Abstract:Filamentous fungi can secrete large amounts of proteins, glycosylate proteins and grow faster than plant, insect or mammalian cells. With the advances in fungal molecular genetics, strain improvement, and especially fungal genomics, filamentous fungi are developed as microbial cell factories for the production of heterologous proteins. This review focuses on recent developments of filamentous fungi as production hosts, such as protein quality control mechanisms, the secretion pathways, protein modification, strain stability, and most importantly the application of functional genomics in protein expression. At the same time, the strategies for improving heterologous protein production were also discussed in details.

Abstract:currently, transgenic crops create huge economic, social and ecological benefits with the development of its commercial production. For China, the speed of development and commercialization of transgenic crops is a strategic issue for the sustainable agriculture development and the international competitiveness of our agricultural products. In this paper, we compared and analyzed the status of commercialization of transgenic crops in China and world-wide.

Abstract:To improve the efficacy of peptide P277 in preventing autoimmune diabetes, heat shock protein 65 kD (HSP65) of Mybobacterium tuberculosis var. bovis was fused with linear polypeptide epitope of P277 and expressed as soluble protein in Escherichia coli. The fusion protein HSP65-P277 was purified by anion exchange column chromatography and then used to immunize prediabetic NOD mice with three ip inoculations in absence of adjuvants. Serum samples from the immunized mice were collected monthly and the concentration of blood glucose was measured. The study showed that administration of HSP65-P277 to NOD mice could prevent the development of diabetes more efficiently than the peptide P277 itself or HSP65. Fused to heat shock protein 65 of Mycobacterium tuberculosis could improve the efficacy of diabetes prevention of P277 in nonobese diabetic mice. The results suggest the fusion protein of HSP65-P277 would be useful for treating insulin-dependent diabetes mellitus.

Abstract:The actions of four organic acids in Radix isatidis, a traditional Chinese medicinal (TCM) herb, on Escherichia coli, Staphylococcus aureus and Shigella dysenteriae growth were investigated by microcalorimetry. The four organic acids were syringic acid, 2-amino-benzoic acid, salicylic acid and benzoic acid. The power-time curves of Escherichia coli, Staphylococcus aureus and Shigella dysenteriae growth with and without organic acids were acquired, meanwhile the extent and duration of inhibitory effects on the metabolism were evaluated by growth rate constants (k1, k2), maximum heat-output power (Pm) and peak time (tm). The inhibitory activity varied with different drugs. The sequences of anti-microbial activity of the four organic acids on Escherichia coli, Staphylococcus aureus and Shigella dysenteriae were all: syringic acid > 2-amino-benzoic acid > salicylic acid > benzoic acid. And benzoic acid promoted the growth of Staphylococcus aureus and Shigella dysenteriae. This study provides a basis for the further study on Radix Isatidis.

Abstract:Outer membrane proteins (OMPs) are embedded in the outer membrane of Gram-negative bacteria, mitochondria, and chloroplasts. The cellular location and functional diversity of OMPs makes them an important protein class. Researches on prediction of OMPs by bioinformatics methods can bring helpful methodologies for identifying OMPs from genomic sequences and for the successful prediction of their secondary and tertiary structures. In this paper, three feature classes were calculated from protein sequences: amino acid compositions, dipeptide compositions and weighted amino acid index correlation coefficients. Then, three feature classes were combined and inputted into a support vector machine (SVM) based predictor to identify OMPs from other folding types of proteins. The results of discrimination using several combined features including four amino acid index categories were calculated, and the influence on discrimination accuracy using different correlation coefficients with different orders and weights was discussed. In cross-validated tests and independent tests for identifying OMPs from a dataset of 1087 proteins belonging to all different types of globular and membrane proteins, the method using combined features obtains an overall accuracy of 96.96% and 97.33% respectively. And these results outperform that of other methods in the literature. Using this method, high specificities are shown from the results of identifying OMPs in five bacterial genomes, and over 99% OMPs with known three-dimensional structures in the PDB database are correctly discriminated. These results indicate that the method is a powerful tool for OMPs discrimination in genomes.

