Abstract:Abstract：Rumen of ruminant animals is known as a natural reactor involved in highly efficient lignocellulose degradation. The research on rumen microbes is a subject of keen interest in terms of biofuel development. Traditionally, the studies were dependent on isolating of fibrolytic bacteria, and thereafter the lignocellulose degrading enzymes were analyzed individually. With the development of genomic or metagenomic technologies, a variety of new genes or gene clusters associated with lignocellulose degrading enzymes could be found in rumen, based on which lignocellulose degrading mechanism could be studied and discussed. Previous studies indicate that the lignocellulose degradation process of rumen microorganism is much more complicated, which includes a large number of different rumen microorganisms, enzymes, genes or gene clusters. With the development and maturation of new analysis technologies, much progress on these rumen microbes, enzymes, genes has been made. This review reported the recent progress about these researches.

Abstract:Abstract: γ-Lactamase belongs to the amidase. The (+) γ-lactamses can be applied in the kinetic resolution of racemic γ-lactam, which can produce (-) γ-lactam efficiently. The (-) γ-lactam enantiomer is an important synthon for the synthesis of Carbocyclic Nucleosides. Up to now 7 strains of microbes were reported that produced γ-lactamses. The crystal structure of (-) lactamase from Aureoacterium sp. was resoluted, and the catalytic mechanism based on the structure data analysis was alike with the α/β hydrolase family. However, no structure data and mechanism theory is available for (+) lactamase till now. The future works focus on protein engineering of the γ-lactamase to improve the catalysis of the protein, elucidation of the catalytic mechanism of the γ-lactamase and the functions of the γ-lactamase in vivo.

Abstract:Abstract: [Objective] In order to discover new leader compounds, the diversity and some bioactivities of cultured actinomycetes from Sichuan and Yunnan were studied. [Methods] In total 250 soil samples were collected from forest of Huangjin, Emei and Qingcheng Mountains, Jiuzhaigou in Sichuan, and Xishuangbanna in Yunnan. Actinomycetes in these samples were isolated and identified. Bioactivities of isolated strains were determined. [Results] In total 2676 strains of actinomycetes were isolated from these samples. The 16S rRNA gene sequences of 98 selected strains were determined, and the phylogenetic analysis was carried out. 13, 5, 9 and 20 genera were identified from Forest of Huangjin, Emei and Qingcheng Mountains (one sample area), Jiuzhaigou, and Xishuangbanna respectively. The diversity of Xishuangbanna was the richest. That of Emei and Qingcheng Mountains was monotone, and only five genera were isolated. Antimicrobial activities of 169 selected strains against 11 bacteria and fungi were tested using agar well diffusion method, and genes of type I and II polyketide synthases (PKS I, PKS II), non-ribosomal peptide synthase (NRPS) and polygene cytochrome P450 hydroxylase (CYP) were detected by PCR. High rate of antimicrobial activity and the synthesis genes of four antibiotic existed in these actinomyctes. [Conclusion] In sample-collecting areas, the poorer human beings disturbance, the richer the diversity of actinomycetes. For isolation of actinomycetes, author advocate using of "extreme" conditions, although it may got small number of actinomycetes, but the proportion of unknown actinomycetes was greater. Gram-negative bacteria and fungi could be inhibited by adding inhibitors in media.

Abstract:Abstract: [Objective] Peganum harmala, a famous traditional Chinese drug, contains a variety of alkaloids and toxic for many animals. Camels mainly live in desert or semi-desert areas, with the robust gastro- intestine system in digesting various feed including toxic plants without disease symptoms. [Methods] Camel rumen content was used as the inoculant to inoculate medium M98-5 which contains 100 mg?L-1 harmin and cultivated for 5 days. Upto 5 subculturings, strains that could degrading or tolerant harmine were isolated. Their conversion activity was determined by thin-layer chromatography. The taxonomic position of the strains were identified based on 16S rRNA sequences analysis. [Results] 15 out of the 29 isolates have harmine degrading activity. Most of the isolates are identified as the members of the Genera Lactobacillus (16 strains, 55%), Shigella (7 strains, 24%) and Bacillus (4 strains, 13.8%). Only one strain belong to genus Enterococcus and one belong to genus Megasphaera. [Conclusion] The results indicated that the harmine tolerance/degrading communities of camel rumen are limited and only Lactobacillus have harmine-degrading activity.

