Abstract:At present，the genetic engineering technology has become a hot research topic to modify enzyme molecule. Fusion enzyme technique based on the fusion protein design has been used in the construction of multi-functional enzymes and has shown the important potential application and theory values. As an indispensable component of recombinant fusion proteins，linker peptides have been shown to be important in the stability and bioactivity of fusion proteins. According to the latest research progress in recent years，the general properties of linkers derived from naturally-occurring multi-domain proteins，the classification and the advantageous of the linkers are summarized in this review. Finally，the key problems in the design of the linker and perspectives of this field were also discussed.

Abstract:L-Asparaginase catalyzes the hydrolysis of L-asparagine to L-aspartic acid and ammonia，which can hydrolyze free L-Asn to aspartic acid and decrease acrylamide formation during food processingd. In order to meet the high requirements and complicated procedures of food processing，the activity and stability of L-Asparaginase need to beimproved. In this study，we successfully improved the enzyme activity and stability of L-asparaginase （BsAII） from Bacillus subtilis B11-06 by site-directed mutagenesis. Five residues of BsAII were selected and used to construct the mutants by sequence alignment and homology modeling. The enzyme activity assayshowed that the enzyme activity of S299Nansz and P348Aaansz were increased by 28% and 32%，respectively，as compared to the BsAII. The thermal stability and pH stability of P348Aansz were significantly improved compared to that of BsAII. This study showed that residues 299 and 348 in amino acid sequence of BsAII had a great influence on the catalytic action，which provided a basis for the study of the catalytic mechanism of the enzyme，and extended the application in food industry.

Abstract:Transglutaminase（EC 2.3.2.13，TGase） can catalyze the acyl transfer reaction and leads to the crosslinking among proteins. It has been widely used in food，textile，leather，and pharmaceutical industry. In order to improve the specific activity and thermal stability，a recombinant Streptomyces hygroscopicus TGase chemically was modified by phthalic anhydride（PA） in this study. The optimal modification condition of the cultivation was at pH 8.0，20 ℃ for 2 h，and the molar ratio of surface-amine to phthalic anhydride 1∶2. In the presence of 10 mmol/L of cystamine，which is a competitive inhibitor of the TGase，the thermal stability of TGase was increased 31.3% after the PA modification，whereas specific activity of the TGase increased 48.3% in the absence of the cystamine. The modeling crystal structure showed that 21 lysine residues existed in surface of S. hygroscopicus TGase，and five of them located at region close the active site. The analysis of surface hydrophobicity indicated that PA modification reduced the surface hydrophobicity from 9.46% to 21.13%. Thus，PA may improve the specific activity and thermal stability of TGase by modifying the surface hydrophobicity at different regions. These results suggested that PA modification was an effective way to improve the catalytic properties of TGase，which provided a theoretical reference for molecular modification.

Abstract:Using the fused gene-gbs3s2 composed of the gbss（1-261），ssⅢ（2164-2407） and ssⅡ（161-441） gene cDNA fragments as target，we constructed ihRNAi expression vector pART-GF regulated by GBSS of potato tuber specificity promoter. The inverted repeat construct was transformed to elite potato cultivars NHD3，NF and NC by Agrobacterium LBA4404-mediated transformation，PCR technology was used to detect transgenic plants，and the ratio of amylose eand amylopectin in transgenic mini-potato were determined by dual-wavelength spectrophotometry. Ten regenerated plants with kanamycin resistance were obtained through resistance screening and PCR detection. Among them，two was NC plants，three was NF plants，and five was NHD3 plants，respectively. The ratio of amylose and amylopectin in transgenic mini-potato were detected to decrease significantly. IhRNAi expression vector pART-GF regulated by GBSS of potato tuber specificity promoter was successfully constructed and breeding material of transgenic potato was obtained.

Abstract:Beta-1，3-endoglucanase has important applications in food，agriculture and biotechnology. To enhance the productivity of β-1，3-endoglucanase and its ratio in total β-1，3-glucanase activity by Trichoderma harzianum Rifai GIM 3.442，the effects of pH and agitation speeds were investigated in a 7 L fermentator. It was shown that the mono-control of pH or agitation speed could not satisfactorily offer higher biomass and β-1，3-endoglucanase production simultaneously. A two-stage pH and agitation speed control strategy was therefore employed，i.e.，pH and agitation speed were controlled at 7.0 and 100 r/min in the first stage of fermentation（0~24 h） and then switched to pH 5.0 and 200 r/min in the second stage（24~168 h）. On account of this， β-1，3-endoglucanase activity and its productivity reached 407.8 U/mL and 3.09 U/（mL?h），320.0 % and 398.4 % higher than that of uncontrolled condition，respectively. The ratio of β-1，3-endoglucanase to total β-glucanases activity was 0.71，increasing 65.1 % than that of uncontrolled condition.

