Abstract:Experiments have confirmed that tea is effective in preventing cancer, and catechins play the important role, among which EGCG has strong anti-oxidation to inhibit cancer cell proliferation and induce cancer cell apoptosis. EGCG is also one of the most potential natural products for cancer chemoprevention. In this paper, the biological activities of catechins were reviewed. Synergistic mechanism of EGCG with natural anticancer products and clinical reagents was summarized, to provide a reference for further research on cancer chemoprevention mechanism.

Abstract:Microbialtransglutaminase (mTGase), as a high-performance cross-linking enzyme, is often used to improve protein's techno-functional properties, however, more attention should be paid to the potential health problems caused by its protein cross-linking products and its own. Due to the formation of isopeptide bonds, mTGase cross-linking can cause the changes of protein digestibility and immunogenicity. mTGase may act as an autoantigen in patients with celiac disease, causing autoimmune diseases. Therefore, this paper mainly analyzes and discusses the health effects of mTGase cross-linking and the corresponding detection methods, in order to enrich and expand the research field of mTGase and provide reference for the safety application of mTGase.

Abstract:Yeast β-glucan is a component of cell wall structural polysaccharide with various biological functions. However, the poor water solubility of natural yeast β-glucan has limited its application in medical, health food and cosmetic industries. The low molecule β-glucan was prepared to improve the solubility by degradation of yeast β-glucan using β-1,3-glucanase Gly5m and β-1,6-glucanase BT3312. Heterologous expression of β-1,3-glucanase Gly5m and β-1,6-glucanase BT3312 were expressed by Escherichia coli. After induced with 0.2 mmol/L IPTG at 25 ℃, the highest enzyme activities of Gly5m and BT3312 were 1 086 U/mL and 2 355 U/mL, respectively. The solubility of yeast β-glucan separately hydrolyzed by Gly5m or BT3312 increased by 2.6 or 2.4 times. The solubility of yeast β-glucan co-hydrolyzed by Gly5m and BT3312 increased by 3.2 times, and the concentration of water-soluble yeast glucan reached 12.5 g/L. Therefore, Gly5m and BT3312 have important application value in improving the water solubility of yeast glucan and preparing small molecule yeast glucan.

Abstract:The biological coupling method was used in tandem with the enzyme reaction process to synthesize (3R,5S)-6-chloro-2,4,6-trideoxy-erythro-hexonolactone, the statin side-chain intermediate, in 'one-step' bio-synthesis. The recombinant plasmids of Streptococcus suis deoxyribose aldolase gene and Lodderomyces elongisporus alcohol dehydrogenase gene were co-transformedinto the same cell of Escherichia coli BL21 to realize their soluble co-expression.(3R,5S)-6-chloro-2,4,6-trideoxy-erythro-hexonolactone was obtained by the whole cell co-catalysis of 400 mmol/L acetaldehyde and 200 mmol/L chloroacetaldehyde using recombinant E. coli was used as the biocatalysts. In the absence of standard products, mass spectrometry and 1H nuclear magnetic resonance were performed to identifythe products. The final product was determined as the statin side-chain intermediate (3R,5S)-6-chloro-2,4,6-trideoxy-erythro-hexonolactone.

Abstract:Coenzyme Q ₁₀(CoQ₁₀) is a natural antioxidant and cell metabolic activator, possessing several important biological properties, such as eliminating free radicals, maintaining cell membrane permeability, and improving immune function. It is of great value to develop high-yield CoQ₁₀ strain and realize high-efficient synthesis of functional compounds by fermentation. In this work, a potent CoQ₁₀-producing strain YLL-13 was isolated from the silt of a sewage treatment plant. Based on the morphological observation, biochemical characteristics analysis and 16S rDNA sequence alignment, the strain was identified as Rhodobacter sphaeroides and named as R. sphaeroides YLL-13. In further, a high-yield mutant strain was screened by nitrosoguanidine chemical mutagenesisusing R. sphaeroides Yll-13 as the starting strainand Vitamin K3 and p-hydroxybenzoic acid(pHBA) as the compound screening markers. A genetically stable CoQ₁₀mutant with high yield of CoQ₁₀, R. sphaeroides YLL-13-T,was achieved by mutagenesis. This strain had relatively low carotenoid synthesis, high growth rate and high CoQ10 accumulation characteristics. In the studied fermentation system, the biomass, CoQ₁₀ concentration and specific CoQ₁₀ content of the mutant strain YLL-13-T after 120 h fermentation were 83.5 g/L, 1.04 g/L and 12.46 mg/g(DCW) respectively, which were 12.3%, 42.5% and 23.6% higher than those of original wild-type strain, showing a good prospect of industrial application.

