Abstract:L-asparaginase（EC 3.5.1.1，L-ASNase）has been widely used in the fields of food and medicine. In order to achieve the efficient production of L-ASNase，the effects of stirring speed and fed-batch fermentation conditions on the cell growth and enzyme production of /Bacillus subtilis//ASNΔ25/B2 were investigated in 3 L fermenter. After the optimization，conditions used for fed-batch fermentation were determined as follows：the stirring speed was set at 700 r/min for the first 8 h and shifted to 900 r/min till the end of the fermentation；for fermentation at 16~28 h，sucrose solution（800 g/L）and nitrogen sources（yeast peptone：200 g/L；corn syrup：80 g/L）were fed at 18.75 mL/h and 32 mL/h，respectively. Based on this fermentation condition，the L-ASNase yield reached 1 413.6 U/mL at 48 h，which was 66.2% higher than that of batch fermentation. The final production intensity obtained here was 24.6% higher than that of the latter. The results provide the basic data for the industrial production of the recombinant L-ASNase.

Abstract:The increase of thermostability of aminopeptidase by creating hydrophobic interactions in the substrate binding region was exploited in Bacillus subtilis 168. The hydrophilic residue N385 was targeted and substituted by hydrophobic residue L385. Based on thermostability analysis，N385L mutant was found to retain 20% while the relative activity of the wild type was reduced to 0% at the same temperature（80 ℃）in 30 min. Furthermore，the affinity and catalytic efficiency of the mutant N385L for the subtrate of L-Leu-pNA increased by 47.4% and 28.5%，respectively，compared to wild type. This study will inspire the production of thermostable aminopeptidase for industrial application.

Abstract:A mutant D-6 of MTSase from /Sulfolobus acidocaldarius ATCC 33909 was obtained through directed evolution using error-prone PCR and high-throughput screening. Gene analysis showed that there were two mutations G432D/G586D with the G586D on the loop of Aβ8 and AEα14. The enzymatic properties of wild-type MTSase and mutant D-6 was determined. The results showed that the specific activity of mutant D-6 was 1.22 times that of wild-type MTSase. The /K_m value of the mutant D-6 was 2.77 mmol/L while that of wild-type was 4.74 mmol/L. Thus, the substrate affinity of mutant increased compared to the wild-type.

Abstract:L-malate is an important C4 compound，which is widely used in food，chemical and pharmaceutical industries. In this study，the effects of nitrogen source，concentration of CaCO₃，agitation speed and aeration rate on morphology of Aspergillus oryzae and L-malate production were investigated. The optimum requirements for L-malate production were as follows: nitrogen source of tryptone；CaCO₃ concentration of 80 g/L；agitation speed of 600 r/min and aeration rate of 2 vvm. Furthermore，the relationship between the morphology and L-malate production was analyzed， and when the total volume of the pellet in the unit volume fermentation broth（V value） was 76.4 mm3/mL， L-malate had the highest production of 109.9 g/L.

Abstract:The gene coding aspartic protease App was cloned from the genome of Aspergillus pseudoglaucus. Its self propeptide gene pro，the propeptide gene CP from Candida tropicalis candidapepsin，the gene PP from Saccharomyces cerevisiae proteinase A，the gene RP from Rhizomucor miehei proteinase，and the gene SP from Candida albicans aspartic proteinase 2 were cloned at the 5′-terminal of App gene. The different gene fragments including the different propeptides were obtained，and expressed in Escherichia coli. SDS-PAGE analysis showed that App could not expressed in E. coli without any propeptides at 5'-terminal App gene. When casein was used as substate，proApp with its self propeptide showed the highest specific activity of 903 U/mg at 55 ℃ and pH 2.8. Although the App with propeptides of the other proteases were expressed at a high level in E. coli，they did not present enzyme activity towards casein. The results of circular dichroism showed that the different propeptides changed the constituents of the secondary structure of protease，and thus the protein were not correctly folded. With milk protein as the substrate，proApp showed the highest hyhrolytic activity at 55 ℃ and pH 2.2. This work shows the important of the specific propeptide on heterologous expression and hydrolytic function of protease，and provides a good research basis for aspartic protease catalyzing the hydrolysis of milk protein.

