Abstract:In this study，the co-expression of chaperones was used to improve the production of laccase in Pichia pastoris. According to the codon bias of Pichia pastoris，the gene of laccase from Cerrena sp. WR1 was optimized and cloned into pPIC9K. The resulted plasmid was transformed into Pichia pastoris GS115，yielding the strain PP-L. In the PP-L，we co-expressed six chaperones，relating to protein folding（BIP and ERO1），transportation（SEC53 and SEC1） and stress response（HAC1 and GCN4）. The results indicated that the co-expressions of chaperones had no effect on the cell growth of strain PP-L. Expression of BIP increased the extracellular enzyme activities by 359% while ERO1 decreased that by 22%. The other four chaperones increased the extracellular activities by 18%~53%. In contrast to strain PP-L，simultaneous co-expression of BIP and HAC1（strain P1） increased 602% of the extracellular activities，reaching 3 896 U/L. It was suggested that the overexpression of BIP in endoplasmic reticulum improved the ability of protein folding，thus improving the secretion efficiency of laccase. The results obtained here may improve the industrialization of laccase.

Abstract:The thermostability and catalytic efficiency of Bacillus licheniformis α-amylase have been improved at high temperature and acidic conditions using site-directed mutations in our previous study. To achieve highly efficient extracellular production of this mutant α-amylase，the Bacillus licheniformis α-amylase was co-expressed with Thermobifida fusca cutinase in Escherichia coli and the induction strategy was also optimized. First，the co-expression system was compared with individual expression system，which expressed α-amylase individually，in shake flask and fermenter. The results showed that co-expression system has advantages over individual expression system in extracellular production of α-amylase. Then，the extracellular production of recombinant α-amylase in co-expression system was optimized using different induction strategies. Using the optimal induction strategy of 32 ℃ and combined use of 0.15 μmol/L IPTG and 0.5 g/（L·h） lactose，the co-expression system achieved a maximum extracellular α-amylase activity of 6.05×104 U/mL（8.92 g/L） at 32 h，which was 28.3 fold to that of achieved by individual expression system in shake flask. In addition，the extracellular α-amylase activity occupied 93.2% of total α-amylase activity.

Abstract:α-Phosphogluconate，an important enzyme in polysaccharide synthesis pathway in Ganaderma lucidum，was overexpressed to determine polysaccharide production in submerged fermentation. In this study，the homologous pgm gene was transformed into G.lucidum protoplasts by Agrobacterium tumefaciens mediated method. The intracellular polysaccharides（IPS） and extracellular polysaccharides（EPS） production of G.lucidum transformant was 21.02 mg/100 mg mycelium and 0.71 g/L，respectively. The overexpression of pgmin G. lucidum effectively enhanced IPS（9.1%） and EPS（39.2%） production comparing with wild strain. Moreover，the transcription levels of pgm and pgi gene in polysaccharide synthesis pathway were up-regulated，and intracellular activities of these two enzymes were also significantly enhanced in comparison to wild strain.

Abstract:Saccharomyces cerevisiae possesses cell wall-localized proteins（CWPs），which are essential for the growth. The majority of CWPs are initially synthesized as glycosylphosphatidylinositol（GPI）-anchored proteins，followed by transfer of the protein part to the β-glucan. Two GPI-anchored proteins localized at the plasma membrane，Dfg5 and Dcw1，have been proposed to participate in the transfer reaction，however，molecular functions of these homologous proteins are still unclear. In this study，we characterized the Dfg5 using a conditional mutant strain in which DFG5 was disrupted and DCW1 expression was controlled under MET3 promoter. We found that down-regulation of DCW1 expression in the mutant strain caused a great accumulation of our model CWP（Cwp1） in the plasma membrane. Site-directed mutagenesis of Dfg5 revealed that three residues conserved in glycoside hydrolase family 76 were necessary to complement the lethality of the mutant strain. In contrast，the growth defect was complemented by the expression of soluble form of Dfg5，which was secreted in culture medium due to a lack of its pro-peptide sequence. Taken together，these results suggested that at least Dfg5 acts as an enzyme to release CWPs from GPI anchor at plasma membrane.