Abstract:SSU rRNA librarywas carried out to investigate the microbial diversity in fermented yellow water of traditional intense flavor liquor. All 151 bacterial sequences recovered from fermented yellow water were classfied into Proteobacteria，Firmicutes，Bacteroidetes，Lentisphaerae，Actinobacteria，Tenericutes and an unclassied domain，respectively.The Firmicutes and Proteobacteria were the dominant in yellow water. The Clostridium （25.8%），Lactobacillus （15.9%） and Serratia （14.6%） were the dominant genus. All 37 archae clones belonged to the Euryarchaeota，Methanomicrobia，and the archea community in yellow water mostly consist of genera Methanosarcina （59.5%） and Methanoculleus（35.1%）. The diversity index of bacterial and archea communities were 2.75 and 0.83，respectively. The dominance index were 12.88 and 2.14，and coverage were 99.9% and 100%，respectively. All the results indicated that the microbes involved in yellow water play a key role in metabiolizing nutrients and producing flavor components such as alcohol，esters，acerbity，ketone，aldehyde et al.

Abstract:An analytical method was developed to determine the contents of N-nitrosamine compounds in pickled vegetables by solid-phase extraction（SPE） combining gas chromatography-mass spectrometry（GC-MS）. In this study，different extraction methods and the ratios of the eluents in SPE were investigated. The results showed that solid phase extraction got not only the higher recoveries but also the cleaner spectrum figure peaks compared with ultrasonic extraction. Moreover，by eluting with 90% methanol，the solid phase extraction obtained the recoveries more than 90% as well as reducing the organic solvent usage，the linear correlation coefficients of two N-nitrosamine compounds were up to 0.994 and 0.999 respectively，within 0.1~10.0 mg/L. The detection limits were 0.02 mg/L and the recoveries were 90%~112%. The reproducibility was good with the relative standard deviations（RSD） less than 2.3%. This method is convenient and it is suitable for the determination of N-nitrosamine compounds in the pickled vegetables.

Abstract:In this paper，we developed an easy and efficient method for Agrobacterium-mediated transformation of the mycelia of Pleurotus ostreatus. Using nested plates，the mycelia of Pleurotus ostreatus were grown on the inner plate. When the mycelia grew to the edge of the plate，the induced Agrobacterium tumeifaciens were mixed with co-cultivation medium（CCM） and then were added to the space between the inner and outer plate，the above mixtures further were co-cultivated with the growing mycelia. The average conversion rate of the transformations obtained by the usage of resistance screening to the hygromycin B is about 33%. The advantages of this method are simple operation，short term，and easy to expand the scale of experiments. The results may provide reference for the molecular breeding of Pleurotus ostreatus and the genetic transformation of the other filamentous fungi.

Abstract:For the heterologous expression of phospholipase A1，the gene of phospholipase A1 from Citrobacter youngae（CICC No.21596） was cloned into the expression vector pET28a（+） to construct the recombinant plasmid pET28a（+）-pla1.Then the recombinant plasmid was transformed into E.coli BL21（DE3） to achieve the recombinant strain pET28a（+）-pla1/DE3. After subsequent induction by isopropyl β-D-1-thiogalactopyranoside （IPTG），an approximately 33 000 protein was detected in the cell lysate supernatant by SDS-PAGE. The PLA1 activity was detected in an egg yolk agar plate，indicating that the PLA1 gene was expressed in E.coli. The best induction conditions for PLA1 expression were achieved by the optimization of fermentation condition and it was showed as follows，4% of inoculum amount，2 h of initial culture，8 h of induced culture in the presence of 0.4 mmol/L of IPTG，and growth at 37 ℃. Under the optimized conditions，the maximum PLA1 activity in the supernatant was （5.6±0.2） U/mL.

Abstract:L-asparaginase（Asn） can catalyzes the hydrolysis of L-asparagine to L-aspartic acid and ammonia. It is mainly applied in pharmaceutical and food industry. In this study，to improve the characteristic of Asn，we fused six self-assembling amphipathic peptides（SAPs） to its N-terminal and expressed the recombinant Asns in E. coli，respectively. Compared to wide-type Asn，the SAP4-Asn activity increased by 30%，the value of Km decreased by 7.5% and the Vmax increased by 10%. The reaction kinetics analysis showed that the affinity ability and catalytic efficiency of the fusion enzyme towards L-asparagine improved compared to wide-type Asn.

Abstract:With 1∶1 buckwheat rice and glutinous rice as raw materials，the impacts of fermentation time，the mass fraction of jiuyao and wheat starter，proportion of water added into fermenter，fermentation temperature on buckwheat dry rice wine fermentation during the main fermentation process were studied by single factor experiment. The alcoholic strength of main fermentation was used as the response value to optimize main fermentation technology for buckwheat dry rice wine by adopt response surface method. The results indicated that under the condition of 0.6% jiuyao，3.74% wheat starter，1.2 mL/g proportion of water added into raw material，29.0 ℃ fermentation temperature，and 8 d fermenting time，the alcoholic concentration up to 17.0%（20 ℃），the product is aroma and has a unique taste style of buckwheat dry rice wine.

