Abstract:The rare monosaccharide D-allulose is a C-3 epimer of D-fructose, which is mainly produced through the isomerization of D-fructose by D-tagatose 3-epimerases (DTEases) or D-allulose 3-epimerases (DAEases). D-Allulose can not only be used as a food ingredient and dietary supplement, but also exhibits a variety of physiological functions and health benefits such as insulin resistance improvement, antioxidant enhancement and hypoglycemic controls. Therefore, D-allulose is of great value in development and research as a healthy alternative for conventional high-energy sugars such as sucrose and fructose. This review provides a systematic analysis of physicochemical property, existing sources, metabolism in vivo, physiological functions, applications and recent processing technologies of D-allulose. The existing problems and proposed solutions in the production of D-allulose are discussed. A green and recyclable process technology for D-allulose production is also proposed for low waste generation, low energy consumption and high conversion.

Abstract:Food safety problems have emerged in endlessly in recent years. Foodborne pathogens are one of the main factors causing food safety outbreaks, which seriously endanger human health. The common pathogens causing foodborne diseases are Escherichia coli, Salmonella spp, Staphylococcus aureus, Listeria monocytogenes and so on. In the past few decades, due to the excessive use of antibiotics in medical, aquaculture and other fields, antimicrobial resistance has become increasingly serious and has further aggravated the potential risk of foodborne pathogens. Although there is a surveillance network for the antimicrobial resistance of foodborne pathogens in China at the national level, it is not available at the provincial one. Therefore, this paper aims to summarize the antimicrobial resistance of foodborne pathogens in Shaanxi Province, in order to provide a basis for antimicrobial resistance monitoring of foodborne pathogens.

Abstract:Red beet is a kind of edible vegetable with high nutritional value, which contains a large amount of betalains. Betalains are water-soluble, nitrogen-containing quaternary amine alkaloid with a core structure of betalamic acid. According to the chemical structure, betalains are divided into betacyanins and betaxanthins. As a kind of edible natural pigment, betalains have been applied to a lot of foods, however, their applications in some foods, nutritional health foods and cosmetics are limited owing to their poor stability. The composition and structure of betalains were reviewed, the effects of pH, light, temperature, oxygen and degrading enzymes on the content and color of betalains were discussed in details. And it was also summarized that the chemical changes occurred at easy degradable positions of betacyanins for deglycosylation, decarboxylation, dehydrogenation, aldimine bond hydrolysis and isomerization. This review aims to provide the references for further study on the stability of betalains and to expand the application fields of red beet and betalains.

Abstract:Ectoine is widely used in medicine, cosmetics and other fields. This study aimed to construct recombinant Escherichia coli strain to synthesize ectoine to solve the dependence of wild-type strain on high salt environment. Ectoine synthesis encoding gene cluster ectABC was cloned from Halomonas elongata ATCC 33173 and reconstructed in Escherichia coli. In order to confirm a suitable host, four commonly used E. coli strains, i.e., E. coli W3110, E. coli DH5α and E. coli (asp) and E. coli BL21(DE3) were compared, and E. coli BL21(DE3) was found to be the best host for ectoine synthesis(185.23 mg/L). Further analysis of the effect of expression systems with different copy numbers on ectoine synthesis was carried out, and the results showed that pRSFDuet-1 with high copy number generated the highest titer of ectoine(267.3 mg/L). On this basis, the ribosomal binding site optimization strategy was introduced to optimize the expression of three enzymes including EctA, EctB and EctC, producing ectoine to 521.24 mg/L. To increase the supply of precursors aspartate and aspartate semialdehyde, aspartate kinase, aspartate hemialdehyde dehydrogenase, phosphoenolpyruvate carboxylase, aspartate aminotransferase and aspartate lyase were overexpressed. The results demonstrated that the single overexpression of aspartate kinase was beneficial to ectoine biosynthesis and the production was eventually enhanced to 551.24 mg/L. The present study provides a new strategy to construct ectoine-producing strain.

