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A hyperthermostable xylanase XYN10B from Thermotoga maritima (PDB code 1VBR, GenBank accession number KR078269) was subjected to site-directed and error-prone PCR mutagenesis. From the selected five mutants, the two site-directed mutants (F806H and F806V) showed a 3.3-3.5-fold improved enzyme half-life at 100 °C. The mutant XYNA generated by error-prone PCR showed slightly improved stability at 100 °C and a lower K

Mannanases catalyze the cleavage of β-1,4-mannosidic linkages in mannans and have various applications in different biotechnological industries. In this study, a new β-mannanase from Verrucomicrobi...

Mannanases catalyze the cleavage of β-1,4-mannosidic linkages in mannans and have various applications in different biotechnological industries. In this study, a new β-mannanase from

In this study, the first xylantic enzyme from the family Marinifilaceae, XynSPP2, was identified from Marinifilaceae bacterium strain SPP2. Amino acid sequence analysis revealed that XynSPP2 is a rare Fn3-fused xylanase, consisting of a signal peptide, a fibronectin type-III domain (Fn3), and a C-terminal catalytic domain belonging to glycoside hydrolase family 10 (GH10). The catalytic domain shared 17–46% identities to those of biochemically characterized GH10 xylanases. Structural analysis revealed that the conserved asparagine and glutamine at the glycone −2/−3 subsite of GH10 xylanases are substituted by a tryptophan and a serine, respectively, in XynSPP2. Full-length XynSPP2 and its Fn3-deleted variant (XynSPP2ΔFn3) were overexpressed in Escherichia coli and purified by Ni-affinity chromatography. The optimum temperature and pH for both recombinant enzymes were 50°C and 6, respectively. The enzymes were stable under alkaline condition and at temperature lower than 50°C. With beechwood xylan as the substrate, XynSPP2 showed 2.8 times the catalytic efficiency of XynSPP2ΔFn3, indicating that the Fn3 module promotes xylanase activity. XynSPP2 was active toward xylooligosaccharides (XOSs) longer than xylotriose. Such a substrate preference can be explained by the unique −2/−3 subsite composition in the enzyme which provides new insight into subsite interaction within the GH10 family. XynSPP2 hydrolyzed beechwood xylan into small XOSs (xylotriose and xylotetraose as major products). No monosaccharide was detected by thin-layer chromatography which may be ascribed to putative transxylosylation activity of XynSPP2. Preferring long XOS substrate and lack of monosaccharide production suggest its potential in probiotic XOS manufacture.

Cellulophaga algicola DSM 14237, isolated from the Eastern Antarctic coastal zone, was found to be able to hydrolyze several types of polysaccharide materials. In this study, a predicted &beta, agarase (CaAga1) from C. algicola was heterologously expressed in Escherichia coli. The purified recombinant CaAga1 showed specific activities of 29.39, 20.20, 14.12, and 8.99 U/mg toward agarose, pure agar, and crude agars from Gracilaria lemaneiformis and Porphyra haitanensis, respectively. CaAga1 exhibited an optimal temperature and pH of 40 oC and 7, respectively. CaAga1 was stable over a wide pH range from 4 to 11. The recombinant enzyme showed an unusual thermostability, that is, it was stable at temperature below or equal to 40oC and around 70 oC, but was thermolabile at about 50 oC. With the agarose as the substrate, the Km and Vmax values for CaAga1 were 1.19 mg/mL and 36.21 U/mg, respectively. The reducing reagent (dithiothreitol) enhanced the activity of CaAga1 by more than one fold. In addition, CaAga1 was salt-tolerant given that it retained approximately 70% of the maximum activity in the presence of 2 M NaCl. The thin layer chromatography results indicated that CaAga1 is an endo-type &beta, agarase and efficiently hydrolyzed agarose into neoagarotetraose (NA4) and neoagarohexaose (NA6). A structural model of CaAga1 in complex with neoagarooctaose (NA8) was built by homology modeling and explained the hydrolysis pattern of CaAga1.

The 5′ untranslated region (5′UTR) plays a key role in post-transcriptional regulation, but interaction between nucleotides and directed evolution of 5′UTRs as synthetic regulatory elements remain unclear. By constructing a library of synthesized random 5′UTRs of 24 nucleotides in Saccharomyces cerevisiae, we observed strong epistatic interactions among bases from different positions in the 5′UTR. Taking into account these base interactions, we constructed a mathematical model to predict protein abundance with a precision of R2 = 0.60. On the basis of this model, we developed an approach to engineer 5′UTRs according to nucleotide sequence activity relationships (NuSAR), in which 5′UTRs were engineered stepwise through repeated cycles of backbone design, directed screening, and model reconstruction. After three rounds of NuSAR, the predictive accuracy of our model was improved to R2 = 0.71, and a strong 5′UTR was obtained with 5-fold higher protein abundance than the starting 5′UTR. Our findings provide ne...

Glycoside hydrolase family 10 (GH10) xylanases are responsible for enzymatic cleavage of the internal glycosidic linkages of the xylan backbone, to generate xylooligosaccharides (XOS) and xyloses. The topologies of active-site cleft determine the substrate preferences and product profiles of xylanases. In this study, positional bindings and substrate interactions of TmxB, one of the most thermostable xylanases characterized from Thermotoga maritima to date, was investigated by docking simulations. XOS with backbone lengths of two to five (X2&ndash, X5) were docked into the active-site cleft of TmxB by AutoDock The modeled complex structures provided a series of snapshots of the interactions between XOS and TmxB. Changes in binding energy with the length of the XOS backbone indicated the existence of four effective subsites in TmxB. The interaction patterns at subsites &minus, 2 to +1 in TmxB were conserved among GH10 xylanases whereas those at distal aglycone subsite +2, consisting of the hydrogen bond network, was unique for TmxB. This work helps in obtaining an in-depth understanding of the substrate-binding property of TmxB and provides a basis for rational design of mutants with desired product profiles.

