Zhongyuan Li

Rapid biodegradation of aflatoxin B1 by metabolites of Fusarium sp. WCQ3361 with broad working temperature range and excellent thermostability.

BACKGROUNDnContamination of food and feed by aflatoxin B1 (AFB1) poses serious economic and health problems worldwide, so the development of biological methods for effective AFB1 degradation is strongly required.nnnRESULTSnAmong three AFB1-degrading microorganisms isolated from moldy peanut, Fusarium sp. WCQ3361 could remove AFB1 extremely effectively, with a degradation ratio of 70.20% after 1u2009min and 95.38% after 24u2009h. Its degradation ratio was not much affected by temperature change (0-90u2009°C) and it also displayed excellent thermostability, maintaining 99.40% residual activity after boiling for 10u2009min. Since protease K could reduce the AFB1 degradation ratio by 55.15%, it is proposed that the effective component for AFB1 degradation is a protein. The AFB1 degradation ability of Fusarium sp. WCQ3361 was further verified by feed stock detoxification and the MTT test with HepG2 cells. In addition, no degradation products were detected by preliminary liquid chromatography/mass spectrometry, suggesting that AFB1 might be metabolized to products with different chemical characteristics from AFB1.nnnCONCLUSIONnFusarium sp. WCQ3361 is the first reported AFB1 degradation fungus belonging to the genus Fusarium with broad working temperature range, excellent thermostability and high activity, which provides a potential highly useful solution for dealing with AFB1 contamination in the human diet and animal feed.

Gene expression pattern analysis of a recombinant Escherichia coli strain possessing high growth and lycopene production capability when using fructose as carbon source

ObjectiveEscherichia coli K12f-pACLYC has a high capability for growth and lycopene production when using fructose as carbon source and the transcription of genes involved was compared in glucose-grown and fructose-grown cells.ResultsEscherichia coli K12f-pACLYC was grown on 10xa0gxa0fructosexa0l−1 and reached 4.6xa0gxa0DCWxa0l−1 with lycopene at 192xa0mgxa0gxa0DCW−1, values that are 3-fold and 7-fold higher than when growing on glucose. Gene transcription profiles of fructose-grown and glucose-grown cells were compared. 384 differentially expressed genes (DEGs) with fold changesxa0≥4 were identified, and the transcription of genes involved in fructose uptake and metabolism, pyruvate catabolism, tricarboxylic acid cycle and oxidative phosphorylation varied significantly. These changes enhanced the metabolic flux into the Embden–Meyerhof–Parnas pathway and the tricarboxylic acid cylcle and coupled to oxidative phosphorylation. These enhanced activities provide more precursors, cofactors and energy needed for growth lycopene production.ConclusionThe high capability of E. coli K12f-pACLYC for growth and lycopene production when growing on fructose is due to transcriptional regulation, and the relevant genes were identified.

Cloning and Expression of a Novel Xylanase Xyn11-1 from Alkaline Soil

A novel xylanase of family 11 (Xyn11-1) was obtained from the metagenomic DNA of alkaline soil by touchdown-PCR and thermal asymmetric interlaced (TAIL) PCR methods. Xyn11-1 is composed of 645 nucleotides, which encodes a signal peptide of 19 amino acids and a mature protein of 196 amino acids. It is a novel GH11 xylanase sharing the highest identity (79 %) with the reported GH11 xylanase (XP_008721536) in GenBank database. In order to detect its biological activity, the recombinant plasmid xyn11-1-pET28a(+) was constructed and recombinant protein was successfully expressed in heterologous hosts Escherichia coli BL21 (DE3) induced by isopropy-beta-d-thiogalactopyranoside (IPTG). Using the 3,5-dinitrosalicylic acid (DNS) method, the xylanase activity of crude intracellular protein is 11.32 U/mL. The optimal inducing IPTG concentration and inducing temperature was examined at 0.2 mM and 15 °C, respectively.

