Abstract:By construction of small fragments genome bank, several different fragments from chromosomal DNA of streptomyces production strain HS007 were cloned into cloning vector pSP-SIM. According to the sequencing results, five of them, which were 3- to 7- kb in size with single restriction sites in the middle, were cut down and re-cloned into bacte-ria-streptomyces shuttle vector pHJL400. Subsequently, the marker cassette, composed of special labeling sequence, oriT, 2 FLP recognition target sites and apramycin resistance gene, was inserted into the single restriction sites to create re-combinant plasmids pHJL02AFOH, pHJL07AFOH, pHJL08AFOH, pHJL10AFOH and pHJL12AFOH. These recombinant plasmids were then transformed into target production strain HS007 by conjugal transfer method, and corresponding marked mutants named 02-72, 07-44, 08-02, 10-81 and 12-58 were screened. Two of these mutants, 02-72 and 12-58, did not show changes in fermentation ability, whereas the others lost partially or completely fermentation ability. The apra-mycin resistance gene and oriT, flanked by two FLP recognition target sites, were then removed by FLP-mediated deletion from recombinant plasmid pSP02AFOH to give pSP02F, whereas the special labeling sequence was still reserved in pSP02F because of being located out of the two FLP recognition target sites. Finally, replacement plasmid pGH02FH was constructed by replacing the vector part of pSP02F with bacteria-streptomyces shuttle vector pGH112 and transformed into mutant 02-72. By selecting apramycin sensitive colonies, marked mutant 02-72-36, whose chromosome was inserted by special labeling sequence without apramycin resistance gene, was screened. Fermentation confirmed that its production ability was not reduced. Such genome labeling technique might be used in other strains of streptomyces to protect the property marks.

Abstract:The ATH1 gene encoded acid trehalase in Saccharomyces cerevisiae. The gene disruption cassette combined the heterologous dominant kanr resistance marker with a Cre/loxP-mediated marker removal procedure. The gene disruption cassette was produced by PCR using the same long oligonucleotides comprising 50 nucleotides that annealed to sites upstream or downstream of the genomic target sequence to be deleted. After transformation of the linear disruption cassettes with a Cre/loxP-mediated marker into the cells of Saccharomyces cerevisiae BY-6, selected transformants were checked by PCR for correct the integration of the cassette and concurrent deletion of the chromosomal target sequence. The copper-resistance gene (CUP1-MT1) was cloned into pSH47, which yielded pSH-CUP. The recombinant plasmid pSH-CUP was transformed into the cells of Saccharomyces cerevisiae BY-6(ΔATH1, G418r), and transformants were selected for copper resistance. Upon expression of the Cre recombinase results in removal of the kanr gene, leaving behind a single loxP site at the chromosomal locus. Construction of the recombinant plasmid pSH-CUP avoided inserting non-yeast gene and made the loxP - kanMX - loxP gene disruption cassette more conventional for eukaryotic organism gene disruption.

Abstract:A thermophilic bacterial strain MS-1 was isolated from the water let from an oil well, and identified as Bacillus sp. by its 16S rDNA sequence and morphological characteristics. The bacterium was able to grow at 60℃ and produce extracellular polymeric substance (EPS). Analysis of the EPS showed that there was 48.3%~54.5% polysaccharide, which was composed of mannose:glucose:galactose at a ratio of 2.04︰1.00︰0.89. The protein fraction was 37.2%~42.4% in EPS, containing methionine, leucine, aspartic acid, alanine, histidine and serine.Transmission Electron Microscopy and Environmental Scanning Electron Microscopy were used to observe the extracellular polymeric substances. The strain is useful in microbial profile modification in high temperature oil fields.

