A thermophilic chitinase 1602 from the marine bacterium Microbulbifer sp. BN3 and its high-level expression in Pichia pastoris.

Chitinases play an important role in many industrial processes, including the preparation of oligosaccharides with potential applications. In the present study, a 1,713 bp gene of Chi1602, derived from a marine bacterium Microbulbifer sp. BN3, encoding a GH18 family chitinase, was expressed at high levels in Pichia pastoris. Distinct from most of the marine chitinases, the recombinant chitinase 1602 exhibited maximal activity at 60 °C and over a broad pH range between 5.0 and 9.0, and was stable at 50 °C and over the pH range 4.0-9.0. The hydrolytic products derived from colloidal chitins comprised mainly (GlcNAc)2 and GlcNAc, indicating that rChi1602 is a GH18 processive chitinase in conformity with its hypothetical structure. However, rChi1602 showed traces of ß-N-acetylglucosaminidase activity on substrates such as powder chitin, chitosan, and ethylene glycol chitin. The thermophilic rChi1602, which manifests adaptation to a wide pH range and can be expressed at a high level in P. pastoris, is advantageous for applications in industrial processes.

Highly efficient expression and characterization of a ß-mannanase from Bacillus subtilis in Pichia pastoris.

A ß-mannanase gene (Man5) from Bacillus subtilis BS5 was cloned by PCR and integrated into the genome of Pichia pastoris GS115 via pPIC9 vector. The recombinant Man5 with a molecular mass of 43 kDa was successfully expressed and secreted into the culture medium. After methanol induction in a shake flask for 96 H, the recombinant Man5 protein reached 375 µg/mL in concentration, with an enzyme activity of 892 U/mL. The recombinant Man5 was purified 3.35-fold with 60% yield by using HiTrap DEAE FF and HiTrap Phenyl FF columns. The specific activity of the purified enzyme was 7,978 U/mg. The optimum temperature and pH of the recombinant Man5 were 50 °C and 6.0, respectively. Studies of substrate specificity showed that the optimum substrate for the Man5 was konjac flour, suggesting that it has great potential as an effective additive in the food industry.

Expression and biochemical characterization of a novel chitinase ChiT-7 from the metagenome in the soil of a mangrove tidal flat in China.

Chitinases play an important role in the process of chitin bioavailability. In this study, we cloned a new chitinase gene and characterized its recombinant protein. The new 1251 bp gene of chitinase (ChiT-7) was cloned from the metagenome of the mangrove tidal flat soil in the city of Zhangzhou in Fujian Province (China) by genome walking. The gene encoded a mature protein with 381 amino acids, which manifested certain sequence similarity (59% identity) to characterized GH18 chitinases. The mature protein of ChiT-7 was successfully expressed in E. coli BL21 (DE3). After purification, the specific activity of the recombinant enzyme was 0.63 U/mg at the optimal pH of 6.0 and the optimal temperature of 45 °C. The rChiT-7 was active over a wide pH range, and the residual enzyme activity reached 80% or higher at 30 °C-50 °C. rChiT-7 hydrolyzed colloidal chitin with (GlcNAc)2 and GlcNAc as the main final products. Structural analysis of ChiT-7 indicated that ChiT-7 could be a processive chitinase. rChiT-7 manifested characteristics analogous to those of fungi and actinomycetes and exhibited sequence homology.

A novel GH16 beta-agarase isolated from a marine bacterium, Microbulbifer sp. BN3 and its characterization and high-level expression in Pichia pastoris.

An agar-degrading bacterium, strain BN3, was isolated from a coastal soil sample collected in Taiwan Strait, China and identified to be a novel species of the genus Microbulbifer. The gene (N3-1) encoding for a novel ß-agarase from the isolate was cloned and sequenced. It encoded a mature protein with 274 amino acids and a calculated molecular mass of 34.3 kDa. The deduced amino acid sequence manifested sequence similarity (61-84% identity) to characterized ß-agarases in the glycoside hydrolase family 16. The recombinant agarase was hyper-produced extracellularly using Pichia pastoris as the host. After induction in a shake flask for 96 h, the yield of recombinant N3-1 protein reached 0.406 mg/mL, and the enzyme activity attained 502.1 U/mL. The enzyme purified by ion exchange chromatography displayed a specific activity of 6447 U/mg at pH 6.0 and 50 °C. The optimal pH and temperature for agarase activity were approximately 6 and 50 °C, respectively. The pattern of agarose hydrolysis showed that the enzyme was an endo-type ß-agarase, capable of hydrolyzing agarose and Gracilaria lemaneiformis, with neoagarobiose and neoagarotetraose as the final main products.

