Preparation of pectate lyase/Cu3(PO4)2 hybrid nanoflower and its catalytic performance as an immobilized enzyme

Biocatalysts could substitute conventional chemical reagents in textile industrial process for green-production process as well as lowering the costs. Alkaline pectate lyases (Pels) are enzymes that could be used in scouring and degumming process, in which the biochemical properties of Pels with high activity and stability under process conditions are of great interests. In our previous studies, an alkaline pectate lyase PEL168 derived from Bacillus subtilis 168 was engineered with improved enzymatic performance. The obtained Pel3 mutant presented an increased specific activity of 4.3-fold and extended T50 to 330 min. Here, we introduce a facile and rapid method of preparing an immobilized enzyme Pel3-inorganic hybrid nanoflower to increase its biocatalytic efficiency. After evaluating four divalent ions, including Mn2+, Ca2+, Zn2+ and Cu2+ as inorganic part with PBS buffer, Pel3/Cu3(PO4)2 hybrid nanoflower was obtained with improved biocatalytic properties. The optimum temperature and pH of Pel3/Cu3(PO4)2 hybrid nanoflower were determined to be 55℃ and pH 9, respectively, exhibiting subtle difference from the free Pel3. However, the Pel3/Cu3(PO4)2 hybrid nanoflower maintained 33% total activity after treated at 55℃ in 24 h, while the free Pel3 completely lost its activity in 18 h. Furthermore, the residual activity of the Pel3/Cu3(PO4)2 hybrid nanoflowers remain over 50% even after four times of repeatitive utilization, demonstrating its promising stability for practical application

Access tunnel engineering to optimize the catalytic cycle of carbohydrate hydrolases with buried active site

The active site of many enzymes is buried inside the protein core and is connected with the surrounding solvent by access tunnels. An emerging approach to optimize these enzymes properties is the engineering of structural features governing the exchange of ligands between the active sites and bulk solvent. However, it is still challenging to redesign the access tunnels of enzymes catalyzing biopolymers like carbohydrate hydrolases because of the extremely complicated substrate structure. In this study, structure-guided saturated mutagenesis was performed to reconstruct all three access tunnels of xylanase S7-xyl from Bacillus halodurans S7, which results in a mutant 254-RL1 with 3.4-fold increase in specific activity. Structural comparison and kinetic analysis revealed that products egress is the rate-limiting step in the catalytic cycle of S7-xyl. The products release tunnel in S7-xyl was experimentally validated, and not the tunnel radius but the length determining the products release efficiency. Application assessment showed that relieving the inhibition of reducing sugars on mutant 254-RL1 could accelerate the hydrolysis efficiency of cellulase on different pretreated lignocellulose materials, representing a good candidate in enzyme cocktails for lignocellulose biodegradation. In addition, the same strategy was successfully utilized to improve the specific activities of three other xylanases with buried active site, suggesting the general application of tunnel engineering to optimize carbohydrate hydrolases with buried active site

Case-control association analysis of rheumatoid arthritis with candidate genes using related cases

We performed a case-control association analysis of rheumatoid arthritis (RA) for several candidate genes using the North American Rheumatoid Arthritis Consortium (NARAC) data provided in Genetic Analysis Workshop 15. We conducted the case-control association analysis using all related cases and unrelated controls and compared the results with those from the analysis of samples using only one randomly selected case from each family and all unrelated controls. For both analyses we used a weighted composite likelihood ratio test based on single-nucleotide polymorphism (SNP) markers or haplotypes accounting for the correlation among samples within a family. Several SNPs, including R620W in the candidate gene PTPN22, showed an association with RA status, which confirmed previously reported results. Several other SNPs in the candidate genes, such as CTLA4, HAVCR1, and SUMO4, also had rather small p-values (<0.05), suggesting the associations between them and RA. Our results showed that the p-values obtained from the analysis including all related cases were generally smaller than those obtained from the analysis including only one randomly selected case per family. These results, together with the results, based on simulated data, showed that higher power could be achieved using all related cases

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

BACKGROUND: The 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. RESULTS: The 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 K(m) of 0.09 mg/ml and a V(max) of 18.13 μmol/min. K(+), Li(+), Ni(2+) and Sr(2+) showed little or no inhibitory effect on PEL168P activity, and Ca(2+) 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. CONCLUSIONS: Pectate 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

Prompting Fab Yeast Surface Display Efficiency by ER Retention and Molecular Chaperon Co-expression.

For antibody discovery and engineering, yeast surface display (YSD) of antigen-binding fragments (Fabs) and coupled fluorescence activated cell sorting (FACS) provide intact paratopic conformations and quantitative analysis at the monoclonal level, and thus holding great promises for numerous applications. Using anti-TNFα mAbs Infliximab, Adalimumab, and its variants as model Fabs, this study systematically characterized complementary approaches for the optimization of Fab YSD. Results suggested that by using divergent promoter GAL1-GAL10 and endoplasmic reticulum (ER) signal peptides for co-expression of light chain and heavy chain-Aga2 fusion, assembled Fabs were functionally displayed on yeast cell surface with sigmoidal binding responses toward TNFα. Co-expression of a Hsp70 family molecular chaperone Kar2p and/or protein-disulfide isomerase (Pdi1p) significantly improved efficiency of functional display (defined as the ratio of cells displaying functional Fab over cells displaying assembled Fab). Moreover, fusing ER retention sequences (ERSs) with light chain also enhanced Fab display quality at the expense of display quantity, and the degree of improvements was correlated with the strength of ERSs and was more significant for Infliximab than Adalimumab. The feasibility of affinity maturation was further demonstrated by isolating a high affinity Fab clone from 1:103 or 1:105 spiked libraries

