Abstract:Multilocus house-keeping gene sequence analysis is a hotspot of taxonomy and phylogeny of prokaryotes. In this research, we used atpD and glnⅡ gene sequences to analyze the phylogeny of nine rhizobia strains of Albizia spp., Acacia spp. and Leucaena leucocephala and compared the results to that of 16S rDNA. The phylogenetic relationships based on the sequence analysis of these three genes were congruent at the genera level. CCBAU43060 and CCBAU 61139 were located in the branch of Rhizobium-Agrobacterium. CCBAU51471, CCBAU35220, CCBAU51276 and CCBAU61158 belonged to the genera of Mesorhizobium. CCBAU35234, CCBAU61178 and CCBAU35085 were assigned to Bradyrhizobium. Differences were found for some strains, for example CCBAU 61158, CCBAU43060, CCBAU61178, at the species level. Insertion fragment and mosaic gene were also found in some isolates. These results indicated that there was recombination between species in the same genera. It is reliable to determine the taxonomy status at genera levels based on the sequence analysis of 16S rDNA. If the relationships between strains belonging to the same genera were studied using the phylogeny meth-ods, researches should be carried out with more than one house-keeping genes.

Abstract:Molecular diversities of rumen archaea of Jinnan (South Shanxi Province, China) cattle was analyzed and compared by 16S rRNA gene sequencing from three clone libraries generated using three different archaea-specific primer sets, respectively. DNA from rumen of 4 Jinnan cattle was extracted, and methanogen 16S rRNA gene was amplified using archaea-specific primer sets. Three clone libraries were generated by using vector pGEM-T and cloning into E.coli JM109. One hundred clones were randomly picked up for each library and RFLP was analyzed for each clone to obtain OTUs. Sequences from each OTU were analyzed and compared with available sequences in GenBank. The first library, generated with primers Arch f364/1386, produced four groups of sequences, affiliated with 4 Methanobrevibacter strains, 1Y (61% of clones), SM9 (23% of clones), NT7 (14% of clones), and AK-87(2% of clones). The second library, generated with primers 1 Af/1100Ar, two groups of sequences, one affiliated with Methanobacterium aarhusense(72% of clones) and the other with Methanosphaera stadtmanae DSM 3091 (28% of clones). The third library, generated with primers Met86F/Met1340R, produced a high degree of diversity. It included the sequence groups found in the first and the second libraries, as well as sequences affiliated with the Methanomicrobium mobile (2% of clones) and uncultured euryarchaeote sequences (7% of clones). The phylogenetic analysis indicated that archaea found in the three libraries were clustered in Methanobrevibacter, Methanobacterium, Methanosphaera, Methanomicrobium, and unidentified euryarchaeote of the Euryarcharota. There were 25 unidentified sequences belonged to Euryarchaeota. This suggests the existence of novel methanogens in the rumen of Jinnan cattle.

Abstract:The largest detected plasmid pBMB165 from Bacillus thuringiensis subsp. tenebrionis strain YBT-1765 (H8ab) was cloned and its physical map was analyzed. For the cloning, two BAC libraries were constructed with their plasmid DNA and genomic DNA, respectively. The plasmid DNA BAC library was obtained by partially digesting plasmid DNA with BamHI and then cloning to pBeloBAC11 vector, whereas the genomic DNA BAC library was done with HindIII partial digestion. With the chromosome walking strategy, the plasmid BAC library was initially screened by the primers designed according the sequence coding replication protein Rep165 on a previously identified 3.6kb DNA fragment (pBMB165-F4A). Finally, 5 clones covering the most of plasmid pBMB165 were obtained. When screening the genomic DNA BAC library, 8 clones covering whole plasmid pBMB165 were isolated. By restriction analysis of these 13 BAC clones, the physical map and the linear linkage map of plasmid pBMB165 were constructed and the size of pBMB165 was calculated to be 82kb. Based on the DNA sequence of the BAC insertion ends and a previously published 20kb fragment on recombinant plasmid pBMB165A2, there were redundant transposable elements appeared on this large plasmid. This study provided a novel way to clone large plasmid from B. thuringiensis, to draw the physical map by construction of BAC library, and to dissolve the problem in cloning large plasmid from B. thuringiensis.

