Semiholoenzyme optimizes activity and stability of a hyperthermostable iron-superoxide dismutase.

Metal ion coordination is an essential step for the maturation of metalloenzymes. Generally, the metal coordination sites are thought to be fully occupied to achieve the maximum activity and stability. In this research, we compared the structural features, activity and stability of the apo-, semiholo- and holo-forms of a hyperthermostable tetrameric Fe-superoxide dismutase (SOD). Strikingly, the three forms of enzymes had similar compact tetrameric structures. Removal of iron ions destabilized subunit-subunit interactions during guanidine hydrochloride-induced unfolding. The partially metalized semiholoenzyme possessed most of the activity and identical hyperthermostability of the holoenzyme, but weaker propensity to aggregate. Furthermore, both of the iron content and activity of the semiholoenzyme were unaffected by a 200-fold excess iron ions in solutions, suggesting that conformation of the apo-subunits were forced to the close state by the iron-containing subunits. These observations suggest that fully metalized enzyme is probably nonessential for multimeric metalloenzymes and the semiholoenzyme may be a better choice. The unique properties of semiholoenzyme also provide the organisms a compromised solution to survival under metal deficiency conditions.

Characterization of FM2382 from Fulvimarina manganoxydans sp. Nov. 8047 with potential enzymatic decontamination of sulfur mustard.

Sulfur mustard (SM) can be hydrolyzed by haloalkane dehalogenases such as DhaA, LinB and DmbA. However, the low resistance to the elevated temperatures limited the practical application of haloalkane dehalogenases. Here we reported a new thermotolerant dehalogenase FM2382 from Fulvimarina manganoxydans sp. nov. 8047. The specific activity of FM2382 to SM is 0.6 U/mg. FM2382 possessed high heat stability (45 °C) in slight alkali environment (pH 7.5) and retained approximately 50% activity after incubation at 70 °C for 40 min. The catalytic activity of FM2382 was activated by Co2+ and Mg2+, and inhibited by Zn2+, Cu2+ and Fe3+. Furthermore, site-specific mutagenesis proved that D34, K207 D232, D237 were amino acid residues related to the catalytic activity of SM. In conclusion, we found a thermostable haloacid dehalogenases (HAD) family dehalogenase showing SM-degradation activity, which may be useful for practical application in the future.

Injured vertebra pedicle screww fixation versus short-segment pedicle instrumentation for thoracolumbar fracture:a meta-analysis / 中国组织工程研究

BACKGROUND: Posterior internal fixation is one of the most common methods for thoracolumbar fractures. There is a lack of systematic evaluation about the efficacy of injured vertebra pedicle screw fixation(IVPSF)versus short-segment pedicle instrumentation (SSPI) for thoracolumbar fracture. OBJECTIVE: To compare the clinical outcomes of IVPSF and SSPI for single thoracolumbar fracture through a METHODS: A computer-based on-line research of PubMed, Medline, Embase, Cochrane Library, CNKI, and WanFang databases was performed for the studies regarding IVPSF versus SSPI for thoracolumbar fracture from 1990 to 2016. meta-analysis. The randomized controlled trials and cohort studies were collected based on the strict criteria of inclusion and exclusion. A meta-analysis was conducted on Revman5.3 sofeware. RESULTS AND CONCLUSION: (1) Eleven articles were enrolled, including 5 English and 6 Chinese ones, involving 689 patients (328 cases for IVPSF and 361 cases for SSPI). (2) The meta-analysis indicated that the operation time, blood loss and mean hospital stay showed no significant differences between two groups. IVPSF showed more effective than SSPI in the kyphotic angle correction and anterior vertebral height recovery at postoperation and 1-5 years of follow-up. Moreover, the incidence of postoperative fixation failure in IVPSF was lower than that in SSPI. (3) These findings suggest that IVPSF that reduces the postoperative fixation failure rate for thoracolumbar fractures provides better kyphosis correction and restoration of anterior vertebral height at post-operation and 1-5 years of follow-up.