Abstract:Abstract: Streptomyces strains were isolated from soil samples of Tibet, five small linear plasmids were detected by pulsed-field gel electrophoresis. [Objective] Cloning, sequencing and analysis of telomeres of these plasmids. [Methods] The telomeres were cloned by a modified procedure — “alkaline treatment and enzyme digestion in gels”. [Results] Telomeres of five linear plasmids were cloned and sequenced. Compared with the typical Streptomyces telomeres, the newly identified telomeres contained multiple palindromes, but some could not “fold-back” of their first conserved palindrome I with the internal palindromes to form a “super-hairpin”, and palindromes of some telomeres did not contain the 3-nt “loop”. [Conclusion] New telomere sequences were cloned by a modified procedure. Both folding-back of the telomere palindromes and 3-nt loop of palindromes varied among telomeres.

Abstract:Abstract: [Objective] To optimize the production of tiacumicin B in Dactylosporangium aurantiacum NRRL 18085, we developed a genetic modification system by disrupting genes involved in tiacumicin biosynthesis. [Methods] We developed a method of conjugation to transfer exotic DNA pSET152 into D. aurantiacum NRRL 18085. Using the PCR-targeting system, we disrupted a putative tiacumicin halogenase gene in vitro by “in-frame deletion” in E. coli, and then the resulting cosmid was transferred into D. aurantiacum NRRL 18085 by conjugation. [Results] The putative tiacumicin halogenase gene in D. aurantiacum NRRL 18085 was disrupted by in-frame deletion from a double-crossover recombination event. The resulting mutant strain lost the ability to produce tiacumicin B. [Conclusion] We developed a genetic manipulation system for D. aurantiacum NRRL 18085, enabling the functional characterization of tiacumicin biosynthetic genes in vivo, and we offer a positive example for other Actinobacteria lacking an appropriate genetic manipulation system.

Abstract:Abstract: [Objective] To explore the secondary metabolites of fungus Aspergillus ochraceus LCJ11-102 associated with the coral Dichotella gemmacea under environmental stress and to obtain characteristic compounds with biological activities. [Methods] A nutrient-deprived culture medium (biomimetic culture) and a high salt culture medium were used for fermentation. Fingerprints of HPLC of the fermentation broth were used to investigate the diversity of secondary metabolites. Compounds were isolated by column chromatography on silica gel, Sephadex LH-20, and preparative HPLC. Their structures were identified by spectroscopic analyses and the modified Mosher’s method. [Results] Different secondary metabolites were produced by A. ochraceus LCJ11-102 under two different culture conditions. (R)-mellein (1), (5,6-trans, 8,9-threo-)-9-chloro-8-hydroxy-8,9-deoxyaspyrone (2), (5,6-erythro-, 8,9-threo-)-9-chloro-8-hydroxy-8,9-deoxyasperlactone (3), and (5S,6R,9S)-dihydroaspyrone (4) were identified from the biomimetic cultures, and R(+)-semi-vioxanthin (5) was identified from the high salt cultures, respectively. [Conclusion] Environmental stress obviously induces microbes to produce different secondary metabolites. And biomimetic culture is an effective approach to obtain active chloro compounds from marine microorganisms.