Abstract:In this study， Artemisia argyi essential oils were extracted by simultaneous distillation（SDE）with dichloromethane and n-hexane，respectively. To explore the differences of antimicrobial activities，components of the oil were analyzed by GC-MS，and scanning electron microscope（SEM）was used to explore the mechanism of antimicrobial activity. Compared with the steam distillation，SDE saved one hour and had a three times higher yield（1.5%). SDE（n-hexane）oil had a higher antimicrobial activity with the minimum inhibitory concentration（MIC） being 6.25 μL/mL. GC-MS results revealed that the SDE（n-hexane）oil had 92.95% similarity in the chemical composition with the oils extracted with steam distillation and SDE（dichloromethane). SEM results showed that the antibacterial activity of Artemisia argyi essential oil could be achieved by disruption of cell wall.

Abstract:Mannosylation in the endoplasmic reticulum（ER）lumen is common to four glycosylation pathways，and involves N-linked glycosylation，glycosylphosphatidylinositol（GPI)-anchor and protein O- and C-mannosylation. Thus，mannosylation in the ER lumen is vital for eukaryotes. The donor for mannosylation in the ER lumen is dolichol-phosphate-mannose（Dol-P-Man)，thus the synthesis of Dol-P-Man is important for understanding different kinds of glycosylation modification. Dol-P-Man synthase catalyzes the transfer of mannose from GDP-mannose（GDP-Man）to dolichol-phosphate（Dol-P） to produce Dol-P-Man. This study expressed yeast Dol-P-Man synthase（Dpm1）in Escherichia coli，and purified it. The conserved DXD motif in Dpm1 playsed crucial roles for these activities. We used phytanol with a short lipid chain to substitute for its dolichol homologue with a long lipid chain，and synthesized phytanol-phosphate mannose（Phy-P-Man）using purified Dpm1. Furthermore，Phy-P-Man could be used as a substrate for Alg3 for N-glycosylation research.

Abstract:For the research of amino acid transporter system，it is important to construct a rational amino acid production strain. In this study， Escherichia coli 3110 mutants with MetD methionine absorption system pertinent genes metI，metN or metQ deleted were constructed to confirm the main factor in extracellular methionine absorption. Then genes yjeH and yeaS of methionine secretion system were overexpressed by plasmids and genomic integration to investigate the comprehensive effects on extracellular methionine accumulation and biomass. Compared with wild type strain W3110，the methionine uptake capacity of metI gene damaged strain decreased 16.7%. Combining yjeH and yeaS overexpression through episomal plasmid，the methionine accumulation could reach 0.21 g/L and 0.18 g/L，and the concentration increased 128% and 38.9% after yjeH and yeaS overexpression through genomic integration，reached 0.48 g/L and 0.25 g/L，respectively. In conclusion，to improve amino acid secretion and absorption simultaneously is effective for extracellular methionine accumulation and this mode can regarded as a strategy for constructing rational amino acid production strains.

Abstract:α-Ketoglutaric acid is widely used in food，chemical and pharmaceutical industries as an important organic acid. Fermentative production of α-ketoglutaric acid by Yarrowia lipolytica is currently a competitive method. The accumulation of pyruvic acid is unavoidable during the production of α-ketoglutaric acid by Y. lipolytica. The two keto acids have similar physical and chemical properties，which brings big challenge for the downstream separation process. In this study，effects of three different calcium salts on the separation of keto acids in the fermentation broth were compared. An effective precipitation method was established for the separation of α-ketoglutaric acid from fermentation broth. CaCO ₃ was the optimum precipitant of α-ketoglutaric acid. The recovery and purity rate of α-ketoglutaric acid were 82.5% and 98.89%，respectively. Meanwhile，the precipitation method could not be used for the separation of pyruvic acid，because pyruvic acid was unstable under the partial neutrality and high temperature. This study can thus provide an important theoretical reference for the separation and extraction of keto-acid.