Galactomannan Degrading Enzymes from the Mannan Utilization Gene Cluster of Alkaliphilic Bacillus sp. N16-5 and Their Synergy on Galactomannan Degradation

Two glycoside hydrolases encoded by the mannan utilization gene cluster of alkaliphilic Bacillus sp. N16-5 were studied. The recombinant Gal27A (rGal27A) hydrolyzed both galactomannans and oligo-galactomannans to release galactose, while the recombinant Man113A (rMan113A) showed poor activity toward galactomannans, but it hydrolyzed manno-oligosaccharides to release mannose and mannobiose. rGal27A showed synergistic interactions with rMan113A and recombinant β-mannanase ManA (rManA), which is also from Bacillus sp. N16-5, in galactomannan degradation. The synergy degree of rGal27A and rManA on hydrolysis of locust bean gum and guar gum was 1.13 and 2.21, respectively, and that of rGal27A and rMan113A reached 2.00 and 2.68. The main products of galactomannan hydrolyzed by rGal27A and rManA simultaneously were galactose, mannose, mannobiose, and mannotriose, while those of galactomannan hydrolyzed by rGal27A and rMan113A were galactose and mannose. The yields of mannose, mannobiose, and mannotriose dramatically increased compared with the hydrolysis in the presence of rManA or rMan113A alone.

Revealing the influence of microbiota on the quality of Pu-erh tea during fermentation process by shotgun metagenomic and metabolomic analysis

Multispecies microbial community in natural solid-state fermentation (SSF) is crucial for the formation of Chinese Pu-erh teas unique quality. However, the association between microbiota and tea quality are still poorly understood. Herein, shotgun metagenomic and metabolomic analysis showed that significant variations in composition of microbiota, collective functional genes, and flavour compounds occurred during SSF process. Furthermore, the formation pathways of the dominant flavours including theabrownin, methoxy-phenolic compound, alcohol and carvone were proposed. Moreover, biological interaction networks analysis among functional core microbiota, functional genes, and dominant flavours indicated Aspergillus was the main flavour-producing microorganism in the early SSF, while many other genera including Bacillus, Rasamsonia, Lichtheimia, Debaryomyces were determined as the functional core microorganism for flavours production in the late SSF. This study provides a perspective for bridging the gap between the microbiota and quality in Pu-erh tea, and benefited for further optimizing production efficiency and product quality.

Expression and bioactivity analysis of TNF30, a TNFα nanobody, in Escherichia coli

ABSTRACT Antibodies and antagonists targeting tumour necrosis factor alpha (TNFα) have become a number of the best-selling anti-inflammatory drugs. The nanobody is a single domain antibody derived from the variable domain of a heavy-chain antibody. However, so far, there are still no reports on the recombinant expression of TNFα nanobodies. Here, TNF30, the key anti-TNFα nanobody domain of ozoralizumab, was cloned, expressed in Escherichia coli, and then purified with the Ni2+-chelating affinity chromatography. The western blot results showed that the recombinant TNF30 has strong binding capability with TNFα. Furtermore, the pharmacological and histopathological analysis in the carrageenan-induced mice paw oedema demonstrated that the recombinant nanobodies had potential anti-inflammatory activity in vivo. These works provided a novel approach for the production of TNF30 in E. coli, and might establish a foundation for the industrial production of the drug and the development of more active derivatives against TNFα in the future.

Bioconversion of conjugated linoleic acid by Lactobacillus plantarum CGMCC8198 supplemented with Acer truncatum bunge seeds oil

Conjugated linoleic acid (CLA) isomers, c9, t11-CLA and t10, c12-CLA, have been proved to exhibit excellent biomedical properties for potential use in anti-cancer applications and in reducing obesity. Acer truncatum Bunge (ATB), which is rich in unsaturated fatty acids, including oleic acid, linoleic acid, and nervonic acid, is a new resource for edible oil. In the present study, we developed a new method for producing two CLA isomers from ATB-seed oil by fermentation using Lactobacillus plantarum CGMCC8198 (LP8198), a novel probiotics strain. Polymerase chain reaction results showed that there was a conserved linoleate isomerase (LIase) gene in LP8198, and its transcription could be induced by ATB-seed oil. Analyses by gas chromatography–mass spectrometry showed that the concentration of c9, t11-CLA and t10, c12-CLA in ATB-seed oil could be increased by about 9- and 2.25-fold, respectively, after being fermented by LP8198.