Abstract:Abstract: [Objective] The deletion of pyruvate decarboxylase-like enzyme gene (YDL080C gene) in industrial Saccharomyces cerevisiae haploids a-8 and α-22 were respectively constructed, and the effect of YDL080C gene deletion on the production of higher alcohols, especially isoamyl alcohol, was investigated in high gravity ethanol fermentation. [Methods] Upper and down stream fragments YA (460 bp) and YB (630 bp) of YDL080C gene were amplified by PCR using the genomic DNA of S. cerevisiae haploids a-8 and α-22, respectively; KanMX marker for G418 resistance from plasmid pUG6 was cloned by PCR. Fragments YA, YB and KanMX were respectively inserted into the same plasmid pUC19 using EcoRI and KpnI, KpnI and BamHI, and KpnI sites in the order of YA-KanMX-YB to construct the recombined vector pUC-YABK. The recombinant cassette YA-KanMX-YB was cloned from plasmid pUC-YABK by PCR and respectively transformed into industrial yeast haploid a-8 and α-22. By the double homologous recombination, YDL080C gene deletion mutants were constructed and selected by the growth extent on YEPD agar plates containing 600 μg/mL G418. At the end of high gravity ethanol fermentation, fermentation performance and higher alcohols production of parental haploids and their mutants were determined. [Results] YDL080C deletion mutants were respectively selected from their corresponding parental haploid a-8 and α-22. The alcohol fermentation results showed that higher alcohols, especially isoamyl alcohol, were almost invariable among the mutants and their corresponding parental haploids. However, mutants yielded more amount of ethanol of 0.6 (%, v/v) and 0.4 (%, v/v) over its parental haploid, respectively. [Conclusion] Deletion of YDL080C gene in S. cerevisiae haploids has no noticeable effect on decreasing the production of higher alcohols, especially isoamyl alcohol, but it seems to somehow increase the production of ethanol.

Abstract:Abstract: [Objective] Endophytic fungi in medicinal plants produce a variety of bioactivity compounds. In this study, an endophytic fungus zjqy610 with antifungal activity was isolated from Polygonatum cyrtonema in Zhejiang Qingyuan Baishanzu Mountain nature reserve. [Methods] Strain zjqy610 was identified as Penicillium canescens based on the morphology and its rDNA sequence analysis. Three antifungal compounds were isolated from the fermentation broth of zjqy610 through normal-phase silica gel column chromatography and gel (Sephadex LH-20) column chromatography, traced by ultraviolet light or iodine vapor with bioassay-guided fractionation. [Results] These three compounds were elucidated as o-acetylbenzeneamidinocarboxylic acid (zjqy610B-g-3), griseofulvin (zjqy610D-4) and naphtho [1,2-b] furan-3 -carboxylic acid, 4-hydroxy-5-methoxy-2-methyl-(zjqy610F-2) based on mass spectrometry and nuclear magnetic resonance spectroscopy. The antifungal activity assays showed that the three compounds had inhibitory to variety of plant pathogenic fungi. Compound zjqy610D-4 had strong antifungal activity against Botrytis cinerea, Colletotrichum orbiculare, Didymella bryoniae and Sclerotinia sclerotiorum. EC50 was 0.68, 0.38, 0.91 and 0.61 mg/L, respectively. [Conclusion] Zjqy610D-4 is deserved to develop an agricultural antibiotics.

Abstract:Abstract: [Objective] Antibacterial and cytotoxic activities were screened for marine bacteria which have been isolated from Karlodinium micrum, in order to obtain potential strains with antibacterial and cytotoxic activities. [Methods] In total 38 bacteria isolated from Karlodinium micrum were screened by agar-screening and MTT methods. The 16S rRNA genes were amplified from the genome DNA of those bacteria positive for both antibacterial and cytotoxic activities, which were cloned into pMD18-T vector for sequencing analysis. [Results] Twenty-five isolates had antimicrobial activity and 5 isolates (W-14-2, W-2-2, W-12, E-8-2 and W-4) had cytotoxicity. Molecular phylogenetic analysis of marine bacteria with cytotoxicity based on 16S rRNA sequences indicated that they exhibited the highest similarity (98%, 99%, 99%, 98% and 99%, respectively) to the 16S rRNA fragments of Alteromonas alvinellae, Stappia aggregata, Pelagibaca bermudensis, Marinobacter kribbensis and Maribacter dokdonensis. [Conclusion] The bacteria with bioactivity in Karlodinium micrum were abundant. We obtained five strains positive for both antibacterial and cytotoxic activities, which provide a clue to understanding the mechanism of toxin biosynthesis in Karlodinium micrum based on epiphytic and endophytic bacteria.