Abstract:A β-xylosidase Xln-DT has been cloned and heterologously expressed from the thermophilic bacterium Dictyoglomus thermophilum in the previous studies. A mutant of β-xylosidase Xln-DT with high enzyme activity and excellent enzymatic properties was obtained, in order to improve its vital application value in bio-fuel, food and medicine industries. The key amino acid mutation sites of β-xylosidase Xln-DT were obtained through bioinformatics methods to improve the specific enzyme activity. Amino acids HIS/LEU, PHE/LEU and TRP were introduced at sites 161, 202 and 231, respectively, and the constructed mutant enzymes Xln-DT-202PHE and Xln-DT-202LEU obtained significantly increased β-xylosidase activity. In addition, the enzymatic properties before and after the mutation were analyzed and compared. The expressed enzyme activities of Xln-DT-202PHE and Xln-DT-202LEU were 3.28 and 2.97 times higher than that of Xln-DT, respectively, while their specific activities were 2.86 and 2.54 times higher than that of Xln-DT, respectively. The optimal pH of mutant enzyme Xln-DT-202PHE and Xln-DT-202LEU decreased significantly from pH 6.0 to pH 4.5 and pH 5.0, respectively. The enzyme activity of Xln-DT-202LEU remained unchanged when incubated at 75 ℃ for 2 h, which indicated significantly improved thermal stability compared with that of Xln-DT under 75 ℃. Xln-DT-202PHE and Xln-DT-202LEU could maintain more than 80% of the residual enzyme activity in the range of pH 4.0~7.0 after 24 h incubation. The mutant enzyme not only significantly improved the enzyme activity, but also greatly improved its thermal stability, laying a foundation for its wide application in food thermal processing and other fields.

Abstract:PAPS is the only active sulfonic acid group donor for sulfation in living organisms. Adenylyl-sulfate kinase catalyzes the formation of PAPS and ADP from APS and ATP. In order to achieve the enzymatic synthesis of PAPS, the adenylyl-sulfate kinase encoding gene from Mycobacterium tuberculosis AUSMDU00018547 was codon-optimized and heterologously co-expressed with thioredoxin TrxB to achieve the active and high-level heterologous expression of adenosine phosphorylsulfate kinase (APK) in Escherichia coli BL21(DE3). His-tagged recombinant adenylyl-sulfate kinase was successfully purified. Further enzymatic property analysis results showed that the optimal temperature, pH and the specific enzyme activity of recombinant adenylyl-sulfate kinase were 35 ℃, pH 7.5 and (1.26±0.08) U/mg, respectively. Heterologous expression of adenylyl-sulfate kinase and enzymatic property analysis lay a foundation for the enzymatic synthesis of PAPS in the future.

Abstract:A P.pastoris strain for efficient endo-β-1,3-glucanase expression was constructed to hydrolyze curdlan and produce curdlan oligosaccarides. Firstly, error-prone PCR technology was implemented on endo-β-1,3-glucanase gene from Trichoderma harzianum to construct a mutation library of BGN13.1a. High-throughput screening technology and Congo red plate screening were then employed to obtain high expression P. pastoris strains. Finally, TLC and MALDI-TOF MS were used to analyze the curdlan hydrolysate and their enzymatic properties. The results showed that the activity of endo-β-1,3-glucanase from the successfully screened mutant P. pastoris K827 was improved by 25% compared with that of the original enzyme. The expressed endo-β-1,3-glucanase hydrolyzed curdlan to produce oligosaccharides at 1.492 g/L with a polymerization degree of 2~10. The optimal reaction temperature and pH for endo-β-1,3-glucanase were 50 ℃ and pH 5.5, respectively. The pH stability and temperature stability of mutant K827 were significantly improved compared with the original enzyme. This study could provide experimental basis for the industrialized production and application of endo-β-1,3-glucanase.