An Ime2-like mitogen-activated protein kinase is involved in cellulase expression in the filamentous fungus Trichoderma reesei.

OBJECTIVES: Eukaryotic mitogen-activated protein kinases (MAPKs) play crucial roles in transducing environmental and developmental signals inside the cell and regulating gene expression, however, the roles of MAPKs remain largely unknown in Trichoderma reesei. RESULTS: T. reesei ime2 (TrIme2) encodes an Ime2-like MAPK in T. reesei. The deletion of the TrIme2 gene led to 90% increase in cellulase activity against filter paper during earlier period time of cellulase induction as well as the extracellular protein production. Compared to the parent strain, the transcriptional levels of the three major cellulase genes cbh1,cbh2, egl1 were increased by about 9 times, 4 times, 2 times, respectively, at 8 h after cellulase induction in the ΔTrIme2 mutant. In addition, the disruption of TrIme2 caused over 50% reduction of the transcript levels of cellulase transcriptional regulators cre1 and xyr1. CONCLUSION: TrIme2 functions in regulation of the expression of cellulase gene in T.reesei, and is a good candidate for genetically engineering of T. reesei for higher cellulase production.

High production of ectoine from aspartate and glycerol by use of whole-cell biocatalysis in recombinant Escherichia coli.

BACKGROUND: Recently, the compatible solute 1, 4, 5, 6-tetrahydro-2-methyl-4-pyrimidinecarboxylic acid (ectoine) has attracted considerable interest due to its great potential as a protecting agent. To overcome the drawbacks of high salinity in the traditional bioprocess of ectoine using halophilic bacteria, various attempts have been made to engineer ectoine biosynthesis in nonhalophilic bacteria. Unfortunately, the yields of ectoine in these producers are still low and hardly meet the demands of large scale production. In this paper, the whole-cell biocatalytic process using aspartate and glycerol as substrates was tried for high production of ectoine in nonhalophilic bacteria. RESULTS: The ectoine genes ectABC from the halophilic bacterium Halomonas elongata were successfully introduced into Escherichia coli K-12 strain BW25113 under the arabinose-inducible promoter. To our delight, a large amount of ectoine was synthesized and excreted into the medium during the course of whole-cell biocatalysis, when using aspartate and glycerol as the direct substrates. At the low cell density of 5 OD/mL in flask, under the optimal conditions (100 mM sodium phosphate buffer (pH 7.0), 100 mM sodium aspartate, 100 mM KCl and 100 mM glycerol), the concentration of extracellular ectoine was increased to 2.67 mg/mL. At the high cell density of 20 OD/mL in fermentor, a maximum titre of 25.1 g/L ectoine was achieved in 24 h. Meanwhile, the biomass productivity of ectoine is as high as 4048 mg per gram dry cell weight (g DCW)(-1), which is the highest value ever reported. Furthermore, it was demonstrated that the same batch of cells could be used for at least three rounds. Finally, a total yield of 63.4 g ectoine was obtained using one litre cells. CONCLUSION: Using aspartate and glycerol as the direct substrates, high production of ectoine was achieved by the whole-cell biocatalysis in recombinant E. coli. Multiple rounds of whole-cell biocatalysis were established to further improve the production of ectoine. Our study herein provided a feasible biosynthesis process of ectoine with potential applications in large-scale industrial production.

A novel major facilitator transporter TrSTR1 is essential for pentose utilization and involved in xylanase induction in Trichoderma reesei.

Trichoderma reesei (teleomorph Hypocrea jecorina) is an industrially important filamentous fungus for glycoside hydrolases production, with its xylanolytic enzymes widely applied in many areas. However, the molecular mechanisms underlying xylanase expression are still insufficiently understood. In particular, the effect of sugar transporter on the induction of xylanase expression is unclear. In this work, we identified a novel major facilitator transporter TrSTR1 that is capable of transporting xylose by using a xylose utilization system in Saccharomyces cerevisiae. In T. reesei, TrSTR1 is essential for the utilization of d-xylose, l-arabinose, and even their downstream metabolites D-xylitol and L-arabitol. TrSTR1 is also involved in the induction of xylanase expression since both the xylanase activity and extracellular protein concentration in the Tu6△str1 strain were decreased, which further confirmed by a qRT-PCR analysis of the transcript levels of the key transcriptional regulators. Our observations provide new insights into connections between pentose utilization and xylanase production in T. reesei.

