G. Q. Zhang

Effects of salts on activity of halophilic cellulase with glucomannanase activity isolated from alkaliphilic and halophilic Bacillus sp. BG-CS10

Alkaliphilic and halophilic Bacillus sp. BG-CS10 was isolated from Zabuye Salt Lake, Tibet. The gene celB, encoding a halophilic cellulase was identified from the genomic library of BG-CS10. CelB belongs to the cellulase superfamily and DUF291 superfamily, with an unknown function domain and less than 58% identity to other cellulases in GenBank. The purified recombinant protein (molecular weight: 62xa0kDa) can hydrolyze soluble cellulose substrates containing beta-1,4-linkages, such as carboxylmethyl cellulose and konjac glucomannan, but has no exoglucanase and β-glucosidase activities. Thus, CelB is a cellulase with an endo mode of action and glucomannanase activity. Interestingly, the enzyme activity was increased approximately tenfold with 2.5xa0M NaCl or 3xa0M KCl. Furthermore, the optimal temperatures were 55°C with 2.5xa0M NaCl and 35°C without NaCl, respectively. This indicates that NaCl can improve enzyme thermostability. The Km and kcat values of CelB for CMC with 2.5xa0M NaCl were 3.18xa0mgxa0mL−1 and 26xa0s−1, while the Km and kcat values of CelB without NaCl were 6.6xa0mgxa0mL−1 and 2.1xa0s−1. Thus, this thermo-stable, salt and pH-tolerant cellulase is a promising candidate for industrial applications, and provides a new model to study salt effects on the structure of protein.

The alkaline pectate lyase PEL168 of Bacillus subtilis heterologously expressed in Pichia pastoris is more stable and efficient for degumming ramie fiber

BackgroundThe conventional degumming process of ramie with alkaline treatment at high temperature causes severe environmental pollution. Pectate lyases can be used to remove pectin from ramie in a degumming process with reduced environmental pollution and energy consumption. Pectate lyase PEL168 from Bacillus subtilis has been previously characterized and the protein structure was resolved. However, Bacillus is not a suitable host for pectate lyases during the degumming process since most Bacillus produce cellulases endogenously with a detrimental effect to the fiber. Pichia pastoris, which does not express endogenous cellulases and has high secretion capability, will be an ideal host for the expression. No previous work was reported concerning the heterologous expression of pectate lyase PEL168 in P. pastoris with an aim for industrial application in ramie bio-degumming.ResultsThe gene pel168 was expressed in P. pastoris in this study. The recombinant protein PEL168 in P. pastoris (PEL168P) showed two bands of 48.6 kDa and 51.4 kDa on SDS-PAGE whereas the enzyme expressed in E. coli (PEL168E) was the same as predicted with a band of 46 kDa. Deglycosylation digestion suggested that PEL168P was glycosylated. The optimum reaction temperature of the two PEL168s was 50°C, and the optimum pH 9.5. After preincubation at 60°C for 20 min, PEL168E completely lost its activity, whereas PEL168P kept 26% of the residual activity. PEL168P had a specific activity of 1320 U/mg with a Km of 0.09 mg/ml and a Vmax of 18.13 μmol/min. K+, Li+, Ni2+ and Sr2+ showed little or no inhibitory effect on PEL168P activity, and Ca2+ enhanced enzyme activity by 38%. PEL168P can remove the pectin from ramie effectively in a degumming process. A 1.5 fold increase of PEL168 enzyme expression in P. pastoris was achieved by further codon optimization.ConclusionsPectate lyase PEL168 with an available protein structure can be heterologously expressed in P. pastoris. The characterized recombinant PEL168P can be used to remove pectin from ramie efficiently and the expression level of PEL168 in P. pastoris was increased markedly by codon optimization. Therefore, PEL168 is an ideal candidate for further optimization and engineering for bio-degumming.

Characterization of a thermostable xylanase from an alkaliphilic Bacillus sp.

