Abstract:As an essential amino acid for mammals，L-threonine has a wide application in the food，feeds，pharmaceutical and cosmetics industries. To date，L-threonine is almost exclusively produced through microbial fermentation. Metabolic engineering provides an effective means to strain development and thus to enhancing the L-threonine production. In this article，the pathway and regulation of L-threonine in the major industrial strains，Corynebacterium glutamicum and Escherichia coli are summarized，and advances on metabolic engineering to increase L-threonine production are reviewed.

Abstract:As one of the key enzyme in the phenylalanine metabolism pathway，tyrosine ammonia-lyase（TAL） catalyzes the formation of p-coumaric acid from L-tyrosine. p-Coumaric acid serves as a precursor of a wide array of anti-oxidative and anti-aging natural phenylpropanoid products，such as resveratrol and naringenin. In this study，the tal gene from Rhodotorula glutinis was selected，codon optimized and overexpressed in Escherichia coli BL21（DE3）. After treatment with ammonium sulfate precipitation，the recombinant TAL was purified with anion exchange chromatography and gel filtration chromatography，resulting in specific activity of 1.78 U/mg enzyme. Under the same culture conditions，different expression vectors were constructed to get different productions of p-coumaric acid. Among them，the strain with pET-32a（+） as expression vector gained the highest yield of p-coumaric acid，up to 196.3 mg/L after 24 h fermentation from L-tyrosine. The optimized tal gene can facilitate the construction of phenylalanine metabolism pathway. Different expression vectors used in this study also offered more options for biological production of p-coumaric acid.

Abstract:By analyzing the active site of Klebsiella variicola SHN-1 pullulanase and alignment of the amino acid sequences of the pullulanases from K. pneumoniae，Bacillus acidopullulyticus and B.naganoensis，eight site-directed mutagenesis including H626D，R756E，H852D，R908E，I619N，F723Y，S850T，and K851S were subjected to improve the acid resistance of K. variicola pullulanase. After analysis of enzymatic properties，the optimum pH of H852D was shifted from 5.0 to 4.7，and its stability was obviously improved when stored at pH 4.5. By the analysis of homology modeling and structure of the pullulanase，the formation of two hydrogen bonds by single-site substitution was supposed to be responsible for the improvement of stability at low pH，and the change of pKa value in the active center of pullulanase caused the optimum pH migration.

Abstract:The poly-γ-glutamic acid（γ-PGA） synthase gene pgsBCA of a glutamic acid-independent γ-PGA producing strain Bacillus methylotrophicus SK19.001 was cloned and expressed in the Escherichia coli. The pET-28a-pgs vector harboring pgsBCA genes were constructed and transformed into E.coli BL21. E. coli BL21 could synthesize γ-PGA when cultivated in the flask culture of LB plus glucose and L-glutamate，respectively. The pgsBCA genes were proved be essential for the synthesis of γ-PGA in B. methylotrophicus SK19.001. The blast result of deduced amino acid sequence from the pgsBCA genes in SK19.001 comparing other reported strains showed that the pgsB and pgsC gene were relatively conservative gene for pgsBCA.

Abstract:Aiming to improve the catalytic activity and oxidative stability of glucose oxidase from Aspergillus niger. This work conducted site-directed mutagenesis based on an analysis of the protein structure. Four methionines（M582L，M524L，M556L and M305L） were selected as the mutation sites and individually replaced with leucine. The native GOD and the mutated GOD were separately expressed in P. pastoris GS115. In the presence of H2O2（10 mM，20 mM，50 mM，100 mM，and 500 mM） at 35 ℃ for 2 h，the oxidative stability of the mutants increased compared to the wild-type under most of the circumstances. Meanwhile，M556L showed a higher（2 folds than the wild-type） oxidative stability of all in the presence of both 100 mM and 500 mM H2O2 for 2 h. Compared to the wild-type enzyme，the kcat/Km value of M556L increased（2.08 folds than the wild-type）. The thermol stability of the mutants remained unchanged while the stable pH range of M524L was extended from 4.0-6.0 to 3.0-7.0 compared to the wild-type. It is a fact that a more stable enzyme with higher oxidative stability is an ideal choice in most of the industrial application of GOD.

Abstract:To study the effect of microwave irradiation on the catalytic activity of carbon-based solid acid catalyst made from mung-bean，the catalyst was successfully prepared by carbonization after with sulfonation process and used in methyl esterification of oleic acid by the self-made microwave reactor in laboratory. To select the appropriate reaction conditions，the single factor，including microwave power，reaction time，temperature，catalyst amount and the molar ratio of methanol and oleic acid was studied and based on this result，the orthogonal test was designed to optimize the process parameter. At last，the optimized process condition was microwave power 400 w，temperature 65 ℃，the molar ratio of methanol and oleic acid 11∶1，reaction time 30 min and catalyst amount 5% with 93.97% methyl ester yield.

