Abstract:In order to prepare a stable and efficient immobilized enzyme to produce fumarate from maleate，maleate cis-trans isomerase（MaiA） from Serratia marcescens was used as a model enzyme in which a silica-precipitating peptide R5 were fused to its N-terminal. The specific activity of the purified fusion enzyme（R5-MaiA） could reach 42 U/mg. The optimum conditions for preparing the immobilized enzyme could be summarized as：cell crude extract was precipitated with 40%-saturation ammonia sulfate followed by cross-linking with 0.1% glutaraldehyde for 1 h at room temperature；then the cross-linking enzyme aggregates（CLEAs） was encapsulated through biosilicification by rapidly mixing with 1 mol/L hydrolyzed tetramethoxysilane（TMOS）. The immobilized enzyme Si-CLEAs retained 60% specific activity of the free enzyme. Si-CLEAs had higher thermostability（t1/2=4 h） than the free enzyme（t1/2=1.5 h） at 55 ℃. Si-CLEAs retained about 78% of the initial activities after eight catalytic batches. When loaded in a packed bed reactor，the packed-bed reactor showed high stability and conversion ratio of 95% after reused for 10 times. The study will be useful for industrial applications of fumarate synthesis using immobilized maleate cis-trans isomerase.

Abstract:A strain with high feruloyl esterases activity was screened out. Its fermentation characteristics and application in Chinese rice wine was investigated. Through preliminary and secondary screening，colonial morphology，physio-biochemical tests and rDNA ITS1-5.8S-ITS2 sequence analysis were adopted to determine the phylogenetic position of the isolated strain. Ferulic acid content was used as measurable indicator，Temperature，Initial pH，Wheat bran content，Alcohol content was chosen as variables，the production of ferulic acid in the fermentation process was studied. Strain was added to rice wine brewing process after enrichment cultivation. A high feruloyl esterases activity strain was screened and identified as Cladosporium cladosporioides strain. The strain was preserved in CCTCC with the accession number CCTCC NO：M2015549. Aften cultivating for 72 h under 30 ℃，pH 4.8，5% Wheat bran，9% alcohol content，the activity peak value of feruloyl esterase enzyme produced by this strain was 175.4 U/L. When the strain content was 0.010%，ferulic acid content increased to 150%. A Cladosporium cladosporioides strain with high enzyme activity which could produce feruloyl esterase was first screened out. It has positive effect on the improvement of ferulic acid content when adding to the brewing process. It has proved that the strain has great potential in production of feruloyl esterases and development of function food rich with ferulic acid.

Abstract:In order to improve the level of lycopene biosynthesis，this study use atmospheric and room temperature pressure plasma to mutate Blakeslea trispora negative bacteria for lycopene efficient production. The standard curve of lycopene content analysis and the lethal rate curve were established. By constructing the "saddle shaped" lethal rate curve，the suitable mutation treatment time was determined. After several rounds of screening，a mutant strain ARTP-3 was obtained with the lycopene production of 0.85±0.04 g/L，which was increased by 51.4% than that of the original strain. Furthermore，the subculture experiments showed that the ycopene production of ARTP-3 was much stable. In this study，we have not only obtained a high efficient lycopene producer with high economic value，but also can provide useful reference for the breeding of other industrial microorganisms，especially for of filamentous fungi.

Abstract:The inhibition effect on biofilm production of Pseudomonas aeruginosa PAO1 by the cinnamon extract was studied under the minimum inhibitory concentrations （MIC）. Firstly，it was studied the inhibition effect of the cinnamon extract on the purple pigment production by C. violaceum CVO26 as screening bacteria. Biofilm formation by P. aeruginosa PAO1 was quantitatively determined based on Microplate reader. The profile of the biofilm of PAO1 was observed under light microscope. The results showed that the cinnamon extract under MIC inhibited the purple pigment production of C. violaceum CVO26 and biofilm formation by P. aeruginosa PAO1，and the inhibition rate increased with the increase of the concentration of cinnamon extract. Also，it had obvious influence on the formation of P. aeruginosa PAO1 biofilm profile characteristics. Therefore，the cinnamon extract under MIC had inhibitory effect on the biofilm formation of P. aeruginosa PAO1.