Abstract:Prediction of RNA secondary structures including pseudoknots is a difficult topic in RNA field. Current predicting methods usually have relatively low accuracy and high complexity. Considering that the stacking of adjacent base pairs is a common feature of RNA secondary structure, here we present a method for predicting pseudoknots based on covariance with stacking and minimum free energy. A new score scheme, which combined stacked covariance with free energy, was used to assess the evaluation of base pair in our method. Based on this score scheme, we utilized an iterative procedure to compute the optimized RNA secondary structure with minimum score approximately. In each interaction, helix of high covariance and low free energy was selected until the sequences didn’t form helix, so two crossing helixes which were selected from different iterations could form a pseudoknot. We test our method on data sets of ClustalW alignments and structural alignments downloaded from RNA databases. Experimental results show that our method can correctly predict the major portion of pseudoknots. Our method has both higher average sensitivity and specificity than the reference algorithms, and performs much better for structural alignments than for ClustalW alignments. Finally, we discuss the influence on the performance by the factor of covariance weight, and conclude that the best performance is achieved when l1 : l 2=5 : 1.

Abstract:The expression changes of genes associated with adipose metabolism and regulation of cell growth in backfat tissue at five growth stages (1, 2, 3, 4 and 5 months) of Landrace (a leaner Western breed) and Taihu pigs (a fatty indigenous Chinese breed) were measured by using pathway-focused Oligo microarray. The comparative results between two pig breeds of change ratios showed that the fold changes of 10, 6, 11, 8 and 19 genes were over two times at same month from 1 to 5 months respectively. Variance analysis (ANOVA) revealed that 25 genes in Landrace pigs showed significantly altered in expression among five growth stages (P<0.05). Principal components analysis (PCA) revealed that angiopoietin-like 4 (ANGPTL4), cathepsin K (CTSK), isocitrate dehydrogenase 2(NADP+) (IDH2), lipoprotein lipase (LPL), malic enzyme 1 (ME1), stearoyl-CoA desaturase (SCD) and uncoupling protein 2 (UCP2) displayed distinctive expression patterns from other genes, which suggested that these genes may be subject to special regulation during the period from 1 to 5 months. K-means clustering analysis revealed that genes in Landrace pigs which showed up-regulation were mainly related to the positive regulation of fatty acid metabolism and genes in Taihu pigs which showed slight fluctuation were mainly related to the regulation of cell growth. Quantitative real-time RT-PCR was used to verify the microarray data for five modulated genes, and a good positive correlation between the two measures of expression was observed for both two pig breeds at different growth stages. These results highlight some possible candidate genes for porcine carcass and meat quality traits, reveal the expressional disciplinarian of correlative genes and provide some data on which to base further study of the dynamic balance process for fatty acid biogynthesis and hydrolyzation.

Abstract:Short-chain esters play a significant role in the food industry as flavor and aroma constituents. Candida antarctica lipase B (CALB) is one of the most effective catalysts for organic synthesis. We constructed a CALB-displaying yeast whole-cell biocatalyst and applied it to esterification from caproic acid and ethanol. CALB was fused with the α-agglutinin C-terminal and the signal peptide of Glucoamylase in pICAS, a yeast surface display vector, to construct plasmid pICAS-CALB. An extremely Asn-rich linker, named celAL was inserted in the Xho I of pICAS-CALB to construct plasmid pICAS-celAL-CALB. The fused gene was under the control of GAPDH promoter. After incubated at 30oC for 96 h the lipase hydrolytic activity of the yeast whole cells reached a plateau, 26.26 u/(g·dry cell). In nonaqeous media, the yield of 98.0% ethyl hexanoate was obtained after 24 h esterification from caproic acid and ethanol (the molar ratio of caproic acid : ethanol = 1 : 1.25)using lyophilized CALB displaying yeast whole cells.