Abstract:Terahertz spectroscopy technique was used to test industrial gelatin in order to qualitative and quantitative analysis and it was shown that characteristic absorption peak appear in absorption spectra. The characteristic absorption peak was 1.55 THz. In addition，there was significant absorbtion when the concertration of industrial gelatin was 1.57%. The lowest absorbtion baseline sample was selected as basement with 0%，50% and 70% PE powder to produce positive samples by adding different proportion industrial gelatin. The results show that there was best absorbtion peak of samples mixed with 70% PE powder due to the lowest absorbtion baseline. Moreover，there was significant absorbtion when the concertration of industrial gelatin was above 5%. There is a significant linear relationship between the peak height of characteristic absorption peak and the concentration of industrial gelatin. Therefore，terahertz spectroscopy can be used for qualitative and quantitative analysis of industrial gelatin.

Abstract:To enhance the yield of trans-4-Hydroxyproline from the engineering strain E. coli JM109ΔargB/pUC19-BH，optimization of fermentation medium and culture condition was carried out with a single-factor and orthogonal design experiment. Further，a recombinant E. coli JM109ΔargB/pUC19-BHV，containing the vitrescilla hemoglobin gene VHB was constructed to be desirable for high-density fermentation. Results showed that the fermentation medium was optimized as follows：maltose 10 g/L，glycerol 5 g/L，trypsin 22 g/L，K₂HPO₄ 4 g/L，FeSO₄：VC 5 mmol/L，MgSO₄ 1.7 g/L，NaCl 3 g/L and CaCl2 0.005 g/L. The optimal fermentation conditions were as below：inoculum size 4%（v/v），initial pH 8.5 and culture temperature 30 ℃. At 24 h of the fermentation，the yield of P4H reached 1 602 mg/L，which was about 1.4 times of that before the optimization. In a 7 L fermenter，the recombinant E. coli JM109ΔargB/pUC19-BHV yielded 18.3 g/L of P4H，13.2% higher than that of the parental strain，indicating that the introduction of VHB gene was beneficial for the high-density production of trans-4-Hydroxyproline.

Abstract:Endo-β-N-Acetylglucosaminidase belonging to glycoside hydrolase family 85（ENGase） exhibits transglycosylation activity，which is a useful tool for the synthesis of glycoconjugates with a defied structure of N-glycan. In the present study，We constructed a cell-based screening system to obtain mutant ENGase. As a substrate for ENGase，we expressed monomer enhanced green fluorescent protein（mEGFP） with N-glycan in the Golgi of Chinese hamster ovary cells. Cells expressing N-glycosylated mEGFP provided 10 times lower fluorescence than those expressing mEGFP. Inhibition of protein glycosylation by tunicamycin caused further decrease of fluorescence from the N-glycosylated mEGFP. Our results suggest that this cell line can be used for the high throughput screening of mutant ENGase.

Abstract:To investigate the ameliorative effects of n-Hexane extract Fraction 1（Fr.1） from the flower of Edgeworthia gardneri（Wall. ） Meisn on insulin resistance（IR），we established IR cell model of C2C12 by using palmitic acid（PA）. Glucose consumption，glucose uptake，relative mRNA and protein expression were detected by the glucose oxidase method，2-NBDG uptake，qRT-PCR and western blot analyses. 0.75 mmol/L PA and 16 hours were the optimal concentration and time to induce IR in C2C12 cells. In the range of non-cytotoxic concentration（12.5~100 μg/mL），Fr.1 promoted glucose consumption and uptake in C2C12/IR. Fr.1 also significantly upregulated Insulin receptors（IRs），Insulin receptor substrate-1（IRS-1），Glut4，PI3K and AMPKα mRNA expression and increased p-IRs，p-IRS，Glut4，p-AMPKα，p-ACC and p-AKT protein expression. Fr.1 from the flower of E. gardneri ameliorated IR through modulating IRs/IRS-1/PI3K/Akt/Glut4 and AMPK/ACC signaling pathways in C2C12 cells.

Abstract:Lactobacillus reutrei can product reuterin with safety and high efficiency. The objective of present study was to investigate the optimal conditions on the biotransformation of glycerol to enhance the yield of reuterin. The biotransformation conditions were optimized by orthogonal tests as follows：glycerol concentration 400 mmol/L，cell concentration 110 g/L，conversion time 2 h，temperature 30 ℃ and pH 6.2. An average yield of reuterin was 241.2±3.4 mmol/L under optimal conditions.