Soluble expression and purification of heparinase I in Escherichia coli using a hexahistidine-tagged small ubiquitin-like modifier as a fusion partner

ABSTRACT Heparinase has an important application in the preparation of low-molecular-weight heparins and the deheparinization of heparin-treated blood. To increase the soluble expression of heparinase I (HepI) in recombinant Escherichia coli, the hepA gene (coding for HepI) from Flavobacterium heparinum was obtained through chemical synthesis and fused with the gene encoding hexahistidine, small ubiquitin-like modifier (SUMO), a flexible peptide linker (G4S) and a bovine enterokinase site (D4K). The constructed fusion protein (SUMO–HepI) was expressed in E. coli BL21 (DE3), and then purified with Ni2+-chelating affinity chromatography. The fermentation conditions were optimized and the enzymatic properties were also analyzed. As the results showed, the recombinant SUMO–HepI was successfully expressed in E. coli BL21 (DE3) and purified with affinity chromatography. The recombinant protein reached the highest soluble expression when the expression strain was induced by 0.6 mmol/L isopropyl β-D-thiogalactoside and grown at 30 °C with a shaking speed of 150 r/min for 9 h. The fusion protein could exhibit high enzyme activity without requirements of in vitro refolding and SUMO-tag releasing process, and the optimum enzyme activity was obtained at 30 °C, pH 7.0 and 10 mmol/L Ca2+ in the reaction buffer. This work provided a novel simple approach for the soluble expression of HepI in E. coli, and might establish a foundation for the following production and application of HepI in the industry.

A Novel GH10 Xylanase Xyn13-3 from Alkaline Soil: Gene Cloning and Heterogenous Expression

Xylanase, as critical hemicellulose degrading enzyme, it could degrade xylan into oligosaccharides and has been widely used in medicine, paper-making, animal forage, marine functional foods and textile industry. So exploring new xylanase with excellent enzymatic characteristic will be benefit for improving production efficiency and reducing cost. In this study, a novel xylanase of family 10 (Xyn13-3) was obtained from the genomic DNA of alkaline soil by touchdown-PCR and thermal asymmetric interlaced (TAIL) PCR methods, which shares the highest identity (61%) with the reported GH10 xylanase (XP_018659952.1) in GenBank database. Xyn13-3 contained a catalytic motif of GH10 (339 residues). The recombinant plasmid pPIC9-Xyn13-3 was constructed and the recombinant protein was successfully expressed in Pichia pastoris GS115. The special activity of recombinant xylanase was 135.24 U/mg. Exploring the use of novel enzymes from different organisms, with distinct characteristics, could contribute to the development of a highly efficient system for lignocellulose conversion.

Comparative Quantitative Analysis of Probiotic Bacteria During Puer Tea Pile Fermentation

To explore the function of probiotic bacteria in puer tea pile fermentation which was implemented by a symbiosis of many fermenting microbes and enzymes, the species, abundances and dynamics of probiotic bacteria were quantitatively investigated. Using the real-time quantitative PCR (qPCR) primers specifically targeting different genera bacteria, the results showed that Enterococcus spp., Lactococcus spp., Lactobacillus group and Bacillus spp. were all found at 0, 15, 30, and 45 days during the fermentation, but Bifidobacterium spp. was not involved throughout the fermentation. Furthermore, among these four genera, Lactococcus spp. was the predominant probiotic with population of 1.68 × 109–2.78 × 109 copy g−1 in the fermentation, followed by Enterococcus spp. harboring 3.43 × 104–6.25 × 105 copy g−1, and Lactobacillus group and Bacillus spp. had 4.26 × 103–5.17 × 104 and 2.21 × 103–2.31 × 105 copy g−1, respectively. In addition, the dynamics of four genera probiotic bacteria and universal bacteria were explored to show completely different and complex change trends with no significant correlation with pH of tea and each other. Population dynamic studies of probiotic bacteria revealed their ecological feature and importance for puer tea pile fermentation, and also facilitated further exploration of probiotic resources and health benefits of puer tea.