Abstract:Genes of zwf1 and zwf2 encode two isozymes of glucose-6-phosphate dehydrogenase (G6PDH) of Streptomyces, respectively. G6PDH is the first enzyme in the oxidative pentose phosphate pathway (PPP) and the key enzyme for NADPH generation.Based on thermal sensitive plasmid pKC1139, a recombinant plasmid pKC1139-zwf2′was constructed and verified with restriction enzyme digestion. The plasmid pKC1139-zwf2′was electropolated into competent Streptmyces rimosus M4018 cells after it was demethylated by E.coli GM2929. Transformants grown on TSA plate containing 500ug/mL apramycin were selected, and identified using dot hybridization analysis and PCR amplification with apramycin resistant gene as primers.A positive clone was then selected and designated M4018-△zwf2. With parent strain S. rimosus M4018 as control, mutant M4018-△zwf2 was cultured in shaking flask. Specific acitivity of G6PDH of M4018-△zwf2 was only half of that of parent strain whereas yield of oxytetracycline (OTC) of mutant was 27% higher. to the mutant had a similar biomass profileto that of the control Oxytetracycline biosynthesis started when the growth entered stationary phase on the 4th day. However, specific oxytetracycline production of mutant was 31% higher thanthat of the parent strain, indicating that zwf2 disruption could enhance oxytetracycline biosynthesis in S. rimosus M4018-△zwf2.

Abstract:Endocystic metabolism of energy and material is a complex matrix. We do not know the consequence of replacing the genes controlling the metabolic fluxes. Single gene(add) deletion cannot change the direction of metabolic fluxes of adenosine. Through deleting 3 genes, namely add (encoding adenosine deaminase [EC:3.5.4.4]), deoD (encoding purine-nucleoside phosphorylase [EC:2.4.2.1]) and amn (encoding AMP nucleosidase [EC:3.2.2.4]), and introducing ado1 gene(encoding adenosine kinase in S. cerevisiae[EC:2.7.1.20]), we modified the salvage pathway of adenine metabolism, and constructed a strain named J991. Extract of J991 was analyzed by HPLC. The endocystic concentration of ATP, ADP and AMP raised 2-fold as the control, and the metabolic fluxes of adenosine are also changed. It is a new way for ATP stimulation. Multi-gene manipulation is more effective than single-gene manipulation in salvage pathway of adenine.

Abstract:Actinomycete strain Z314 with the capability of converting compactin to pravastatin was isolated from soil samples. Based on the results of morphological, physiological, chemotaxonomic characteristics and 16S rRNA analysis, strain Z314 was placed within the genus Streptomyces and identified as the species Streptomyces xan-thochromogenes. The production of pravastatin reached 1580mg/L under optimized feeding of compactin, and the conversion rate was 49.45%. With the efficient purification system by fibrin column, macroporous adsorption resin and high performance liquid chromatography, pravastatin was purified from the culture supernatant with an overall recovery of 45.06% and purity of 99.02%. It is the first report in the world to produce pravastatin by con-verting compactin using Streptomyces xanthochromogenes under optimized conditions, the conversion of compactin to pravastatin was higher than other methods reported. The recovery and purity were improved by the three-step purification of pravastatin from the culture supernatant.

Abstract:Galacto-oligosaccharides (GOS) are promising non-digestible oligosaccharides recognized as prebiotics. Commercial GOS containing galactose as subunit, are synthesized from lactose using the galactosyl-transferase activity of β-galactosidase. A strain producing β-galactosidase with transglycosylation activity was screened from the soil. Phenotypic analysis including morphology and physiology characteristics and 16S rDNA sequence analysis were carried out. Based on taxonomy results , the strain was identified as Enterobacter agglomerans B1. Medium and fermentation conditions were optimized by single factor and orthogonal experiments. The enzyme reached 9.7 U/mL in the medium (pH 7.5) containing 1% lactose, 1% yeast extract, and 0.5% peptone when cultured at 25℃ for 26 h. Effects of pH, temperature, lactose concentration, and reaction time on transgalactosylation by whole cells were studied. Yield of GOS reached 40.7% in 30% lactose (pH 7.5) at 50℃for 12 h, as analyzed by HPLC and TLC. The results of an MS analysis showed that GOS were composed of di, tri-, and tetrasaccharides.