Abstract:This work aimed to construct the Escherichia coli expression system, obtain the optimum expression conditions and establish a robust purification method of single-chain antibody PD0721 against epidermal growth factor receptor variant type Ⅲ(EGFRvⅢ). The recombinant plasmid PD0721-pET-22(+) was constructed and transformed into Escherichia coli BL 21.The optimum concentration of isopropyl-β-D-thiogalactoside (IPTG), induction temperature,induction time and cell concentration (OD₆₀₀) of the PD0721 expression were investigated by SDS-PAGE method. Ni²⁺ affinity chromatography was employed to purify PD0721 protein. Western Blotting and N-terminal sequencing were used to identify PD0721 protein. Colony PCR and agarose gel electrophoresis confirmed the successful construction of PD0721-pET-22b(+) recombinant plasmid and PD0721 recombinant Escherichia coli BL 21. The optimum IPTG concentration, temperature, induction time and OD₆₀₀ of PD0721 expression were 0.6 μmol/L, 15 ℃, 12 h and 0.6. PD0721 protein of high purity could be obtained when the concentration of imidazole was 150 mmol/L during the Ni²⁺ affinity purification. SDS-PAGE and Western Blotting results showed that the molecular weight of the recombinant target protein was about 31 000, consistent with the theoretical molecular weight. N-terminal sequencing of the protein was also consistent with the theoretical sequence of PD0721. In summary, the in vitro prokaryotic expression system of anti-EGFRvⅢ antibody PD0721 was successfully constructed. The optimum expression conditions were obtained, and a feasible method for purifying PD0721 protein was established.

Abstract:Mycobacterium neoaurum JC-12 is an important strain to transform phytosterolsto steroid hormones. Traditional gene editing methods are inefficient, long cycles, complicated to manipulate and resistance labels required. The successful application of CRISPR(clustered regular interspaced short palindromic repeats)/Cas9 system in many non-model strains provides a referencefor this study. A CRISPR-Cas system for genome editing of Mycobacterium neoaurum JC-12 was constructed by combining non-homologous end joining(NHEJ). Targeted sgRNAs were respectively designed for the knockout and transcriptional activation of anthrone-dehydrogenase(KSDD) synthesizing androst-1,4-diene-3,17-dione(ADD) by catalyzing 4-androstene-3,17-dione(AD)on the Mycobacterium neoaurum JC-12 genome. The results showed that the ksdd gene was successfully knocked out and the enzyme activity of KSDD decreased by 80%. The AD yield of the mutant strain after 96 h was 30% higher than that of the wild-type strains, and no ADD was produced. Furthermore, the transcriptional regulation results indicated that the transcriptional level of ksdd at -29 target site decreased by 38% compared to that of the control, while the transcriptional level of target-83 and-141 increased 2.24 and 3.23 times, respectively. However, the corresponding AD and ADD yields did not change significantly when the -29 site was activated. Notably, there was 26% and 25% increase in the yields of AD and ADD respectively at the -83 site, and 29.5% and 36% increase at the -141 site. This study provides amore extensive and convenient way to further implement genome level metabolic engineering in Mycobacterium neoaurum.

Abstract:A immunoassay platform for Cd²⁺ was constructed based on fluorescence resonance energy transfer (FRET) between the up-conversion nanoparticles (UCNPs) and the gold nanoparticles (AuNPs) and the recognition of antigen and antibody. Water-soluble UCNPs modified with Cd-antigen were prepared as the energy donor probe, and AuNPs modified by Cd-antibody were synthesized as the energy receptor probe. The immune binding of antigen-antibody triggered FRET process of UCNPs and AuNPs, causing fluorescence quenching of UCNPs. In the presence of Cd²⁺, Cd²⁺ competitively bound to AuNPs-antibody with UCNPs-antigen, which inhibited the FRET process, and the fluorescence signal value enhanced with the increase of Cd²⁺ concentration. The results showed that the detection range was from 0.01 ng/mL to 10 ng/mL, and the detection limit was 0.01 ng/mL. The method was applied to the detection of Cd²⁺ in tap water samples. At the spiked level of 0.1, 1, 10 ng/mL, the recoveries for Cd²⁺ ranged from 98% to 109% with the relative standard deviations (RSDs) of 3.4% to 4.1%.