Abstract:Pyruvate phosphate dikinase (PPDK; EC 2.7.9.1) is found in certain microorganisms and plants, and catalyzes the conversion of AMP, PPi and phosphoenolpyruvate (PEP) to ATP, Pi and pyruvate. Using the genomic DNA of Microbispora rosea subsp. aerata as the template, a DNA fragment encoding the gene PPDK was amplified by PCR and inserted into the expression vector pET28a(+), yielding pET28a (+)-PPDK. The E. coli BL21 (DE3) was transformed with the pET28a (+)-PPDK. After inducing with IPTG, the E. coli BL21 (DE3) [pET28a (+)-PPDK] expressed recombinant PPDK fused to an N-terminal sequence of 6-His Tag. The molecular weight of PPDK was estimated to be 101 kD by SDS-PAGE. The PPDK was purified by His ? Bind Resin affinity chromatography and ultrafiltration using 10 kD cut-off membrane. The successful application of PPDK in pyrosequencing was also demonstrated.

Abstract:HIV-1 gp41 has been successfully anchored on the cell surface of yeast Saccharomyces cerevisiae by yeast cell-surface display systems using His-tag for the detection of protein expression. Gp41 activity has been detected by gp41 monoclonal antibody. The vector for gp41 yeast display has been constructed as follows: the gene-encoding gp41 was amplified by PCR using pMD18T-gp41 as a template, and then inserted into shuttle vector pICAS-His by restriction enzyme digestion. Next, the vectors were introduced into Saccharomyces cerevisiae MT8-1. After cultivation, recombinant cells were immunofluorescence labelled. The bright green cells were observed by the microscopy indicating the proteins have been displayed on the cell surface successfully, flow cytometry convinced that gp41 has been folded correctly on the cell surface. Then different concentrations of initial glucose were used to enhance the expression of protein. gp41 has been expressed by 82.46% yeast cells as the concentration of glucose was 1%. Protein expression was depressed when the concentration was increased.

Abstract:Several antigen epitopes were designed according to the sequences of Swine influenza virus hemagglutinin (HA) genes and lined with the interval. The gene was amplified by PCR and sub cloned into pET30a (+) vector. The fusion protein was expressed in E. coli BL21 (DE3) by induced with IPTG and purified by affinity chromatography. The molecular weight of the protein was about 20 kD in SDS-PAGE. Immunological activity of the fusion protein was analyzed by Western blot. The results showed that the fusion protein could interact with anti-His antibody and the rabbit antiserum against SIV. The immunized mouse can produced antibodies against the target peptide and HI antibody against SIV H1N1 or H3N2. This study provides a new vaccine candidate for SIV.

Abstract:Using a modified TAIL-PCR technique, the 5′-flanking region of the X gene in wheat was successfully isolated. Two novel modifications of the TAIL-PCR were introduced here: using a battery of random 10-mers as the short arbitrary primers instead of three degenerate 16-mers; using 29°C instead of 44°C as the annealing temperature for the low-stringency cycle; increasing five high-stringency cycles and reducing five low-stringency cycles; and using single primers for the third round of product identification. Isolated 5′-flanking region was fused to the GUS gene, and tested for expression in Arabidopsis plants. Histochemical analysis of the transgenic plants showed the report gene was driven by isolated 5′-flanking region. Modified TAIL-PCR technique could isolate rapidly the promoter of any gene from organisms with large genomes.

Abstract:To establish a model of inactivation adenosine A2A receptors in brain tissues of mice, we transplanted bone marrow cells (BMCs) from wild type (WT) C57BL/6 mice into A2A receptor knockout (A2A KO) C57BL/6 mice which were previously fractionated total body irradiation of 6.2 Gy×2. Six weeks later, we identified and evaluated the model. The results showed that the sexual chromagene pattern on white blood cells of recipient mice changed from female pattern to male pattern and there were 95.9% of A2AR+ cells in peripheral white blood cells of recipient mice, whereas there was no significant difference of A2AR mRNA level in brains between these recipient mice and A2AR KO mice. Furthermore, there was no significant difference of breathing frequency, brain water content and level of glutamate between the model mice and WT mice. These results indicated that we established successfully a mouse model of inactivation adenosine A2A receptors in brain tissues. This may provide a new and efficient strategy to study the effect of adenosine A2A receptors in disease and injuries of central nervous system.