Characterization and homologous overexpression of an N-acetylglucosaminidase Nag1 from Trichoderma reesei.

Trichoderma reesei is thought to be a promising recombinant host for the production and secretion of complex proteins due to its ability to secrete large amounts of proteins. In this study we identified a functional N-acetyl-ß-glucosaminidase (NAGase) gene Nag1 in T. reesei. Nag1, a putative gene encoding a GH 20 family NAGase in T. reesei, was cloned and homologous overexpressed in the T. reesei RutC30ΔU3 with a strong cellobiohydrolase1 gene (cbh1) promoter. Nag1 was secreted in its active form and the highest expression level was around 499.85IU/ml. Nag1 has a molecular mass of 80kDa. The optimum pH and temperature were 4.0 and 60°C, respectively.

Formation of high-order oligomers by a hyperthemostable Fe-superoxide dismutase (tcSOD).

Hyperthermostable proteins are highly resistant to various extreme conditions. Many factors have been proposed to contribute to their ultrahigh structural stability. Some thermostable proteins have larger oligomeric size when compared to their mesophilic homologues. The formation of compact oligomers can minimize the solvent accessible surface area and increase the changes of Gibbs free energy for unfolding. Similar to mesophilic proteins, hyperthermostable proteins also face the problem of unproductive aggregation. In this research, we investigated the role of high-order oligomerization in the fight against aggregation by a hyperthermostable superoxide dismutase identified from Tengchong, China (tcSOD). Besides the predominant tetramers, tcSOD could also form active high-order oligomers containing at least eight subunits. The dynamic equilibrium between tetramers and high-order oligomers was not significantly affected by pH, salt concentration or moderate temperature. The secondary and tertiary structures of tcSOD remained unchanged during heating, while cross-linking experiments showed that there were conformational changes or structural fluctuations at high temperatures. Mutational analysis indicated that the last helix at the C-terminus was involved in the formation of high-order oligomers, probably via domain swapping. Based on these results, we proposed that the reversible conversion between the active tetramers and high-order oligomers might provide a buffering system for tcSOD to fight against the irreversible protein aggregation pathway. The formation of active high-order oligomers not only increases the energy barrier between the native state and unfolded/aggregated state, but also provides the enzyme the ability to reproduce the predominant oligomers from the active high-order oligomers.

Enhanced production of N-acetyl-D-neuraminic acid by multi-approach whole-cell biocatalyst.

N-Acetyl-D-neuraminic acid (Neu5Ac) has attracted considerable interest due to its promising potential applications in medicine. Significant efforts have been made in whole-cell biocatalyst for Neu5Ac production, but the processes often result in suboptimal performance due to poor expression of enzymes, imbalances of pathway components, disturbance of competing pathways, and barriers of mass transport. In this study, we engineered Escherichia coli strains capable of producing Neu5Ac by assembling a two-step heterologous pathway consisting of N-acetyl-D-glucosamine 2-epimerase (AGE) and Neu5Ac aldolase (NanA). Multiple approaches were used to improve the efficiency of the engineered pathway and process for enhanced Neu5Ac production. Firstly, we identified that NanA was the rate-controlling enzyme in this pathway. With increased expression of NanA, a ninefold increase in Neu5Ac production (65 mM) was observed. Secondly, knocking out nanTEK genes blocked Neu5Ac uptake and the competing pathway, which kept the reactions to the synthetic direction as the final product went outside of the cells and enhanced the Neu5Ac production by threefold, resulting in 173.8 mM of Neu5Ac. Thirdly, we improved the performance of the system by promoting substrate transport and optimizing concentrations of substrates. An overall whole-cell biocatalytic process was developed and a maximum titer of 240 mM Neu5Ac (74.2 g/L) was achieved, with productivity of 6.2 g Neu5Ac/L/h and conversion yield of 40 % from GlcNAc. The engineered strain could be reused for at least five cycles with a productivity of >6 g/L/h. It is a cost-effective process for Neu5Ac production with potential applications in large-scale industrial production.