Abstract:A bacterium capable of producing Coenzyme Q10 (CoQ10, was isolated from soil. Based on analysis of the 16S rDNA sequence, traditional physiological characteristics, and Biolog-GN, the strain was belonging to the genus Sphingomonas and named as Sphingomonas sp. ZUTEO3. The optimum fermentation condition of CoQ10 production was as following: glucose 15 g/L, (NH4)2SO4 10 g/L, original pH 8.0 and at 25℃. The addition of solanesol could improve CoQ10 production. The optimal condition for the bioconservation from solanesol to CoQ10 was as following: adding 0.75 g/L of raw solanesol after the first 12 h fermentation and continued for another 12 h fermentation. The maximal yield of CoQ10 reached 96.88 mg/L.

Abstract:In this study, we found that many metal ions could affect the antibacterial activity of chitosan, such as K+, Na+, Mg2+ and Ca2+. High concentration (about 0.5%) of metal ions could make chitosan completely lose its antibacterial activity, except for the effect of Na+ on the antibacterial activity against Staphylococcus aureus. We also found that chitosan could weaken the barrier function of the outer membrane of different microorganisms. Large amount of K+ and ATP leakages were observed during exposure of Staphylococcus aureus and Candida albicans to chitosan. Both 50kDa chitosan and 5kDa chitosan had good antibacterial activity against Staphylococcus aureus and Candida albicans, but the 50kDa chitosan could make nearly two to four times amount of K+ and ATP leakages than the 5kDa chitosan. There might have been different antibacterial mechanisms of high-molecular-weight and low-molecular-weight chitosan.

Abstract:Two thermophilic bacterial isolates, strain 173 and strain 174, with raw starch-digesting activities were selected from thermophilic bacteria growing in hot spring of Tengchong County, Yunnan Province, China. By amplification, sequencing and alignment analysis of 16S ribosomal DNAs, they were identified as members of genus Geobacillus. In shaker flask culture Geobacillus sp. 173 produced 14.5 U/mL amylase and for Geobacillus sp. 174 with 12.9 U/mL . Both amylases were purified by starch absorption-desorption and chromatograph. The amylases from strain 173 and strain 174 purified to about 50 and 29 folds were respectively achieved with an overall yield of 34% and 41%. The maximum gelati-nized-starch-digesting activity of the purified amylases were at 70°C and pH 5.0~5.5. The high raw-starch-digesting ac-tivity of these enzymes were observed at 50°C~60°C ( from strain 173) and 40°C ~60°C (from strain 174). Both enzymes were not sensitive to ions including mental ions (Na+, K+, Mg2+, Ca2+, Mn2+, Zn2+) and others (EDTA, Citrate), but were slightly inhibited by ions such as Co2+, Cu2+ for amylase from strain 173 and Cu2+ for amylase from strain 174. Both en-zyme had specificity of starch substrates.

Abstract:Docosahexaenoic acid (DHA C22:6n-3), a typical long chain polyunsaturated fatty acids (PUFAs) has many positive effects on diseases such as artherosclerosis, hypertriglyceridemia, hypertension and cancers. Marine fungi, especially Thraustochytrium spp. producing much DHA can serve as model organisms for explaining the mechanism on the biosynthesis of PUFA. We described two elongase genes (TFD6 and TFD5) involved in the biosynthesis of DHA in Thraustochytrium sp. FJN-10 was cloned by using reverse transcription PCR and rapid amplification of cDNA ends. TFD6 cDNA was 816 bp in length and encoded a protein of 271 amino acids. TFD5 cDNA was 831 bp in length and encoded a protein of 276 amino acids. Transmembrane analysis revealed that TFD6 contained five transmembrane domains while TFD5 contained seven. Tertiary structures of TFD6, TFD5 elongases were predicted by HHMMSTR (Hidden markov model for local sequence-structure) model and Rosetta program. Alignment of TFD6, TFD5 with other elongases showed that both of them shared an HXXHH conserved histidine-rich motif. Phylogenetic analysis showed that TFD6 was the closest to Thraustochytrium Δ6 elongase, while TFD5 was the closest to Thraustochytrium sp. Δ5 elongase. TFD6 and TFD5 were subcloned into the Hind Ⅲ/Xba Ⅰrestriction site of pYES2 vector respectively. Recombined plasmids were transformed into Saccharomyces cerevisiae using lithium acetate method. Gas chromatography analysis showed that TFD6 could elongate C18:3n-3 to C20:3n-3 while TFD5 could elongate C20:5n-3 to C22:5n-3.

