C) and at 1 h after the onset of acid stress (pH = 1). Acid stress at pH 1 for 25 and 60 min led to the DnaJ expression levels 2.1 times and 35.7 times above that of the control, respectively. In response to cold stress at 0 °C, the DnaJ expression level decreased drastically to 0.04 times that of the control level after 1 h. The expression patterns of DnaJ in response to the stress conditions shown here explained the heat and acidity endurance of A. acidoterrestris. PRACTICAL APPLICATION: This study directly addresses the role of the DnaJ gene in temperature and acid endurance in A. acidoterrestris. This provides a basis for the development of genetic and molecular techniques that may minimize the adverse effects of A. acidoterrestris in fruit juice production. This study also sheds light on the design of heat- and acid-tolerant recombinases and the understanding of the molecular mechanisms underlying heat and acid resistance in A. acidoterrestris.

High level expression of extracellular secretion of a ß-glucosidase gene (PtBglu3) from Paecilomyces thermophila in Pichia pastoris.

A novel ß-glucosidase gene (designated PtBglu3) from Paecilomyces thermophila was cloned and sequenced. PtBglu3 has an open reading frame of 2,557 bp, encoding 858 amino acids with a calculated molecular mass of 90.9 kDa. The amino acid sequence of the mature polypeptide shared the highest identity (70%) to a glycoside hydrolase (GH) family 3 characterized ß-glucosidase from Penicillium purpurogenum. PtBglu3 without the signal peptides was cloned into pPIC9K vector and successfully expressed in Pichia pastoris as an active extracellular ß-glucosidase (PtBglu3). High activity of 274.4 U/ml was obtained by high cell-density fermentation, which is by far the highest reported yield for ß-glucosidase. The recombinant enzyme was purified to homogeneity with 3.3-fold purification and a recovery of 68.5%. The molecular mass of the enzyme was estimated to be 116 kDa by SDS-PAGE, and 198.2 kDa by gel filtration, indicating that it was a dimer. Optimal activity of the purified enzyme was observed at pH 6.0 and 65 °C, and it was stable up to 60 °C. The enzyme exhibited high specific activity toward pNP-ß-D-glucopyranoside, cellooligosaccharides, gentiobiose, amygdalin and salicin, and relatively lower activity against lichenan and laminarin. The present results should contribute to improving industrial production of ß-glucosidase.

Cloning and expression of the endo-1,3(4)-ß-glucanase gene from Paecilomyces sp. FLH30 and characterization of the recombinant enzyme.

The cDNA encoding ß-1,3(4)-glucanase, named PsBg16A, from Paecilomyces sp. FLH30 was cloned, sequenced, and over expressed in Pichia pastoris, with a yield of about 61,754 U mL⁻¹ in a 5-L fermentor. PsBg16A has an open reading frame of 951 bp encoding 316 amino acids, and the deduced amino acid sequence of PsBg16A revealed that it belongs to glycoside hydrolase family 16. The purified recombinant PsBg16A had a pH optimum at 7.0 and a temperature optimum at 70 °C, and randomly hydrolyzed barley ß-glucan, lichenin, and laminarin, suggesting that it is a typical endo-1,3(4)-ß-glucanase (EC 3.2.1.6) with broad substrate specificity for ß-glucans.

High-level expression of a specific beta-1,3-1,4-glucanase from the thermophilic fungus Paecilomyces thermophila in Pichia pastoris.

In this study, a novel beta-1,3-1,4-glucanase gene (designated as PtLic16A) from Paecilomyces thermophila was cloned and sequenced. PtLic16A has an open reading frame of 945 bp, encoding 314 amino acids. The deduced amino acid sequence shares the highest identity (61%) with the putative endo-1,3(4)-beta-glucanase from Neosartorya fischeri NRRL 181. PtLic16A was cloned into a vector pPIC9K and was expressed successfully in Pichia pastoris as active extracellular beta-1,3-1,4-glucanase. The recombinant beta-1,3-1,4-glucanase (PtLic16A) was secreted predominantly into the medium which comprised up to 85% of the total extracellular proteins and reached a protein concentration of 9.1 g l(-1) with an activity of 55,300 U ml(-1) in 5-l fermentor culture. The enzyme was then purified using two steps, ion exchange chromatography, and gel filtration chromatography. The purified enzyme had a molecular mass of 38.5 kDa on SDS-PAGE. It was optimally active at pH 7.0 and a temperature of 70 degrees C. Furthermore, the enzyme exhibited strict specificity for beta-1,3-1,4-D: -glucans. This is the first report on the cloning and expression of a beta-1,3-1,4-glucanase gene from Paecilomyces sp.

Cloning and expression of a Paecilomyces thermophila xylanase gene in E. coli and characterization of the recombinant xylanase.

A cDNA library of Paecilomyces thermophila was constructed, and the gene encoding xylanase (designated Pt xynA) was isolated from the library. Pt xynA consisted of 681 bp, and the translated protein encoded 226 amino acids. This is the first functional gene cloned from P. thermophila. The gene was successfully expressed in Escherichia coli BL21 and the recombinant xylanase (XynA) was purified to homogeneity by Ni-NTA and Sephadex G50. XynA showed an optimum activity at 75 degrees C and pH 7.0. Its residual activity was more than 60% after being treated at 85 degrees C for 30 min. K(m) values of XynA for birchwood xylan, beechwood xylan and oat-spelt xylan were 4.4, 3.6 and 9.7 mg ml(-1), respectively. The enzyme has an endohydrolytic mode of action and can hydrolyse xylotriose to xylobiose through transglycosylation. These results indicate the XynA is a thermostable enzyme and has great potential in various industries.

Crystallization and preliminary X-ray analysis of a 1,3-1,4-beta-glucanase from Paecilomyces thermophila.

In this study, the crystallization and preliminary X-ray analysis of a thermostable 1,3-1,4-beta-glucanase produced by Paecilomyces thermophila is described. The purified 1,3-1,4-beta-glucanase was crystallized using the hanging-drop vapour-diffusion method. The crystal belongs to the hexagonal space group P6(3)22, with unit-cell parameters a = b = 154.54, c = 87.62 A. X-ray diffraction data were collected to a resolution of 2.54 A and gave a data set with an overall R(merge) of 7.3% and a completeness of 94.6%. The Matthews coefficient (V(M)) and the solvent content are 2.38 A(3) Da(-1) and 48%, respectively.