Abstract:Previously，a recombinant Brevibacillus sp. producing the Bacillus stearothermophilus cyclodextrin glycosyltransferase was constructed. In this study，the strain was cultured in shake flask for 48 h，it showed that the highest extracellular enzyme activity was 49 U/mL. The supernatant of the medium was used as crude enzyme for the production of β-cyclodextrin. Then the reaction conditions，such as initial pH，reaction temperature，starch concentration，CGTase dosage，pullulanase dosage and reaction time，on the yields of β-cyclodextrin was investigated. The optimized conditions for the conversion as the following：the initial pH was 6.0，the reaction temperature was 50 ℃，initial starch concentration was 150 g/L ，CGTase concentration was 13 U/mL，pullulanase concentration was 50 U/g starch. Under this conditions，the reaction was continued for 18 h，thecyclodextrin yield is 81.4% and the proportion of β-cyclodextrin is 97.7%.

Abstract:IFT25 is one of the important components of IFT-B complexes，which are necessary to maintain cilia movement and perception. To explore IFT25 mechanism especially in the model organism Chlamydomonas reinhardtii plays an important role. This experiment adopts economic and simple method to prepare the rabbit polyclonal antibody of the Chlamydomonas reinhardtii IFT25.The target gene ift25 was cloned into vectors of pMAL-c2X- and pET-28a（+）-，respectively. Then the plasmids were transformed into E. coli BL21（DE3） to induce highly expressedby IPTG. After purified by affinity adsorption purification，the purity of fusion protein is both higher than 90%.Through the back of the neck skin repeatedly immune eight weeks of the New Zealand white rabbit to prepare polyclonal antibody. After the fifth immune antiserum titer was determined by ELISA，which titer more than 12 800. Polyclonal antibodies were then successively prepared by Protein A affinity adsorption purification and nitrocellulose membrane purification. And the specificity of polyclonal antibodies was detected by Western blotting. Results showed that the polyclonal antibody prepared could specifically and precisely bind IFT25 in Chlamydomonas reinhardtii，which could provide important theoretical and methodological supports to clarify mechanisms of the action of IFT25 and diagnose diseases associated with cilia.

Abstract:Due to the unsatisfactory effects of deflavination from sarcosine oxidase（SOX），the SOX inclusion protein without cofactor was dialysed and renatured for testing the reconstruction effects between SOX apoprotein and the coenzymes. The comparatively suitable conditions for the binding of flavoprotein to SOX apoprotein were shown as following：the concentration of enzyme protein 0.3 g/L， pH 8.5， temperature 4 ℃， the optimal redox state（GSH / GSSG ） GSH / GSSG with the value 2，and the performing time was 24 h. The reconstitution effects of SOX protein binding to different natural flavins were compared. Through CD，FTIR，and DSC analysis，the secondary structure，enzyme specific activity and stability of the reconstituted SOX were also studied.

Abstract:To establish an efficient genetic transformation protocol for Desert Chlorella. It was optimized methods for electroporation conditions and related parameters. Human lactoferrin gene regard as reference gene of optimization of electro-transformation conditions. The various parameters of cell viability and transfection efficiency were determined with single factor experiments.Then，the optimal electroporation conditions were obtained through orthogonal test. Results shows the optimal composition of electroporation conditions：breakdown voltage 1 400 V，cell cycle 8day，osmosis buffer 0.2 mol/L，osmosis time 0 h，plasmid 6 g/mL，electroporation buffer 0.4 mol/L. Under the optimal composition of electroporation conditions，the detected results of cell viability is 51.30%，and fluorescence positive rate is 52.54%，and PCR positive rate is 42.30%.

Abstract:The effects of spontaneous sourdoughs started with pear and navel orange fermentations on the staling of breads，including the hardness，moisture，water activity and starch retrogradation enthalpy. The influence of the isolates on the staling characteristics of the bread was also investigated，and the mechanism of the dominant microorganisms on the bread staling was explored. The results indicated that sourdough fermentation slowed the rate of moisture loss of the bread and inhibited the retrogradation of starch during storage. The starch retrogradation enthalpy of pear sourdough bread （PSB） was 76.01% of that of control （CB），and navel orange sourdough bread （OSB） was 80.66%. In addition，LAB had a positive effect on the reduction of moisture loss and the inhibition of starch retrogradation.

Abstract:In the fermentation process，it is difficult to implement a good online control of residual sugar concentration with delayed measurements. Due to the inherit characteristics of biomass growth，the field operator can achieve an effective estimation on the residual sugar concentration according to operating experience or knowledge. However，it is ineffective in the large fluctuation of the residual sugar concentration. Based on the operating experience or knowledge，this paper introduces a feedback correction idea to compensate the experience estimation algorithm. Moreover，considering the inaccuracy model of the residual sugar concentration，this paper proposes a fuzzy predictive estimation algorithm，a fuzzy predictive estimation algorithm with compensation combining fuzzy intelligent technology. Afterwards，the residual sugar concentration is forecast by these two predictive estimation algorithms. The accuracy and effectiveness of the fuzzy model and compensation algorithm are verified by a real experiment.