Abstract:The complete dehydrochlorinase gene linA of a γ-hexachlorocyclohexane (γ-HCH) degrading strain Sphingomonas sp. BHC-A, containing promoter and Shine-Dalgarno sequence (SD sequence), was amplified by PCR. The linA gene was inserted into NotI-cut transposon vector pUT/mini-Tn5 (Kmr) to get a novel transposon vector pUT/mini-Tn5-linA. With the helper plasmid RK600, the transposon vector pUT/mini-Tn5-linA was introduced into one carbendazim degrading gram-positive strain Rhodococcus sp. DJL-6 by triparental conjugation and then the dehydrochlorinase gene linA was integrated into the chromosome of Rhodococcus sp. DJL-6 by the transposon mini-Tn5. The selected multifunctional genetically engineered strain DJL-6A could degrade γ-HCH and carbendazim simultaneously. The dehydrochlorinase activity of DJL-6A was as strong as that of Sphingomonas sp. BHC-A in 0.05 and 5 μg/mL initial γ-HCH concentration. The linA of the strain DJL-6A was genetically stabile after successive plating DJL-6A for 30 days on nonselective media.

Abstract:Phosphate-dissolving microorganisms can be applied for better use of insoluble phosphorus as fertilizer. , A phosphate-dissolving strain P21was isolated from soil samples in China. The isolate was identified as Erwinia herbicola var. ananas, based on its 16Sr DNA sequence and physiological characteristics. Its activity was measured in solid media as well as liquid media using different phosphate sources including tricalium phosphate, hydroxyapatite, ferric phosphate, aluminium phosphate, zinc phosphate, and rock phosphates. E. herbicola could strongly dissolve 1206.20mg tricalium phosphate and 529.67mg hydroxyapatite in per liter liquid media. The strain showed high phosphate-dissolving ability for rock phosphates from Jinning and Kunyang in Yunnan province, Yaan in Sichuan province and Jinping in Guizhou province with the capacity of 6.64 mg, 78.46 mg, 67.07 mg and 65.24 mg soluble phosphate respectively per liter medium, whereas the phosphate-dissolving ability to the rest of the eight rock phosphates was weak. According to the experiments, the phosphate-dissolving ability of E. herbicola was specific to different rock phosphates, and phosphate-dissolving ability of E. herbicola was not directly related to pH reduction of liquid media.

Abstract:Deinococcus radiodurans R1 has extraordinary resistance to radiation. DRB0099 might play an important role in protecting the bacterium against radiation. To verify the inference, we deleted drb0099 and constructed the mutant. Comparing with the wild type, the mutant grew more slowly in the beginning cultivation stage (0~16 h) under normal conditions. After being cultivated for 16 h, the mutant grew faster than the wild type. The biomass concentration of the wild type was always higher than that of the mutant. The mutant cell’s fission during the growing phase might be blocked. When treated with UV, although Deinococcus radiodurans R1 cultures’ survival fraction was lower with UV treatment time increased, the survival fraction was much higher than that of⊿drb0099. The wild type could repair DNA double strands breaks better than the mutant. The gene drb0099 might directly relate with the DNA repair system. The mutant was more sensitive to H2O2 than the wild type. The wild type could better protect protein and DNA against reactive oxygen species (ROS) or in DNA repair. When treated with low concentration of H2O2, although the survival fraction of both R1 and the mutant decreased, the difference was small. However, with the concentration of H2O2 increased, the difference value increased. The mutant without drb0099 was more easily injured than the wild type with ROS increased. Under UV or H2O2 treatment, DRB0099 could protect protein and DNA from oxidation.

