Abstract:With the establishment and improvement of food traceability and security system，it becomes popular that using exogenous DNA as internal standards for food tracing and fake detection. However，due to nucleic acid damaging factors in food products，such as nuclease and acidic environment，low stability of DNA strongly restricts its application as food tracing standards. In this study，the stability of DNA-polycation complexes was investigated in 8 kinds of liquid foods. The results showed that polycations，especially chitosan，significantly improved the stability of DNA in alcoholic drinks，milk and sour drinks. This study indicates that DNA-chitosan complex is potential to show broad application prospect as internal standards in food traceability system.

Abstract:To improve the thermal stability of creatinase（EC 3.5.3.3，CRE)，the "hot-spot" Lys195 in Arthrobacter nicotianae 23710 CRE is identified by sequence alignment，and saturation mutagenesis is conducted at this site. In contrast to wild-type enzyme，the mutants K195V，K195T，K195C，and K195L exhibited 260%，230%，60%，and 20% increase in half-life at 50 ℃，respectively；the specific activity of K195V and K195C are respectively increased by 80.7% and 88.2%. Furthermore，the catalytic efficiency（k_cat/K_m）of mutants K195V，K195T，K195C，and K195L are increased by 131%，218%，83% and 100%，respectively. As indicated by structure analysis，the number of hydrogen bonds of the K195V，K195T，and K195L are increased by 7，12 and 13 in comparison with the wild-type enzyme，respectively. The results indicated mutations at Lys195 could affect the thermal stability and catalytic efficiency of CRE，and the increase in hydrogen bonds may account for the improved thermal stability of CRE.

Abstract:The piggyBac（PB） transposon is a TTAA-specific transposon isolated from the cabbage looper moth，Trichoplusia ni. The PB transposon system works in various eukaryotic cells，and is used as a tool for insertional mutagenesis. We here developed the PB transposon screening system for in vivo mutagenesis in the budding yeast Saccharomyces cerevisiae. We established a system for PB-based mutagenesis consisting of a yeast strain containing a PB element，which was inserted in the ADE2 gene coding sequence，and expressing the PB transposase gene under a galactose-inducible promoter. The PB element was precisely excised from and re-inserted into the genome by PBtransposase expression in the strain. The PB element keeps one copy during transposition，and only one insertion site was found in each mutant cell. The DNA region containing the inserted PB elements can be easily identified. This study suggests our PB-based screening system is useful for isolating genes required for phenotypes of interest in budding yeast.

Abstract:Based on the potential hazards of lipoxygenase（LOX） activity to host strain，three antioxidases（two superoxide dismutases and a catalase） are respectively co-expressed to improve the expression of lipoxygenase expression in Escherichia coli Rosetta（DE3）. The superoxide dismutase genes（sodB and sodM） from P. aeruginosa BBE and catalase gene（katE） from E. coli are cloned into expression plasmid pRSFDuet-1，yielding the plasmids pRSF-sodB，pRSF-sodM and pRSF-katE. The recombinant plasmids are transformed into N6（a LOX expressing strain generated by E. coli） to obtain strains N6-B，N6-M and N6-K，respectively. Under the induction with 1 mmol/mL ITPG at 20 ℃，the total LOX activities of N6-B，N6-M and N6-K reach 21.6，28.1 and 7.1 U/mL respectively. The yield of LOX in N6-B and N6-M is increased by 83% and 138% in contrast to N6（11.8 U/mL），respectively. The optimized induction by using orthogonal test is as follow：OD₆₀₀=2.5，IPTG=2 mmol/mL，and inducing temperature is 20 ℃. The results show that the co-expression of superoxide dismutases could effectively improve the expression of LOX in E. coli，which provides a new idea for efficient heterologous expression of this enzyme.

Abstract:To improve the production of cordycepin，the experiments were conducted to optimize the production of cordycepin by solid-state fermentation of Cordyceps militaris. Through a series of single-factor experiments，rice was the fermentation substrate，glucose and soybean flour were the optimum carbon source and nitrogen source respectively，the optimum medium compositions and culture conditions were：rice 30 g（particle size 0.90~1.25 mm），solid-liquid ratio（m/v）1∶1.5，glucose 3%（based on substrate，the same below），soybean flour 2%，wheat bran 1%，inoculation amount was 6 mL，seed age was 2 d，fermentation time was 12 d. The optimization results are that cordycepin production reached 4.69%，which is about 6.34 times compared with the initial level（0.74%）.