Abstract:The water content，oil content，crispness，the values of L，a and b were used as indexes to study the effect of NaCl soaking treatment at different concentrations on the drying efficiency and quality of the Pleurotus eryngii using microwave-assisted vacuum frying and vacuum frying in this study. The results showed that the product using microwave-assisted vacuum frying had a lower oil content，a better crispness and a desirable color as compared to that using vacuum frying. Moreover，higher NaCl concentration significantly reduced the oil content ，which helps to get the healthier frying food.

Abstract:Cellulose is one of the most popular-used biomass resource and widely applied in the bioethanol production，medicine，and food industry. The β-glucosidase（BGL） is of interest as the key enzyme in conversion of cellulose to glucose. In this study，glucosidase from Aspergillus niger in bean dregs broth was purified and characterized. The optimum temperature was 55 ℃，and the optimum pH was 2.0~2.5. BGL was stable at pH 2.0~7.5，The Km value was 8.4×10-4 mmol/L，the kcat value was 16 s-1 and the catalytic efficiency（kcat/Km） was 1.92×104 L/（mmol?s） in the optimum condition. This study could promote the theoretical research and the production and application of BGL.

Abstract:Five strains of bacteria producing chitinase were separated from Jianmen harbour seafloor mud in Taizhou of Zhejiang Province，China. One of strains with higher enzyme activity was further identified by morphology，physiology and biochemistry，composition and content of fatty acid，content of（G+C） mol%，types of quinones，and molecular biological characteristics. The results showed that this strain belonged to the genus Blastobacter，named as Bradyrhizobiaceae blastobacter SYBC-H2. Moreover，the results of the Plackett-Burman Deisign experiments showed that chitin，glucose and corn steep powder were the key nutritional factors to produce chitinase. Composite Design Contral was applied to optimize the fermentative production of chitinase. As a result，the maximum chitinase activity was up to 5.70 U/mL when the culture medium was composed of 2.7 g/L chitin，0.85 g/L glucose and 2.64 g/L corn steep powder.

Abstract:In this study，the survival microorganism and quality of Ganoderma lucidum spore powder affected by electron beam and γ-rays was studied. The results showed that the microorganism and quality affected by electron beam was accordance with that of γ-rays. The D10 values of electron beam and γ-rays irradiation for aerobic bacterial count were 2.167 kGy and 2.172 kGy respectively，whereas the D10 values of mould and yeasts were 2.646 kGy and 2.452 kGy，respectively. When the irradiation dose was lower than 8 kGy，ganodermic polysaccharide of Ganoderma Lucidum spore powder was not significant difference irradiated by this two models. However，when the irradiation dose was higher than 8 kGy，the significant difference of ganodermic polysaccharide of Ganoderma lucidum spore powder was found. The irradiation dose of Ganoderma lucidum spore powder was best to lower than 8 kGy.

Abstract:In this paper，the streak plate separation method was used to classify and identify the primary bacterial flora in the coating solution of paper-making reconstituted tobacco sheet according to colony shape，bacterial characteristics，physio-biochemical tests as well as 16S rDNA sequence analysis. The results indicated that the spoilage bacteria in the coating solution of paper-making reconstituted tobacco sheet were Bacillus pumilus，Bacillus licheniformis and Klebsiella pneumonia. Among them，Klebsiella pneumoniae was the main spoilage bacteria. Moreover，the antibacterial effects of five food preservatives[potassium sorbate，sodium diacetate，butylparaben sodium，EDTA-2Na and ε-polylysine（ε-PL）] on the spoilage bacteria were evaluated. The results revealed that antibacterial activities of these five preservatives ranked as follows：ε-PL> EDTA-2Na > sodium diacetate > potassium sorbate > butylparaben sodium. The minimal inhibitory concentration of ε-PL on Klebsiella pneumoniae was 0.075 mg/mL.

Abstract:The antioxidant activity and DNA damage protective effect of the essential oil from the roots of Rubia cordifolia（REO） by hydrodistillation were studied. Five different antioxidant systems，evaluated by DPPH radical method，?OH and O2-? scavenging activity，reducing power assay and the FRAP assay were employed in order to evaluate the antioxidant activities of the essential oil as compared with natural and synthetic antioxidants. The DNA damage protective activity of essential oil was evaluated by pBR322 plasmid induced by ?OH radicals. The results revealed that the oil exhibited varying degrees of efficacy in each assay and a dose-dependent free-radical-scavenging activity against DPPH radical，superoxide anions and hydroxyl radical，as well as the oil exerted more potent radical scavenging activity against DPPH and ?OH radicals than that against O2-?radical. The essential oil exhibited significant protective activity against ?OH radical induced DNA damage. The mechanism may be originated from the superior ability of essential oil to scavenge ?OH radicals.