This study aimed to study the phylogenetic diversity and community structure of bacteria in petroleum contaminated soils from Karamay oil field, and to analyze the relationship between the community variation and the environment parameters, to provide a reference for bioremediation of petroleum contaminated soils.We collected samples from petroleum contaminated soils in 5 cm, 20 cm and 50 cm depth layers, and measured the environment parameters subsequently. We constructed three 16S rRNA gene clone libraries of these soil samples, and then determined the operation taxonomy units (OTUs) restriction fragment length polymorphism method, and finally sequenced the representative clones of every OUT. The diversity, richness and evenness index of the bacteria communities were calculated by using Biodap software. Neighbor-Joining phylogenetic tree was constructed based on 16S rRNA gene sequences of bacteria from Karamay oil field and the references from related environments. Canonial correspondence analysis (CCA) was used to analyze the relationship between environment parameters and species by using CANOCO 4.5 software.Environment parameters showed that 50 cm deep soil contained the highest amount of total nitrogen (TN) and total phosphorus (TP), whereas the 20 cm depth soil contained the lowest amount. The 5 cm depth soil contained the highest amount of total organic carbon (TOC), whereas the 50 cm depth soil contained the lowest amount. Among the 3 layers, 20 cm depth had the highest diversity and richness of bacteria, whereas the bacteria in 50 cm depth was the lowest. Phylogenic analyses suggested that the bacteria in Karamay oil field could be distributed into five groups at the level of phylum, Cluster I to V, respectively belong to Proteobacteria, Actinobacteria, Firmicute, Bacteroidetes, Planctomycetes. Cluster I accounts for 78.57% of all tested communities. CCA results showed that TN, TP, TOC significantly affected the bacteria community structure. Especially, TOC content is significantly related to the distribution of Pseudomonas.The petroleum-contaminated soil inhabited abundant of bacteria. The diversity index and spatial distribution of these communities were affected by the environment parameters in the soil.

Sediment bacteria are the important biological factors for remediating of eutrophic environments. To enrich our understanding of the bacteria communities in eutrophic urban lake sediments for better environment protection and pollution control in urban lake eco-systems, we resolved the composition of bacteria communities and their spatial variation in the sediments of a middle-size eutrophic urban lake, East Lake.We used 16S rRNA gene RFLP and sequencing methods to generate the phylogeny information of the bacteria community, used principal coordinates analysis (PCoA) and canonical correspondence analysis (CCA) methods to resolve the relationship between East Lake and other lakes, and the relationship between environmental factors and the bacteria communities.Sediments inhabited 13 phyla and 2 unclassified clusters. PCoA further revealed that the bacteria communities in three sub-lakes of East Lake sediments were closely related to the communities in similar eutropic lake environments, and divergent from the hypereutrophic sub-lake Miao Lake, which was also found to inhabit a relative abundant amount of Thermogymnomonas-type archaea. CCA further revealed that the distribution of bacteria was closely correlated with the carbon, nitrogen and phosphate contents in the sediments.The environment factors regulated the bacteria community composition and distribution. The results of this study providereference to the research, protection and pollution control on urban lake eco-systems.

Biotechnological application of xylanolytic enzymes is normally hindered by their temperature-dependent catalytic property. To satisfy the industrial demands, xylanases that can perform catalysis under cold condition are attracting attention. In this study, the biochemical properties of a predicted xylanase (laXynA) encoded in the genome of marine bacterium Luteimonas abyssi XH031T were characterized. Structure modeling and structure-based sequence alignment indicated that laXynA belongs to the glycoside hydrolase family 10, and it is 20–26% identical to other characterized cold-active xylanases in the same family. Recombinant laXynA was successfully produced in Escherichia coli system by autoinduction and purified by Ni-affinity chromatography. The isolated enzyme showed an optimum temperature of 30 °C toward beechwood xylan and retained important percentage of optimal activity at low temperatures (64, 55, and 29% at 10, 5, and 0 °C, respectively). A remarkable characteristic of laXynA was extreme halophilicity as demonstrated by fourfold enhancement on xylanase activity at 0.5 M NaCl and by maintaining nearly 100% activity at 4 M NaCl. Thin layer chromatography analysis demonstrated that laXynA is an endo xylanase. This study is the first to report the over-expression and characterization of a cold-active xylanase from Luteimonas species. The enzymatic property revealed the cold-active nature of laXynA. The enzyme is a promising candidate in saline food processing application.

1,6-Anhydro-N-acetylmuramic acid kinase (AnmK) is the unique enzyme that marks the recycling of the cell wall of Escherichia coli. Here, 81 fungal AnmK-like kinase sequences from 57 fungal species were searched in the NCBI database and a phylogenetic tree was constructed. The three-dimensional structure of an AnmK-like kinase, levoglucosan kinase (LGK) of the yeast Lipomyces starkeyi, was modeled; molecular docking revealed that AnmK and LGK are conserved proteins, and 187Asp, 212Asp are enzymatic residues, respectively. Analysis suggests that 1,6-anhydro-N-acetylglucosamine (anhGlcNAc) and/or 1,6-anhydro-β-d-glucosamine (anhGlcN) would be the appropriate substrates of AnmK-like kinases. Also, the counterparts of other characteristic enzymes of cell wall recycling of bacteria were found in fungi. Taken together, it is proposed that a putative recycling of anhGlcNAc/anhGlcN, which is associated with the hydrolysis of cell walls, exists in fungi. This computational analysis will provide new insights into the metabolism of fungal cell walls.

Atlantis II and Discovery are two hydrothermal and hypersaline deep-sea pools in the Red Sea rift that are characterized by strong thermohalo-stratification and temperatures steadily peaking near the bottom. We conducted comprehensive vertical profiling of the microbial populations in both pools and highlighted the influential environmental factors. Pyrosequencing of the 16S rRNA genes revealed shifts in community structures vis-à-vis depth. High diversity and low abundance were features of the deepest convective layers despite the low cell density. Surprisingly, the brine interfaces had significantly higher cell counts than the overlying deep-sea water, yet they were lowest in diversity. Vertical stratification of the bacterial populations was apparent as we moved from the Alphaproteobacteria -dominated deep sea to the Planctomycetaceae - or Deferribacteres -dominated interfaces to the Gammaproteobacteria- dominated brine layers. Archaeal marine group I was dominant in the deep-sea water and interfaces, while several euryarchaeotic groups increased in the brine. Across sites, microbial phylotypes and abundances varied substantially in the brine interface of Discovery compared with Atlantis II, despite the near-identical populations in the overlying deep-sea waters. The lowest convective layers harbored interestingly similar microbial communities, even though temperature and heavy metal concentrations were very different. Multivariate analysis indicated that temperature and salinity were the major influences shaping the communities. The harsh conditions and the low-abundance phylotypes could explain the observed correlation in the brine pools.