Abstract:Performance and fouling of forward osmosis（FO） membranes were investigated in an anaerobic osmotic membrane bioreactor assisted with microfiltration membrane（AnMF-OMBR） treating the synthetic domestic wastewater. The results indicated that the salinity in the reactor could effectively controlled at a level of about 3 mS/cm with the help of MF membrane. The thin-film composite polyamide FO（TFC-FO） membrane had an excellent rejection for organic matters and phosphorus，while had a limited rejection for ammonia nitrogen. The water flux of TFC-FO membrane declined from an initial value of 7.94 LMH to a final value of 2 LMH during 30 days operation of the AnMF-OMBR. Based on the stable salinity in the reactor，the flux decline was mainly due to the membrane fouling，which was composed of organic fouling，inorganic fouling and biofouling. Compared to the inorganic fouling，organic fouling and biofouling played a more important role in the fouling of TFC-FO membrane. The analyses of confocal laser scanning microscope（CLSM） indicated that total cells，proteins and β-D-glucopyranose polysaccharides were the dominant foulants. Moreover，the physical backwashing cannot recover the permeability of the fouled TFC-FO membrane，implying that the irreversible fouling was the main membrane fouling.

Abstract:In this paper，the D-mannose-modified amphiphilic β-cyclodextrin（C₃-CD-Man₇） and a three-arm adamantine-conjugated BODIPY photosensitizer（BTA） are synthesized. Because of the presence of supramolecular host-guest interactions between β-cyclodextrin and adamantane，BTA is loaded into the hydrophobic cavity of micelles self-assembled from C3-CD-Man₇，leading to the formation of BTA-loaded micelles（BTA@C₃-CD-Man₇）. The size，morphology，and stability of BTA@C₃-CD-Man₇are characterized by transmission electron microscopy and dynamic light scattering. The cellular uptake，PDT effect，and phototoxicity of BTA@C₃-CD-Man₇ are investigated using the MTT assay. The results show that BTA@C₃-CD-Man₇ has uniform size distribution and excellent stability in aqueous solution. More importantly，BTA@C₃-CD-Man₇ could be specifically internalized by MDA-MB-231 breast cancer cells that overexpress mannose receptors on cell surfaces. Thus，with 665 nm LED irradiation，the enhanced phototoxicity of BTA@C3-CD-Man7 showed their potential for targeted photodynamic therapy applications.

Abstract:The effects of zinc-resistant strain Penicillium janthinellum BC109-2 on the promoting of zinc uptake in soil by Indian mustard were evaluated. The promoting solubility of insoluble zinc was analyzed. The abilities of producing siderophore and indole acetic acid（IAA），activities of amino cyclopropane-1-carboxylic acid（ACC） deaminase and the ability of phosphate dissolution were determined. Through the root elongation experiment of Indian mustard seed and pot experiment，the effects of zinc resistant strain BC109-2 on plant growth and the efficiency of zinc uptake were evaluated. The results showed that the strain BC109-2 had the obvious ability of dissolving zinc carbonate. Compared with the control treatment，the strain BC109-2 made the water-soluble Zn content in nutrient solution increase by 213% and the pH values in nutrient solution decrease from 7.0 to 5.7. In addition，this strain could produce IAA and siderophore and had ACC deaminase activity and phosphorus dissolving ability，which indicated that strain BC109-2 was a plant growth promoting rhizobacteria（PGPR）. The strain promoted the root elongation of Indian mustard. The root elongation ratio was between 4.14%~44.83%. The strain induced the increase of India mustard biomass in different degrees. The zinc accumulation in India mustard inoculated with the strain BC109-2 increased by 1.01~1.46 times compared with the control. This study demonstrated that Penicillium janthinellum BC109-2 possessed a variety of growth promoting properties and had good growth-promoting effect on India mustard. The PGPR BC109-2 might be used for enhancing remediation efficiency for the soil polluted by zinc.

