Yajian Song

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

BACKGROUND Contamination 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. RESULTS Among three AFB1-degrading microorganisms isolated from moldy peanut, Fusarium sp. WCQ3361 could remove AFB1 extremely effectively, with a degradation ratio of 70.20% after 1 min and 95.38% after 24 h. Its degradation ratio was not much affected by temperature change (0-90 °C) and it also displayed excellent thermostability, maintaining 99.40% residual activity after boiling for 10 min. 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. CONCLUSION Fusarium 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.

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.

Transcriptional regulation of the mannan utilization genes in the alkaliphilic Bacillus sp. N16-5

&NA; Bacillus sp. N16‐5 is an alkaliphile with a great ability to utilize mannan. Its mannan utilization gene cluster has been identified in a previous study. The ManR protein encoded by the cluster was predicted to be a LacI family regulator, and the transcription level of the mannan utilization gene cluster was upregulated after the manR gene was deleted, indicating that ManR is the repressor of this cluster. The transcription of the related genes was downregulated when manH, encoding the extracellular substrate‐binding domain of the manno‐oligosaccharide transporter, was deleted. Furthermore, isothermal titration calorimetry revealed that mannotetraose and mannopentose are ligands of ManR. These results all corroborate the hypothesis that the mannan utilization gene cluster is repressed by the transcription regulator ManR, and that the repression is removed when it binds to manno‐oligosaccharides, which are generated by mannan degradation and transported into the cell by a specific transporter. &NA; Graphical Abstract Figure. Mannan utilization gene cluster of Bacillus sp. N16‐5 is repressed by the transcription regulator ManR whose ligands are manno‐oligosaccharides.

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.

Molecular and biochemical characterization of a novel cold-active and metal ion-tolerant GH10 xylanase from frozen soil

ABSTRACT A novel xylanase gene of family 10 (xyn27) was obtained from the genomic DNA of frozen soil of Daxinganling in China by Touch-down polymerase chain reaction (PCR) and thermal asymmetric interlaced (TAIL) PCR methods. Xyn27 contained a putative signal peptide (20 residues) and a catalytic motif of GH10 (327 residues) and shared the highest similarity (83%) with the reported GH10 xylanase (XM_003662144). The recombinant xyn27 was successfully expressed in Pichia pastoris GS115 and the optimal induction condition was 30 °C for 48 h. Xyn27 was demonstrated to be a cold-active xylanase, which shows its highest activity at 35 °C and still had 60.25%, 38.70% and 10.8% relative activity at 20 °C, 10 °C and 0 °C. Further analysis showed that Xyn27 has fewer arginine and more alanine residues compared with its mesophilic or thermophilic counterparts. The optimal pH of Xyn27 was 7.0, and it was stable after incubating under the pH range from 3.0 to 9.0 for 1 h. Besides, Xyn27 exhibited superior metal ion tolerance than other GH10 cold-active xylanases, being tolerant to most metal ions and organic solvents, and significantly enhanced by Ca2+, Mn2+ and Zn2+ metal ions. In addition, the Km, Vmax, kcat and kcat Km−1 against beechwood xylan were 13.42 mg mL−1, 9.07 μmol min−1 mg−1, 192.98 min−1 and 14.38 mL min−1 mg−1, and Xyn27 could completely degrade xylan into xylobiose. The features of cold activity and metal-ion tolerance suggested that xylanase Xyn27 could have potential application in basic research and various industries like food possessing.

Analysis on Acid, Bile, and Heat Tolerance of Probiotics Strains in Maca-Probiotics Granule

Maca and probiotics are benefit to people. In order to combine the functions of maca and probiotics, a maca-probiotics compound granule was prepared in our previous study. However, whether the probiotics could survive well in vivo has been remained unclear. To address this problem, the present study analyzed the acid resistance, salt tolerance and heat tolerance of the five probiotics strains in the compound granule. The results showed that the number of viable bacteria would be decreased when the maca-probiotics compound granule was treated with low pH (pH ≤ 3.5) for 4 h or 0.05–0.3% bile salts for 2 h, but there still have a certain survivors could resist these rigorous conditions. The number of survivors began to decrease after 2 h at pH 3.0. Besides, when the product was treated with different temperature (25–52 °C) for 10 min, the reduction in the number of viable bacteria would be accelerated significantly when the temperature exceed 47 °C. These results suggested that the probiotics strains in the maca-probiotics compound granule still have acceptable resistance ability to acid, bile salts and heat, indicating that they could exhibite expected functions after oral administration. In addition, maca-probiotics compound granule should be stored at low temperature and be taken after meals with water below 47 °C.

Isolation and 16SrDNA Identification of Bacteria from Traditional Kazak Dairy Products

In this study, we collected 6 dairy samples from Xinyuan County, Yili Kazakh Autonomous Prefecture, Xinjiang Uygur Autonomous Region, China. These samples are all traditional Kazak dairy product including ghee, dry yogurt, butter, yogurt milk cheese and red knots. Total 22 strains were isolated and identified by 16SrDNA sequencing and their phylogenetic tree was constructed according to the 16SrDNA sequences. These strains belong to 5 genera, including Lactobacillus, Enterococcus, Leuconostoc, Acetobacter, and Bacillus. Among them Lactobacillus and Enterococcus contain most abundant strains, and L. plantarum and A. orientalis were the most widespread strains. L. plantarum A1-3-15 was found to exhibit good bile salt and acid tolerance. This study indicated that the traditional Kazak dairy products exhibit good biodiversity and are good resources of probiotics.

Construction of L-tert-Leucine Producing Strain by Expressing Heterologous Leucine Dehydrogenase and Formate Dehydrogenase in Escherichia coli

L-tert-Leucine is an unnatural amino acid that is a key intermediate for the synthesis of several important drugs. The L-tert-Leucine synthesis can be performed continuously by the collaboration of leucine dehydrogenase and formate dehydrogenase. In this study, recombinant strains of Escherichia coli expressing leucine dehydrogenase (LeuDH) and formate dehydrogenase (FDH), respectively, and the strain co-expressing the two enzymes were constructed. The activity for the two enzymes of the cell extraction from different recombinant strains was determined. L-tert-Leucine was successfully synthesized by the recombinant strains, and the yield in different conditions was compared. The production of L-tert-Leucine was the highest when cell extraction of strains containing pLeuDH and pFDH, 1 mL cells extract could produce 4.5 mg L-tert-Leucine, while 1 mL whole cells could only produce 1.05 mg L-tert-Leucine. The yield of L-tert-Leucine was 3.375 mg/mL cell extraction of the strain containing pLeuDHFDH when NAD was added, while the yield fell to 1.635 mg/mL when the whole cell was used.