A xylanase gene (xyn10) from alkaliphilic Bacillus sp. N16-5 was cloned and expressed in Pichia pastoris. The deduced amino acid sequence has 85% identity with xylanase xyn10A from B. halodurans and contains two potential N-glycosylation sites. The glycosylated Xyn10 with MW 48xa0kDa can hydrolyze birchwood and oatspelt xylan. The enzyme had optimum activity at pH 7 and 70°C, with the specific activity of 92.5U/mg. The Xyn10 retained over 90% residual activity at 60°C for 30xa0min but lost all activity at 80°C over 15xa0min. Most tested ions showed no or slight inhibition effects on enzyme activity.

Efficient production of polymer-grade L-lactate by an alkaliphilic Exiguobacterium sp. strain under nonsterile open fermentation conditions.

In this study, a newly isolated alkaliphile Exiguobacterium sp. strain 8-11-1 was used to produce optically pure L-lactate. With an initial glucose concentration of 80 g/L, a high overall L-lactic acid productivity of 8.15 g/L/h was achieved using NaOH as a neutralizing agent. The fed-batch fermentations were carried out under both sterile and nonsterile conditions. Under the nonsterile condition, 125 g/L L-lactic acid was obtained with a high percent yield and average productivity of 98.33% and 3.79 g/L/h, respectively. These values were consistent with the results from sterile condition. No d-isomers of lactic acid were detected, resulting in an optical purity of 100% in both conditions. The high levels of optically pure L-lactic acid produced by Exiguobacterium sp. 8-11-1, combined with the ease of handling and low costs associated with the open fermentation strategy, provide a novel and potentially important approach for L-lactic acid production in the future.

Different mechanism of two-proton emission from proton-rich nuclei 23 Al and 22 Mg

Two-proton relative momentum (q(pp)) and opening angle (theta(pp)) distributions from the three-body decay of two excited proton-rich nuclei, namely Al-23 --> p + p + Na-21 and Mg-22 --> p + p + Ne-20, have been measured with the projectile fragment separator (RIPS) at the RIKEN RI Beam Factory. An evident peak at q(pp) similar to 20 MeV/c as well as a peak in theta(pp) around 30 degrees are seen in the two-proton break-up channel from a highly-excited Mg-22. In contrast, such peaks are absent for the Al-23 case. It is concluded that the two-proton emission mechanism of excited Mg-22 is quite different from the Al-23 case, with the former having a favorable diproton emission component at a highly excited state and the latter dominated by the sequential decay process

Giant Dipole Resonance as a Fingerprint of a Clustering Configurations in C-12 and O-16

It is studied how the α cluster degrees of freedom, such as α clustering configurations close to the α decay threshold in (12)C and (16)O, including the linear chain, triangle, square, kite, and tetrahedron, affect nuclear collective vibrations with a microscopic dynamical approach, which can describe properties of nuclear ground states well across the nuclide chart and reproduce the standard giant dipole resonance (GDR) of (16)O quite nicely. It is found that the GDR spectrum is highly fragmented into several apparent peaks due to the α structure. The different α cluster configurations in (12)C and (16)O have corresponding characteristic spectra of GDR. The number and centroid energies of peaks in the GDR spectra can be reasonably explained by the geometrical and dynamical symmetries of α clustering configurations. Therefore, the GDR can be regarded as a very effective probe to diagnose the different α cluster configurations in light nuclei.

Truncation of the unique N-terminal domain improved the thermos-stability and specific activity of alkaline α-amylase Amy703

High pH condition is of special interest for the potential applications of alkaline α-amylase in textile and detergent industries. Thus, there is a continuous demand to improve the amylase’s properties to meet the requirements set by specific applications. Here we reported the systematic study of modular domain engineering to improve the specific activity and stability of the alkaline α-amylase from Bacillus pseudofirmus 703. The specific activity of the N-terminal domain truncated mutant (N-Amy) increased by ~35-fold with a significantly improved thermo-stability. Kinetic analysis demonstrated that the Kcat and Kcat/Kmof N-Amy were enhanced by 1300-fold and 425.7-fold, respectively, representing the largest catalytic activity improvement of the engineered α-amylases through the methods of domain deletion, fusion or swapping. In addition, different from the wild-type Amy703, no exogenous Ca2+ were required for N-Amy to maintain its full catalytic activity, implying its superior potential for many industrial processes. Circular dichroism analysis and structure modeling revealed that the increased compactness and α-helical content were the main contributors for the improved thermo-stability of N-Amy, while the improved catalytic efficiency was mainly attributed by the increased conformational flexibility around the active center.

