Polyphenol oxidases exhibit promiscuous proteolytic activity

Tyrosinases are an industrially significant class of polyphenol oxidase. Here, two tyrosinases are shown to cleave a specific peptide bond in a carboxylesterase, yielding a truncated product with higher catalytic activity than the full-length enzyme

Enzymatic surface hydrolysis of PET : effect of structural diversity on kinetic properties of cutinases from thermobifida

In this study cutinases from Thermobifida cellulosilytica DSM44535 (Thc_Cut1 and Thc_Cut2) and Thermobifida fusca DSM44342 (Thf42_Cut1) hydrolyzing poly(ethylene terephthalate) (PET) were successfully cloned and expressed in E.coli BL21-Gold(DE3). Their ability to hydrolyze PET was compared with other enzymes hydrolyzing natural polyesters, including the PHA depolymerase (ePhaZmcl) from Pseudomonas fluorescens and two cutinases from T. fusca KW3. The three isolated Thermobifida cutinases are very similar (only a maximum of 18 amino acid differences) but yet had different kinetic parameters on soluble substrates. Their kcat and Km values on pNP–acetate were in the ranges 2.4–211.9 s–1 and 127–200 μM while on pNP–butyrate they showed kcat and Km values between 5.3 and 195.1 s–1 and between 1483 and 2133 μM. Thc_Cut1 released highest amounts of MHET and terephthalic acid from PET and bis(benzoyloxyethyl) terephthalate (3PET) with the highest concomitant increase in PET hydrophilicity as indicated by water contact angle (WCA) decreases. FTIR-ATR analysis revealed an increase in the crystallinity index A1340/A1410 upon enzyme treatment and an increase of the amount of carboxylic and hydroxylic was measured using derivatization with 2-(bromomethyl)naphthalene. Modeling the covalently bound tetrahedral intermediate consisting of cutinase and 3PET indicated that the active site His-209 is in the proximity of the O of the substrate thus allowing hydrolysis. On the other hand, the models indicated that regions of Thc_Cut1 and Thc_Cut2 which differed in electrostatic and in hydrophobic surface properties were able to reach/interact with PET which may explain their different hydrolysis efficiencies.This study was performed within the Austrian Centre of Industrial Biotechnology ACIB, the MacroFun project and COST Action 868. This work has been supported by the Federal Ministry of Economy, Family and Youth (BMWFJ), the Federal Ministry of Traffic, Innovation and Technology (bmvit), the Styrian Business Promotion Agency SFG, the Standortagentur Tirol and ZIT - Technology Agency of the City of Vienna through the COMET-Funding Program managed by the Austrian Research Promotion Agency FFG. Financial support was also given from Sachsisches Staatsministerium fur Umwelt und Landwirtschaft, Germany. PET was kindly provided by Dr. Vincent Nierstrasz from Ghent University

Structural insight into molecular mechanism of poly (ethylene terephthalate) degradation

Plastics, including poly(ethylene terephthalate) (PET), possess many desirable characteristics and thus are widely used in daily life. However, non-biodegradability, once thought to be an advantage offered by plastics, is causing major environmental problem. Recently, a PET-degrading bacterium, Ideonella sakaiensis, was identified and suggested for possible use in degradation and/or recycling of PET. However, the molecular mechanism of PET degradation is not known. Here we report the crystal structure of I. sakaiensis PETase (IsPETase) at 1.5 angstrom resolution. IsPETase has a Ser-His-Asp catalytic triad at its active site and contains an optimal substrate binding site to accommodate four monohydroxyethyl terephthalate (MHET) moieties of PET. Based on structural and site-directed mutagenesis experiments, the detailed process of PET degradation into MHET, terephthalic acid, and ethylene glycol is suggested. Moreover, other PETase candidates potentially having high PET-degrading activities are suggested based on phylogenetic tree analysis of 69 PETase-like proteins

Intravenous alteplase for stroke with unknown time of onset guided by advanced imaging: systematic review and meta-analysis of individual patient data