Hydrothermal ecosystems have a wide distribution on Earth and many can be found in the basin of the Red Sea. Production of aromatic compounds occurs in a temperature window of ∼60–150 °C by utilizing organic debris. In the past 50 years, the temperature of the Atlantis II Deep brine pool in the Red Sea has increased from 56 to 68 °C, whereas the temperature at the nearby Discovery Deep brine pool has remained relatively stable at about 44 °C. In this report, we confirmed the presence of aromatic compounds in the Atlantis II brine pool as expected. The presence of the aromatic compounds might have disturbed the microbes in the Atlantis II. To show shifted microbial communities and their metabolisms, we sequenced the metagenomes of the microbes from both brine pools. Classification based on metareads and the 16S rRNA gene sequences from clones showed a strong divergence of dominant bacterial species between the pools. Bacteria capable of aromatic degradation were present in the Atlantis II brine pool. A comparison of the metabolic pathways showed that several aromatic degradation pathways were significantly enriched in the Atlantis II brine pool, suggesting the presence of aromatic compounds. Pathways utilizing metabolites derived from aromatic degradation were also significantly affected. In the Discovery brine pool, the most abundant genes from the microbes were related to sugar metabolism pathways and DNA synthesis and repair, suggesting a different strategy for the utilization of carbon and energy sources between the Discovery brine pool and the Atlantis II brine pool.

To gain insight into the species richness and phylogeny of the microbial communities associated with sponges in mangroves, we performed an extensive phylogenetic analysis, based on terminal restriction fragment length polymorphism profiling and 16S ribosomal RNA gene sequences, of the 4 sponge species Aplysina fulva, Haliclona hogarthi, Tedania ignis and Ircinia strobilina as well as of ambient seawater. The sponge-associated bacterial communities contained 13 phyla, including Poribacteria and an unclassified group not found in the ambient seawater commu- nity, 98% of which comprised Proteobacteria, Cyanobacteria and Bacteroidetes. Although the sponges themselves were phylogenetically distant and bacterial community variation within the host species was observed, microbial phyla such as Proteobacteria, Acidobacteria, Chloroflexi and the unclassified group were consistently observed as the dominant populations within the communities. The sponge-associated bacterial communities resident in the Caribbean Sea mangroves are phyloge- netically similar but significantly distinct from communities found in other biogeographical sites such as the deep-water environments of the Caribbean Sea, the South China Sea and Australia. The inter- specific variation within the host species and the distinct biogeographical characteristics that the sponge-associated bacteria exhibited indicate that the acquisition, establishment and formation of functional sponge-associated bacterial communities may initially be the product of both vertical and horizontal transmission, and is then shaped by the internal environment created by the sponge species and certain external environmental factors.

Marine sponges are associated with a remarkable array of microorganisms. Using a tag pyrosequencing technology, this study was the first to investigate in depth the microbial communities associated with three Red Sea sponges, Hyrtios erectus, Stylissa carteri and Xestospongia testudinaria. We revealed highly diverse sponge-associated bacterial communities with up to 1000 microbial operational taxonomic units (OTUs) and richness estimates of up to 2000 species. Altogether, 26 bacterial phyla were detected from the Red Sea sponges, 11 of which were absent from the surrounding sea water and 4 were recorded in sponges for the first time. Up to 100 OTUs with richness estimates of up to 300 archaeal species were revealed from a single sponge species. This is by far the highest archaeal diversity ever recorded for sponges. A non-negligible proportion of unclassified reads was observed in sponges. Our results demonstrated that the sponge-associated microbial communities remained highly consistent in the same sponge species from different locations, although they varied at different degrees among different sponge species. A significant proportion of the tag sequences from the sponges could be assigned to one of the sponge-specific clusters previously defined. In addition, the sponge-associated microbial communities were consistently divergent from those present in the surrounding sea water. Our results suggest that the Red Sea sponges possess highly sponge-specific or even sponge-species-specific microbial communities that are resistant to environmental disturbance, and much of their microbial diversity remains to be explored.

Aspergillus niger lipases are important biocatalysts for a broad range of industrial applications. To enhance the expression level of a newly cloned lipase gene lip2 of A. niger in Pichia pastoris, we applied codon optimization and synthesized the full length codon-optimized gene by a two-step gene synthesis strategy. This strategy consists of an assembly PCR for several small DNA fragments and enzymatic digestion and ligation steps to ligate these fragments into the full-length gene. First, the full-length lip2 gene was divided into three fragments F1 (237 bp), F2 (238 bp) and F3 (422 bp) with the additions of proper restriction sites, and separately amplified by assembly PCR reactions. Second, three PCR amplified fragments were digested and ligated into the full-length lip2 gene. In the two-step gene synthesis, synthesis of smaller DNA fragments resulted in a significant lower level of nonspecific mismatching among oligonucleotides and a very low mutational rate of the PCR products, demonstrating the superiority of the method. When compared with the originally cloned lip2 gene of A. niger, the new codon optimized lip2 gene expressed at a significantly higher level in yeasts after methanol induction for 72 h, and both the enzyme activity and protein content reached maximal levels of 191 U/ml and 154 mg/1, with 11.6- and 5.3-fold increases, respectively.

Bioimprinting and sol–gel encapsulation of lipases by silane precursors are efficient methods of enhancing lipase performance in non-aqueous medium. The correlation between bioimprinting, the alkyl-chain length of silane precursors, and the catalytic activity of gel-encapsulated lipase was investigated using a series of silane precursors: methyltrimethoxysilane (MTMS), vinyltrimethoxysilane (VTMOS), vinyltriethoxysilane (VTEOS), and n-octyltrimethoxysilane (OTMOS). The optimal parameters for lipase immobilization were also determined. Both bioimprinting and increasing the chain-length of alkyl groups, apparently by increasing hydrophobicity, significantly improved the specific activity and the total activity of the immobilized lipase. Compared to a non-imprinted MTMS/TMOS gel, the specific activity of an imprinted OTMOS/TMOS gel improved 14.4-fold, and the total activity improved 6.8-fold. Nitrogen adsorption–desorption assays and gel matrix surface characterization showed that the bioimprinting molecule and the hydrophobic alkyl groups of silane triggered lipase to change from the closed to the open conformation, and contributed to creating sol–gel matrices that were more porous and with less mass transfer resistance structure, apparently improving the activity of encapsulated lipase.

A novel lipase gene, lipJ08, was cloned from Candida rugosa ATCC14830, along with the already reported five lipase genes (lip1-lip5). Nucleotide sequencing indicated that the lipJ08 gene contains a 1650 bp open reading frame (ORF) without introns. The deduced amino acid sequence corresponds to 534 amino acid residues, including a putative signal sequence of 15 amino acid residues. Seventeen of the non-universal serine codons (CTG) of lipJ08 were converted into universal serine codons (TCT) by PCR-based mutagenesis. The native and codon-optimized lipJ08 genes were expressed in Pichia pastoris. The hydrolytic activity of the recombinant LIPJ08 was 4.7 U/ml, whereas the activity of the recombinant wild-type lipase could not be detected.