Abstract:By means of bioinformatics, we aligned nucleotide sequence of reported lipase gene from Geotrichum. Primers were designed based on the conservative nucleotide sequence, and the lipase gene of G. candidum Y162 was cloned for the first time in China. Nucleotide sequencing revealed that the open reading frame has 1692 nucleotides without any introns, encoding 563 amino acid residues including a signal sequence of 19 amino acid residues, which is 86% identical to lipase I of G. fermentans. Subsequently, we cloned the lipase gene into expression vector pPIC9K, and then transformed into Pichia pastoris GS115. Cultures of recombined P. pastoris accumulated active enzyme in the supernatant to levels of 55 U/mL after induction for 96 hours in shake flasks. The purified lipase exhibited maximum activity at 50°C and pH 8.0, and was stable between pH 6.0 and 10.0 and below 60°C. Lipase activity was compatible with the presence of organic solvents such as methanol, n-heptane, hexane, cyclohexane, glycerol, benzene and diethyl ether. Lipase showed hydrolysis prefer-ence for triacylglycerol substrates containing cis-9 unsaturated fatty acid. The results suggest that the lipase could be a candidate for industrial applications.

Abstract:We cloned the NS1 gene of BmDNV-3 into the prokaryotic expression vector pET-30a after we amplified the NS1 gene by PCR, and then we transformed the pET-30a-NS1 plasmid into BL21 star to express BmDNV-3 NS1. After we induced NS1 expression by IPTG, we used Western blot analysis to indentify the recombinant protein, the result indicated that the recombinant protein was BmDNV-3 NS1. After purification, we used NS1 to immunize New Zealand white rabbits following standard protocol to harvest anti-NS1 anti serum. On the other hand, we cloned the BmDNV-3 NS1 into pFastBacHTb-eGFP vector, and then transformed the pFastBacHTb-NS1-eGfp into BmDH10BAC to harvest recombinant bacmid genome. We obtained the recombinant virus from the cells, which was transfected by the recombinant bacmid genome using liposomes. We used the virus genome to infect Bombyx mori larvae. We observed the fluorescence in the cells and silkworm larvae at 2 days post infection, and then we used SDS-PAGE and fluorescence image analysis to identify the fusion protein. The result showed that the size of the fusion protein was not consistent with the expected size of NS1-eGFP, indicating the fusion protein was degraded randomly by the intrinsic digestive protease. To further confirm the fusion protein, we used Western blot with an anti-NS1 antibody.

Abstract:Neisserial surface protein A (NspA) of Neisseria gonorrhoeae is a potential anti-gonorrhea vaccine, and the heat-labile enterotoxin subunit B (LTB) of Escherichia coli is a kind of mucosal adjuvant that can assist mucosal immune response. We constructed a prokaryotic expression vector of the fusion gene ltB-nspA, and expressed and identified the fusion protein LTB-NspA. The nspA gene and the ltB gene were amplified from the genomic DNA of standard strains of Neisseria gonorrhoeae and Escherichia coli respectively by polymerase chain reaction(PCR). Two fragments were ob-tained by agarose gel electrophoresis. One was about 525bp of nspA gene, and the other was about 372bp of ltB gene. Fragment nspA and fragment ltB were fused with a linker encoding 6 amino acids (Asp-Pro-Arg-Val-Pro-Ser) by recombination PCR and a 900bp fragment of the fusion gene ltB-nspA was found on the gel. The fusion gene ltB-nspA was cloned into the prokaryotic expression vector pET-30a after digestion with BamH Ⅰ and Hind III. Recombinants were selected by enzyme digestion and sequencing. The recombinant plasmid with ltB-nspA gene was then transformed into E.coli BL21 with IPTG to induce and express the fusion protein. SDS-PAGE analysis showed that the relative mo-lecular weight of the expressed recombinant protein was about 39 kDa equivalent to the theoretically predicted value. The specificity of the expressed fusion protein was confirmed by Western-blot. The prokaryotic expression vector was constructed correctly and the fusion protein LTB-NspA was successfully expressed, which provided a basis of investi-gating its biological functions and developing of a Neisseria gonorrhoeae mucosal vaccine.