Abstract:Saccharomyces cerevisiae, which has vegetative yeast state and spore state under different nutrient conditions, is an important probiotics for human beings and animals. In order to investigate the effect of yeast spores on immune system, the response mechanism of macrophages to yeast spores was preliminarily explored. By comparing the endocytic efficiency of macrophages on vegetative yeasts and wild type spores, it was found that the endocytosis of macrophage to wild type spores was more efficient. Syk or PI3K inhibitor inhibition experiments revealed that the endocytosis of wild type yeast spores and vegetative yeasts were both dependent on the Syk pathway. The endocytosis of wild type yeast spores was more dependent on the PI3K pathway, indicating that the endocytosis of yeast spore and vegetative yeasts was both receptor mediated phagocytosis, however, their phagocytosis pathways were different. High salt and protease treatment experiments demonstrated that the ligands of wild type spores were tightly bound to the spore wall, and the ligands were not protein. Sugar inhibitors competition experiments showed that the macrophages phagocytosis of vegetative yeast, not wild type spores, was inhibited by glucan. The spore phagocytosis of wild-type spores and mutants further showed that the phagocytosis of wild type spores was related to the structure of dityrosine layer. This study demonstrated that the signaling pathway and ligand molecules related to phagocytosis of wild type yeast spores were different from those of vegetative yeasts, which could be regarded as a theoretical reference for the follow-up immune application of Saccharomyces cerevisiae.

Abstract:(S)-N-Boc-3-hydroxypiperidine ((S)-NBHP) is a key chiral intermediate for the synthesis of ibrutinib treating mantle cell lymphoma. Asymmetric reduction of N-Boc-3-piperidone (NBPO) catalyzed by alcohol dehydrogenase is the most promising method for (S)-NBHP synthesis. After screening of the alcohol dehydrogenase library, KpADH from Kluyveromyces polyspora showed excellent catalytic efficiency on NBPO, with specific activity of pure enzyme as high as 83.9 U/mg and the product enantiomeric excess value of 97.0% (S). Coupled cofactor regeneration reaction was established by introducing glucose dehydrogenase, and the effects of monoaquous phase, biphasic phase, ionic liquids and different substrate concentrations on the conversion were explored. The results indicated that high concentration product (S)-NBHP had inhibitory effect on KpADH, and the inhibitory kinetic analysis showed that it belonged to non-competitive inhibition. The inhibition of (S)-NBHP on KpADH catalytic reaction could be reduced and the conversion efficiency could be improved by using single monoaquous phase catalysis. The inhibition of asymmetric reduction of NBPO catalyzed by alcohol dehydrogenase was studied, which could provide guidance for the enzymatic preparation of (S)-NBHP.

Abstract:Type II restriction endonuclease NcoI derived from Nocardia corallina is one of the enzymes commonly used in genetic engineering. However, the three-dimensional structure of NcoI has not been determined, and there is no theoretical guidance for the genetic modification of NcoI. To analyze the crystal structure of NcoI, wild-type NNcoI and its selenoprotein Se-NcoI with purity > 95% were obtained by high efficient recombinant expression and purification using E. coli system. Mass spectrometric analysis showed that all sulfur atoms in Se-NcoI were successfully substituted by selenium atoms. Enzyme digestion experiments showed that selenoproteins had similar restriction enzyme activity with the wild-type. The crystallization conditions of wild type NcoI were screened by sitting-drop method. Needle-like crystals were grown in three conditions and granular crystals were grown in another condition. A set of data with a resolution of 0.8 nm was collected by preliminary X-ray diffraction analysis, which provided a basis for further analysis of the three-dimensional structure of NcoI.