Abstract:Ubiquitin conjugating enzyme functions as the second enzyme required for protein ubiquitination and plays an important role in ubiquitin transferring and substrate specific recognition. UBE2W, a newly described member of E2 family, was formerly reported probably involving in phototransduction or retinal degeneration in Drosophila. In this study, we report that murine UBE2W harbors a typical UBC domain and is highly conserved in different vertebrate homologues. GST-tagged UBE2W was expressed in E. coli BL21 (DE3) and purified with GST affinity chromatography. Using this antigen, we generated and further separated rabbit polyclonal antibody of UBE2W, of which the activity and specificity were confirmed by immunoblotting of transiently expressed myc-UBE2W fusion protein. Wide expression of UBE2W was found in brain, muscle, heart, lung, liver, spleen, kidney and testis of mouse with the generated antibody, indicating the functional importance of this novel protein. Furthermore, the UBE2W highly expression was confined to the adult testis and was developmental stage-specific.

Abstract:The amino acid sequence (1-301aa) coding the human PTP1B catalytic domain (PTP1Bc) was obtained from the GenBank. The PTP1Bc gene was constructed by overlapping PCR, then was inserted into vector pET-22b(+) and expressed efficiently in E. coli BL21(DE3) under optimum condition after IPTG induction. The proteins were expressed mainly as inclusion bodies with the yield of more than 30% of total bacterial proteins. The expressed products were purified through Ni2+-affinity chromatographic column. After purification, the purity of the proteins was more than 95%. Western blotting analysis suggested that the purified proteins could specially combine with anti-PTP1B antibody. Enzyme activity assay showed that the protein has phosphatase activities.

Abstract:The cDNA of Insulin-like growth factor binding protein 3 was subcloned into a eukaryotic secretory expression vector pSectagA to construct pSectag-IGFBP3. Human renal cell carcinoma (RCC) 786-O cells were transfected with pSectag-IGFBP3 using lipofectamine 2000. After 48 h, the secretory IGFBP-3 was tested and identified by western blotting. Meanwhile, Annexin V-EGFP stain was used to analyze the apoptosis of 786-O cells induced by IGFBP-3. Secretory IGFBP-3 protein could express successfully in the 786-O cells and the expressed IGFBP-3 directly displayed an apoptotic effect on the host cells. This work provides a basis for further study on the apoptosis-inducing mechanism of IGFBP-3 and the development of a new anti-tumor drug.

Abstract:Self-biting is a chronic disease, which cause wound to take effect on mink growth and pelt quality. In this study, we firstly adopted RAPD (random amplification polymorphism DNA) technique based on the reproducible 26 polymorphism primers screened from 100 random primers to analyze hereditary constitution of the samples from healthy minks and self-biting minks, respectively, at molecular level to aim to discuss the causes of self-biting. The results showed that 29 straps showed polymorphism among amplified 105 straps, of which the polymorphism rate is 27.62%. Between healthy and sick mink groups, the amplified DNA fragment through different primers indicated different distribution frequency. The similarity coefficient of mink groups is 0.8471 and genetic distance (variation) index is 0.1529. Through primer S356 (whose sequence is CTGCTTAGGG), we amplified different straps between healthy and sick mink. The amplified 1000 bp DNA fragment in the sick mink groups can preliminarily serve as molecular genetic label to distinguish from healthy and sick mink groups to gradually remove the mink individual of self-biting, achieve to purify mink groups and reduce economy loss of mink breeding industry. This work provide theoretical basis for further study on molecular breeding and disease prevention of mink.