Abstract:Hyalurondiases（HAase） are a kind of hydrolases with medicine values. In order to improve the expression of HAase in the recombinant Pichia pastoris，the effect of induction temperature on HAase expressed was investigated in the recombinant P. pastoris. The result showed that the production of HAase could be improved with the decrease of induction temperatures，especially，two-stage temperature induction strategy. In this strategy，temperature was controlled at 25 ℃ during the induction stage 0~60 h，and then shifted to 22 ℃ after 60 h. HAase activity reached to 1.18×106 U/mL，2.6 fold improvement compared to induction temperature at 30 ℃（4.53×105 U/mL）. In addition，two-stage temperature induction strategy had an advantage on cell viability and alcohol oxidase activity，which offered a new strategy for expression of exogenous protein in the recombinant P. pastoris.

Abstract:In order to investigate the parameters affecting the bioaccessibility of β-carotene in emulsion system，in vitro digestion model and three special digestion models（one without starch hydrolases，one without lipase and one without bile salts） were established to investigate the bioaccessibility of β-carotene emulsions stabilized by octenyl succinic anhydride（OSA） modified starches. The results showed that the bioaccessibility of β-carotene in four digestion models reduced in the following order：the complete model > the model without starch hydrolases > the model without bile salts > the model without lipase. It could be concluded that the bioaccessibility of β-carotene would decrease when one of the three processes of starch digestion，lipolysis or micellization of bile salts was inhibited. And the influence degree of these three factors on β-carotene bioaccessibility decreased in the following order：lipolysis > micellization > digestion of emulsifiers.

Abstract:In order to provide the theory basis for prevention and treatment the disease caused by Aeromonas hydrophila in the process of Hypophthalmichthys molitrix breeding，the strains which have the strong and stable abilities against A. hydrophila from H. molitrix intestinal tract were screened and their growth and culture conditions were studied. Antagonistic strains against A. hydrophila were separated and screened from H. molitrix intestinal tract. Then，the physiological and biochemical，molecular identification and the optimization of growth conditions of the stable antagonistic bacterium were determined. After many times of screening，15 antagonistic strains were got. Among them，the more stable antagonistic bacterium is number S4，which was an alkaline Gram-negative bacillus brevis bacteria and belonged to Aeromonas genus. Orthogonal test indicated that the optimal growth conditions were 1.5 g/dL starch，1.5 g/dL beef extract，1 g/dL CaCL2，32 ℃，pH 8.5 and inoculation quantity 3%. The results provide the theoretical basis for the prevention of A. hydrophila and the application of antagonistic bacterium S4.

Abstract:Hydantoinases are important enzymes with industrial potential in producing D-amino acids. In this study，6 strains with hydantoinase activity were selected from 23 wildtype microbial strains using 5'-monosubstituted hydantoin derivatives as induce substrates. Four 5'-monosubstituted hydantoins were chemically synthesized and verified with 1H-NMR. From 6 hydantoinase-producing strains，strain Pseudomonas fluorescenswas successfully identified with the highest enantioselectivity，desired substrate specificity and high activity in the hydrolysis of 5'-monosubstituted hydantoins andwas selected for further study. After induction with hydantoin substrates at 30 ℃ for 12 h，the D-hydantoinase activity could reach 92.1 U/L. The reaction condition was optimized to be 60 ℃ and pH 8.5，which was in favor of the spontaneous racemization of substrate. In a 50 mL whole-cell biocatalytic system，20 mmol/L D，L-p-hydroxyphenyl hydantoin was enantioselectively hydrolyzed by 0.25 g dry cells with a conversion ratioof 98% within 3 hours.

Abstract:To analyze the main factors causing the loss of viability and the physiological damage mechanism of Lactobacillus plantarum D5-5，in this study，the survival rates of bacteria was investigated during different treatments concentrations of ethanol. In addition，the activity changes of lactate dehydrogenase（LDH），ATPases，the extracellular relative β-galactosidase and the cell membrane with stress treatments were determined relatively. The results showed that the survival rate of Lactobacillus plantarum D5-5 was decreased by 44.07%、61.81%、69.79% with 6%~8%ethanol concentration treatments，and the activities of LDH and ATPase were significantly affected，but the extracellular relative β-galactosidase activity was increased. Meanwhile，the membrane integrity and cell ultrastructure was injured in the presence of ethanol stress treatment. The above results indicated that LDH and ATPase are the key enzymes resulting the activity loss by ethanol stress and could be used to evaluate the effect of ethanol stress on viability and metabolic activities of Lactobacillus plantarum D5-5 during different treatments concentrations of ethanol.