Abstract:Cholesterol can be converted by Burkholderia cepacia into a series of steroid hormone prodrugs. A conversion product of cholesterol with higher medicinal value was identified as cholest-4-ene-3,6-dione by mass spectrometry, infrared spectrum and nuclear magnetic resonance spectroscopy, however, the hydrophobicity of cholesterol resulted in a relatively low conversion yield. Cyclodextrin was used to increase the conversion yield of the target product, and the effects of cyclodextrin on cell growth and substrate embedding were investigated. Multiple cyclodextrins were added to the conversion system and the molar ratio of methyl-β-cyclodextrin to cholesterol was 1∶1.The results showed that the molar conversion yield of cholest-4-ene-3,6-dione was significantly improved when methyl-β-cyclodextrin or hydroxyethyl-β-cyclodextrin was added, which was 27.55 times and 37.95 times higher than the control group, respectively. The addition ratio of hydroxyethyl-β-cyclodextrin and cholesterol was further optimized. And it was found that when the molar ratio of the two substances was 2∶1, the conversion yield of cholest-4-ene-3,6-dione was 1.59 times as high as that achieved at the molar ratio of 1∶1. Infrared spectroscopy was used to characterize the inclusion complexation of 2 mol hydroxyethyl-β-cyclodextrin with 1 mol substrate cholesterol. It was found that the A ring of the steroid nucleus, 26-C and 27-C of the side chain of cholesterol could be effectively wrapped by 2 mol of hydroxyethyl-β-cyclodextrin, respectively, to form a stable inclusion. Cholesterol could be effectively coated by hydroxyethyl-β-cyclodextrin, and the yield of cholester-4-ene-3,6-dione was improved, which could be used as a reference for the microbial transformation of cholest-4-ene-3,6-dione.

Abstract:The effects of soybean peptides from soybean protein isolate(SPI) hydrolyzed by the recombinant Aspergillus sojae alkaline protease (rAp) on immunity function and antioxidationin mice was explored. Soybean protein isolate (SPI) was hydrolyzed by induced expression of purified rAp, and then the hydrolysate was administered to mice. The effects of its hydrolysate on immune organ index, serum lysozyme activity and serum anti-bacterial activity of mice were determined. The antioxidant activity of GSH-PX and CAT, and the content of MDA in mice were measured based on the anti-oxidation test carried out in mice. Furthermore, the effect of soybean peptides on the activity of GSH-PX, CAT and the content of MDA in mice blood were determined. The results indicated that soybean peptide had no significant effect on immune organ index of mice, but it had significant improvement on serum lysozyme activity and serum anti-bacterial activity of mice. It had no significant effect on serum GSH-PX activity of mice, however, CAT activity and MDA content in serum were significantly increased. In conclusion, the soy peptides hydrolyzed by rAp could enhance the immune function and antioxidant activity of mice, providing experimental basis for high value utilization of soybean in health food industry.

Abstract:The mutant of Saccharomyces cerevisiae XM2-9 has been used for the production of Se-methylselenocysteine by fed-batch fermentation. First, the optimal medium and sulfur concentration were obtained by batch fermentation. The specific growth rate of cell at different initial disodium selenite concentrations was calculated. Meanwhile, the specific selenium consumption rate (SSCR) was calculated under different concentrations of magnesium sulfate, and the highest SSCR of 0.47 mg/(g·h) was achieved. Afterward, Se-methylselenocysteine production was carried out using a fed-batch fermentation mode. At first, the glucose medium was used as the feeding material. Then three flow rates were selected to carry out the constant flow test according to the calculation of the specific growth rate of the yeast cell. Simultaneously, the flow rate of disodium selenite solution was controlled via SSCR. The highest organic selenium productivity of 10.16 mg/(L·h) was achieved when the flow rate of glucose medium was 0.8 L/h. It was found that the exponential flow mode was better than the constant speed flow mode based on the study on glucose flow. The experimental results showed that the exponential flow rate set at μ=0.08 h^-1 resulted in a maximum production intensity of organic selenium, which reached 12.55 mg/(L·h). Under the optimized conditions, the final concentration of yeast cell was 35.6 g/L, and the organic selenium content in the cell was 4 937 μg/g. Among them, 72% of total selenium compounds were in the form of Se-methylselenocystein (3 555 μg/g). The final concentration of Se-methylselenocystein reached 8 189 μg/g based on the molecular weight conversion.