Abstract:Wolbachia are a group of maternally inherited bacteria harbored by a variety of arthropods and can manipulate the reproductivity of their hosts. Eighteen populations of whiteflies were collected from Hebei, Xinjiang, Beijing, Shandong, Zhejiang, Guangxi, Hainan, Guangzhou and Fujian provinces, China. These whiteflies were molecularly identified using internal transcribed spacer 1 ITS1 rDNA sequences. They were then detected for Wolbachia infection using Wolbachia-specific primers designed based on 16S rDNA and wsp gene sequences. The results showed that almost all populations were detected positive for Wolbachia infection. Whiteflies of B / Q group mainly carried Wolbachia belonging to supergroup A, while non- B / Q whiteflies were commonly detected for Wolbachia superinfection. This study indicated that the infection rate of Wolbachia in natural populations of B. tabaci might be much higher than expected, and the threshold of detection methods may be one of the key factors influencing detection of Wolbachia infection.

Abstract:Streptococcus suis serotype 2 (SS2) is an economically important, zoonotic agent causing death and disease in both human and swine. According to published SS2 European strain P1/7 complete genomic sequence, primers for detection and amplification autolysin were designed. Autolysin gene was detected by PCR with genomic DNA of HA9801 (Jiangsu isolate), ZY05719 (Sichuan isolate), ATCC43765 (reference strain), other SS2 isolates from different regions, and strains of other different serotypes (such as serotype 1, 1/2, 7 and 9). PCR results showed that all 27 SS2 virulent isolates harbored gene autolysin, but not in non-virulent SS2 strain. Among other serotypes, only serotype 7 strain had this gene. The complete autolysin genes of HA9801 and ZY05719 were respectively amplified by PCR and sequenced. Their putative protein sequences were analyzed through online software, results showed both had six repeated domain “GBS_Bsp-like” and one domain “N-acetylmuramoyl-L-alanine amidase”. In addition, sequence similarity analysis demonstrated that autolysins of the two strains (HA9801 and ZY05719) showed high homologue to that of European strain P1/7(99.8%), but obviously differed from Canada strain 89/1589, the latter lacks one domain “GBS Bsp-like”. Software DNASTAR was used to analyze autolysin protein sequence and predicted its putative antigenicity. Then partial antigentic segment of autolysin was amplified, cloned and inserted into expression vector pET30a(+), and induced by IPTG to express recombinant autolysin. Its reactivity was analyzed by SDS-PAGE and western blot with swine convalescent sera, blot result showed that the recombinant protein had good reactivity and could be considered as a vaccine candidate.

Abstract:Actinobacillus pleuropneumoniae is a very important respiratory pathogen for swine and causes great economic losses in pig industry worldwide. Signature-tagged mutagenesis is an effective method to identify virulence genes in bacteria. In this study, we selected nalidixic acid-resistant strains of APP serotypes 1 and 3 by in vitro cultivation, and used as receipt strains for constructing transposon mutants by mating with E. coli CC118 λpir or S17-1 λpir containing mini-Tn10 tag plasmids pLOF/TAG1-48, with or without the help of E. coli DH5α (pRK2073). We screened mutant strains by antibiotics selection, PCR and Southern blot identification. Our data revealed that nalidixic acid-resistance of APP strains could easily be induced in vitro and the resistance was due to the mutation in the DNA gyrase A subunit gene gyrA. In the mating experiments, the bi-parental mating was more effective and easier than tri-parental mating. Different APP strains showed a different mating and transposon efficiency in the bi-parental mating, with the strains of serotype 1 much higher than serotype 3 and the reference strain of serotype 3 higher than the field strains. These data were helpful for the construction of STM mutants and pickup of virulence genes of APP.

