Identification and characterization of alkaline serine protease from goat skin surface metagenome

Metagenomic DNA isolated from goat skin surface was used to construct plasmid DNA library in Escherichia coli DH10B. Recombinant clones were screened for functional protease activity on skim milk agar plates. Upon screening 70,000 clones, a clone carrying recombinant plasmid pSP1 exhibited protease activity. In vitro transposon mutagenesis and sequencing of the insert DNA in this clone revealed an ORF of 1890 bp encoding a protein with 630 amino acids which showed significant sequence homology to the peptidase S8 and S53 subtilisin kexin sedolisin of Shewanella sp. This ORF was cloned in pET30b and expressed in E. coli BL21 (DE3). Although the cloned Alkaline Serine protease (AS-protease) was overexpressed, it was inactive as a result of forming inclusion bodies. After solubilisation, the protease was purified using Ni-NTA chromatography and then refolded properly to retain protease activity. The purified AS-protease with a molecular mass of ~63 kDa required a divalent cation (Co2+ or Mn2+) for its improved activity. The pH and temperature optima for this protease were 10.5 and 42°C respectively

Superantigen profiles of emm and emm-like typeable and nontypeable pharyngeal streptococcal isolates of South India

<p>Abstract</p> <p>Background</p> <p>The major virulence factors determining the pathogenicity of streptococcal strains include M protein encoded by <it>emm </it>and <it>emm</it>-like (<it>emmL</it>) genes and superantigens. In this study, the distribution of <it>emm, emmL </it>and superantigen genes was analyzed among the streptococcal strains isolated from the patients of acute pharyngitis.</p> <p>Methods</p> <p>The streptococcal strains were isolated from the throat swabs of 1040 patients of acute pharyngitis. The <it>emm </it>and <it>emmL </it>genes were PCR amplified from each strain and sequenced to determine the <it>emm </it>types. The dot-blot hybridization was performed to confirm the pathogens as true <it>emm </it>nontypeable strains. The presence of eleven currently known superantigens was determined in all the strains by multiplex PCR.</p> <p>Results</p> <p>Totally, 124 beta-hemolytic streptococcal strains were isolated and they were classified as group A streptococcus (GAS) [15.3% (19/124)], group C streptococcus (GCS) [59.7% (74/124)] and group G streptococcus (GGS) [25.0% (31/124)]. Among 124 strains, only 35 strains were <it>emm </it>typeable and the remaining 89 strains were <it>emm </it>nontypeable. All GAS isolates were typeable, whereas most of the GCS and GGS strains were nontypeable. These nontypeable strains belong to <it>S. anginosus </it>[75.3% (67/89)] and <it>S. dysgalactiae </it>subsp. <it>equisimilis </it>[24.7% (22/89)]. The <it>emm </it>and <it>emmL </it>types identified in this study include <it>emm12.0 </it>(28.6%), <it>stG643.0 </it>(28.6%), <it>stC46.0 </it>(17.0%), <it>emm30.11 </it>(8.5%), <it>emm3.0 </it>(2.9%), <it>emm48.0 </it>(5.7%), <it>st3343.0 </it>(2.9%), <it>emm107.0 </it>(2.9%) and <it>stS104.2 </it>(2.9%). Various superantigen profiles were observed in typeable as well as nontypeable strains.</p> <p>Conclusions</p> <p>Multiplex PCR analysis revealed the presence of superantigens in all the typeable strains irrespective of their <it>emm </it>types. However, the presence of superantigen genes in <it>emm </it>and <it>emmL </it>nontypeable strains has not been previously reported. In this study, presence of at least one or a combination of superantigen coding genes was identified in all the <it>emm </it>and <it>emmL </it>nontypeable strains. Thus, the superantigens may inevitably play an important role in the pathogenesis of these nontypeable strains in the absence of the primary virulence factor, M protein.</p

Modifying the Substrate Specificity of Carcinoscorpius rotundicauda Serine Protease Inhibitor Domain 1 to Target Thrombin

Protease inhibitors play a decisive role in maintaining homeostasis and eliciting antimicrobial activities. Invertebrates like the horseshoe crab have developed unique modalities with serine protease inhibitors to detect and respond to microbial and host proteases. Two isoforms of an immunomodulatory two-domain Kazal-like serine protease inhibitor, CrSPI-1 and CrSPI-2, have been recently identified in the hepatopancreas of the horseshoe crab, Carcinoscorpius rotundicauda. Full length and domain 2 of CrSPI-1 display powerful inhibitory activities against subtilisin. However, the structure and function of CrSPI-1 domain-1 (D1) remain unknown. Here, we report the crystal structure of CrSPI-1-D1 refined up to 2.0 Å resolution. Despite the close structural homology of CrSPI-1-D1 to rhodniin-D1 (a known thrombin inhibitor), the CrSPI-1-D1 does not inhibit thrombin. This prompted us to modify the selectivity of CrSPI-1-D1 specifically towards thrombin. We illustrate the use of structural information of CrSPI-1-D1 to modify this domain into a potent thrombin inhibitor with IC50 of 26.3 nM. In addition, these studies demonstrate that, besides the rigid conformation of the reactive site loop of the inhibitor, the sequence is the most important determinant of the specificity of the inhibitor. This study will lead to the significant application to modify a multi-domain inhibitor protein to target several proteases