Abstract:Abstract: [Objective] The aim of this study is to identify of genes involved in long-chain (LC) alkane degradation in Alcanivorax hongdengensis A-11-3. [Methods] PCR was applied to obtain Flavin-binding monooxygenase genes, then quantitative real-time polymerase chain reaction (Q-RT-PCR) and RT-PCR were applied to analyze gene expression in response to different LC-alkanes and pristane. [Results] Two homologues, almA1and almA2, were obtained. They showed 58.6% and 53.2% similarities with almA of Acinetobacter sp. Strain DSM 17874, respectively, at amino acid level. Enhanced expression of almA1 genes was observed when strain A-11-3 grew with long chain alkanes (C28 to C 32), in sodium acetate medium. However, the induction expression was not observed in the case of C9-C22 alkanes. Similarly, almA2 was induced by long chain alkanes (C24 to C 34). In addition, it was also induced by the branched alkane pristane. [Conclusion] AlmA genes were mostly responsible for the degradation of long-chain alkanes and pristane in strain A-11-3.

Abstract:Abstract: [Objective] Extracellular feruloyl esterase (EC 3.1.1.73) from Penicillium citrinum culture filtrates was studied. The effect of feruloyl esterases on the enzymatic hydrolysis of brewer′s spent grain was also investigated. [Methods] Feruloyl esterase was purified to homogeneity by ammonium sulfate precipitation, ion-exchange chromatography with a DEAE-Sepharose Fast Flow column, and column chromatography with a Phenyl-Sepharose 6 Fast Flow column. [Results] The purified homogeneous preparation of native feruloyl esterase had a molecular mass of 58 kDa by native-PAGE and a subunit molecular mass of 31 kDa by SDS-PAGE. The optimum enzymatic activity was achieved at pH 6.0 and 45-65°C. The enzyme was stable at pH from 5.0 to 6.0 and temperature from 25 to 55°C. The enzymatic activity was slightly enhanced by Mg2+, Fe2+, Mn2+, Ca2+, and Na+, whereas it was slightly inhibited by Zn2+, strongly inhibited by Cu2+, and completely inhibited by Hg2+ and phenylmethanesulfonyl fluoride. EDTA had a slight influence on the enzymatic activity. The determination of kcat/Km revealed that the enzyme hydrolyzed methyl p-coumarate, methyl sinapate, methyl ferulate, methyl caffeate, and the values were 823, 416, 103, and 0, respectively. The kcat/Km values showed that the enzyme hydrolyzed MpCA faster and more efficiently than all the other substrates. When the crude feruloyl esterase was used to hydrolyze the brewer’s spent grain, about 7.2% of the alkaline-extractable ferulic acid could be released, with the concentration of 5 u feruloyl esterase /g. [Conclusion] A feruloyl esterase was discovered. Its biochemical characteristics were different from what has been reported in literature. This provided an important basis for the exploitation of a feruloyl esterase.

Abstract:Abstract: [Objective] In order to investigate the effect of environmental factors on the bacterial composition of Lake Dongping sediment. [Methods] We set six sampling points in Lake Dongping and sampled once in July and once October. Terminal Restriction Fragment Length Polymorphism (T-RFLP) method was used to analyze the bacterial diversity. Ammonium nitrogen (NH4+-N), nitrate nitrogen (NO3--N), total nitrogen (TN), total phosphorus (TP), total organic carbon (TOC) and water depth were measured. [Results] T-RFLP profiles showed high similarity among samples. However, bacterial diversity indices were significant difference in various samples. The richness, diversity, and evenness in samples which were collected in dry season (October) were generally higher than those in wet season (July), and the bacterial species dominance was higher in wet season than that in dry season. Principal Component Analysis showed that the structure of bacterial communities in sample 2B was marked different from the other samples. The results of Canonical Correspondence Analysis suggested that the abundance of 558 bp T-RF was negatively correlated with NH4+-N, NO3--N, TN, TP and TOC, but positively correlated with TOC/TN and the water depth; the abundance of 64.5, 164, 509, and 543 bp T-RFs were positively correlated with NH4+-N, NO3--N, TN, TOC, TOC/TN and the water depth. The dominant bacteria in Lake Dongping sediments were Firmicutes and Proteobacteria. [Conclusion] Environmental factors affect bacterial diversity of Lake Dongping sediments, although affect less on indigenous bacteria.