Abstract:AIM: To analyze the localization of attenuated Salmonella typhimurium after oral immunization. METHODS: Prokaryotic expression plasmid pYA33-DsRed, carrying the RFP gene, was constructed and electro-transformed into an attenuated strain X4550 of Salmonella typhimurium, the recombinant bacteria were named as X4550(33-DsRed). The macrophage cell line RAW264.7 and bone marrow dendritic cell (BMDC) were invaded by X4550(33-DsRed) in vitro. Furthermore, BALB/c mice were immunized with recombinant bacteria orally. RFP positive cells (RFP+ cells) were detected by Flow Cytometry (FACS) from spleen, liver, Mesenteric lymp node (MLN), Peyer’s patch (PP), Inguinal lymph node (ILN). RESULTS: The invasion rate increased when the multiplicity of infection(MOI) were improved in this two kinds of cells respectively. After oral immunization with X4550(33-DsRed), RFP+ cells were detected by FACS on 1d, 2d, 3d, 5d, 7d in spleen, liver, MLN, PP, ILN cells. The first day, RFP+ cells were detected in MLN and PP, and in PP at a higher rate of 1.4% than that of MLN. 0.4% RFP+ cells were detected the next day in ILN. On 3th day, the rates of RFP+ cells were increased in all of above tissues or organs and decreased on the 5th day. At the 7th day, RFP+ cells couldn’t be detected in all tissues or organs tested. CONCLUSIONS: It is suggested that the invasion ability and the transfer througy mucosal pathway and targeting to recognize immune tissue or organs are favor of the research in mucosal vaccine and the vaccine efficiency.

Abstract:As porcine circovirus 2 (PCV2) ORF2 encodes the major structural protein (capsid) that is closely related to the pathogenesis, the capsid (Cap) protein could be used as a target antigen for serological analysis. The immunoactivities of the truncated capsid proteins containing immunogenic epitopes of PCV2 (Cap2s) or PCV1 (Cap1s) expressed in Escherichia coli were described, as well as the characteristic of their polyclonal antibodies in diagnosis of PCV2 infection. Western blot analysis revealed that both Cap2s and Cap1s gave strong signals on nitrocellulose membranes to their corresponding polyclonal antibody. Furthermore, either PCV2-positive sera from PMWS cases or PCV1-positive swine sera could only recognize its relative polyclonal antibody. There was also no cross-reactivity between the two polyclonal antibodies when reacted with natural Cap proteins of viral particles on cells by immunofluorescence assay (IFA). Thus, an ELISA was then developed using PCV2 Cap as coating antigen to evaluate the sero-prevalence of PCV2 infection in pigs. The PCV2-positive rate ranged from 48.28% to 100% among different herds (n=13) with an average of 80.69% (209/259). These results indicate that Cap2s was type-specific and could be used as a discriminative antigen for monitoring PCV2 antibody in serum. The polyclonal antibodies were also useful for differential identification of PCV1 and PCV2 infection by immunohistochemistry.

Abstract:We previously showed some differences in Marek’s disease virus (MDV) VP22 gene between virulent and avirulent strains, in the deletion from 201aa to 206aa, namely 201TKSERT206. In this study, VP22 genes were amplified from strains: CVI988/Rispens and GA. And then the fragments were subcloned into pcDNA3.1/zeo(+), respectively, which were co-expressed with an enhancer green fluorescent protein (EGFP) after transfection into COS-1 cells. As with both human herpesvirus 1 and bovine herpesvirus 1 VP22-EGFP fusion proteins, the subcellular localization of the three MDV EGFP-VP22 products revealed few differences, which bind to microtubules and nucleus membrane, and then to heterochromatin. In addition, VP22s also bind to centrosomes and inter-membrane. During mitosis, EGFP-VP22s bind to sister chromatids, but dissociates from the centrosomes and the microtubules of the mitotic spindle. In truncated fragments’ transfection experiments, stained with the specific monoclonal antibody against VP22, it concluded that the full length of VP22 was required for protein transduction, and N1-18aa was essential to VP22 translocating from cytoplasm to nucleus as a potential nucleus localization site in the absence of other viral factors in MDV-1.