Background: Patients who have had a stroke with unknown time of onset have been previously excluded from thrombolysis. We aimed to establish whether intravenous alteplase is safe and effective in such patients when salvageable tissue has been identified with imaging biomarkers. Methods: We did a systematic review and meta-analysis of individual patient data for trials published before Sept 21, 2020. Randomised trials of intravenous alteplase versus standard of care or placebo in adults with stroke with unknown time of onset with perfusion-diffusion MRI, perfusion CT, or MRI with diffusion weighted imaging-fluid attenuated inversion recovery (DWI-FLAIR) mismatch were eligible. The primary outcome was favourable functional outcome (score of 0–1 on the modified Rankin Scale [mRS]) at 90 days indicating no disability using an unconditional mixed-effect logistic-regression model fitted to estimate the treatment effect. Secondary outcomes were mRS shift towards a better functional outcome and independent outcome (mRS 0–2) at 90 days. Safety outcomes included death, severe disability or death (mRS score 4–6), and symptomatic intracranial haemorrhage. This study is registered with PROSPERO, CRD42020166903. Findings: Of 249 identified abstracts, four trials met our eligibility criteria for inclusion: WAKE-UP, EXTEND, THAWS, and ECASS-4. The four trials provided individual patient data for 843 individuals, of whom 429 (51%) were assigned to alteplase and 414 (49%) to placebo or standard care. A favourable outcome occurred in 199 (47%) of 420 patients with alteplase and in 160 (39%) of 409 patients among controls (adjusted odds ratio [OR] 1·49 [95% CI 1·10–2·03]; p=0·011), with low heterogeneity across studies (I2=27%). Alteplase was associated with a significant shift towards better functional outcome (adjusted common OR 1·38 [95% CI 1·05–1·80]; p=0·019), and a higher odds of independent outcome (adjusted OR 1·50 [1·06–2·12]; p=0·022). In the alteplase group, 90 (21%) patients were severely disabled or died (mRS score 4–6), compared with 102 (25%) patients in the control group (adjusted OR 0·76 [0·52–1·11]; p=0·15). 27 (6%) patients died in the alteplase group and 14 (3%) patients died among controls (adjusted OR 2·06 [1·03–4·09]; p=0·040). The prevalence of symptomatic intracranial haemorrhage was higher in the alteplase group than among controls (11 [3%] vs two [<1%], adjusted OR 5·58 [1·22–25·50]; p=0·024). Interpretation: In patients who have had a stroke with unknown time of onset with a DWI-FLAIR or perfusion mismatch, intravenous alteplase resulted in better functional outcome at 90 days than placebo or standard care. A net benefit was observed for all functional outcomes despite an increased risk of symptomatic intracranial haemorrhage. Although there were more deaths with alteplase than placebo, there were fewer cases of severe disability or death. Funding: None

Kemijska modifikacija in karakterizacija površine polisulfonskih membran

Asymmetric porous membranes were prepared from polysulfone by a wet-phase separation procedure. Different chemical modifications were applied to the upper membrane surface, i.e. the surface which was exposed to interaction with nonsolvent (water) in the coagulation bath during the membrane formation. The membrane surface was modified by the series of Friedel-Crafts electrophilic substitutions of aromatic rings in the polysulfone molecules. As a reagent 1-chlorodecane or propylene oxide dissolved in hexane and $AlCl_3$ as a catalyst were used. In the former case a hydrophobic and in the latter one a hydrophilic coating was achieved, respectively. The membrane surface was also modified by sulfonization with sulfuric (VI) acid water solutionin this way negative charges were introduced at the membrane surface. The membranes, unmodified and chemically modified, were characterized by measuring the membrane thickness, the deionized water flux through the membrane, the zeta potential and contact angle. The specific chemical modifications of the membrane surface affect the water flux, the zeta potential and contact angle values whereas the membrane thickness remains unchanged. These effects are interpreted in terms of hydrophilicity and hydrophobicity, changes of the membrane surface charge and the thickness of shear layer at the membrane surface, all with respect to the particular modification applied. Reaction with 1-chlorodecane gave a hydrophobic surface by nonpolar $–(CH_2)_9-CH_3$ groups and reaction with propylene oxide gave a hydrophilic surface with polar group $-CH(CH_3)-CH_2-OH$. The surface of sulfonized polysulfone membranes contained ionizable ($-SO_3H$) functional groups.Površino asimetričnih poroznih membran iz polisulfona, pripravljenih po postopku mokre fazne separacije, smo kemijsko modificirali s Friedel-Crafts-ovo elektrofilno substitucijo aromatskih delov molekule polisulfona, kjer smo kot reagent uporabili 1-klorodekan in propilen oksid ob prisotnosti $AlCl_3$ kot katalizatorja. Na površino smo vezali nepolarne in polarne skupine, ki tvorijo hidrofobno oziroma hidrofilno oblogo. Površino membrane smo kemijsko modificirali tudi s sulfoniranjem z žveplovo(VI) kislino in s tem vezali na površino negativni naboj. Nemodificirane in kemijsko modificirane membrane smo karakterizirali z merjenjem debelin, pretokov za deionizirano vodo, potenciala zeta in omočitvenega kota. Kemijske modifikacije površine membrane so vplivale na pretok za deionizirano vodo, na potencial zeta in omočitveni kot, debelina pa je ostala nespremenjena. Te spremembe smo kvalitativno pojasnili s spremembami hidrofilnosti in hidrofobnosti ter spremembami naboja na površini in debeline mejnega sloja ob površini membrane. Pri reakciji z 1-klorodekanom tvorijo nepolarne $–(CH_2)_9-CH_3$ skupine hidrofobno površino, pri reakciji s propilen oksidom pa tvorijo polarne $–CH(CH_3)-CH_2-OH$ skupine hidrofilno površino. Površina membrane, modificirana z žveplovo(VI) kislino, pa vsebuje ionizabilne $–SO_3H$ funkcionalne skupine