Geotrichum sp. lipase with enhanced activity and operational stability was prepared for use in non-aqueous media. A combined strategy comprising bioimprinting with dual imprint molecules and a co-solvent coupled to pH tuning, KCl salt activation, lecithin coating and immobilization on macroporous resin effectively enhanced the activity and operational stability of Geotrichum sp. lipase. The modified lipase exhibited 18.4-fold enhanced esterification activity towards methyl oleate synthesis, and retained 90% activity following repeated use in 10 cycles. The combined strategy exhibited a significant synergistic effect and was suitable for lipase modification, dramatically enhancing the enzyme activity and operational stability. This approach is applicable to the preparation of other enzyme biocatalysts, since the methods are effective for upgrading crude enzyme to a refined product with high activity and stability for use in non-aqueous media.

Bioimprinting is a promising, though relatively unexplored, approach to improving the performance of enzymes. In this study, bioimprinting with substrate analogues of fatty acids was systematically conducted to improve the esterification activity of Burkholderia cepacia lipase that had undergone a sol–gel immobilization procedure with methyltrimethoxysilane (MTMS) and tetramethoxysilane (TMOS) as the precursors. The specific activity of the bioimprinted lipases was 3682.0 μmol h−1 mg protein, which was a 47.9- and 2.5-fold increase over the free and non-imprinted immobilized lipases, respectively. Compared to the free and non-imprinted immobilized lipases, bioimprinted lipases exhibited better thermal stability, and their activity did not change after being incubated at 60 °C for 12 h. Bioimprinted lipases were more easily affected by alcohol than the non-imprinted ones, whose specific activity could be markedly enhanced by ethanol, isopropanol and n-butanol by factors of 1.23-, 1.28- and 1.12-fold, respectively. The reasons for the improvement of imprinted enzyme activity are also discussed based on the surface structure, specific surface area and average pore diameter of the silane particles.

Although the lipase of Geotrichum candidum has been extensively reported, little attention has been focused on molecular genetic and biochemical characterizations of Galactomyces geotrichum lipases. A lipase gene from G. geotrichum Y05 was cloned from both genomic DNA and cDNA sources. Nucleotide sequencing revealed that the ggl gene has an ORF of 1692 bp without any introns, encoding a protein of 563 amino acid residues, including a potential signal sequence of 19 amino acid residues. The amino acid sequence of this lipase showed 86% identity to lipase of Trichosporon fermentans WU-C12. The mature lipase gene was subcloned into pPIC9K vector, and overexpressed in methylotrophic Pichia pastoris GS115. Active lipase was accumulated to the level of 100.0 U/ml (0.4 mg/ml) in the shake-flask culture, 10.4-fold higher than the activity of the original strain (9.6 U/ml). This yield dramatically exceeds that previously reported with 23–50 U/ml, 0.06 mg/ml and 0.2 mg/ml. The purified lipase exhibited several properties of significant industrial importance, such as pH and temperature stability, wide organic solvent tolerance and broad hydrolysis on vegetable oils. Such a combination of properties makes it a promising candidate for its application in non-aqueous biocatalysis, such as biodiesel production, selective hydrolysis or esterification for enrichment of PUFAs and oil-contaminated biodegradation, which have been drawn considerable attention currently.

A lipase gene lipA and its chaperone gene lipB were cloned from Burkholderia cepacia strain G63. The lipA was composed of 1092 bp, encoding 363 amino acid residues, and the lipB composed of 1035 bp, corresponding to 344 amino acid residues. The significant amino acid similarity with Pseudomonas cepacia lipase revealed that this enzyme could be classified into the lipolytic subfamily I.2. The lipA and lipB genes were cloned into pBBR1Tp vector and conjugated into B. cepacia strains G63 with the help of pRK2013. The recombinant strain was fermented in 10 l bioreactor and the lipase was purified by a combination of ammonium sulfate fractionation, DEAE ion-exchange chromatography and gel filtration. The purified lipase kept stable at a temperature range of 40–70 °C. After incubated at 70 °C, the optimal temperature of this enzyme, for 10 h it remained 86.1% of its activity. The enzyme was also highly tolerant to a series of organic solution. Incubated in 50% methanol solution up to 48 h, the enzyme still kept 98.3% of its activity. The transesterification activity of soybean oil to fatty acid methyl esters (FAMEs) reached 87.8% after 72 h, indicating that it is a potential biocatalyzer for biodiesel production.

Microbial associations with corals are common and are most likely symbiotic, although their diversity and relationships with environmental factors and host species remain unclear. In this study, we adopted a 16S rRNA gene tag-pyrosequencing technique to investigate the bacterial communities associated with three stony Scleractinea and two soft Octocorallia corals from three locations in the Red Sea. Our results revealed highly diverse bacterial communities in the Red Sea corals, with more than 600 ribotypes detected and up to 1,000 species estimated from a single coral species. Altogether, 21 bacterial phyla were recovered from the corals, of which Gammaproteobacteria was the most dominant group, and Chloroflexi , Chlamydiae , and the candidate phylum WS3 were reported in corals for the first time. The associated bacterial communities varied greatly with location, where environmental conditions differed significantly. Corals from disturbed areas appeared to share more similar bacterial communities, but larger variations in community structures were observed between different coral species from pristine waters. Ordination methods identified salinity and depth as the most influential parameters affecting the abundance of Vibrio , Pseudoalteromonas , Serratia , Stenotrophomonas , Pseudomonas , and Achromobacter in the corals. On the other hand, bacteria such as Chloracidobacterium and Endozoicomonas were more sensitive to the coral species, suggesting that the host species type may be influential in the associated bacterial community, as well. The combined influences of the coral host and environmental factors on the associated microbial communities are discussed. This study represents the first comparative study using tag-pyrosequencing technology to investigate the bacterial communities in Red Sea corals.

Rhizopus oryzae lipase (ROL) is not only a biocatalyst used in a broad range of biotechnological fields, but also a model to investigate the function of intramolecular chaperone in the post-translational processing of lipase. In this study, we cloned and expressed the mature lipase gene (m-ROL) containing the pre-sequence (pro-ROL) of R. oryzae HU3005 in Pichia pastoris GS115 and characterized their enzymatic activities. m-ROL exhibited higher hydrolysis activity towards middle-chain substrates (C10 and C12) at pH 9.0, whereas pro-ROL preferred short-chain substrates (C4) and displayed maximal activity at pH 8.0. Moreover, pro-ROL possessed better thermal stability than m-ROL. This enzymatic discrepancy between m-ROL and p-ROL may be due to the pre-sequence that affects the folding and conformation of the mature lipase domain. To improve the expression level of m-ROL in P. pastoris, overlap extension PCR was conducted to substitute eight less-frequently used codons of m-ROL with frequently used codons. After methanol-induced expression for 72 h, the activity and protein content of the codon optimized m-ROL reached 132.7 U/mL and 50.4 mg/L, while the activity of the parental m-ROL and pro-ROL are 28.7 U/mL and 14.4 mg/L, 29.6 U/mL and 14.1 mg/L, respectively.