Improved production of heterologous lipase in Trichoderma reesei by RNAi mediated gene silencing of an endogenic highly expressed gene.

A lipase gene (Lip) of the Aspergillus niger was de novo synthesized and expressed in the Trichoderma reesei under the promoter of the cellobiohydrolase I gene (cbh1). RNAi-mediated gene silencing was successfully used to further improve the recombinant lipase production via down-regulation of CBHI which comprised more than 60% of the total extracellular proteins in T. reesei. The gene and protein expression of CBHI and recombinant lipase were analyzed by real-time PCR, SDS-PAGE and activity assay. The results demonstrated that RNAi-mediated gene silencing could effectively suppress cbh1 gene expression and the reduction of CBHI could result in obvious improvement of heterologous lipase production. The reconstructed strains with decreased CBHI production exhibited 1.8- to 3.2-fold increase in lipase activity than that of parental strain. The study herein provided a feasible and advantageous method of increasing heterologous target gene expression in T. reesei through preventing the high expression of a specific endogenenous gene by RNA interference.

Distinct symmetry and limited peptide refolding activity of the thermosomes from the acidothermophilic archaea Acidianus tengchongensis S5(T).

Recombinant thermosomes from the Acidianus tengchongensis strain S5(T) were purified to homogeneity and assembled in vitro into homo-oligomers (rATcpnalpha or rATcpnbeta) and hetero-oligomers (rATcpnalphabeta). The symmetries of these complexes were determined by electron microscopy and image analysis. The rATcpnalpha homo-oligomer was shown to possess 8-fold symmetry while both rATcpnbeta and rATcpnalphabeta oligomers adopted 9-fold symmetry. rATcpnalphabeta oligomers were shown to contain the alpha and beta subunits in a 1:2 ratio. All of the complexes prevented the irreversible inactivation of yeast alcohol dehydrogenase at 55 degrees C and completely prevented the formation of aggregates during thermal inactivation of citrate synthase at 45 degrees C. All rATcpn complexes showed trace ATP hydrolysis activity. Furthermore, rATcpnbeta sequestered fully chemically denatured substrates (GFP and thermophilic malic dehydrogenase) in vitro without refolding them in an ATP-dependent manner. This property is similar to previously reported properties of chaperonins from Sulfolobus tokodaii and Sulfolobus acidocaldarius. These features are consistent with the slow growth rates of these species of archaea in their native environment.

Genetic characteristics of entervirus 71 isolated in Ningxia Hui Autonomous region in 2009 / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To analyze the genetic characteristics of EV71 strains isolated from HFMD cases in Ningxia Hui Autonomous Region in 2009.</p><p><b>METHODS</b>In 2009, totally 385 specimens from 344 HFMD cases were collected from Ningxia. Enterovirus isolation was performed in RD cell line from all the specimens. EV71 isolates were identified by specific RT-PCR from the positive cultures, and sequences of complete EV71 VP1 encoding region were determined for farther analyses.</p><p><b>RESULTS</b>Totally from 126 EV strains isolated in this study, 58 EV71 strains (46%) were identified. And complete VP1 sequences of 46 EV71 strains were determined, and genetic analyses were performed. It was showed that the nucleotide identity of 46 Ningxia strains with the representatives of A and B genotypes were 81.7%-82.8% and 83.1%-85.2%, and the amino acid identity were 93.9%-95.9% and 96. 2%-97.9% respectively. The nucleotide identity of NingXia EV71 isolates with representatives of subgenotype C1, C2, C3, C4a, C4b, and C5 were 88.3%-90.6% (97.9%-99.6%), 88.3%-90.1% (97.9%-99.3%), 87.8%-89.0% (97.6%-98.9%), 94.2%-98.9% (97.9%-100%), 91.8%-94.1% (98.6%-99.6%), and 86.7%-89.1% (97.9%-98.9%). Phylogenetic tree analysis revealed that 46 stains were clustered with reference stains of subgenotype C4 and the Ningxia EV71 isolates were belonged to subgenotype C4a.</p><p><b>CONCLUSION</b>EV71 of subgenotype C4a had spread widely in Ningxia in 2009, which was absolutely predominant type in Ningxia in 2009 and also as the predominant type in China mainland since 2005.</p>

