Improving thermostability of Bacillus amyloliquefaciens alpha-amylase by multipoint mutations.

The medium-temperature alpha-amylase of Bacillus amyloliquefaciens is widely used in the food and washing process. Enhancing the thermostability of alpha-amylases and investigating the mechanism of stability are important for enzyme industry development. The optimal temperature and pH of the wild-type BAA and mutant MuBAA (D28E/V118A/S187D/K370 N) were all 60 °C and 6.0, respectively. The mutant MuBAA showed better thermostability at 50 °C and 60 °C, with a specific activity of 206.61 U/mg, which was 99.1% greater than that of the wild-type. By analyzing predicted structures, the improving thermostability of the mutant MuBAA was mainly related to enhanced stabilization of a loop region in domain B via more calcium-binding sites and intramolecular interactions around Asp187. Furthermore, additional intramolecular interactions around sites 28 and 370 in domain A were also beneficial for improving thermostability. Additionally, the decrease of steric hindrance at the active cavity increased the specific activity of the mutant MuBAA. Improving the thermostability of BAA has theoretical reference values for the modification of alpha-amylases.

Considerations regarding affinity determinants for aflatoxin B1 in binding cavity of fungal laccase based on in silico mutational and in vitro verification studies.

Laccase, a multicopper oxidase, is well known for its industrial potentials to remove environmental pollutants due to its low substrate specificity to oxidize phenols and thus catalytic versatility. Many efforts focused on the metabolic mechanism, yet to decipher the structural determinants responsible for the differentiation between substrates. Aflatoxin B1 (AFB1), a new substrate for laccase, is a mycotoxin with a formidable environmental threat to public health and food safety. In the present study, we combined biochemical, in silico mutational and molecular-docking data to gain an insight to the function of key residues in the active cavity close to the T1 copper site in a characterized recombinant laccase from Cerrena unicolor (rCuL). Kinetic data for computer-assisted virtual mutants established the binding affinity of hydrogen bonds and residues (Asn336, Asp207, Val391, and Thr165) in rCuL to AFB1. The augmented binding affinity to AFB1 may be related to the conformational rearrangements of the laccase and its ability to hydrogen-bond with the substrate. Furthermore, the optimal pH and temperature for rCuL and variants mediated AFB1 degradation may depend on their pH stability and thermostability. Our findings reinforce the importance of the structure-function relationship of fungal laccases in degrading AFB1, providing mechanistic guidance for future biocatalyst and bioengineering applications.

Flow-through enzymatic reactors using polymer monoliths: From motivation to application.

The application of monolithic materials as carriers for enzymes has rapidly expanded to the realization of flow-through analysis and bioconversion processes. This expansion is partly attributed to the absence from diffusion limitation in many monoliths-based enzyme reactors. Particularly, the relatively ease of introducing functional groups renders polymer monoliths attractive as enzyme carriers. After summarizing the motivation to develop enzymatic reactors using polymer monoliths, this review reports the most recent applications of such reactors. Besides, the present review focuses on the crucial characteristics of polymer monoliths affecting the immobilization of enzymes and the processing parameters dictating the performance of the resulting enzymatic reactors. This review is intended to provide a guideline for designing and applying flow-through enzymatic reactors using polymer monoliths.

A New Laccase of Lac 2 from the White Rot Fungus Cerrena unicolor 6884 and Lac 2-Mediated Degradation of Aflatoxin B1.

Aflatoxin B1 (AFB1) is a known toxic human carcinogen and can be detoxified by laccases, which are multicopper oxidases that convert several environmental pollutants and toxins. In this study, a new laccase that could catalyze AFB1 degradation was purified and identified from the white-rot fungus Cerrena unicolor 6884. The laccase was purified using (NH4)2SO4 precipitation and anion exchange chromatography, and then identified as Lac 2 through zymogram and UHPLC-MS/MS based on the Illumina transcriptome analysis of C. unicolor 6884. Six putative laccase protein sequences were obtained via functional annotation. The lac 2 cDNA encoding a full-length protein of 512 amino acids was cloned and sequenced to expand the fungus laccase gene library for AFB1 detoxification. AFB1 degradation by Lac 2 was conducted in vitro at pH 7.0 and 45 °C for 24 h. The half-life of AFB1 degradation catalyzed by Lac 2 was 5.16 h. Acetosyringone (AS), Syrinagaldehyde (SA) and [2,2' -azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid)] (ABTS) at 1 mM concentration seemed to be similar mediators for strongly enhancing AFB1 degradation by Lac 2. The product of AFB1 degradation catalyzed by Lac 2 was traced and identified to be Aflatoxin Q1 (AFQ1) based on mass spectrometry data. These findings are promising for a possible application of Lac 2 as a new aflatoxin oxidase in degrading AFB1 present in food and feeds.

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.

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.

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.

[Enzymatic hydrolysis of antler and properties of hydrolysates].

Lyophylized antler powder was hydrolyzed by pepsin and trypsin separately and also simultaneously to give hydrolysates with special physical activities. Complete hydrolysis peptides with MW lower than 1 x 10(3) were collected for assay of angiotensin I-converting enzyme (ACE) inhibitory activity, antioxidant activity and proliferative activity toward UMR-106 osteoblast cells. The results of the experiments revealed that all hydrolysates exhibited potent hydroxyl radical scavenging activity with an IC50 value less than 1 mg/ml which was much lower than the value of 5.5 g x L(-1) for vitamin C. The peptic and peptic tryptic hydrolysates demonstrated strong angiotensin I-converting enzyme (ACE) inhibitory activity. The tryptic hydrolysate increased the proliferation of the UMR-106 cells by 73.43%. The results verified the traditional use of antler in bone-strengthening, anti-aging. The exploratory studies on the ACE inhibitory activity of antler hydrolysates indicated that the hydrolysates might be potentially useful in prevention and treatment of hypertension. Further purification of peptides contributing to the antioxidant activity, angiotensin I-converting enzyme-inhibitory activity and proliferative activity toward osteoblasts from antler hydrolysates is warranted.

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.

[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.