The study aims to investigate the phylogeny diversity of the denitrification bacteria communities in the sediments of the eutrophic East Lake, Wuhan based on nitrite reductase gene (nirS) restriction fragment length polymorphism (RFLP) method and sequencing analysis, and to analyse community variation according to the environment parameters.We collected the sediment samples from the four typical sub-lake of East Lake in Wuhan, Guozheng Lake, Tangling Lake, Tuan Lake and Miao Lake, and measured the environmental parameters appropriately. After extracted the genomic DNA from the sediment, four nirS gene clone libraries were successfully constructed. The operation taxonomy units (OTUs) were determined by RFLP method and the representative fragment of every OTU was sequenced. The diversity, richness and evenness statistics of the NirS-like communities were calculated by using DOTUR software. Neighbor-joining phylogenetic tree was constructed basing on the amino acid sequences of NirS from the East Lake sediments and reference sequences retrieved from the GenBank database. The relationship between tested NirS communities and the references from different environments was also discussed.Environmental parameters showed that Miao Lake sediment contains the highest amount of total nitrogen (TN) and NH4+ -N, while the Tuan Lake sediments contains the lowest amount. Among the four sub-lakes, Tuan Lake harbours the highest diversity and richness of NirS-like denitrifiers, while the denitrifiers in Miao Lake was the lowest. Phylogenic analyses suggested that sedimentary NirS-like denitrifiers in the East Lake could be distributed into three groups, Group I to III. Group I accounts for 67.7% of all tested communities. Eighty-one percent of sequences from Guozheng Lake were clustered into Group I, while 67.7% of sequences from Miao Lake were clustered into Group II. Comparative analysis of communities from East Lake and artificial wetland found there are phylogenetically related.There are diverse and abundant NirS-like denitrifiers inhabited in the sediments of East Lake, Wuhan. The diversity indices and spatial distribution of these communities are affected by the content of TN, NH4- and NO3- nutrients in the sediments.

Penicillium digitatum Sterol 14α-demethylase (PdCYP51), a prime enzyme in membrance sterol biosynthesis, is a key target of antifungal drugs for citrus disease. Based on the recently determined X-ray crystal structure human CYP51, a three-dimensional structure model of PdCYP51 was built through homology modeling. After molecular dynamics (MD) simulation, the refined model was assessed by Verify-3D and Procheck, which confirmed that the refined model was reliable. Further evaluation on the model quality was performed by investigating the interaction of some sterol 14α-demethylase inhibitors (DMIs) with the modeled enzyme. Molecular docking program was employed to determine such interactions. The binding pattern predicted by the docking revealed that DMI inhibitor interacted with PdCYP51 mainly through hydrogen-bonding and hydrophobic interactions. Moreover, the results are compatible with the spectra assay data in the laboratory. The docking complex provided further refinement of the DMI binding interaction that may be used as a basis for virtual screening and for novel design to discover more potent compounds.

Aspergillus niger lipases are important biocatalysts widely used in industries for food processing and pharmaceutical preparation. High-level expression recombinants can lead to cost effective lipase large scale production. Total gene synthesis is an efficient method to enhance the expression level of the gene. According to the mature peptide sequence of A. niger lipase and codons of preference in Pichia pastoris, 26 primers with 20bp overlaps at both 57 and 37 ends between adjacent oligonucleotides were designed and synthesized. Fragments lipA1(300bp), lipA2(237bp), lipA3(234bp) and lipA4(210bp) were separately synthesized by assembly PCR, and then they were used as the template to get the full length gene. The synthetic gene was cloned into pPIC9K secretory expression vector. The expression efficiency was verified with analysis of mRNA secondary structure using RNA Structure 4.5.

The cowpea ( Vigna unguiculata L.), peanut ( Arachis hypogaea L.), and mung bean ( Vigna radiata L.) belong to a group of plants known as the “cowpea miscellany” plants, which are widely cultivated throughout the tropic and subtropical zones of Africa and Asia. However, the phylogeny of the rhizobial strains that nodulate these plants is poorly understood. Previous studies have isolated a diversity of rhizobial strains from cowpea miscellany hosts and have suggested that, phylogenetically, they are from different species. In this work, the phylogeny of 42 slow-growing rhizobial strains, isolated from root nodules of cowpea, peanut, and mung bean from different geographical regions of China, was investigated using sequences from the 16S rRNA, atpD and glnII genes, and the 16S–23S rRNA intergenic spacer. The indigenous rhizobial strains from the cowpea miscellany could all be placed in the genus Bradyrhizobium , and Bradyrhizobium liaoningense and Bradyrhizobium yuanmingense were the main species. Phylogenies derived from housekeeping genes were consistent with phylogenies generated from the ribosomal gene. Mung bean rhizobia clustered only into B. liaoningense and B. yuanmingense and were phylogenetically less diverse than cowpea and peanut rhizobia. Geographical origin was significantly reflected in the phylogeny of mung bean rhizobia. Most cowpea rhizobia were more closely related to the 3 major groups B. liaoningense, B. yuanmingense, and Bradyrhizobium elkanii than to the minor groups Bradyrhizobium japonicum or Bradyrhizobium canariense . However, most peanut rhizobia were more closely related to the 2 major groups B. liaoningense and B. yuanmingense than to the minor group B. elkanii.

To achieve fast, safe and stable expression of Burkholderia cepacia lipase in Pichia pastoris.We first amplified B. cepacia lipase gene, and then analyzed the codon usage of B. cepacia and Pichia, lipase gene signal peptide with bioinformatics methods. On this basis, we applied the overlap PCR to modify the lipase gene and finally got the optimized gene with Pichia codon usage and lower G + C content. Subsequently, we cloned the optimized and wild lipase gene into vector pGAPZalpha and pPIC9K, respectively. As a result, constitutive expression vector pGAPlipW, pGAPlipO and inducible expression vector pPIClipW, pPIClipO were obtained. Finally, we electroporated these expression vectors into GS115, and therefore, got a series of engineering strains. After fermentation and NTA resin purification, the enzymatic properties of lipase were studied.The lipase activities of pPIClipW, pPIClipO, pGAPlipW and pGAPlipO were 37.8 U/mL, 129.5 U/mL, 40.2 U/mL, and 184.3 U/mL, respectively. The optimized lipase activity increased 4.6-fold. Enzymatic properties study showed that the optimal temperature and pH was 60 degrees C and 9.0, respectively. The lipase was rather stable at 40 degrees C - 65 degrees C and pH 6.0-pH10.0.After overlap PCR modification, the lipase expression efficiency in Pichia was significantly increased, which indicates that the overlap PCR modification is a potential strategy for lipase overexpression. The GAP promoter is more appropriate than the AOX1 promoter for the B. cepacia lipase expression. Additionally, the recombinant lipase whose enzymatic properties were identical to the wild type satisfies the needs of industrial application.