Abstract:The glutaminase gene（ylaM）was amplified from Bacillus subtilis by PCR.Construction of pMA5-ylaM and expressed in 168.The specific activity of purified glutaminase was 939.48 U/mg. The optimization activity of recombinant enzyme was at pH 7.5 and 55 ℃. La³⁺、Zn²⁺、Fe³⁺ and A^l3+ inhibited the glutaminase activity. More than 50% of the maximum activity was retained in the presence of 15%~17.5% NaCl.The highest production of glutaminase of fed-batch fermentation was 215.06 U/mL in the 5 L fermentation.

Abstract:L-theanine，is a nonproteic amino acid which recently has an increasing demand as it is applied in food and pharmaceutical industries. In this study，a GRAS（Generally Recognized As Safe） strain Bacillus subtilis 168 was used as the host to produce a novel γ-glutamyl transferase（GGT） from B. pumilus ML413. The enzyme produced was applied to L-theanine synthesis. Subsequently，glutamine conversion efficiency with the GGT was optimized. Finally，for the improved L-theanine production，a fed-batch strategy was introduced and the maximum titer of L-theanine reached 50.8 g/L at 16 h. This titer could be comparable to the efficient but toxic GGT producer recombinant E.coli.

Abstract:In order to explore the effect of silencing opaque2 gene on the kafirin content in sorghum，the suppressed expression vector of opaque2 gene is constructed by RNAi technology，and then transformed to young embryos by Agrobacterium tumefaciens-mediated transformation system. The kafirin content in these transgenic seeds is determined and indentified by SDS-PAGE，and also the changes of protein bodies in these endosperm cells are observed by transmission electron microscope. The results show that the kafirin content of four fifths of transgenic plants is significantly lower than that of the wild type（P<0.05）. Those of the protein bands between the wild type and all transgenic plants are similar. The starch granules in the endosperm cells of the wild type arranged closely and most of them are fused into large blocks，and large numbers of protein bodies are indicated with large volumes. While the starch granules in the transgenic plants arrayed loosely and indicated many gaps in the endosperm cells，and while those numbers and volumes of the protein bodies are less and smaller than the wild type，respectively. In conclusion，opaque2 gene silencing induced changes in number and volume of protein bodies in endosperm cells，which additionally affects the internal structure of endosperm cells and the kafirin content in sorghum.

Abstract:Lactosucrose is a rare trisaccharide with prebiotic effect，which can be formed from lactose and sucrose by levansucrase. In this study，levansucrase expression was efficiently carried out by comparing two different promoters and making them tandem. Six different strains with different promoters had been successfully constructed，including WB-PH，WB-P，WB-H，1A-PH，1A-P，1A-H. Amongst them，WB-PH exhibited a higher production level. The effect of growing condition on WB-PH was studied. Using soy peptone as nitrogen source and a higher dissolved oxygen level can enhance levansucrase expression. The enzyme activity reached 108.34 U/mL，which was 25.92 times higher than it before optimization.

Abstract:An integrative expression vector applied for Kluyveromyces lactis was constructed in this study. To verify its application，the adenylate deaminase gene（AMPD） derived from Streptomyces murinus was sucessfully expressed by this vector. The recombinant vector pTRGA-amdS that based on the plasmid pMD 19T-simple was established containing K. lactis-derived 18S rDNA sequence for homologous recombination，the Saccharomyces cerevisiae-derived galactosidase gene promoter（pScGAL7） for expression of heterologous genes，the acetamidase gene（amdS） as selection marker，and AMPD as reporter gene. The recombinant expression vector was linearized by Sac II，removing E.coli ori and Amp resistant gene，then electransformed into K. lactis GG799 to obtain transformants. Real-time quantitative PCR analysis indicated that the copy number of the integrated AMPD gene ranged from 1 to 3 and positively correlated with AMP deaminase activity. When the inducer galactose was used as carbon source，the AMP deaminase activity in the culture supernant of the recombinant with three AMPD gene copies reached to 590±13.33 U/mL，improved 32.6%. The integrants were mitoically stable for 58 generations under the non-selective pressure condition（98.58%）. We constructed a new vector with the stable expression ability of heterologous gene and confirmed the 18S rDNA as a suitable recombination site. These research results lay a foundation for further molecular modification of K. lactis.