Abstract:Abstract: [Objective] In order to search novel nematicidal protease genes, a metagenomic fosmid library was constructed and screened by uncultured method. [Methods] Density gradient centrifugation was used to extract and purify total greenhouse soil microbial DNA. After end-repair, ligation, packing and transformation, metagenomic fosmid library was constructed. At the same time, in order to screen the library, function-driven screening was used as a potential strategy, skim milk was served as substrate and root-knot nematodes as targets. [Results] The library contained 31,008 clones with the average insert fragment of 36.5 kb, including 1.13Gbp microbial genetic information, so it was suitable for large-scale microbial functional gene screening. By the function-driven screening, fosmid clone pro12 which contained the nematicidal protease gene was screened. Then, subclones were constructed and screened. A subclone named espro124a5 was screened. After analysis of gene structure, espro124a5 is a secreted extracellular protease and a database search for homologies revealed it possessed 45% identities with peptidase S15 from Maricaulis maris MCS10 (accession no. YP_756822 at NCBI). It is a novel serine protease. Besides these, it has the serine protease-conserved catalytic triad residues,Asp469,His541 and the catalytic nucleophile Ser348. [Conclusion] DNA obtained from the method of Nycodenz density gradient centrifugation had high purity, long fragment, and can meet the requirements of constructing metagenomic fosmid library. At the same time, the metagenomic fosmid library contains a lot of microbial genetic information, which is suitable for the screening of the other microbial genetic resources.

Abstract:Abstract: [Objective] Targeted at the important enzyme in human glucose metabolic pathway, the purpose of this paper is to establish α-glucosidase inhibitors high throughput screening model. [Methods] Pichia pastoris expression system was used to clone and express the human α-maltase glucosidase. Using the catalytic properties of enzyme to establish α-glucosidase inhibitor screening model. This model was applied in screening of actinomycete metabolites library. The taxonomic status of positive strains were analyzed by constructing 16S rRNA phylogenetic tree. [Results] The N-terminal catalytic domain of human α-maltase glucosidase was successfully cloned and expressed for the first time. The high-throughput screening model of α-glucosidase inhibitors was established. A natural product library containing metabolites from nearly 2000 actinomycetes was screened, 20 α-maltase glucosidase inhibitor producing strains were obtained finally, of which, 19 strains initially identified as Streptomyces, and showed taxonomically rich diversity. [Conclusion] The α-glucosidase inhibitor high-throughput screening model has high practical value, this work laid the foundation for developing new hypoglycemic drugs.

Abstract:Abstract: [Objective] We investigated the advantages and disadvantages of six methods to detect classical swine fever virus (CSFV). [Methods] We used six methods, including the virus isolation, colloidal gold immunochromatographic assay (CGIA), antigen-capture ELISA (AC-ELISA), reverse transcription?polymerase chain reaction (RT-PCR), TaqMan real-time RT-PCR (RT-qPCR) and reverse transcription?loop-mediated isothermal amplification assay (RT-LAMP), to detect CSFV in 50 samples parallelly. [Results] The results showed that 13 samples were detected positive by RT-qPCR and RT-LAMP, 11 by PCR, 10 by virus isolation, 9 by AC-ELISA and 8 by CGIA, and 8 samples were detected positive and 37 samples negative by the six methods. [Conclusion] These results indicated that the 3 RNA-amplification assays could be used as the first choice for detection of CSFV due to the high sensitivity, while they were vulnerable to false positive results arising from sample to sample contaminations or from other contaminated sources. Although the virus isolation was time-consuming, it was still considered the “gold standard” and was indispensable for confirming CSF outbreaks. The rest two methods, AC-ELISA and CGIA, yielded the results in a short time yet their performance was hampered by a low sensitivity. Therefore, they were mainly used for herd diagnosis and not suitable for individual test for CSFV infection.