Abstract:The molecular weight，structure and antioxidant activity of extracellular polysaccharide（EPS） from Phellinus vaninii Ljup was investigated in this study. The EPS was isolated and purified，and the molecular weight of EPS was determined by gel filtration. The type of anomeric carbon in the EPS structure was investigated by FT-IR，the carbohydrate composition of the EPS was determined by GC/MS，the molecular conformation of EPS was analyzed by SEC/MALLS binding viscosity method，the antioxidant activity of EPS was determined according to OH and DPPH radical scavenging activity. The results showed that：two EPS fractions（Fr-I and Fr-II） were obtained after separation and purification，and the relative molecular weight of Fr-I and Fr-II were 627 500 and 55 000 respectively. FTIR results showed that the Fr-I was coexistence of α- and β-configuration of mannopyranoside acidic polysaccharides，while Fr-II α-mannopyranoside acidic polysaccharides. GC/MS results showed that the monosaccharide components of Fr-I and Fr-II contained ribose，xylose，glucuronic acid，galactose，glucose，mannose and galactose；SEC/MALLS results showed that the dispersion of the EPS is very low，and the EPS kept spherical conformation in aqueous solution. The EPS is a highly compact aggregates with branch. The antioxidant activity tests showed that the ·OH scavenging rate was 38.58% when EPS concentration was 10 mg/mL，and the

Abstract:The nucleotide and amino acid sequences of starch synthase gene（SS） of six plants（Solanum tuberosum，Impomoea batatas，Triticum aestivum，Sorghum bicolor，Cucurbita moschata，Oryza sativa） were analysed using bioinformatic method and the physical and chemical properties and functional domains were predicted. The results showed that SS cDNAs of S. tuberosum，I. batatas，T. aestivum and S. bicolor contained methylation site. The average length of SS was 1 847 bp and the average length of the open reading frame was 877 bp. The start codons were all ATG，and the stop codon consists of 3 kinds. The average number of amino acid residues was 286，and the average relative molecular mass was 31 915.88，the average isoelectric point was 8.2645，the average charge was -0.598 1 in neutral solution. The average number of hydrophilic amino acid was 63，and the average number of hydrophobic amino acid was 102. All the SS of these six plants had two transmembrane domains.The irregular curl and extension chain were found the main component of all the SS secondary structures. Throngh starch synthase by phylogenetic analysis，T.aestivum was similar with S. bicolor in degree of evolution ，but far away from C. moschata.

Abstract:Glucaric acid is a kind of organic acid with important biological values. In order to achieve high-yield production of glucaric acid，a glucaric acid synthetic pathway was constructed in Pichia pastoris by co-expression of the mouse MIOX gene and the udh gene from Pseudomonas putida KT2440，which led to accumulation of glucaric acid. By fermentation optimization in shake flasks，the results showed the optimum carbon source was glucose（60 g/L was the best initial concentration），the optimum initial pH was 5.5，the optimum seed time was 24 h，and the optimum inoculation amount was 25%. The glucaric acid production was increased from （566.36±16.98） mg/L to （967.60±3.90） mg/L after optimization. According to the optimized conditions，the production of glucaric acid was substantially increased to （2.60±0.04） g/L in a 3 L fermentor.

Abstract:Atmospheric and room temperature plasma（ARTP） was used to mutagenize Alternaria alternate which is a naringinase-producing strain. The naringinase activity of the mutagenized stain SK37.002 reached 327.32 U/mL，which was 208% higher than that of original strain.The optimum carbon sources for naringinase-producing is sucrose. The most suitable inducer is naringin and nitrogen sources being NaNO3. According to Plackett-Burman design，NaNO3，CaCl2 and K2HPO4 were screened out of 8 factors as main affecting variables of naringinase production. Then steepest ascent method was used to approach themaximum response regions and it was followed by Box-Behnken and response surface analysis. As a result，the optimum medium formula（g/L） determined was composed of naringin 1，sucrose 5，NaNO3 5，K2HPO4 0.8，CaCl2 0.7，KCl 0.5，MgCl2 0.5. The resulting naringinase activity in fermentation broth reached up to 624.73±30.33 U/mL and was 90% higher than before which was consistent with the predicted maximum value.

Abstract:In order to investigate the antimicrobial mechanism of Ginkgo biloba leaf extracts （GBLE） against specific spoilage bacteria in aquatic products，minimal inhibitory concentration （MIC） of GBLE against Saprophytic staphylococcus was measured by method of broth dilution combined with plate counting in this paper. The impact of GBLE on the growth of bacteria，the permeability of cell membrane and cell wall were also investigated by the methods，such as growth curve，content of alkaline phosphatase （AKP），electrical conductivity and content of protein. The structure of the Saprophytic staphylococcus cells was observed by scanning electron microscope （SEM）. The results showed that the MIC was 100 mg/mL. The inhibitory rates of MIC and 2MIC concentration of GBLE were 60.58% and 81.34% respectively. The growth of bacteria was inhibited and the permeability of cell membrane increased significantly. The result of observation with SEM revealed that the structure of Saprophytic staphylococcus cells were destroyed and the bacteria began to be adhesive with each other. The inhibition effect of GBLE against Saprophytic staphylococcus was related with the damage of cell membrane and cell wall.