Abstract:The bacterial ghost (BG) system is a novel vaccine delivery system endowed with intrinsic adjuvant properties. Bacterial ghosts are nonliving Gram-negative bacterial cell envelopes devoid of cytoplasmic contents while maintaining their cellular morphology and native surface antigenic structures. They are produced by PhiX174 protein E-mediated lysis of Gram-negative bacteria, and can induce humoral and cellular immune response, including mucosal immune responses. Plasmid pElysis consisting E gene was transformed into AhJ-1. Through shifting the culture temperature from 28℃ to 42℃, A. hydrophila J-1 (pElysis) was induced to lyse and the OD600 value of culture media was measured every 15 minutes during the induction. The lysed bacteria were observed by scanning electron microscopy (SEM). The A. hydrophila ghosts (AHG) used as oral vaccine were also investigated. The OD600 value of A. hydrophila J-1(pElysis) began to decline after 30min of induction, and after 75min of induction, the OD600 value decline speed become slowly. The efficiency of ghost induction in non-lyophilized A.hydrophila was 99.99%, 16 hours post induced, no live bacteria can be detected in culture. Scanning electron microscopy observation proved that most lysed bacteria were emptied. Fish vaccination experiments shows that the antibody evoked highest degree after 5 weeks by oral administration of bacterial ghost vaccine and the agglutination antibody titer reached 27 and continued two weeks, while the agglutination antibody titer of formalin killed vaccine only reached 26 and only maintained one week. After challenged with the parent strain J-1, the survival rate of bacterial ghost vaccinated fish was higher than the control group and formalin killed vac-cine group, the relative percent survival (RPS) was 78.95% (16/20), but the RPS of formalin killed vaccine group was 57.9% (12/20). This suggests that the bacterial ghost vaccine has higher potential to induce protective adaptive immu-nity than normal vaccine.

Abstract:The spaA gene was amplified by PCR from the genomic DNA of Erysipelothrix rhusiopathiae C43311 strain, and inserted into the pMD18-T vector and then sequenced. The N-terminal protective domain of the spaA gene was amplified by PCR from the recombinant plasmid pMD18-spaA, then cloned into the prokaryotic expression vector pGEX-6p-2 and expressed in E. coli BL21 (DE3) by IPTG induction. The expressed protein was identified by SDS-PAGE and Western blot. The sequence analyses showed that the coding region of the spaA gene of C43311 strain was 1881bp in length, and the nucleotide sequence homology of the spaA genes between the C43311 strain and the pre-viously reported different serotype strains of E.rhusiopathiae was 93 to 99%. The SDS-PAGE analyses revealed a single fusion protein band with a molecular weight of 64kDa, and the Western blot results showed that the GST-SpaA-N fusion protein was recognized specifically by an antiserum against the SpaA protein of C43311 strain, suggesting that the fu-sion protein of GST-SpaA-N possessed high immunoreactivity.