Abstract:Anti-AFB1 Monoclonal antibody secreted by the differeent hybridoma cell lines have different sensitivity，three different hybridoma cell lines named 2C10，2E6 and 2H1 were screened and studied，the result of enzyme-linked immunosorbent（ELISA） test showed that the sequence of the sensitivity of monoclonal antibodies secreted by 2C10 ，2E6 and 2H1 was 2C10>2E6>2H1. Three anti-AFB1 scFvs from different hybridoma cell lines were constructed and expressed in E. coli BL21（DE3） respectively in order to better show and understanding the relationship between antigen and antibody and improve the sensitivity of recombinant antibodies at the greatest extent. The ELISA method was used to detect the sensitivity relationship between the different monoclonal antibodies and anti-AFB1 scFvs after protein expression and purification. The result showed that the sequence of the sensitivity of anti-AFB1 scFvs was scFv-2C10>scFv-2E6>scFv-2H1，and showed that the sensitivity of monoclonal antibodies were much better than the corresponding anti-AFB1 scFvs.

Abstract:Since the broth of Bacillus mucilaginosus SM-01 was of high viscosity and hard to be separated，the diatomite sorption-filtration method was applied to improve the extraction efficiency of exopolysaccharide by optimizing the process and ultrafiltration conditions. The bacteria and protein of the broth can be removed facilely by filtration at room temperature under the optimal conditions included：the addition of NaCl is 3 g/dL，the dilution ratio of the broth is 3 times，and the amount of diatomite is 10 g/L. The filtrate can be purified by ultrafiltration（relative molecular-weight cut-off of 200 000） at 0.1 MPa. The recovery of exopolysaccharide was up to 73.2%.

Abstract:In response to nutrient limitation，diploid cells of the budding yeast Saccharomyces cerevisiae undergo meiosis and form spores. Sporulation is a developmental process that do not happen in mammals. However，the process includes several biological events which are conserved in eukaryotic cells. This study developed a method to screen human genes which specifically inhibits yeast sporulation. In this screening，human genes are individually expressed in yeast cells and monitored their effects on sporulation. In the present study，49 genes has been examined. Among them，three genes strongly inhibited sporulation. Expression of these genes in yeast cells did not affect on the vegetative growth. Interestingly，one of them did not interfere with the meiotic progression very well，suggesting that it prevents the post-meiotic events in sporulation. These results demonstrate that our method can identify human genes which can antagonize yeast sporulation without compromising the vegetative growth. Such genes are possibly involved in sporulation specific events such as nutrient sensing，meiosis，and re-organization of membrane trafficking. Thus，this screening would be useful to find novel functions of human genes.

Abstract:The hepatocyte growth factor（HGF）plays an important role in the treatment of liver fibrosis，but the instability and short half-life it is not stabl in blood circulation limit its application. Thus，great efforts have been invested to improve the stability of HGF. Based on the preliminary research，this study is devoted to the study of the anti-liver fibrosis activity in vivo of human serum albumin（HSA）fusion protein HSA-HGF. Firstly，the mice were randomly divided into the following groups：（1）control；（2）liver fibrosis model；（3）positive control；（4）HSA group；（5）the treatment group of HGF；（6）the treatment group of HSA-HGF. And the mice liver fibrosis models were constructed by CCl4 induction. Secondly，the anti-fibrosis effects were determined by hematoxylin eosin staining，masson staining，serum liver function index activity detection，qRT-PCR detection of α-SMA and COL I，and TRFIA detection of Half-Life in vivo. Finally，the results indicated that：1）the fusion protein and its monomer drug could both significantly promote liver recovery of the mice liver fibrosis model；2）Compared with the monomer drug，the fusion protein still maintained the significant anti-fibrosis effect even at low injection frequency；3）the half-life of this fusion protein remarkably increased from 3 min to 3 h in vivo. Our study indicated that the HSA-HGF fusion protein could provide the capability for liver protection and anti-fibrosis，and the fusion protein shows higher stability and a longer half-life than the monomer drug. In general，the HSA-HGF fusion protein would be a potential drug for the treatment of liver fibrosis.