Preparation of bioactive neoagaroligosaccharides through hydrolysis of Gracilaria lemaneiformis agar: A comparative study.

Hydrolysis of Gracilaria lemaneiformis agar by ß-agarase was compared with HCl hydrolysis. The results showed that optimum catalysis conditions for the ß-agarase were pH 7.0 at 45°C. Mass spectroscopy, thin-layer chromatography and GPC results showed that the polymerization degrees of the hydrolysis products by the ß-agarase were mainly four, six and eight (more specific than the hydrolysate by HCl). The enzymatic degradation products of agar were distinctly different from those of HCl hydrolysis in the ratios among galactose and 3,6-anhydro-galactose and sulfate group contents. The NMR spectrometry proved that the products of ß-agarase were neoagaroligosaccharides, which was not found in the agarolytic products by HCl. The neoagarotetraose inhibited tyrosinase activity competitively with the KI value of 16.0mg/ml. Hydroxyl radical-scavenging ability of neoagaroligosaccharides was much greater than that of agar HCl hydrolysate. This work suggests that neoagaroligosaccharide products produced by our ß-agarase could be more effective in function than products from acid hydrolysis.

Quantification of cholesterol in foods using non-aqueous capillary electrophoresis.

A simple method for the rapid quantification of cholesterol in egg yolk and milk by non-aqueous capillary electrophoresis (NACE) is described in this paper. The samples were treated with saponification and then quantified by NACE, in which 100 mM sodium acetate-acetic acid in methanol was employed as the running buffer. The correlation coefficient between the cholesterol concentration and the corresponding peak area was 0.999. The detection limit of cholesterol was 5 microg/ml (twice the signal-to-noise ratio). This method can be used as a routine method for the rapid and sensitive determination of cholesterol in foods.

High-level expression of a sika deer (Cervus nippon) Cu/Zn superoxide dismutase in Pichia pastoris and its characterization.

Production of a sika deer Cu/Zn-SOD was achieved in Pichia pastoris after the reconstituted expression vector pPIC9K was transformed into the strain GS115. By employing Saccharomyces cerevisiae secretion signal peptide (α-factor) under the regulation of the methanol-inducible promoter of the gene of alcohol oxidase 1 (AOX1), sika deer Cu/Zn-SOD with a molecular mass of 16kDa was expressed while recombinant sika deer Cu/Zn-SOD with an activity of 3500U/mL was obtained from a 5L bioreactor. After two successive steps of chromatography on DEAE-650C and Superdex75, recombinant sika deer Cu/Zn-SOD was obtained with 13.8% yield, 14.5-fold purification, and a specific activity of 3447U/mg. Its optimum temperature and optimum pH were 40°C and 7.0, respectively.

[Rapid separation and determination of enterotoxigenic E. coli by capillary zone electrophoresis].

Rapid separation and determination of enterotoxigenic E. coli K88, K99 and 987P with intact cells using capillary zone electrophoresis (CZE) are described. The CZE was performed in a running buffer of 0.05 mol/L Na2CO3-NaHCO3 (pH 9.9), under applied voltage of 14.1 kV and detected by an on-line UV at 210 nm. Results show that K88, K99 and 987P were separated completely. Moreover, each strain presented a characteristic peak and a reproducible retention time (RSD < or = 0.9%). The successful analysis of enterotoxigenic E. coli in the excrement of diarrhea piglets is also discussed. K88 and K99 were detected in the excrement of diarrhea piglets born in 5 d - 6 d, and 30 d - 35 d respectively, and nothing was detected in the excrement of diarrhea piglets born in about 60 d.