The ecosystems of the Red Sea are among the least-explored microbial habitats in the marine environment. In this study, we investigated the microbial communities in the water column overlying the Atlantis II Deep and Discovery Deep in the Red Sea. Taxonomic classification of pyrosequencing reads of the 16S rRNA gene amplicons showed vertical stratification of microbial diversity from the surface water to 1500 m below the surface. Significant differences in both bacterial and archaeal diversity were observed in the upper (20 [corrected] and 50 m) and deeper layers (200 and 1500 m). There were no obvious differences in community structure at the same depth for the two sampling stations. The bacterial community in the upper layer was dominated by Cyanobacteria whereas the deeper layer harbored a large proportion of Proteobacteria. Among Archaea, Euryarchaeota, especially Halobacteriales, were dominant in the upper layer but diminished drastically in the deeper layer where Desulfurococcales belonging to Crenarchaeota became the dominant group. The results of our study indicate that the microbial communities sampled in this study are different from those identified in water column in other parts of the world. The depth-wise compositional variation in the microbial communities is attributable to their adaptations to the various environments in the Red Sea.

15 bacterial strains with a capacity of poly-phosphate were isolated from enrichment culture of the soil sample collected from the phosphate fertilizer factory of Honghu City in Hubei province, of which one strain named as HS-P7 has the strongest effects of poly-phosphate. Ammonium molybdate spectrophotometric method was used to measure its ability of poly-phosphate. The results showed that phosphorus removal rate about the strain HS-P7 reached to 90.08% when it was cultivated in the phosphorus-rich medium in 30℃ during 72 hours. The homology between the strain HS-P7 and Pseudomonas cf. Monteilii is 98% by comparing their 16S rDNA. Based on the characteristics of morphological, physiological and biochemical, the strain HS-P7 was identified as Pseudomonas sp. The effects of different temperatures, pH, carbon sources and nitrogen sources on the growth of the strain HS-P7 were investigated and the optimum growth condition was determined as pH 7.0 and 30℃.

CYP51 of Fungi, known as sterol 14-α-demethylase (P450 14DM) is a key enzyme in the biosynthesis of membrane ergosterol. It will lead to the destruction and loss of function of membrane structure and ultimately to fungal death without CYP51. Based on the 5′-end nucleotide sequence of cyp51, the upstream region of cyp51 was obtained by chromosome walking technique to investigate the regulatory mechanism of cyp51 gene expression. The upstream fragment of 490bp was successfully cloned and sequenced. Analyzed by bioinformatics software, the transcription initiation site of cyp51 was predicted by the NNPP software is located at 134bp in front of ATG, while several cis-acting elements are also analyzed by TFSEARCH 1.3 software. The current study was benefit to further investigating the regulation of cyp51 over-expression and molecular mechanism involved in fungicides resistance of Ustilago maydis in future.

Biodiesel production catalyzed by Novozyme 435 was optimized using Plaekett-Burman design and response surface methodology. Firstly, Plaekett-Burman was employed to screen the main efficient parameters on conversion rate from enzyme dosage, reaction temperature, tert-butanol amount, oil/alcohol molar ratio and molecular sleves added amount. Then, the steepest ascent experiments were carried out to approach the optimal conditions. Finally, the optimized conditions were addressed with three-factor-three-level response surface methodology. The results showed that the optimized conditions were: lipase dosage 3 wt% (based on oil weight), temperature 40 °C, tert-butanol added amount 38.7 % (based on oil volume, v/v), molecular sleves added amount 11.9 wt% (based on oil weight) and molar ratio of methanol to oil 4.77∶1. Under the optimized conditions, the biodiesel yield was estimated to be 97.69 %. The proposed model on conversion rate had a satisfactory coefficient of R2 (= 98.34 %) and was experimentally verified. The Novozyme 435 exhibited high operational stability in non-aqueous. After 10 cycles (160 h) successively, the activity of combined lipases maintained 90 % of its original activity.

To clone Penicillum expansum CICC 40356 lipase (PEL) gene cDNA and to over-express active lipase in Pichia pastoris GS115.Primers were designed according to the nucleotide sequence of reported lipase gene from Penicillum. Ten rare codons of PEL and nine of the alpha-signal peptide were optimized by PCR. The native and codon-optimized PEL genes were respectively cloned into pPIC9K, pPIC9KM, and pPIC3.5K vectors. The properties of recombinant lipase were also determined.Nucleotide sequence analysis revealed that the PEL cDNA contained an 858 bp open reading frame. The deduced amino acid sequence corresponds to 286 amino acid residues, including a potential signal peptide sequence of 20 amino acid residues. The hydrolysis activity of PEL was enhanced with codon-optimization. Its optimal temperature and pH were 35 degrees C and 9.5. It favored medium chain esters (C8-C12) and showed the maximal activity toward C8 acyl-chains. It could be stimulated by Ca2+ and Mg2+, but strongly inhibited by EDTA and slightly repressed by Fe2+, Zn2+ and Cu2+.The activity of PEL was improved 2.3-2.5 folds compared to that of the wild type, suggesting that the codon optimization is an efficient measure to produce the active PEL in P. pastoris system.

We have cloned a novel lipase gene, lip2, from Aspergillus niger and expressed it in Escherichia coli. Upon purification of the recombinant Lip2 protein, its properties were characterized. In comparison with a previously identified lipase Lip1, both enzymes are acid lipases (optimal pH

We cloned, expressed and characterized a novel lipase gene lipB from Aspergillus niger F044, to facilitate the large scale production and application of that enzyme.We cloned lipB gene and the cDNA sequence by PCR and RT-PCR, and then cloned the open reading frame of lipB into pET28a vector and expressed by isopropyl beta-D-1-thiogalactopyranoside (IPTG) induction. After Ni-agarose purification, the characteristics were determined and the conformation change was checked by circular dichroism methods.The novel lipase genes cDNA of lipB were cloned from Aspergillus niger F044 (GenBank: FJ536287, FJ536288) and expressed in Escherichia coli. The molecular weight of LipB was about 43 kDa. The optimal substrate of this enzyme is 4-nitrophenyl octanoate (pNPC-C8) with Km = 5.98 mmol/L. The optimal temperature and pH was 50 degrees C and pH 6.0. The enzyme was stable below 40 degrees C. After incubated at 60 degrees C for 1 h, only 18.8% activity remained. After treated by 2 mmol/L Ca2+ for 1 h, the activity improved 2.6-fold.Enzymatic characteristics of LipB determined showed this enzyme might have potential in industrial applications.