Abstract:Trans-4-hydroxy-L-proline is widely distributed in nature and applied in the pharmaceutical，chemical，food and cosmetic products. In order to interrupt the bacterial degradation of proline during the biosynthesis process of trans-4-hydroxy-L-proline，putA gene was deleted using Red/ET homologous recombination system. Wild types and mutants were cultured in GT and GC media，and the trans-4-hydroxy-L-proline yields were compared to obtain the optimal strain and medium. On the other hand，vgb gene was introduced into the cell and its effect on trans-4-hydroxy-L-proline production was studied. The results show that the knockout of putA gene can prevent the degradation of proline，and with an adequate supply of the carbon source，GC medium is better for producing trans-4-hydroxy-L-proline than GT medium. Moreover，all of the consumed proline can be transformed to trans-4-hydroxy-L-proline in putA deletion mutants. By contrast，the presence of vgb gene can significantly improve trans-4-hydroxy-L-proline production.

Abstract:The development of the new generation sequencing technology extends the application field of gene sequencing. High throughput sequencing of PCR products has been widely used in functional gene screening，mutation and methylation detection of tumor related genes，etc. In the high throughput sequencing technology，database and computer sequencing experiments are the key factor to decide the quality of DNA data. Based on Illumina sequencing instruments，the sample preparation method and sequencing running for PCR products were optimized，the results make us clearly understand the primary features of high-throughput sequencing techniques，and provide us important references of sequencing methods for low diversity DNA samples to address biological questions of interest.

Abstract:Fumarate could be produced from maleate with whole cell of recombinant Escherichia coli. However，malate，with a yield of 15.5%，was generated as byproduct by the cellular fumarase，resulting in a decreased fumarate yield and purity. The chromosomal genes，fumA and fumC，in E. coli BL21（DE3） were inactivated，followed by over expression of the maleate cis-trans isomerate. After cultured in a bioreactor，the resulting strain BL21（DE3）△fumA-fumC/pET24a-maiA could generate 64 g/L dry cell weight and maleate cis-trans isomerate of 306 U/mL. With a volumetric ratio of fermentation broth（60 g/L dry cell weight）：fumarate（2 mol/L）=1∶4，fumarate yield of 98.4% and malate yield of 0.7% were produced after incubation at 37 ℃ for 1 h. These results laid a foundation for industrial production of fumarate by whole-cell biocatalyst.

Abstract:The study was carried out to investigate the anti-oxidative　effect　of Microbial Transglutaminase Cross-Linked Fermentation Milk Protein（mTG-FPM） in aging mice induced by D-galactose（D-gal）. Sixty C57BL/6J female mice（12 w old）were divided into 6 groups including the control group，D-gal model group，D-gal-induced mice treated with FPM cross-linked mTG by 0 U，1 U and 3 U/g protein（as D-gal+0 UmTG-FMP，D-gal+1UmTG-FMP and D-gal+3 UmTG-FMP groups），at 1.5 g/kg bw via ig，and D-gal-induced mouse treated with vitamin E as the positive group at 100 mg/kg via ig，respectively. After continuous administration for 8 weeks，the anti-oxidative parameters（CAT，SOD，MDA GSH-Px） in liver，kidney tissues，and serum of mice in each group were determined. The results showed that mice induced by D-gal had lower CAT and GSH-Px activity in liver and serum，lower SOD activity of kidney and serum，and higher MDA content of kidney and serum，when compared with the normal mice（P < 0.01，P < 0.05）. Compared to the model and untreated FPM groups，FPM treated with mTG had higher CAT activities in liver tissue（3 U mTG treatment，P < 0.01） and in serum（1 U mTG treatment，P < 0.05） up to 11.14% and 35.57% respectively，whereas the content of MDA in kidney tissue（P < 0.01） in FPM treated with mTG was reduced 26.41%. The activity of GSH-Px in liver tissue was also sharply higher（22.36% increase） in D-gal+3 U mTG-FPM group than that of in FPM group untreated with mTG（P < 0.01）. It was concluded that the mTG Cross-linked effect in FPM can improve the anti-oxidative ability of aging mice induced by D-gal.

Abstract:Organophosphate（OP） pollution is a major hidden danger to food safety，acetylcholinesterase can be used to detect the residual OP pesticide. Apiscerana cerana is a unique kind of Apoidea which has not been found in other countries except for China，and it has a high sensitivity to pesticides. On the other hand，acetylcholinesterase gene from Apiscerana cerana has not been cloned and applied in food safety detection. In this study，using the total RNA from Apiscerana cerana head tissue as a template，a 1.8kb AChE cDNA sequence was obtained by RACE method，then the target sequence was cloned into the yeast expression vector pPIC3.5K. AChE was expressed and located in Pichia pastoris GS115 after induced with 0.5% methanol. The SDS-PAGE and activity analysis showed that Apiscerana cerana AChE gene was successfully cloned and expressed in Pichia pastoris for the first time. A strain with higher activity AChE was selected to express AChE，and its AChE activity was 10 568 U/mg，which could be used to detect the residual OP pesticide.