Abstract:In order to obtain the recombinant thermostable lactase，a DNA fragment containing the mature lactase gene was amplified from Pyrococcus furious by PCR and cloned into pPICZaA，generating a fusion protein with the alpha factor from baker's yeast and integrated into the genome of Pichia pastoris strain X-33. Recombinant yeast transformants with high-level recombinant lactase production was identified by Western blotting，secreting as much as 125 U/mL induction by methanol. The recombinant thermostability lactase was purified by Ni+-NTA affinity chromatography and SDS-PAGE results shown that the purity was over 95%. The specific activity was about 1 800 U/mg and the optimal temperature was about 105 ℃. It was also showed that the purified lactase from P. pastoris has a highly thermostability and strong ability to hydrolysis lactose.

Abstract:In this paper，the expression of glycerol kinase（GK） from Escherichia coli MG1655 and its application for ketose synthesis were investigated. The gene glpK encoding GK was amplified and cloned into the expression plasmid pET-28a. The recombinant plasmid was transformed into the E.coli Rosetta（DE3） to express the protein induced by IPTG. GK enzyme was purified by Ni2+ chelating affinity column chromatography. The GK enzyme can catalyze cheap substrate glycerol to produce L-glycerol 3-phosphate which is converted into DHAP by glycerol phosphate oxidase（GPO）.The generated DHAP can be coupled with the acceptor D-glyceraldehyde catalyzed by different DHAP-dependent aldolases to produce ketose-1-phosphate，which can be dephosphorylated with acid phosphatase（AP） to get a series of ketoses. The results demonstrated that a series of ketoses including rare sugars were successfully synthesized using one-pot multienzymatic reaction with the cheap substrate glycerol as the precursor. The product yields and ratios were detected by TLC and HPLC. This approach could provide the theory basis for the synthesis of more other rare sugars and their derivatives.

Abstract:In this study，immobilization of Sumo-Hep I-His was investigated using Ni2+ chelating agarose gel as carrier. Under optimal conditions，i.e.，weight ratio of barrier to rude enzyme 30∶1，pH 7.0 and immobilized time 6 h，Sumo-Hep I-His was successfully immobilized with an apparent enzymatic activity of 10.07±0.35 IU/mL carrier. Compared with free enzyme，immobilized Sumo-Hep I-His showed great improvement in thermo stability （the half-live was 8 times of free Hep I at 30 ℃ as well as pH stability. The storage and operation stability of the immobilized enzyme was also significantly improved，as 76% relative enzymatic activity was remained after storing for 60 days at 4 ℃ and 75.8% relative enzymatic activity was remained after 8 reaction cycles. GLK-Gel Ni gel also showed good recycling stability that 88.9% adsorptive capacity was remained after 5 cycles immobilization. Conclusively，immobilized Hep I showed great potential in industrial application.

Abstract:trans-4-hydroxyproline has important applications in many fields. On the basis of the constructed< i> E. coli BL21（DE3）ΔputA strain，sucA gene was knocked out and the optimized trans-4-hydroxylase gene（hyp） was introduced. Then，the constructed plasmid pUC19-< i>ptrp2-hyp-vgb which included a tryptophan tandem promoter and Vitreoscilla hemoglobin（VHb） was imported into the recombinant strain and overexpressed. Then in the flask level，fermentation conditions and medium components were confirmed by single factor optimization and orthogonal experiment. The results showed that，compared with the original bacteria and two single knockout strains containing the same plasmid，the double knockout strain had the highest whole-cell activity，which is about 2.6 times as the original bacteria. After optimizing the fermentation conditions，trans-4-hydroxyproline was accumulated to 1.08 g/L，which increased 2.87 fold.

Abstract:In order to achieve efficient production of the MTSase and MTHase from（< i>Sulfolobus acidocaldarius）ATCC 33909，gene sequence encoding MTSase and MTHase were PCR-amplified using recombined plasmid pET-24a（+）-treY < /i>and pET-24a（+）-treZ which were fused to expressional vector pSVN. The recombined plasmid was transfered into expression host Brevibacillus brevis by electroporation，The enzyme activity of MTSase and MTHase expression in Brevibacillus brevis were 630.0 U/g（wet cell） and 170.0 U/g（wet cell） after cultivated in shake flasks for 48 h. Also presented are the preliminary studies on the reaction conditions of conversion by the enzymes. The optimal conditions for the conversion are as following：the initial pH was 5.5，the reaction temperature was 60 ℃，initial potato starch concentration was 100 g/L，enzymes concentration were MTSase 80 U per gram of substrate、 MTHase 20 U per gram of substrate 、Pullulanase 5 U per gram of substrate and CGTase 15 U per gram of substrate，incubated for 48 h. Under this conditions，the yield of trehalose reached approximately 80.2%.