Contributions of the C-terminal helix to the structural stability of a hyperthermophilic Fe-superoxide dismutase (TcSOD).

Hyperthermophilic superoxide dismutases (SODs) are of particular interest due to their potential industrial importance and scientific merit in studying the molecular mechanisms of protein folding and stability. Compared to the mesophilic SODs, the hyperthermostable Fe-SODs (TcSOD and ApSOD) have an extended C-terminal helix, which forms an additional ion-pairing network. In this research, the role of the extended C-terminus in the structural stability of TcSOD was studied by investigating the properties of two deletion mutants. The results indicated that the ion-pairing network at the C-terminus had limited contributions to the stability of TcSOD against heat- and GdnHCl-induced inactivation. The intactness of the C-terminal helix had dissimilar impact on the two stages of TcSOD unfolding induced by guanidinium chloride. The mutations slightly decreased the Gibbs free energy of the dissociation of the tetrameric enzymes, while greatly affected the stability of the molten globule-like intermediate. These results suggested that the additional ion-pairing network mainly enhanced the structural stability of TcSOD by stabilizing the monomers.

Multistate folding of a hyperthermostable Fe-superoxide dismutase (TcSOD) in guanidinium hydrochloride: The importance of the quaternary structure.

Superoxide dismutases (SODs), which are the first line of cellular defense against the toxic effects of reactive oxygen species, are metalloenzymes that catalyze the disproportionation of superoxide radicals to produce oxygen and hydrogen peroxide. Although much effort has been devoted to the folding mechanisms of Cu/Zn-SODs, little is known about the folding of Fe-SODs. In this research, the equilibrium unfolding and refolding of TcSOD, a tetrameric hyperthermostable Fe-SOD, were investigated by circular dichroism, intrinsic fluorescence, ANS fluorescence, size-exclusion chromatography and cross-linking experiments. The results herein suggested that the guanidine hydrochloride-induced unfolding of TcSOD involved a stable monomeric intermediate and a possible tetrameric intermediate. The Gibbs free energy of TcSOD dissociation was about 3-fold larger than that of the monomeric intermediate unfolding, which suggested that the quaternary structure plays a crucial role in TcSOD stability. A comparison of the thermodynamic parameters between TcSOD and other SODs also suggested that the stability of quaternary structure might be responsible for the hyperthemostability of TcSOD.

[Construction and analysis of rumen bacterial artificial chromosome library from a dairy cow rumen microflora].

The high molecular weight DNA was extracted and purified directly from rumen samples in the study by using culture-independent and pulsed field gel electrophoresis approaches. After digestion with Hind III, DNA fragments ranging from 50-100 kb was collected and ligated to pCC BAC vector. The ligation mixture was transformed into E. coli EPI300 and a rumen metagenomic BAC library with about 15360 clones was constructed. The average insert size is about 54.5 kb, mostly ranging from 50-70 kb, and the capacity of this BAC library is about 837Mb. Several BAC clones with activity of amylase, Cmcellulase had been screened from the BAC library. The clones with Cmcelluase activity were screened further for linchenase, xylanase, cellobioase activity and the result is that 25 of them have at least one kind of other enzyme activity.

Characterization of a hyperthermostable Fe-superoxide dismutase from hot spring.