Abstract:The aim of this study was to construct the complete genome of Marek’s disease virus serotype 814 strain as an infectious bacterial artificial chromosome (BAC). Using self-designed selection marker Eco-gpt (1.3 kb) and BAC vector pBeloBAC11 (7.5 kb), we constructed the transfer plasmid pUAB-gpt-BAC11. The plasmid pUAB-gpt-BAC11 and MDV total-DNA were cotransfected into secondary CEFs; we put the virus-containing cells in selection medium for eight rounds and obtained purified recombinant viruses. Recombinant viral genomes were extracted and electroporated into E. coli, BAC clones were identified by restriction enzyme digestion and PCR analysis. Finally, we obtained 38 BAC clones, DNA from various MDV-1 BACs was transfected into CEFs, and recombinant virus was reconstituted by transfection of MDV-BAC2 DNA. We successfully cloned the complete genome of MDV-1814 strain as an infectious bacterial artificial chromosome. With these cloned genomes, a revolutionary MDV-DNA engineering platform utilizing RED/ET recombination system was constructed successfully, which can help the understanding of MDV gene functions and promote the using of MDV as a vector for expressing foreign genes. In addition, it opens the possibility to generate novel MDV-1 vaccines based on the BACs.

Abstract:To achieve efficient and stable expression of heterologous exogenetic protein or antigen in E. coli chromosome, the luciferase report gene was knocked in lacZ site of chromosome lac operon by using Red recombination system and selection-counterselection kan/sacB technology. The quantitative analysis of exogenous gene expression indicated that the target gene could be efficiently expressed at lacZ site of lac operon. The results confirmed the efficient screening and stable expression of heterologous protein or antigen on chromosome by using the recombinant engineering technique. This study demonstrated that the chromosome could be used as a vector for heterologous protein or antigen and the stable expression of exogenous gene on E. coli chromosome had no side effect on the bacterial growth and propagation.

Abstract:Pseudomonas aernginosa (PA) is one of the most universal pathogens in clinical diagnosis, and conventional detection assay has many disadvantages. In this research, a pair of specific primers and a TaqMan fluorescent probe were designed in the conservative region of ETA gene by the method of bioinformatics analysis, the detection method for PA was successfully developed. Different gradient concentrations of PA DNA and various pathogen DNA were amplified by fluorescence quantitative PCR (FQ-PCR) to confirm the specificity and sensitivity of the developed method. Results showed that the developed detection assay is more sensible and specific by comparison to the conventional FQ-PCR method, and it is valuable for research and application prospects.

Abstract:An isolated virulence Riemerella anatipestifer strain passaged 200 times on TSB agar were used for the virulent to avirulent conversion. The effects of passage on biological properties of outer membrane proteins (OMPs) were investigated using the virulent and avirulent strains. Transmission electron microscopy demonstrated that the avirulent strain produced lower amounts of outer membrane vesicles and the outer membrane decreased, the cytoplasmic appearance jumbled. The OMPs of the virulent strain agglutinated only in RA serotype 2 antisera, whereas the OMPs of the avirulent strain agglutinated in antisera of RA 1, 2, 10 and 11. SDS-PAGE Analysis showed the OMPs profiles of both strains were similar but the immunoblotting profiles were different. The protective immunity against Riemerella anatipestifer infection was investigated by immunizations with OMPs in ducks. ELISA results showed that the OMPs induced the production of antibodies in immunized ducks, but the OMPs of virulence strain induced higher antibody titers than the attenuated strain (P<0.05). RA2 group showed significantly higher survival rates (100%) than RA200 group (0%) after challenged with the homologous virulent strain. The ompA gene of both stains were also amplified by PCR, nucleotide homology was 99.9%. In conclusion, OMPs of virulent RA strain are suitable candidates for vaccine development. Biological properties of OMPs undergoes significant changes during serial passage and suggest that vigilance should be used when extrapolating data obtained from the study of high-passage strains.