Abstract:Chlorhexidine was modified on carrier by cross-linking protocol and the effects of modified carriers on neutral proteases were studied. The reaction conditions were optimized. The additive amount of Chlorhexidine was 0.095 mmol；the activate time of resins was 1 h；the molar ratio of carboxyl to EDC was 1∶3；the addition of neutral protease was 600 U and the addition of EDC was 13.7 mg on the process of activating enzymes. The maximal neutral protease activity was 398 U/g resin. The neutral protease was cross-linked to the affinity carrier through the Fourier transform infrared detection. TheKm values of immobilized，free and randomized immobilized neutral protease were 2.9，1.7 and 7.1 mg/mL，respectively.

Abstract:Beta-amylase and other starch hydrolases were complexed to produce high purity maltose syrup in industry. Compared with plant β-amylase，the microbial β-amylase has the advantages of high purity，little limitation with the raw materials，and the suitability for large-scale production. However，the optimum pH of the microbial β-amylase is 6.0~8.0，which is difficult to complex with other starch hydrolases. In the pvious study，the optimum pH of Bacillus flexus β-amylase was 7.0. In the present study，three B.flexus β-amylse mutants T47K，Y164K，L396K were constructed and the optimal pH were changed from 7.0 to 6.0，4.5 and 5.5 respectively. At the same time，the optimum temperature of L396K was increased from 50 ℃ to 60 ℃. The production conditions of maltose using L396K β-amylase was also optimized. When using potato starch（10% w/v） as the substrate under the condition of pH 5.5 and temperature 60 ℃，the yield of maltose reached 80.2%，which satisfied the production requirement of high purity maltose syrup.

Abstract:A filamentous fungi strain named Talaromyces flavus S1 was isolated from the municipal sludge. The dewaterability of sludge could be improved remarkably when the mycelium was inoculated to sludge. The results show that it is the mycelium itself rather than fungal secretions（Microbial flocculatants） improved the sludge dewaterability by separtating the culture solution of mycelium；The dewaterability of high concentration sludge was improved significantly by adding the fungal mycelium，as well as，it could be increased by 40.58%. When the sludge concentration is low，the dewaterability is even poorer than the raw sludge；The Capillary Suction Time（CST） of the sludge by conditioning of mycelium shows greater reduction compared with the Polyacrylamide（PAM） and polyaluminum ferric chloride（PAFC），but PAM is more effective to the specific resistance of sludge（SRF），while，the role of PAFC is weakest. The sludge dewaterability could be improved by 60% under the synergistic effect of PAM and mycelium. From the results of sludge particulate distribution and Scanning Electron Microscope（SEM），it shows that the dewaterability was advanced by the enlargement of sludge floc and particulate due to the small sludge particulate was wrapped by the mycelium. In a word，a more effective and reliable biological conditioning method is put forward to improve the sludge dewaterability on the basis of using the fungal spores. The results have an important significance to develop a new biological technology on sludge dewatering based on this filamentous fungi.

Abstract:The spore wall of the budding yeast Saccharomyces cerevisiae has a multilaminar structure and its outermost layer，termed the dityrosine layer，is mainly composed of a phenolic compound，N，N′-bisformyl dityrosine. Since many cellular phenolic compounds are capable of scavenging free radicals，we hypothesized that N，N′--bisformyl dityrosine could work as an antioxidant. Thus，we investigated whether the dityrosine layer could protect cells from oxidative stresses and N，N′--bisformyl dityrosine have antioxidant activity. Yeast spores with or without the dityrosine layer were treated with reactive oxygen species and their sensitivities to the stresses were assessed. Additionally，N，N′--bisformyl dityrosine was produced by using vegetative cells and its antioxidant activity was measured. Compared to wild-type spores，idit1Δ spores，which lack the dityrosine layer，were sensitive to free radicals. Thus，the dityrosine layer can protect spores from oxidative stresses. Furthermore，we found that N，N′-bisformyl dityrosine can scavenge free radicals. The results suggest that N，N′--bisformyl dityrosine is an intriguing material as an antioxidant. N，N′-