Abstract:The effect of lipase pretreatment on kitchen grease hydrolysis and its anaerobic digestion performance were studied with batch experiments. Firstly，the influence of enzyme dosage，ratio of water to oil，pH and temperature on the kitchen grease hydrolysis were investigated. Then，the optimized pretreatment condition was determined by response surface analysis. Finally，anaerobic fermentation of the kitchen wastewater with grease were carried out to analyze the impact of lipase pretreatment on the kitche grease digestion. Results showed that the optimized hydrolysis conditions were determined as enzyme dosage 1.15%，ratio of water to oil 0.9，pH 8.0，temperature 42.5 ℃. Under these conditions，the hydrolysis rate was predicted at 86.6% and the repeated experimental result was （88.1±2.2）%，which showed that the response surface model could predict the influence of four factors on the hydrolysis reaction well. For the results from anaerobic fermentation，the cumulative methane yield of the enzyme pretreated kitchen wastewater with grease was the highest with the value of 572.12 mL/g·COD，showed 55.10% and 14.56% increase than the wastewater with grease group（499.47 mL/g·COD） and the actual kitchen wastewater group（368.86 mL/g·COD） respectively. This indicated that grease had high methane production potential，and lipase pretreatment could obviously improve the methanogenesis efficiency. Further investigation demonstrated that enzyme pretreatment had little effect on pH variation，which showed the significant increase in volatile fatty acids（VFAs） and dehydrogenase activity during the early stage of anaerobic digestion. This manifested that pre-hydrolysis could promote substrate supply for the methane production and improve the methane yield efficiency.

Abstract:To explore the role of amino acid in beer brewing，the impact of 4 key wort amino acids（glutamate，proline，valine and lysine） on yeast fermentation performance were studied. Lager brewing yeast（Saccharomyces pastorianus TT-1）was studied with synthetic medium through fermentation experiment of amino acid addition. The effect of key wort amino acids on lager yeast growth and synthesis of flavour substance was further verified with brewing wort. The results showed that glutamate and proline were identified as important determinants for their negative roles in yeast proliferation and lysine can facilitate yeast growth on the other hand. The most significant effect on higher alcohol production was exercised by the content of valine.

Abstract:To achieve high-level expression of cyclodextrin glycosyltransferase from Gebacillius sp.CHB1 in methylotrophic yeast Pichia pastoris GS115，the DNA sequence（CGT1） encoding 680 aminno acids was amplified. The CGT2 was synthesized based on the codon preference of Pichia pastoris. Then CGT1 and CGT2 were respectively fused to the pPICZαA and expressed in Pichia pastoris. Two strains （GS115/pPICZαA-CGT1and GS115/pPICZαA-CGT2）were abtained. After methanol induction for 120 h in a shake flask ，the enzyme activity of CGT2 reached 0.62 U/mL，1.7 times higher than CGT1（0.37 U/mL）. Shake flask experiments were conducted to optimize the fermentation conditions. Highest enzyme activity achieved to 1.26 U/mL by induction for 120 h under the optimal conditions.（pH 6.5，28℃，200 r/min，inoculation amount of 1.5% methanol），two times higher than before.

Abstract:Overexpression of N-acetyl-D-glucosamine 2-epimerase（AGE），as a critical enzyme for N-acetyl-D-neuraminic acid（Neu5Ac） synthesis ，is an efficient strategy for improvement of Neu5Ac production. Based on the information about the slr1975 gene encoding Synechocystis sp. PCC6803 AGE and the codon bias of Escherichia coli，slr1975-co was synthesized and cloned into the pET28a expression vector. The recombinant plasmid pET28-slr was purified，and then transformed into E. coli BL21（DE3） strain. The recombinant enzyme was overexpressed by induction of IPTG and SDS-PAGE showed that the molecular mass of the recombinant AGE protein is about 43 kDa，which was in agreement with their theoretical value. The recombinant enzyme was characterized and the reuslts showed that the optimal temperature is 42 ℃ and the optimal reaction pH was 6.0. Kinetic studies indicated that the Km for N-acetyl-D-glucosamine（GlcNAc） and N-acetyl-D-mannosamine（ManNAc） was 39.0 mmol/L and 46.5 mmol/L，respectively. Moreover，Neu5Ac production was evaluated by the recombinant enzmye AGE and the results demonstrated that recombinant enzmye AGE coupled with Neu5Ac aldolase（NanA） could catalyze N-acetyl-D-glucosamine to produce Neu5Ac effectively. In summary，codon-optimization and overexpression of AGE provide paved a way for efficient Neu5Ac production by whole-cell catalysis.