Abstract:One pair of primers were designed and synthesized on the base of the cDNA sequence encoding Schizosaccharomyces pombe N-glycanase reported on the GenBank. The cDNA sequence encoding Peptide N-glycanase was cloned from the Schizosaccharomyces pombe by RT-PCR. And then the RT-PCR product was cloned into the expression vector pET-15b. The expression vector pET-15b(+)/Png1p was transformed into E. coli BL21(DE3). The results showed that the relative molecular weight of the enzyme was determined to be approximately 39 kD using SDS-PAGE. The expression products after induction and purification can catalyze the cleavage of N-linked oligosaccharides from glycoprotein coped with heat, but have no action on the native glycoprotein with the help of DTT. The percentage of deglycosylated RNase B treated with equate Png1p in different reaction temperature, pH, concentration of DTT and denatured temperature showed that the optimum temperature, the optimum pH is 30°C; the optimum concentration of DTT is 10 mmol/L and the optimum denatured temperature is 100°C.

Abstract:To compare the activity of different promoter in baculovirus-insect system, a series of recombinant baculoviruses were generated harboring the E-GFP reporter gene under the control of one of 5 promoters, including the ie1 promoter of shrimp white spot syndrome virus (WSSV), the truncated ie1 (mie1) promoter, the ETL promoter of the baculovirus, the elongated ETL (mETL) promoter, and the polyhedron promoter (PPH) of the baculovirus. The expression efficiency of the E-GFP reporter gene in the recombinant baculovirus-infected Sf9 cells was determined by flow cytometry. The results showed that both ie1 and mETL promoters had a strong promoter activity at early phase, while PPH showed a strong promoter activity at late phase. The ie1 promoter suggested the strongest promoter activity. The homologous region 1 (hr1) was also found to enhance the ETL promoter activity.

Abstract:Pancreas is an important mixed gland having both endocrine and exocrine functions, and has been proven regeneration after injury. To explore the cell lineage tracing methods in pancreas in vivo and the regenerate cells source, we used pseudo-type retrovirus to transfect adult mouse pancreas which had been partially pancreatectomized by rubbing the kerf using a cotton stick saturated with retrovirus suspension then injecting 100 mL retrovirus suspension into pancreas, injecting 100 mL retrovirus by caudal vein, or interperitoneally injecting retrovirus respectively. The results showed that the method of rubbing the kerf then injection of retrovirus suspension into pancreas could more effectively mark the pancreatic cells than the caudal vein injection and the intraperitoneal injection did in vivo. Furthermore, this study also found that some acinus cells could accept injury stimulus signals to regenerate through resuming mitosis after pancreatic injury. This study establishes a cell lineage tracing method in pancreas in vivo using retrovirus and offers a clue for gene therapy of pancreatic diseases using retrovirus vectors.

Abstract:Poly (3-hydroxybutyrate-co-3-hydroxyhexanoate, PHBHHx) has superior mechanical and biocompatibility that may enable it to meet cardiovascular tissue engineering applications. We developed hybrid materials based on decellularized xenogenic vascular scaffolds that were coated with PHBHHx to investigate the intravascular biocompatibility. The hybrid patches were implanted in the rabbit abdominal aorta (hybrid patch, n=12). Only decellularized xenogenic vascular scaffolds were implanted without coating as control (uncoated patch, n=12). The patches were explanted and examined histologically, and biochemically at 1, 4 and 12 weeks after the surgery. The hybrid patches maintained original shapes, covered by confluent layer of cells and had less calcification than uncoated control. The results indicated that PHBHHx coating reduced calcification, promoted the repopulation of hybrid patch with recipients cells. In conclusion, PHBHHx showed remarkable intravascular biocompatibility and would benefit endothelization which would be a useful candidate for lumen of cardiovascular tissue engineering.