Abstract:Based on the antigenic analysis of duck plague virus (DPV) gB protein, we designed a pair of primers to amplify the gene fragment encoding high antigenic domain of DPV N-terminal gB protein from the DPV genome. The cloned gene was digested with EcoRⅠand Hind Ⅲ and then inserted into pET32a vector to obtain the recombinant pET-gB1 plasmid. The recombinant plasmid was transformed into E. coli BL21, and expressed in very high level after induced with IPTG.. The expressed product was analyzed by SDS- PAGE and Western blotting. The result indicated that the fusion protein (pET-gB1) existed as inclusion body, which was about 42.4kDa and showed specific immunoreactivity with anti-DPV sera. The recombinant gB1 protein was purifiedwith His?Bind resin pro-tein purification procedure. Then an indirect ELISA was established to detect antibody against DPV with the puri-fied gB1 protein as the coating antigen. The result showed that the optimal concentration of coated antigen was 6.5μg/mL and the optimal dilution of serum was 1∶80. The positive criterion of this ELISA assay was ODthe tested serum > 0.4 and ODthe tested serum/ODthe negative serum > 2.0. The ELISA was done on 700 sera that were preserved in Shandong, Jiangsu Provinces, and were detected by igB1-ELISA and iDPV-ELISA with duck plague virus as the coating an-tigen respectively. The agreement ratio between the two methods was 95.6 %.

Abstract:We constructed avian pathogenic Escherichia coli (APEC) specific DNA microarray to analyze the transcriptomes of APEC highly pathogenic strain E058 and low pathogenic strain E526 (both belonging to O2 serotype). For cultures in Luria-Bertani (LB) broth medium, 16 distinctly expressed genes were observed in strain E526, and all of them were down-regulated. For cultures in the serum of chickens, 15 distinctly expressed genes were screened in strain E526, and all of them were also down-regulated. The results suggest that DNA microarray could be used to screen distinctly expressed genes among virulence- and potential virulence-associated genes of APEC. The expression of 11 common virulence- or potential virulence-associated genes were down-regulated for strain E526 compared to those of strain E058 cultured both in LB broth and chicken serum. Meanwhile, 4 potential virulence-associated genes,aes-3,aes-10,aes-13 and aes-15 were down-regulated for strain E526 but not for strain E058, when they grew in the chicken serum. Besides known virulence factors, we observed 10 new potential virulence-related genes including aes-1，aes-2，aes-3, aes-4, aes-6, aes-8, aes-10, aes-13, aes-15 and aes-31.

Abstract:Recently, new insecticides containing 3-chloropyridylmethyl group as a versatile building block have been developed, among which imidachloprid is a promising one. The synthesis of imidachloprid can use 6-Hydroxynicotinic acid, the first intermediate of the bacterial degradation of nicotinic acid, as a starting material. It is difficult to hydroxylate nicotinic acid at C6 position in a chemical synthesis. However, biotransformation can produce 6-hydroxynicotinic acid with industrial application possibility. Therefore, methods for large scale screening of 6-hydroxynicotinic acid-producing microorganisms are urgent. A high through-put screening method for 6-hydroxynicotinic acid transforming strains was established by determining 6-hydroxynicotinic acid based on 96-well Microplate-Multiskan Spectrum. The determination wavelength and the reference wavelength of 6-hydroxynicotinic acid were 251nm and 231nm respectively. Beer's law is obeyed in the range of 0.5-11μg/mL (R2=0.9999) for measuring 6-hydroxynicotinic acid. The average recovery rate was 99.11%-100.81%. The results showed that there was no apparent difference between our microplate method and previous HPLC method in the detecting 6-hydroxynicotinic acid formation. The microplate method is simple, convenient and accurate. It has the potential for large scale (about 2000～5000 reactions/d) screening of 6-hydroxynicotinic acid-producing microorganisms.