Structural Basis for Dual-Inhibition Mechanism of a Non-Classical Kazal-Type Serine Protease Inhibitor from Horseshoe Crab in Complex with Subtilisin

Serine proteases play a crucial role in host-pathogen interactions. In the innate immune system of invertebrates, multi-domain protease inhibitors are important for the regulation of host-pathogen interactions and antimicrobial activities. Serine protease inhibitors, 9.3-kDa CrSPI isoforms 1 and 2, have been identified from the hepatopancreas of the horseshoe crab, Carcinoscorpius rotundicauda. The CrSPIs were biochemically active, especially CrSPI-1, which potently inhibited subtilisin (Ki = 1.43 nM). CrSPI has been grouped with the non-classical Kazal-type inhibitors due to its unusual cysteine distribution. Here we report the crystal structure of CrSPI-1 in complex with subtilisin at 2.6 Å resolution and the results of biophysical interaction studies. The CrSPI-1 molecule has two domains arranged in an extended conformation. These two domains act as heads that independently interact with two separate subtilisin molecules, resulting in the inhibition of subtilisin activity at a ratio of 1:2 (inhibitor to protease). Each subtilisin molecule interacts with the reactive site loop from each domain of CrSPI-1 through a standard canonical binding mode and forms a single ternary complex. In addition, we propose the substrate preferences of each domain of CrSPI-1. Domain 2 is specific towards the bacterial protease subtilisin, while domain 1 is likely to interact with the host protease, Furin. Elucidation of the structure of the CrSPI-1: subtilisin (1∶2) ternary complex increases our understanding of host-pathogen interactions in the innate immune system at the molecular level and provides new strategies for immunomodulation

Optimization of machining parameters on laser beam machining of titanium alloy (Ti 3Al-2.5V) using taguchi method

As titanium alloy (Ti 3Al-2.5V) has high strength and stiffness with excellent corrosion resistance, it finds its application in aerospace and industrial application. Ti grade 9 is hard, and we have adopted CO2 laser beam techniques to machine Ti grade 9. We have chosen oxygen and nitrogen gases as assisted gases. Of the various laser beam energy techniques, carbon dioxide laser beam is found to be one of the emerging trends in machining complicated and circuitous parts with maximum accuracy and in less time. Design of experiment is represented by Taguchi’s L9 orthogonal array. Two sets of experiments were conducted with oxygen and nitrogen as assisted gases. Material removal rate and surface roughness were calculated and optimized using group Taguchi relational analysis. It is found that surface finish and material removal rate are improved by using nitrogen gas for machining titanium alloy (Ti 3Al-2.5V) using CO2 laser

Crystallization of a nonclassical Kazal-type Carcinoscorpius rotundicauda serine protease inhibitor, CrSPI-1, complexed with subtilisin

A recombinant serine protease inhibitor, CrSPI-1, from horseshoe crab, which inhibits subtilisin with a K i of 10−9  M, has been cloned, expressed, purified and cocrystallized with subtilisin. The crystals diffracted to 2.6 Å resolution

Crystallization of a nonclassical Kazal-type Carcinoscorpius rotundicauda serine protease inhibitor, CrSPI-1, complexed with subtilisin. Corrigendum (vol F65, pg 533, 2009)

10.1107/S2053230X20014685ACTA CRYSTALLOGRAPHICA SECTION F-STRUCTURAL BIOLOGY COMMUNICATIONS76Pt 12623-62

Parametric optimization of abrasive water jet machining of beryllium copper using Taguchi grey relational analysis

Abrasive water jet machining is a non-conventional machining process to machine complicated intricate shapes, abrasive water jet machine is most suited as it can cut both conducting and nonconductive material with better finish. Beryllium copper grade C25 is used as a material for machining considering its applications in petroleum nozzle washer used in petroleum products and also in marine, aerospace industries. In this work, the predominant process parameters like pressure, abrasive flow, and standoff distance are varied to obtain optimum values of response parameters like (MRR) material removal rate and surface roughness. Since the multi-response optimisation cannot be performed by conventional Taguchi method, Grey-Taguchi methodology is used. Abrasive flow rate and standoff distance are the most influenced process parameters to obtain higher MRR and also for better surface-roughness. After obtaining the optimised values for machining parameters using Grey-Taguchi Methodology. A confirmatory test is performed to test the integrity of the obtained results