Identification and characterization of a new acid-stable endoglucanase from a metagenomic library.

A new endoglucanase gene cel124 was cloned from a metagenomic library and expressed in Escherichiacoli. Catalytic triad analysis showed that the catalytic triad sites were different from the known endoglucanases. Cel124, a 34 kDa protein, exhibited a specific activity (29.08 U mg(-1)) toward 1% of sodium carboxymethyl cellulose and was stable at 50 °C for 30 min. The optimal temperature and pH for its catalytic activity were 50 °C and pH 5.5 respectively. Cel124 could hydrolyze soluble cellulose, but not insoluble cellulose or other polysaccharides. The kinetic parameters (5.63 mg ml(-1) for Km and 0.0397 mmol min(-1) mg(-1) for Vmax) were measured. 3M NaCl in the system could increase its activity by 2 fold. Site-directed mutation and circular dichroism spectra test suggested that the residue (Glu41) was essential for its activity, might be a potential active site. Based on our data, we proposed that Cel124 might represent a new type of endoglucanase.

Identification and characterization of a novel alkaline α-amylase Amy703 belonging to a new clade from Bacillus pseudofirmus

Alkaline α-amylases are of great interest in desizing processes and detergent industries. Here, an alkaline α-amylase gene amy703 from an alkaliphilic Bacillus pseudofirmus strain was cloned and sequenced. Its encoding product, Amy703, might represent a new clade of α-amylase family, because it shared only 35xa0% highest identity with all amylases characterized up to date and was not clustered into any subfamilies with amylase activity in glycoside hydrolase family 13. Heterologous expression and characterization of Amy703 showed that it is a metalloenzyme with maximal activity at 40xa0°C and pH 9.0. Its activity was significantly enhanced by 2- and 2.48-fold at the presence of 10xa0mM Ca2+ and Mg2+, respectively, while Hg2+ was a strong inhibitor of Amy703. Amy703 has a higher affinity (Kmxa0=xa03.92xa0mg/ml) for soluble starch compared to many other alkaline amylases. The computer modeling of its structure indicated that Amy703 contains typical amylase domains and a loop region appearing to bind the substrates. Site-directed mutagenesis suggested that a conserved residue Glu550 was essential for the activity of Amy703, and proposed it working together with other two residues to constitute a catalytic triad (Asp521, Glu550, and Asp615).

Thermodynamic properties and shear viscosity over entropy-density ratio of the nuclear fireball in a quantum-molecular dynamics model

Thermodynamic and transport properties of nuclear fireball created in the central region of heavy-ion collisions below 400 MeV/nucleon are investigated within the isospin-dependent quantum molecular dynamic (IQMD) model. These properties, including the density, temperature, chemical potential, entropy density (s), and shear viscosity (eta), are calculated by a generalized hot Thomas Fermi formulism and a parametrized function, which was developed by Danielewicz. As the collision goes on, a transient minimal eta/s = 5/4 pi - 10/4 pi occurs in the largest compression stage. Besides, the relationship of eta/s to temperature (T) in the freeze-out stage displays a local minimum which is about 9-20 times 1/4 pi around T = 8-12 MeV, which can be argued as indicative of a liquid gas phase transition. In addition, the influences of nucleon-nucleon (NN) cross section (sigma(NN)) and symmetry energy coefficient (C-s) are also discussed, and it is found that the results are sensitive to sNN but not to C-s.