Abstract:Lipopeptides produced by Bacillus subtilis JA antagonized a broad spectrum of plant fungal pathogens. The purification and identification of the lipopeptide antibiotics plays an important role for further research. Crude lipopeptides were extracted with methanol from the precipitate, which was obtained by adding 6mol/L HCl to the cell-free culture broth and then stored at 4°C overnight. The crude extract was run on reversed-phase HPLC system with a Diamonsil C18 column (250 mm × 4.6 mm, Dikma) to separate the lipopeptides. Two antifungal compounds, which had strong inhibitory activity against various plant fungal pathogens, such as Fusarium graminearum, were purified. The molecular weights of two compounds were determined by electrospray ionization mass spectrometry (ESI/MS). Two compounds, with molecular weights of 1042.4 Da and 1056.5 Da, were homologues differed by a structure of –CH2 (CH2 = 14 Da). ESI collision induced dissociation mass spectrometry analysis was used to sequence the structure of purified compounds. Typical b- and y- type fragments showed that compound 1 (with a molecular weight of 1042.4 Da) had a primary structure of Pro – Asn – Tyr – βAA – Asn – Tyr – Asn – Gln (βAA represented β-amino acid), which was consistent with lipopeptide iturin A. Compound 2 was a homologue of iturin A.

Abstract:Alkaline pectate lyase (PL) from recombinant strain E.coli JM109（pHsh PL）was purified by a three-step process including (NH4)2SO4 precipitation followed by dialysis and chromatography. The purified enzyme appeared homologous on SDS-PAGE. The specific activity of the purified enzyme reached 1079 U/mg. The optimal pH and temperature were in the ranges of pH 9.0 to 10.0 and 50℃ to 66℃. The enzyme was preferable in optimal pH range in enzymatic retting of flax. Enzyme activity slightly increased in the presence of Mg2+ ion, whereas decreased in the presence of other ions, especially Fe2+. The Km of the purified enzyme for polygalacturonic acid was 20.93 mg/L, the Vmax for polygalacturonic acid hydrolysis was 105.3 μmol of unsaturated products per min and Ea was 21.74 kJ/mol. The results of the decay constant (kd ) analysis on condition of PL bonding polygalacturonic acid (kd=0.02 min-1) and PL without polygalacturonic acid ( kd=0.0342 min-1) showed the substrate was helpful to decrease thermal inactivation of PL. The products (unsaturated oligomers) from polygalacturonic acid degraded by PL were analyzed by electrospray ionization mass spectrometry(ESI-MS). The following data were obtained: ESI-MS m/z, 350.82 (unsaturated bigalacturonic acid, uG2), 527.04 (unsaturated trigalacturonic acid, uG3). However, m/z 175 (unsaturated galacturonic acid, uG1) was not found. These results indicate that the final PGA degradation products was a mixture of unsaturated oligo-galacturonides including uG3 and uG2 except for uG1. It suggests that the recombinant PL cannot degrade uG3 and uG2.

Abstract:We review briefly the strategies and processes to identify Ser/Thr protein kinases from Streptomycesspp. and discussed in detail the functions of well studied Ser/Thr protein kinases, such as AfsK. We used bioinformatic methods to predict transmembrane regions in Ser/Thr protein kinases of S. coelicolor A3(2) and S. avermitilis. The complete sets of Ser/Thr protein kinases from genomes of Streptomyces coelicolor A3(2) and Streptomyces avermitilis MA-4680 were categorized based on domain analyses.

Abstract:At present, increasing attentions have been paid to lactic acid bacteria because of their probiotic effects. In the nature, there exists a kind of lactic acid bacteria growing at low temperature with a long history of use. However, they have not been well studied and developed. Most articles about the lactic acid bacteria growing at low temperature focused on meat and fish storage at low temperature, or Kimchi, a kind of fermented vegetable. Many microorganisms studied are Leuconostoc and Lactobacillus species. Nevertheless, a few researches in this field are reported in China. In this paper, we review the living environment, varieties and functions of lactic acid bacteria growing at low temperature, to provide an overview for further studies. We also discuss perspectives of further development and utilization of these lactic acid bacteria.