Abstract:In this study, five fragments of recombinant subunit Pasteurella multocida toxin (PMT) were constructed. Only pET28a-N1518 and pET28b-C2115 could be expressed efficiently in Escherichia coli. The molecular weight of the fusion proteins was 57 kDa and 78 kDa .Western blot confirmed that the two proteins could specifically react with an-tiserum against Pasteurella multocida toxin. No mice died after the intraperitoneal administration of these two proteins with the dose of 200 μg per mouse, but Vero cell was pathologically changed after administration of 896ng/mL rPMT-N. The fusion protein of rPMT-N and rPMT-C was purified, and emulsified with Freund’s adjuvant in equal volumes to get subunit vaccine. Groups of Kunming mice were immunized subcutaneously twice at interval of two weeks. All mice were challenged intraperitoneally with 8.2×105 CFU HN-13 strain of T+Pm. The protection efficiency of rPMT-N, rPMT-C and crude PMT against HN-13 strain were 90%, 50% and 80%, respectively. The data revealed that the fusion protein of rPMT-N had immunogenicity and potential for developing a subunit vaccine against PAR in pigs.

Abstract:The hemagglutinin (HA) gene fragment of swine influenza virus A/Swine/Guangdong/LM/05(H1N1) was amplified with HA gene specific primers and cloned into baculovirus transfer plasmid pFASTBacGP67B. The recombinant plasmid pFastBacGP67B-H1 was identified by restriction enzyme digestion and gene sequencing. Following the transformation of DH10Bac Escherichia coli component cells by pFastBacGP67B-H1, recombinant bacmids rBac-mid-H1 were identified by blue/white selection and PCR analysis. Then recombinant baculovirus rBV-H1 was rescued by lipofectant reagent Cellfectin induced rBacmid-H1 DNA transfection of long-phage sf9 insect cells. The recombinant HA protein was characterized by hemagglutination test, western-blot and immunohistochemistry. An indirect en-zyme-linked immunosorbent assay (ELISA) was assessed to detect in pigs IgG against H1 subtype SIV present in Inner Mongolia, Liaoning and Heilongjiang provinces. Positive was found in 31.15% (29 of 93) serum samples tested from swine reared in commercial herds. However, all irrelevant control sera tested were negative. We conclude, therefore, that ELISA performed with recombinant HA as coating antigen was a better tool for swine influenza surveillance in China.

Abstract:Thirteen prevailed Newcastle-disease viruses (NDV) isolated in China during 2001-2004 were purified by chick embryo fibroblast (CEF) plaque assay and characterized pathotypically and genotypically. The biological tests showed that these viruses were highly virulent. Sequence analysis based on the variable region (nucleotide 47-420) of the F gene indicated that of the 13 NDV isolates 2 belonged to genotypeⅡ, 2 to genotype Ⅸ and 9 to genotype Ⅶ. Isolates with genotype Ⅶ shared 94.6%~99.3% nucleotide (nt) homology with the F gene, whereas for genotype Ⅶ and La Sota was only 82.7%~84.1%. In addition, these NDV isolates all shared 95.2%~100% nt homology with the hemagglutinin-neuraminidase (HN) gene, whereas only 79.1%~84.3% compared these viruses with La Sota. The cross neutralization assays were done using positive serums in specific pathogen free (SPF) chicken embryos respectively. Correlation of the neutralization index in chicken embryo with the homologies of F and HN gene of different NDV isolates were analyzed by SPSS8.0 software. The result showed that the neutralization index was closely correlated with nt sequence (P<0.01, r=0.35) or deduced amino acid sequence (P<0.01, r=0.34) of the HN gene, whereas weekly correlated (P<0.05, r=0.20 or 0.19) with the F gene, and non-correlated with 374 nt segment. This implied that the genetic mutations of HN resulted in antigenic variations of these viruses and the search for new vaccines would be necessary.