Abstract:Sulfated chondroitin is an important glycosaminoglycans，which is widely used in medical，food and cosmetic fields. To improve the production of fructosylated chondroitin，GlcN synthase（GlmM） and GlcN-6-P synthase（GlmS） were overexpressed，the confusion protein was constructed and a recombinant strain E. coli K4-H-glmM-glmS was obtained. Then，the optimal induction conditions including IPTG concentration and induction temperature were estalished. Finally，with the DO-stat feeding strategy in a 5 L fermenter，the yield was up to 3.99 g/L，which was 108.90% higher than that with wild type strain. The study provides a basis for industrial-scale production of fructosylated chondroitin.

Abstract:Effervescent tablets based on barley grass powder were prepared by vacuum freeze-drying and high energy nanoscale impact. The effects of the addition of barley grass powder，the ratio of disintegrating agent（NaHCO₃∶citric acid）and the addition of stevioside on the infusion properties of effervescent tablets were investigated. Orthogonal design was used to optimize the formulation of effervescent tablets based on single factor test. The results showed that the effervescent tablet with 10% barley grass powder，1 g∶0.7 g disintegrating agent and 1% stevioside exhibited the highest sensory score with a good color and strong aroma of wheat. The diameter，pH，gas production and disintegration time of the effervescent tablet were 1 cm，6，7 mL and 2 min，respectively.

Abstract:Myofibril-bound serine proteinase（MBSP）in fish can degrade myofibrils during processing，resulting in gel deterioration of surimi products. A new highly specific shark-derived antibody，single-domain antibodies（sdAbs)，binds directly to antigens and inhibits enzyme activity. In this paper, nurse shark (Ginglymostoma cirratum) was immunized with expressed MBSP to construct the library of single domain antibody, and 31 phagemids specific to MBSP were panned from the library. The results could provide the basis for the expression and identification of specific single domain antibody which was expected to overcome the gel deterioration of the surimi products by inhibiting or eliminating the activity of MBSP.

Abstract:Candida tropicalis is an important industrial strain and can overproduce long chain dicarboxylic acid. However，genetic manipulations were still difficult because of lack of effective expression elements such as promoter and plasmid. Therefore，screening novel promoter is of great significance for metabolic engineering of C. tropicalis. In this study，phosphoglycerate kinase gene (PGK1) promoter was cloned from C. tropicalis ATCC 20336 and its function was investigated. The 846 bp PGK1 promoter was fused into a yeGFP3 reporter gene and transformed into C. tropicalis XZX host. The results showed that promoter could effectively regulate yeGFP3 expression. Then，a series of truncated promoters (including 300，550，750 bp) were cloned and used for expression of yeGFP3 reporter gene in C. tropicalis. Transformants harboring various integrative yeGFP3 reporter gene cassettes were obtained and confirmed. The relative fluorescence intensities of 300，550，750 bp promoters were 4.98%，9.84% and 48.4%，respectively，when using 846 bp PGK1 promoter as a control. Furthermore，the transcriptional levels of yeGFP3 in different transformants were detected by RT-qPCR and the results agreed to the fluorescence intensity evaluations. The results indicated that two upstream activation sequences presumably existed in the PGK1 promoter. In summary，PGK1 promoter would provide a fundamental expression element for engineering C. tropicalis in future.

Abstract:Catalase catalyzes the breakdown of hydrogen peroxide to form water and oxygen. The main role of this enzyme is to protect cell damage against reactive oxygen species（ROS）. Although it is widely used industrially，it is sensitive to high temperature，which limits its use. In this work，a thermo-stable heme-catalase（KatX2） was constructed with high catalytic efficiency from Bacillus pumilus ML413 through site-directed mutagenesis and using PoPMuSiC algorithm to predict and calculate folding free energy of highly potential residues for mutation. Lys117 was selected and genetically engineered to enhance KatX2 thermostability. From the set of mutants，the K117V recombinant mutant showed significant improvement in thermo-stability with the increase of half-life at 60 ℃ by 38 minutes compared to the wild-type. Furthermore，the catalytic efficiency of K117V mutant was increased by 31.7% compared to that of the wild-type. Interestingly，no change of secondary structure was observed after structural analysis. Therefore，this highly stable KatX2 could be used as a potential industrial biocatalyst.