Abstract:To enhance DHA production by Schizochytrium sp.AB-610，several promoting factors on growth and DHA synthesis were selected and compared. Fe²⁺ was found with great enhancing effect on the growth of Schizochytrium sp.，and indolebutyric acid（IBA） enhanced both of growth and synthesis of DHA. Based on the above results，supplement of 0.95mM FeSO4 and 6.5 mg/L IBA in 500 mL flask was brought about by the test of Center Composite Design（CCD）. Fermentation parameters and cell morphology in control group were both tested and observed every 12 h during fed-batch cultivation of Schizochytruim sp. AB-610 in 7.5 L fermentor. The fermentation period was divided into 4 phases：adapt phase，balanced growth phase，lipid accumulation phase and lipid turnover phase. Supplement of both FeSO4 and IBA was also scaled up in NBS 7.5 L fermentor. It showed that adding of both promoting factors improved the growth rate and elongated the stage of cell growth obviously，and the final DHA productivity of Schizochytrium sp. increased by 51.22%.

Abstract:IFT57 is a subunit of the intraflagellar transport protein B complex and plays an important role in cilium assembly. To express ift57 gene，DNA fragments were amplified by PCR and cloned into an in-house modified version of the pET28a vector. The resulting proteins contained a 6×His tag at their N terminus. BL21（DE3） Escherichia coli cells harboring the expression plasmid were grown in lysogeny broth（LB） medium at 37 ℃ and then induced with isopropyl-b-D-thiogalactoside，SDS-PAGE（12%） results showed that the molecular weights of the fusion protein 6×His-IFT57 was 17.2 KDa. After purified by affinity chromategraphy，the fusion protein 6×His-IFT57 was used as antigen to immune rabbits to prepare polyclonal antibody. After 5 hours we collected the immune blood，then separated the serum and identified the antibody activity. The ELISA result showed that the antibody titer of serum was determined to be about 512 000. After the affinity purification of Protein A，we got a high specificity and sensitivity polyclonal antibody. The results of Western blotting showed that the polyclonal antibody was specificity recognition to the IFT57 protein in C. reinhardtii. It provided the certain reference value for expression and purification of difficult soluble protein，and antibody preparation of polyclonal antibody. It laid the foundation to continue to study the functional mechanism of IFT57 in cilium assembly.

Abstract:In order to improve the culturability of lactic acid bacteria during spray drying ，taking Lactobacillus delbrueckii（ATx） as study objects，culturability of lactic acid bacteria have been investigated after heat stress. And the effects of spray drying on the activities of hexokinase，lactate dehydrogenase，cell membrane ATPase，cell viability and acid-producting capacity have been investigated in four kinds of protective agent（20% skim milk（SM），12% skim milk +8% synanthrin（ SMS），12%skim milk +8% trehalose（SMT），12% skim milk+4% synanthrin +4% trehalose（SMST））. The results showed that the optimal heat stress condition of ATx was（50 ℃，15 min），and the number of viable cells only decreased by 0.107 lg CFU / mL（P≥ 0.05）. It was worth mentioning that after spray drying，the activities of ATPase，carbohydrolase and lactate dehydrogenase were significantly decreased（P <0.05），but all were still higher than the control group. Four kinds of bacterial powderin the 12% skim milk had a good acid-producting capacity and curd time was 8-10 h，these indicated that spray drying could damage the cell membrane and the key enzyme of glycometabolism in ATx. In order to improve the culture culturability ，we could add protective agent to improve the resistance of cells to unfavorable environment in spray drying.

Abstract:To develop the process of desalination in the separation and purification of ε-poly-L-lysine（ε-PL），the nanofiltration was used in this study. Firstly，several nanofiltration membranes were evaluated，，then the operation parameters were optimized through one-factor-at-a-time experiment using model solution as object. Finally，the eluent of ion exchange step was desalinated by nanofiltration at the optimal condition. The results showed that SP800 was the best choice used for desalination in terms of permeate flux，ε-PL retention ratio and NaCl removal ratio. Meanwhile，the optimal desalination conditions were as follows：the pH was 9.0，and the maximum ε-PL concentration was concentrated to about 100 g/L，while the permeate conductivity was controlled less than 300 μS/cm through adding deionized water. To evaluate the efficient of the developed desalination process，1 000 L ε-PL eluent of ion exchange step was employed for desalination by nanofiltration at the optimal condition. Finally，the NaCl removal ratio and the ε-PL loss ratio were reached 96.43% and 0.77%，respectively；and the ε-PL purity and ash content were attained 98.21% and 1.02%，respectively. This is the first report on the desalination of ε-PL solution using nanofiltration，it would be helpful to the production of ε-PL in industry.