Abstract:As a key enzymein the N-glycosylation pathway, mannosyltransferase Alg11 catalyzes the addition of 4^th and 5^th mannose residues to the substrate Man3-GlcNAc2-PP-Dol with the α-1,2 linkage, resulting Man5- GlcNAc2-PP-Dol. The mutations in human ALG11 gene will lead to the corresponding congenital disorders of glycosylation(CDG), namely ALG11-CDG. Currently, more attention was paid to the clinical symptoms in the study of ALG11-CDG, rather than the properties and functions of the mutant Alg11 proteins.This study aimed to investigate the expression of ALG11-CDG mutant proteins and test the activity. Firstly, the wild-type and mutant Alg11 proteins were expressed in human embryonic kidney cells(HEK293) and Saccharomyces cerevisiae. While in both expression systems, the mutant proteins showed great differences in expression level, indicating unstable expression. Alg11 and Alg11 mutated proteins were finally stably andsuccessfully expressed in E.coli, and the activity of mutated proteins was quantitatively analyzed by liquid and mass chromatography. The results showed that the activity of Alg11 and ALG11-CDG mutated proteins decreased to different degrees, which was correlated with the severity of ALG11-CDG disease.The results indicats that: 1)the instability of mutant proteins may cause ALG11-CDG, 2)the in vitro quantitative assay of the mutant proteins expressed in E.coli may provide an alternative method to analyze the severity of ALD11-CDG.

Abstract:Scientists are trying to utilize the production of "artificial meat" ,which based on high-tech, to solve the meat crisis that is approaching in the world today: On the one hand, there is a huge gap in the supply of meat products in the future. On the other hand, the current safety issues of meat, such as hazards caused by food-borne diseases, have caused widespread concern. This article analyzes the production logic of artificial meat, with answers to four basic questions about artificial meat production. Through these, this article not only summarizes the product types, process principles and technical bottlenecks of artificial meat production, but also analyzes and discusses its social significance, including "the priority is to judge quantity or quality", "development of the nature of production and risk prevention", "the relationship between standard formulation and the right to speak and related interests", etc. The purpose of this article is to understand the reality of artificial meat production, grasp the logic of artificial meat production, and establish the confidence of the people in artificial meat production.

Abstract:In the N-glycosylation pathway, the bifunctional mannosyltransferase Alg2 protein catalyzes the assembly of two mannose onto dolichol linked oligosaccharide Man1GlcNAc2-PP-Dol by α-1,3/1,6 linkages to form Man3GlcNAc2-PP-Dol. In this study, the function of human Alg2 protein (hAlg2) in S. cerevisiae cells was explored and its activity in vitro after expressed and purified from E. coli was investigated. The growth of constructed yeast strain w303a-GAL1_pr-ALG2 was suppressed by glucose, while the overexpression of hALG2 gene could rescue such growth defect, confirming the functional homology of hALG2 and ScALG2. In addition, TrxA-hAlg2 was successfully purified from E.coli and its activity in vitro was further tested using Man1GlcNAc2-PP-Phy (PPGn2M1), a natural substrate analogue of hAlg2. LC-MS analysis showed that the purified TrxA-hAlg2 was capable of producing PPGn2M2 and PPGn2M3 from PPGn2M1, providing the foundation for in vitro reaction system of hAlg2 purified from E. coli.

Abstract:The development of cheese products with characteristic flavor is an important means to improve the cheese acceptability of Chinese consumers. Fresh milk was the raw material, and the addition amounts of sucrose, cheese starter and rennet were selected as factors. The processing of Cheddar-like cheese with Saccharomyces cerevisiae were then optimized by single factor and orthogonal experiments based on the main testing indexes of cheese yield and sensory evaluation. Taking sensory evaluation as the main testing index, the optimal processing conditions were verified. The optimum processing conditions of Cheddar-like cheese with Saccharomyces cerevisiae were as follows: sucrose concentration of 50 g/L, cheese starter concentration of 0.02 g/L and rennet concentration of 223.5 IMCU/L. Under optimized conditions, the sensory score of fresh cheese was 76.29 and the cheese presented bright color, firm texture as well as the rich and suitable aroma of milk and wine.