Application of imputation methods to the analysis of rheumatoid arthritis data in genome-wide association studies

Most genetic association studies only genotype a small proportion of cataloged single-nucleotide polymorphisms (SNPs) in regions of interest. With the catalogs of high-density SNP data available (e.g., HapMap) to researchers today, it has become possible to impute genotypes at untyped SNPs. This in turn allows us to test those untyped SNPs, the motivation being to increase power in association studies. Several imputation methods and corresponding software packages have been developed for this purpose. The objective of our study is to apply three widely used imputation methods and corresponding software packages to a data from a genome-wide association study of rheumatoid arthritis from the North American Rheumatoid Arthritis Consortium in Genetic Analysis Workshop 16, to compare the performances of the three methods, to evaluate their strengths and weaknesses, and to identify additional susceptibility loci underlying rheumatoid arthritis. The software packages used in this paper included a program for Bayesian imputation-based association mapping (BIMBAM), a program for imputing unobserved genotypes in case-control association studies (IMPUTE), and a program for testing untyped alleles (TUNA). We found some untyped SNP that showed significant association with rheumatoid arthritis. Among them, a few of these were not located near any typed SNP that was found to be significant and thus may be worth further investigation

Testing for differences in distribution tails to test for differences in 'maximum' lifespan

<p>Abstract</p> <p>Background</p> <p>Investigators are actively testing interventions intended to increase lifespan and wish to test whether the interventions increase maximum lifespan. Based on the fact that one cannot be assured of observing population maximum lifespans in finite samples, in previous work, we constructed and validated several tests of difference in the upper parts of lifespan distributions between a treatment group and a control group by testing whether the probabilities that observations are above some threshold defining 'old' or being in the tail of the survival distribution are equal in the two groups. However, a limitation of these tests is that they do not consider <it>how much </it>above the threshold any particular observation is.</p> <p>Methods</p> <p>In this article we propose new methods which improve upon our previous tests by considering not only whether an observation is above some threshold, but also the magnitudes by which observations exceed the threshold.</p> <p>Results</p> <p>Simulations show that the new methods control type I error rates quite well and that the power of the new methods is usually higher than that of the tests we previously proposed. In illustrative analyses of two real datasets involving rodents, when setting the threshold equal to 110 (100) weeks for the first (second) datasets, the new methods detected differences in 'maximum lifespan' between groups at nominal alpha levels of 0.01 (0.05) for the first (second) datasets and provided more significant results than competitor tests.</p> <p>Conclusion</p> <p>The new methods not only have good performance in controlling the type I error rates but also improve the power compared with the tests we previously proposed.</p

Deferiprone and gallium-protoporphyrin have the capacity to potentiate the activity of antibiotics in Staphylococcus aureus small colony variants

Small colony variants (SCVs) of bacteria like Staphylococcus aureus are characterized by a reduced colony size and are linked to increased antibiotic tolerance and resistance. Their altered expression of virulence factors, slow growing properties and their ability to form biofilms make the eradication of SCVs challenging. In the context of biofilm-related infectious diseases involving S. aureus SCVs, a therapy targeting bacterial iron metabolism was evaluated. The combination of the iron-chelator deferiprone (Def) and the heme-analog gallium-protoporphyrin (GaPP), in solution and incorporated in a surgical wound gel, was tested for activity against planktonic and sessile SCVs. To this end, the activity of Def-GaPP was assessed against planktonic S. aureus SCVs, as well as against in vitro and in vivo biofilms in the colony biofilm model, an artificial wound model and a Caenorhabditis elegans infection model. While Def alone failed to show substantial antibacterial activity, GaPP and the combination of Def-GaPP demonstrated concentration- and strain-dependent antibacterial properties. Specifically, the Def-GaPP combination significantly reduced the bacterial load in an artificial wound model and increased the survival of S. aureus SCV infected C. elegans. When Def-GaPP were combined with gentamicin or ciprofloxacin, the triple combinations exceeded the antibiofilm activity of the individual compounds in the colony biofilm model. In targeting bacterial iron metabolism, Def-GaPP showed significant activity against planktonic and sessile SCVs. Moreover, Def-GaPP could potentiate the activity of gentamicin and ciprofloxacin. Delivered in a wound healing gel, Def-GaPP showed promise as a new topical strategy against infections with S. aureus SCVs.Katharina Richter, Nicky Thomas, Guimin Zhang, Clive A. Prestidge, Tom Coenye, Peter-John Wormald and Sarah Vreugd

Efimov States From Triple α Resonances

The Efimov trimers in excited 12C nuclei, which no observation exists yet, are discussed by means of analyzing the experimental data of 70(64)Zn(64Ni) +70(64)Zn(64Ni )reactions at beam energy of E/A=35 MeV/nucleon. In heavy ion collisions, the αs interact with each other and can form complex systems such as 8Be and 12C. For the 3α systems, multi resonance processes give rise to excited levels of 12C. The interaction between any two of the 3α particles provides events with one, two or three 8Be. Their interfering levels are clearly seen in the minimum relative energy distributions. Events of three couple αrelative energies consistent with the ground state of 8Be are observed with the decreasing of the instrumental error at the reconstructed 7.458 MeV excitation energy of 12C, which was suggested as the (Thomas) Efimov state