Red recombinase system of the lambda phage is widely used for recombination of short linear DNA fragments and genome. Using this system, we obtained T7 RNA polymerase (RNAP) substitution mutants in Burkholderia cepacia. To test the expression abilities of the T7 mutants, four different lipase expression vectors were transformed and the lipase activity of these recombinants was evaluated. Our results suggest that 500 nt homology between the unit and the genome is sufficient to generate mutations and this strategy enables the rapid establishment of mutant strains with efficiencies of 85%. After expression and purification, the highest purified lipase activity obtained was 3,990 U/l, nearly triple that of the wild-type organism.

Aspergillus niger lipases are important biocatalysis widely used in industries for food processing and pharmaceutical preparation. High-level expression recombinants can lead to cost effective lipase large scale production. Full length gene synthesis is an efficient measure to enhance the expression level of the gene. In order to reduce the non-specific binding between oligonucleotides and bases mutation caused by the complicate secondary structure of DNA and excessive PCR amplification, a frequently phenomenon in one-step gene synthesis, we used a two-step method including assembly PCR (A-PCR) and digestion-ligation step to synthesis Aspergillus niger lipase gene lipA. Assisted by DNA2.0 and Gene2Oliga software, we optimized the codon usage and secondary structure of RNA and induced enzyme sites Cla I (237 site) and Pst I (475 site) into the gene. In the first step, fragments F1 (237 bp), F2 (238 bp) and F3 (422 bp) were separately synthesized by assembly PCR. In the second step, fragments F1, F2 and F3 were separately digested by Cla I and Pst I, and then ligated into a full length lipA gene. Two-step method efficiently enhanced successful ratio for full-length gene synthesis and dispersed the risk for gene redesign. The synthesized gene was cloned into pPIC9K vector and transferred into Pichia pastoris. After methanol inducement, the expression level of the codon optimized lipA-syn gene reached 176.0 U/mL, 10.8-fold of the original lipA gene (16.3 U/mL) in Pichia pastoris GS1115. The recombinant offers the possibility for lipase large-scale production.

In this paper, a novel organophosphate- degrading bacterium HS-D36 belonging to Pseudomonas stutzeri was reported. This bacterium has a strong ability to hydrolyze methyl parathion and the organophosphate pesticides hydrolase gene (oph) was cloned for the organophosphate hydrolase (OPH) function analysis. The oph gene was expressed in Escherichia coli BL21 (DE3) by using pET-28 expression system. The activity of the recombinant OPH in crude extracts reached 52.5 U ldr ml . Thermal stability experiment showed that the enzyme inactivated little for 60 minutes at temperature below 50degC. Further, the sequence alignment and phylogenetic analysis suggested that the OPH protein from the strain HS-D36 was 99.0% similar to MPD protein from Pseudomonas sp. WBC-3 and may be originated from metallo-beta-lactamases family. The protein structure prediction results suggested that the mature OPH comprise two independent subunits, each is composed of an active metal center (Zn 2+ and Cd 2+ ).

In order to realize over-expression of Burkholderia cepacia (B. cepacia) lipase, we introduced the widely used T7 RAN polymerase expression system into B. cepacia G63 to over-express the lipase gene. By using PCR technique, we amplified the T7 RNA polymerase gene (T7 RNAP) from the BL21 (DE3) and cloned it into the suicide plasmid pJQ200SK. After that, we flanked T7 RNAP with two 500 bp homologous fragments and integrated it into the genomes of B. cepacia by tri-parental mating, so that T7 RNAP was under-controlled by lipase gene (lipA) promoter. Then, we cloned the lipA and its partner gene lipB into the vector pUCPCM and pBBR22b both or separately. Therefore, we got 7 expression plasmids pBBR22blipAB, pBBR22blipA, pUCPCMlipAB, pUCPCMlipA, pUCPCMdeltalipAlipB, pUCPCMdeltalipA, pUCPCMdeltalipB, and then electroporated them into B. cepacia containing T7 RNA. After shake flask culture, we found B. cepacia containing pUCPCMlipAB produced the most quantity of lipase, and lipase activity was up to 607.2 U/mg, 2.8-folds higher than that of the wild strain. Moreover, lipase activities of all engineering strains except the one containing pUCPCMdeltalipB were enhanced to some extent. The specific activities of wild type B. cepacia and B. cepacia containing pUCPCMlipAB were respectively 29 984 U/mg and 30 875 U/mg after ammonium sulfate precipitation and gel filtration chromatography. The T7 RNA polymerase expression system could effectively enhanced lipase expression in B. cepacia, and secretion signal PelB and ribosome-binding site may promote lipase expression in engineering strain.

Aspergillus niger lipase (ANL) is an important biocatalyst in the food processing industry. However, there is no report of its detailed three-dimensional structure because of difficulties in crystallization. In this article, based on experimental data and bioinformational analysis results, the structural features of ANL were simulated. Firstly, two recombinant ANLs expressed in Pichia pastoris were purified to homogeneity and their corresponding secondary structure compositions were determined by circular dichroism spectra. Secondly, the primary structure, the secondary structure and the three-dimensional structure of ANL were modeled by comparison with homologous lipases with known three-dimensional structures using the BioEdit software, lipase engineering database (http://www.led.uni-stuttgart.de/), PSIPRED server and SwissModel server. The predicted molecular structure of ANL presented typical features of the α/β hydrolase fold including positioning of the putative catalytic triad residues and the GXSXG signature motif. Comparison of the predicted three-dimensional structure of ANL with the X-ray three-dimensional structure of A. niger feruloyl esterase showed that the functional difference of interfacial activation between lipase and esterase was concerned with the difference in position of the lid. Our three-dimensional model of ANL helps to modify lipase structure by protein engineering, which will further expand the scope of application of ANL. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009