Abstract:TheFKS family genes which encoded 1，3-β-glucan synthase are important to maintain the integrity of Saccharomyces cerevisiae cell wall. To illustrate the functions of FKSfamily genes on the ability of S. cerevisiae to tolerate the environmental stresses and ethanol production，theFKS1，FKS2，and FKSFKS3 knocked out strains were constructed by homologous recombination，then the physiological properties of wile-type strain and mutant strains were measured and compared. The results showed that the content of 1，3-β-glucan in cell wall of the FKS1 knocked out strain was 60% lower compared to the wild type. The knockout of FKS1 gene also weakened the abilities of growth，stress resistance and ethanol production for S. cerevisiae. Furthermore，the growth and stress resistance abilities of FKS3 gene knocked out strain were similar to those of the wild-type strain，however its abilities to tolerate the fermentation environment and to produce ethanol were better than those of the wild-type strain. Therefore，the FKS1 gene was important for S. cerevisiae to keep the cells activity and to resist the environmental stresses while the FKS3 gene negatively influenced the ability of S. cerevisiae to resist outer stresses.

Abstract:Molecular biology techniques are used to modify the N（I _N） and C（IC） fragment of Npu DnaE（Nostoc punctiforme） intein. An affinity chromatography medium with I _N as the affinity ligand and a fusion protein expression system with IC as self-cleavage affinity tag were constructed. Finally，a protein purification system mediated by split intein was developed. The effect of steric hindrance on the C-terminal cleavage rate of fusion protein was studied by constructing 3 kinds of IN affinity ligands and 2 kinds of IC-GFP fusion proteins. Through the combination of in vitro cleavage and Zn²⁺ inhibition experiment，we found the best combination of F and the new Zn binding site. The I _N affinity chromatography media were successfully prepared by using cysteine at the C-terminal of N3 affinity ligands. The purification effect of it was studied，and the high purity GFP protein was obtained successfully.

Abstract:In this study，bacterial strain preserved in laboratory was chosen with the inhibitory activity against Staphylococcus aureus，it was identified as Bacillus licheniformisJN-814C. The optimal conditions for Bacillus licheniformis JN-814C to produce bacteriocin were as follow：2.5 g/L dextrin，7.5 g/L yeast extract，10 g/L peptone，3.0 g/L NaCl，at pH 7.0 and at 37 ℃ for 22 h，with an inoculum density of 2% and inoculum age of 22 h，with fluid volume 50 mL/250 mL. The bacteriocin was purified through salting-out 40-80% and DEAE-Sepharose-FF anion exchange chromatography respectively. This purified bacteriocin had the inhibitory activity on gram-positive bacterium.It is sensitive to alkaline protease and is not sensitive to temperature or acid or alkali and it have high glutamate content. Therefore，it can be basically determined as an antibacterial peptide.

Abstract:Tyrosol（2-（4-hydroxyphenyl） ethanol） is an attractive phenolic compound that is naturally found in several foods such as olive oil，wines and green tea. Tyrosol has widely used in pharmaceutical，chemical and other industrial fields because of its anti-inflammatory and antioxidant activities. Traditionally，tyrosol is often produced chemically in industrial scale，however complex process，low yield and environmental issues are hampers. On the other hand，it would be hard to purify tyrosol from olive oil at an industrial scale because of its low concentration，absence of effective separation methods. Therefore，biotechnological production of tyrosol has been paid more and more attention. In this study，the phenylpyruvate decarboxylase gene ARO10 was cloned from Saccharomyces cerevisiae and introduced into Escherichia coli to generate a recombinant tyrosol producer. Furthermore，the genes of pheA and feaB encoding prephenate dehydratase and the endogenous phenylacetaldehyde dehydrogenase respectively，were deleted sequentially to improve tyrosol synthesis. Under the optimal fermentation conditions，the recombinant strain overexpressing ARO10 gene produced 4.15 mmol/L tyrosol from 10 g/L glucose in 48 h. Moreover，it shows that adding tyrosine in M9Y medium could increase tyrosol titer. Meanwhile，it also shows that it had several enzymes that converted tyrosine into 4-hydroyvphenylpyruvate. In summary，we successfully engineered a novel metabolic pathway in E. coli capable of producing tyrosol and provided a strategy for microbial tyrosol production in an industrial-scale.