Abstract:The high content of salt in the salted duck egg white restricts its development，the utilization of salted duck egg white is very important，microbial fermentation desalination and its characteristics were studied in this paper. At first，Staphylococcus equorum，a salt-tolerant bacterium，was screened from Jinhua ham，which coulddestroy the structure of duck egg white during the growth of the microorganism，reduce the viscosity，and the release of water took away salt after 48 h fermentation. The results showed that，the content of salt in the precipitate was reduced to about 3%after centrifugation，Theantioxidant activity effectsof products from different ultrafiltration membrane，such as DPPH radical scavenging activity，reducing ability，Fe2+ chelating ability and inhibiting lipid peroxidation，were negatively correlated with their molecular weights，and the product with molecular weight less than 3KDa showed the strongest antioxidant activity.

Abstract:This study focused on the immobilization of Candida rugosa lipase（CRL） onto four kinds of different resins by adsorption. The Bradford method was adopted to determine the immobilization ratio，and GC method was used to measure esterification ratio of geranyl butyrate. Finally，the weak-acidic macroporous cation exchange resin D151 obtained the best results of immobilization. Taking the influence of factors including enzyme concentration，the pH of phosphate buffer and the temperature into consideration，the esterification ratio of geranyl butyrate production catalyzed by the immobilized CRL was set as evaluation index to optimize the immobilization process. The results indicated that the highest esterification ratio of 84% could be achieved by the CRL immobilized on D151 resin under optimized conditions：15 mg/mLCRL ，pH of the phosphate buffer 6.5，and immobilization temperature 45 ℃。

Abstract:The study aimed to investigate the antioxidant，anti-tyrosinase，and hypoglycemic activities of the extracts from Ishige okamurai.The antioxidant activities of the supernatants from water extracting-alcohol precipitation of Ishige okamurai（hereafter，referred to as simply SWEAPI） were determined by the DPPH and hydroxyl radical scavenging system，its anti-tyrosinase activity was determined by the dopachrome method. Using SWEAPI as basic material and soluble starch，sodium cyclamate，and β-cyclodextrine as auxiliary materials，the granules were prepared by the optimization conditions from orthogonal experiment. The hypoglycemic effects of granules were studied by in vitro α-glycosidase enzyme activity inhibition experiment and in vivo alloxan-induced diabetic mice. The results show that EC50 of DPPH scavenging is 1.19 mg/mL，EC50 of hydroxyl radical scavenging is 5.08 mg/mL，and IC50 of tyrosinase inhibition is 1.27 mg/mL. These phenomena are related to its higher polyphenol content（8.30%）. When the granules are made by the optimization conditions with the 30∶60∶1∶7.5 ratio of SWEAPI：soluble starch：sodium cyclamate：β-cyclodextrine，it has the best product quality with content of water（≤10%），total sugar（18.74 %），protein（16.60%），and polyphenol（2.53%）. The granules have strong inhibitory activity against α-glycosidase enzyme with IC50 of 0.37 mg/mL. Compared with the model control group，the granules could significantly reduce the blood glucose level of diabetes mice after 14 days of treatment with oral dose of 350 mg/kg. The granules also could reduce postprandial blood glucose level in a short time by the results of glucose tolerance experiment，which are similar to positive drug metformin at dose of 500 mg/kg. In conclusion，SWEAPI has strong antioxidant，anti-tyrosinase，and hypoglycemic activities with potential applications in skin care products and hypoglycemic functional products.

Abstract:Emulsifier has both hydrophilic andoleophylic properties. It can reduceinterfacial tension between the liquid drops. Hence it can make immiscible liquidseasier to be emulsifiedand maintain stable.Fish meat balls are rich in protein，fat and water. Because of the complex composition，it hadpoor stability，and need emulsifying agents to enhance emulsification effect and reduce the influence of low temperature effectson their quality.In this study，five emulsifiers' effects on fish meat balls qualities were compared. Fish meat ball quality changes during freezing procedures under the five emulsifying agents effects were studied.The obtained ice temperature curve，moisture content，gel strength，salt soluble protein concentration and tyrosine content results showed that fish meat ball with sucrose ester as emulsifier was more stable.The experimental results showed that：0.8 percent was the best addition amount in fish meat ball product. Fish meat ball with this amount of sucrose ester had better water holding capacity，more stable gel strength，and better anti-freeze performance. These properties make the fish meat ball have the best taste among others.