Abstract:Abstract: [Objective] We used ultrasonic to treat soil samples to isolate rare actinomycetes strains. [Methods] We collected and suspended soil samples from mix tropical-rain-forest in Xishuangbanna. Farther, we treated the soil suspension with ultrasound for 10 to 120 s and used the plate dilution method to isolate actinomycetes. After got pure colonies, we sequenced their 16S rRNA gene sequences and calculated to put in phylogenetic analysis. The isolates were identified to the genus. We treated 10 kinds of common and identified Streptomyces with 1-5 min using ultrasonic, and then were cultured to measure their survival rate. [Results] The soil suspensions treated with different times by the ultrasonic, actinomycetes gradual increased in the number and types. Ultrasonic treating of known Streptomyces with 0-5 min, there was no significant effect on the survival number of Streptomyces. [Conclusion] The ultrasonic treating with soil suspensions for 40 s can significantly increase the total number of actinomycetes and the types of rare actinomycetes. Thereby, it is an economic and simple method to apparently increase the types of rare actinomycetes in the isolation.

Abstract:Abstract: Two linear plasmids, pNSL1 and pNSL1, were detected from Rhodocuccus sp. NS1. [Objective] Cloning, sequencing and identification of replication origin of the Rhodococcus linear plasmid pNSL1. [Methods] Large amount of linear plasmid DNA was recovered from pulsed-field gels for shotgun-cloning and sequencing, and identification of its replication locus. [Results] The complete nucleotide sequence of pNSL1 consisted of 117252 bp, including the conserved 1282-bp telomere sequences among Rhodococcus linear plasmids. pNSL1 encoded 103 open reading frames, including functions of replication, maintenance and transfer etc. A locus, pNSL1.038 and upstream 767-bp non-coding sequence, was identified for autonomous replication by cloning in an E. coli vector and introduced by electroporation into Nocardia coralline 4.1040. [Conclusion] Cloning and sequencing of Rhodococcus linear plasmid pNSL1, and identification of its replication origin.

Abstract:Abstract: [Objective] To isolate PQQ biosynthesis gene cluster from Gluconobacter oxydans H24 based on sorbose-dehydrogenase activity. [Methods] A library of Gluconobacter oxydans H24 genomic DNA was constructed with host strains Escherichia coli JM109s, which was integrated of sdh gene at the ptsG site on the chromosome of JM109. By detecting sorbose-dehydrogenase activity, clone of PQQ biosynthesis was isolated and subcloned. [Results] A positive clone was isolated from Gluconobacter oxydans H24 genomic DNA library. Within the 5,400-base-pair DNA fragment five reading frames are presented, corresponding to five of the pqq genes (pqqABCDE). The nucleotide and amino acid sequence showed highly homology to pqq genes of other bacteria. [Conclusion] The pqqABCDE gene cluster was successfully isolated from Gluconobacter oxydans H24 by sorbose dehydrogenase activity.

Abstract:Abstract: [Objective] We xpressed and purified Campylobacter jejuni flagellin FlaA protein to develop monoclonal antibodies (mAbs) against this protein. [Methods] The C. jejuni flaA gene was amplified and inserted into the expression plasmids, pET30a (+) and pGEX-6p-1. The purified rHis-FlaA protein was used as an immunogen in 8-week-old BALB/c mice, and injected subcutaneously. The purified rGST-FlaA protein used as a detecting antigen for screening mAbs against FlaA was prepared by using a denaturation and renaturation technique. The specificity of mAbs was characterized by Dot-ELISA and Western blot assays. [Results] The recombinant expression plasmids, pET30a(+)-flaA and pGEX-6p-1-flaA were obtained. The sizes of the recombinant proteins, rHis-FlaA and rGST-FlaA, were consistent with their predicted size. Specific reaction was found between flaA positive serum and expressed protein by Western-blot assay, confirming its identification as a Campylobacter jejuni immunogen. Three hybridoma cell lines, designated 2D12, 5A12 and 6A9, secreting mAbs against FlaA were obtained. Their immunoglobulin subclasses were IgG2a, IgG1 and IgG1, respectively. The ELISA titers of the ascites fluid were 1:102 400, 1:102 400 and 1:51 200, respectively. Western blot analysis confirmed that the three mAbs reacted with the rHis-FlaA fusion protein but not the His tag. The Dot-ELISA results demonstrated that the three mAbs only with FlaA and not the tags for the expression vectors. [Conclusion] The successful preparation of three mAbs specific for the FlaA protein lays the foundation for further study regarding the biological characteristics of FlaA and the pathogenesis of C. jejuni.