A new gene encoding a thermostable Fe-superoxide dismutase (tcSOD) was identified from a metagenomic library prepared from a hot spring sample. The open reading frame of tcSOD encoded a 211 amino acid protein. The recombinant protein was overexpressed in Escherichia coli and confirmed to be a Fe-SOD with a specific activity of 1,890 U/mg using the pyrogallol method. The enzyme was highly stable at 80 degrees C and retained 50% activity after heat treatment at 95 degrees C for 2 h. It showed striking stability across a wide pH span from 4 to 11. The native form of the enzyme was determined as a homotetramer by analytical ultracentrifugation and gradient native polyacrylamide gel electrophoresis. Fe(2+) was found to be important to SOD activity and to the stability of tcSOD dimer. Comparative modeling analyses of tcSOD tetramer indicate that its high thermostability is mainly due to the presence of a large number of intersubunit ion pairs and hydrogen bonds and to a decrease in solvent accessible hydrophobic surfaces.

[Construction and analysis of a metagenomic library from Tengchong hot spring soil in Yunnan Province].

By a combination of freezing/thawing/ proteinase K-based method and SDS/high-salt/heating treatment, the mixed environmental genomic DNA was isolated directly from a hot spring soil in Tengchong, Yunnan, China. With this method, The DNA yield was up to 1 - 2 microg/g soil. After purification with the Wizard DNA clean up system (Promega, Madison, Wis), the mixed genomic DNA was partially digested with restriction enzyme Pst I. Digested DNA fragments of 3 - 8 kb were recovered from agrose gel and ligated to the pSK (+) vector. The ligation mixture was transformed into DH10B strain, resulting in the construction of a metagenomic library with about 2.5 x 10(4) clones. Restriction enzyme analysis revealed that the average insert is about 4.6 kb. Some novel sequences were identified via sequencing and gene annotation analysis of 30 clones randomly chosen from this library.

[Expression of B-glucosidase gene of Sacchromycopsis fibuligera in Pichia pastoris].

A beta-Glucosidase gene (BGL 1) was amplified with PCR from the total DNA of Sacchromycopsis fibuligera, and was linked with pGEM-T vector. After cut down by restriction enzyme from pGEM-T vector, BGL 1 was inserted into the expression vector pPIC9K of Pichia pastoris in reading frame with alpha-factor secreting signal peptide sequence to construct the recombinant plasmid pSHL9K. The recombinant plasmid pSHL9K was transformed into Pichia pastoris GS115 with electroporation. The recombinant Pichia pastoris strains which could efficiently secret recombinant beta-Glucosidase were selected. The optimum temperature of the recombinant beta-Glucosidase was 50degreesC, and the optimum pH was 5.4. The activity of beta-Glucosidase could reach to 47U/mL in the culture medium.

[Integrative expression of XynB of Aspergillus niger UV-11 in industrial yeast].

The cDNA sequence of beta-xylanase gene (xynB) was cloned from Aspergillus niger UV-11. It was inserted into the yeast expression vector and the recombinant plasmid pAX2 was obtained. The plasmid pAX2 was introduced into an industrial S. cerevisiae 2.346 and integrated into yeast genome by co-transformation of a YEPtype plasmid pBEJ16 carrying G418 resistance. The stable engineered yeast strain XY2 was obtained. It could express and secret extracellular xylanase, and enhance the alcohol production in wheat flour fermentation compared with the host strain S. cerevisiae 2.346.

[Expression of endo-beta-mannanase gene from Trichoderma reesei in Pichia pastoris].

Complete mannanase gene with two introns was cloned from Trichoderrna reesei by PCR. The two introns were then removed by overlap extension PCR. The gene encoding the mature mannanase protein was inserted into the expression vector pPIC9K, downstream of a alpha-factor signal peptide sequence. The resultant recombinant vector was named pM242. After linearized with Sac I , pM242 was transformed to Pichia pastoris GS115 by electroporation. After screening, the recombinant strain Gpmf25 that expresses the secretory protein at high level was obtained. The activity of the recombinant mannanase reached 12.5 IU/mL. Optimum pH and temperature for the recombinant enzyme were 5.0 and 80 degrees C, respectively. The enzyme was stable at pH 5.0-6.0 and maintained over 50% of original activity after incubation at 70 degrees C for 30 min.