Abstract:After cloning the C3d cDNA of AA broilers using the liver mRNA source, a pair of primers were designed to subclone the P29 gene to the pUC19 plasmid. Several tandems of P29 were constructed in the pUC19 plasmid using a pair of isoschizomers-BamH I and Bgl II. The pUC- P29.n was igested to get the gene of P29.n that was then cloned to pCDNA3.1 (+) plasmid. After this, the F Gene of Newcastle Disease Virus was cloned through RT-PCR and inserted into the upstream of the P29.n that was in the pCDNA-P29.n, and the DNA vaccines containing F gene against NDV with C3d-P29 as molecular adjuvant were constructed. Several groups of Specefic Pathogen Free chickens were injected with these recombinant plasmids. The pCDNA-F-P29.4 and pCDNA-F-P29.6 group had higher HI antibody titers than the pCDNA-F group. The pCDNA-F-P29.4 and pCDNA-F-P29.6 group’s HI antibody titers did not achieve titers as high as the inactive vaccine group. However, they all provided protection against the lethal F48E9 virus challenge.

Abstract:Text We designed a pair of primers according to fungal glucanase genes obtained from GenBank and cloned a novel β-1,3-glucanase gene (glu) from Trichoderma virid LTR-2 cDNA by PCR. Then we linked the fragment with pMD18-T vector and sequenced it. The sequence analysis indicated that glu was composed of 2289 nucleotide residues. The fragment contained an Open Reading Frame coding 762 amino acids and was similar to previous reports. Translated amino acids sequence of glu contained two Conservative Districts of β-1,3-glucanase which were RVVYIPPGTY and AASQNKVAYF. By nucleotide blasting in NCBI glu showed high homology to three β-1,3-glucanase genes from Trichoderma sp., especially with T.harzianum bgn3.1 and Hypocrea virens bgn13.1, which the homology reached 93%. The sequence was submitted to GenBank and the Ac-cession Number is EF176582. Then we connected glu gene with the Pichia pastoris shuttle vector-pPIC9K. The recombinant plasmid named pGLU14 was transformed into methylotropic yeast P. pastoris KM71 after linearization. The recombinant strain KGLU14 expressing β-1,3-glucanase at high level was obtained through plate screening. The SDS-PAGE result indicated that molecular weight of the recombinant β-1,3-glucanase was about 80kDa and the activity of the recombinant enzyme could reach 889U/mL in liquid culture.

Abstract:Fifty actinomycete strains isolated from 19 acid soil samples collected from Hunan and Yunnan provinces could grow on media at pH4.5 and pH7.0. Thirty-eight actinomycete strains isolated from 14 alkaline soil samples collected from Xinjiang, Qinghai and Yunnan provinces could grow on media at pH10.0 and pH7.0. Thus, they were moderately acidophilic or moderately alkalophilic actinomycetes. Twelve linear plasmids and 3 circular plasmids were detected among the 88 strains by the pulsed-field gel electrophoresis and standard alkaline-denaturation procedure, respectively.

Abstract:Bombyx mori nucleopolyhedrovirus (BmNPV) orf25 gene was characterized for the first time. The coding sequence of Bm25 was amplified and subcloned into the prokaryotic expression vector pGEX-4T-2 to produce glutathione S-transferase-tagged fusion protein in the BL21 (DE3) cells. The GST-Bm25 fusion protein was expressed efficiently after induction with IPTG. The purified fusion protein was used to immunize New Zealand white rabbits to prepare polyclonal antibody. Temporal expression analysis revealed a 30-kDa protein, which was detected beginning 24 hours post-infection using a polyclonal antibody against GST-Bm25 fusion protein. The transcript of Bm25 was detected by RT-PCR at 18-72 h p.i. In conclusion, the available data suggest that Bm25 encodes a 30kDa protein expressed in the late stage of infection cycle.