In order to display extracellular.lipase Lip2 from Yarrowia lipolytica on the surface of yeast Saccharomyces cerevisiae for whole cell catalysts.The mature Lip2 encoding fragment was amplified from Yarrowia lipolytica total DNA, and was inserted into the 3'terminal of AGA2 to give the plasmid pCTLIP2 for surface display of Lip2. Olive oil, tributyrin and p-nitrophenyl palmitate (pNPP) were used as substrates to measure lipase activity. Moreover, the characterization of displayed lipase and its free form was analyzed.The surface displayed lipase was confirmed to be active towards olive oil, tributyrin and p-nitrophenyl palmitate (pNPP), and reached its highest expression level at 182 U/g dry cell after induced by galactose for 72h. The optimum temperature of cell surface displayed Lip2 was 40 degrees C After incubated at 50 degrees C for 4h, the surface displayed lipase retained 23.2% of its full activity, improved a little compared to free Lip2. The surface displayed lipase showed a preference to medium-chain and long-chain fatty acids p-nitrophenyl esters (C8-C16).The cell surface display system based on a-agglutinin is an effective system for displaying Lip2, and the whole cell EBY100-pCTLIP2 will be probably suited to a different range of applications.

Pseudomonas lipases are important biocatalysts widely used in a variety of industrial fields. An extracellular lipase gene lipA with 1,854-bp open reading frame was cloned from Pseudomonas fluorescens 26-2. The multialignment assay of the putative amino acid and the secondary structure prediction revealed this enzyme could be classified into the lipolytic subfamily I.3 and secreted via adenosine-triphosphate-binding cassette pathway. The lipA gene was integrated into Pichia pastoris GS115, and the methanol-inducible recombinants with Mut(S) and Mut(+) phenotypes were acquired. The characteristics and the transesterification capacity shown by this enzyme suggested it is a useful biocatalyst for biodiesel preparation.

Random mutagenesis on Bacillus pumilus lipase YZ02 gene was conducted by using error-prone PCR strategy. Through two cycles of directed evolution, two optimum mutants BpL1-7 and BpL2-1369 with lipase activity improved 2 folds and 6 folds respectively were screened. The sequence of BpL2-1369 lipase gene showed that four nucleotides substitution, T61C, C147T, A334G and T371A have occurred, and three of them caused amino acid changes. Thus, amine acid Ser21 was changed into Pro21, Arg112 to Gly112, and Leu124 to His124. According to the 3D structure of Bacillus pumilus lipase mimicked by SWISS-MODEL Repository, three mutated amino acids were located at the third amino acid of the first alpha-helix, the turn between the fourth and fifth beta fold, and the first amino acid of the fifth beta fold, respectively. The BpL and BpL2-1369 genes were ligated into pET28a vector, and transferred into E. coli BL21 (DE3). After induced by IPTG the lipases were purified and characterized. The results showed that the specific activity of the evolved lipase was 1.31-fold than that of the wild lipase, and the Km decreased from 8.24 mmol/L to 7.17 mmol/L. The pH stability of the evolved lipase was better than wild lipase when pH8.0.

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 degrees C and pH 8.0, and was stable between pH 6.0 and 10.0 and below 60 degrees 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 preference for triacylglycerol substrates containing cis-9 unsaturated fatty acid. The results suggest that the lipase could be a candidate for industrial applications.

Diversity of 42 isolates from effective nodules of Pisum sativum in different geographical regions of China were studied using 16S rRNA gene RFLP patterns, 16S rRNA sequencing, 16S–23S rRNA intergenic spacer (IGS) region RFLP patterns and G-C rich random amplified polymorphic DNA (RAPD). The isolates were distributed in two groups on the basis of their 16S rRNA gene RFLP patterns. The 16S rRNA gene sequences of strains from 16S rRNA gene RFLP patterns group I were very closely related (identities higher than 99.5%) to Rhizobium leguminosarum USDA 2370. Group II consisting of WzP3 and WzP15 was closely related to Rhizobium etli CFN42. The analysis of the 16S-23S IGS RFLP patterns divided the isolates into 18 genotypes and four groups. Group I was clustered with R. leguminosarum USDA2370. Group II consisted of YcP2, YcP3 and CqP7. The strains of group III were distributed abroad. Group IV consisted of WzP3, WzP15 and R. etli CFN42. RAPD divided the isolates into nine clusters in which group IV only consisted of YcP2 and the strains of group V and IX were from Wenzhou and Xiantao, respectively. This assay demonstrated the geographical effect on genetic diversity of pea rhizobia.

A lipase from Aspergillus niger F044 was purified to homogeneity using ammonium sulfate precipitation, dialysis, DEAE-Sepharose Fast Flow anion exchange chromatography, and Sephadex G-75 gel filtration chromatography. This purification protocol resulted in a 73.71-fold purification of lipase with 33.99 % final yield, and the relative molecular weight of the lipase were determined to be approximately 35-40 kD using SDS-PAGE. The optimal pH and temperature for lipolytic activity of the lipase was 7.0 and 45 °C, respectively. It was stable at temperature up to 60 °C and retained 98.70 % of its original activity for 30 min. The stability declined rapidly as soon as the temperature rose over 65 °C. The lipase was highly stable in the pH range from 2.0 to 9.0 for 4h. Ca 2+ and Mg 2+ ions stimulated its lipolytic activity, whereas Mn 2+ , Fe 2+ , and Zn 2+ ions caused inhibition. The lipase was also relatively stable in methanol, 2-propanol, and methyl acetate at a final concentration of 40 % ( V/V ) for 24 h. The substrate exhibited a broad specificity toward various oils. The values of K m and V max calculated from the Lineweaver-Burk plot using ρ-nitrophenyl palmitate as hydrolysis substrate were 7.37 mmol/L and 25.91 μmol·min 1 ·mg 1 , respectively. The N-terminal amino acid sequence of the lipase was Ser/Glu/His-Val-Ser-Thr-Ser-Thr-Leu-Asp-Glu-Leu-Gln-Leu-Phe-Ala-Gln, which is highly homogeneous with that of lipase.

From the N-terminal amino acid sequence of the lipase from Aspergillus niger F044, a potential homologous gene A84689 to the lipanl (the gene encoding the lipase from Aspergillus niger F044) was identified. A pair of primers were designed according to the nucleotide sequence of A84689, and the lipanl was cloned by PCR. Nucleotide sequencing revealed that the lipanl has an ORF of 1,044 bp, containing three introns. The deduced amino acid sequence corresponds to 297 amino acid residues. The cloned cDNA fragment encoding the mature lipase from Aspergillus niger F044 was over-expressed in Escherichia coli BL21(De3) and the recombinant protein was refolded in vitro by dilution followed by DEAE Sepharose Fast Flow chromatography.