Abstract:Concanavalin A (ConA) is immobilized on a pre-activated chitosan microspheres, and then oriented immobilization of urease is carried out based on the strong interaction between ConA and glycoprotein. The optimum immobilization conditions are as follows: glutaraldehyde concentration is 3.5%, ConA concentration 1mg/mL, ConA pH 7.0 and urease concentration 0.4 mg/mL. For orientedly immobilized urease, the highest activity was allowed at pH 5.0~6.0 and temperature 77°C, and the Michaelis constant (Km) was disclosed to be 11.76 mmol/L by Lineweaver-Burk plot. Compared with the free urease and the randomly immobilized urease, the optimum pH of the orientedly immobilized urease becomes smaller and the pH domain wider. Orientedly immobilized urease presents higher temperature resistance, higher affinity to the substrate, and higher stability of operation.

Abstract:A bromoperoxidase from Gracilaria lemaneiformis was purified to homogeneity using a multi-step process of ammonium sulfate precipitation (AS), dialysis, and DEAE-cellulose 52 anion exchange chromatography. The bromoperoxidase activity was unstable or undetectable in crude extract solution. However, it became stable with electrophoretic purity after this multiple purification process. The anion exchange chromatography purification was a critical step in the purification process, which effectively eliminated the phycobiliprotein and smucilaginous polysaccharides. The purified bromoperoxidase was a monomeric enzyme with the relative molecular masses of 66 kD as determined by denaturing and native gradient gel electrophoresis. The optimal pH for bromoination was 6.0 and bromoperoxidase activity was stable as stored at a broad pH range of 3.0~9.0. Of a range of compounds tested, only vanadium enhanced bromoperoxidase activity. Kinetic studies for the bromination of monochlorodimedone (MCD) showed that the Km values of Br? and H2O2 are 53.5 mmol/L, 38 mmol/L respectively.

Abstract:The oxidative response of Bacillus sp F26 to different forms of reactive oxygen species (ROS) stress including H2O2, O2-·and OH·were investigated by using diverse generating source of ROS, which were characterized by synthesis of antioxidative enzymes. It was shown that the responses of cells to oxidative stress are largely dependent on species, mode (instantaneous and continual) and intensity of stress. Higher synthesis rate of catalase (CAT) is crucial for Bacillus sp F26 to resist H2O2 stress. The damage of H2O2 to cell was minor if CAT can efficiently decompose H2O2 entering into cell, furthermore, the response can stimulate cell growths and sugar consumption. Conversely, cell growth and synthesis of antioxidative enzymes are greatly inhibited when the intensity of H2O2 stress overwhelms the cell capability of clearing H2O2. Due to the difference in mode and effect on cells between O2-·and H2O2, higher synthesis rates of CAT and superoxide dismutase (SOD) couldn’t guarantee cells to eliminate H2O2 and O2-·efficiently. Therefore, the toxicity to cells induced by intracellular O2-· is more severe than H2O2 stress. Unlike response to H2O2 and O2-·, OH· stress significantly inhibited cell growth and synthesis of antioxidative enzymes due to the fact OH·is most active ROS. Our results indicated that Bacillus sp F26 will show diverse biological behaviour in response to H2O2, O2-·and OH·of stress due to the discrepancy in chemical property. In order to survive in oxidative stress, cells will timely adjust their metabolism to adapt to new environment including regulating synthesis level of antioxidative enzymes, changing rates of cells growth and substrate consumption.

Abstract:In order to increase the production of alkaline polygalacturonate lyase (PGL) by recombinant Pichia pastoris GS115, the effect of cell and methanol concentration on the PGL production was carefully investigated by single factor experiment. The optimum conditions were listed as follows: the cell concentration 122 g/L, the methanol concentration 20 g/L, and the ratio of methanol and cell concentration 0.16~0.20 g/g (methanol/cell). With the glycerol and methanol feeding strategies, the ratio of methanol and cell concentration could be controlled at the range of 0.171 to 0.195 g/g. And the highest PGL activity (430 u/mL) and highest PGL productivity (4.34 u/mL/h) were achieved.