Abstract:The wild type avian pathogenic Escherichia coli (APEC) isolate A2 (Serotype O2:K89) was selected as the prototype of the (APEC ) TypeⅠFimbriae. Based on the original sequences of TypeⅠFimbriae operon gene clusters in the GenBank,we generated PCR products by using primers with the homologies extension of fimH gene to be deleted and template plasmid pKD3 carrying selectable antibiotic chloramphenicol resistance(cat) gene that is flanked by FRT(FLP recognition target)sites. By using the PCR products, the A2 △fimH::Cat deletion mutant from A2 isolate was constructed by λRed-mediated recombination system in the flanking homologies. After selection, the resistance gene located in the A2△fimH::Cat mutant was eliminated in the second recombination, by using a helper plasmid pCP20, a tempera-ture-sensitive one encoding and expressing the FLP recombinase, which acts on the directly repeated FRT sites flanking the resistance gene. The A2△fimH mutant obtained was further confirmed by fimH PCR amplification and sequencing. The A2△fimH mutant with the deletion of fimH adhesin in the fim gene cluster lost the ability of agglutination reaction with both guinea pig erythrocytes and yeast cells. The A2△fimH deletion mutant restored the agglutination ability of both binding guinea pig erythrocytes and yeast cells when transfected the compatible recombinant plasmid pBR322-fimH with fimH insert in the fimH complementation assay. Very similar to the wild type A2 isolate, The obtained binding activity from the A2△fimH mutant in the complementation assay was completely inhibited when pretreated with 0.5% mannose solution. Compared with the wild type isolate, the A2△fimH mutant grew slowly during all stages of growth. This work provides the basis for us to study the molecular pathogenesis mechanisms of interaction between the APEC TypeⅠ Fim-briae and susceptible host cells, extra-intestinal infection, prevention and control of the APEC-caused disease.

Abstract:Probiotics are live microorganisms. When they were administered in adequate amounts, they could have beneficial effects on the host. They are generally applied in food fermentation, industrial lactic acid production and medical care. With the appearance of commercial probiotics, more concerns were raised on the safety of probiotics. At present, there were four aspects of safety concerns on probiotics: pathogenicity and infectivity; deleterious metabolic activities; excessive immune response and potential gene transfer. Conventional toxicology and safety evaluation has limited value in the safety assessment of probiotics used in marketing food and drugs nowadays. Instead, multidisciplinary approaches are necessary for the systematical safety evaluation of probiotic strains. It may involve genomics, metabolismics, proteomics, among others. The toxigenic metabolic activity should be considered especially. We reviewed recent developments and research methods on the safety of probiotics. Safety evaluation of the lactic acid bacteria for human consumption must be assessed by proposing criteria standards, guidelines and regulations, for better applications for consumers.

Abstract:Polylactic acid is a high molecular-weight polyester made from renewable resources such as corn or starch. It is a promising biodegradable plastic due to its mechanical properties, biocompatibility and biodegradability. To achieve natural recycling of polylactic acid, relative microorganisms and the underlying mechanisms in the biodegradation has become an important issue in biodegradable materials. Up to date, most isolated microbes capable of degrading polylactic acid belong to actinomycetes. Proteases secreted by these microorganisms are responsible for the degradation. However, subtle differences exist between these polylactic acid degrading enzymes and typical proteases with respect to substrate binding and catalysis. Amino acids relative to catalysis are postulated to be highly plastic allowing their catalytic hydrolysis of polylactic acid. In this paper we reviewed current studies on biodegra-dation of polylactic acid concerning its microbial, enzymatic reactions and the possible mechanisms. We also dis-cussed the probability of biologically recycling PLA by applying highly efficient strains and enzymes.

Abstract:Protein phosphatase-1(PP1) is a member of the Ser/Thr phosphatases and widely distributed in many organisms. The enzyme regulates many important physiological processes, including gene transcription, translation, metabolism, cell growth and division. Three grooves and b12- b13 loop in the molecular surface play important roles for binding inhibitors and substrates. Recent research found that b12-b13 loop is important for structure and character of the whole enzyme molecule, except for binding inhibitors. Functional research proves that PP1 also regulates transcription process of HIV-1, and relates with causes of many diseases, for example Alzheimer’s Disease. This review summarized distribution, mo-lecular structure, enzymatic character, catalytic mechanism and physiological function of the enzyme.