Journal of Molecular Catalysis B-enzymatic最新文献

{"title":"An engineered outer membrane pore enables an efficient oxygenation of aromatics and terpenes","authors":"Anna Joëlle Ruff ,&nbsp;Marcus Arlt ,&nbsp;Maike van Ohlen ,&nbsp;Tsvetan Kardashliev ,&nbsp;Monika Konarzycka-Bessler ,&nbsp;Marco Bocola ,&nbsp;Alexander Dennig ,&nbsp;Vlada B. Urlacher ,&nbsp;Ulrich Schwaneberg","doi":"10.1016/j.molcatb.2016.11.007","DOIUrl":"10.1016/j.molcatb.2016.11.007","url":null,"abstract":"<div><p>Biocatalysis with cytochrome P450 enzymes are important for the industrial production of fine chemicals, pharmaceuticals, fragrance and flavor compounds since chemoselective hydroxylation of aromatics and terpenes are chemically difficult to achieve. A few P450 based industrial processes have been developed based on whole cell catalysis. However, the outer membrane of microbial cells forms an effective barrier, which reduces the uptake of hydrophobic substrates. The coexpression of outer membrane proteins in <em>E. coli</em> such as the ferric hydroxamate uptake protein (FhuA) can provide alternative solutions to chemical or physical methods for increasing compound flux through the outer membrane of <em>E. coli</em> and thereby to boost productivities. In this study we employed an engineered FhuA Δ1-160 variant in which the “cork domain” was removed (first 160 residues are deleted); FhuA Δ1-160 has a cross-section of 39–46<!--> <!-->Å with a “free” inner diameter of about 14 Å that serves as passive diffusion channel. FhuA WT and Δ1-160 were coexpressed on a bicistronic system with two P450 BM3 variants for regiospecific hydroxylation of aromatic compounds toluene and anisole as well as for oxidation of two terpenes (α)-pinene and (<em>R</em>)-(+)-limonene. The presence of FhuA Δ1‐160 resulted in a doubled product concentration for toluene (35<!--> <!-->μ to 50<!--> <!-->μM), anisole (25<!--> <!-->μM to 45<!--> <!-->μM), pinene (12<!--> <!-->μM to 20<!--> <!-->μM) and limonene (12<!--> <!-->μM to 25<!--> <!-->μM) and five times higher for the coumarin derivative BCCE. In order to characterizes and compensate for expression variations a quantification method based on Chromeo546-labled StrepTactinII was developed to quantify the number of FhuA Δ1-160 in the outer <em>E. coli</em> membrane (∼44000 of FhuA Δ1-160 per cell). Morphology studies showed that a 6% <em>E. coli</em> surface coverage can be achieved with FhuA Δ1‐160 without significantly influencing the <em>E. coli</em> rod shape. In summary, FhuA Δ1-160 efficiently increases uptake of hydrophobic aromatics and terpenes for whole-cell biotransformations and can likely be used for other enzymes and/or substrates.</p></div>","PeriodicalId":16416,"journal":{"name":"Journal of Molecular Catalysis B-enzymatic","volume":"134 ","pages":"Pages 285-294"},"PeriodicalIF":0.0,"publicationDate":"2016-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1016/j.molcatb.2016.11.007","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"79947463","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Efficient production of ethyl (R)-4-chloro-3-hydroxybutanoate by a novel alcohol dehydrogenase from Lactobacillus curieae S1L19","authors":"Yiping Zhang,&nbsp;Hualei Wang,&nbsp;Lifeng Chen,&nbsp;Kai Wu,&nbsp;Jingli Xie,&nbsp;Dongzhi Wei","doi":"10.1016/j.molcatb.2016.09.010","DOIUrl":"10.1016/j.molcatb.2016.09.010","url":null,"abstract":"<div><p>Ethyl (<em>R</em>)-4-chloro-3-hydroxybutanoate ester [(<em>R</em>)-CHBE] is an important chiral intermediate for the synthesis of chiral drugs. In this study, a novel short-chain, NADH-dependent dehydrogenase (LCRIII) from <em>Lactobacillus curieae</em> S1L19 was discovered to exhibit high activity and enantioselectivity in the production of (<em>R</em>)-CHBE by reduction of ethyl 4-chloroacetoacetate (COBE). LCRIII was heterologously overexpressed in <em>Escherichia coli</em> and the protein was purified to homogeneity. Characterization of LCRIII showed broad substrate specificity towards a variety of ketones. In addition, an efficient cofactor regeneration system was constructed by co-expressing LCRIII and glucose dehydrogenase (GDH) in <em>E. coli</em> cells. Up to 1.5<!--> <!-->M (246.8<!--> <!-->g/L) COBE could be completely reduced to (<em>R</em>)-CHBE with excellent enantiomeric excess (<!--> <!-->&gt;<!--> <!-->99% <em>ee</em>) in a monophasic aqueous system. Moreover, the process could be performed even without external addition of cofactors. These results demonstrate the great potential of this process in industrial applications.</p></div>","PeriodicalId":16416,"journal":{"name":"Journal of Molecular Catalysis B-enzymatic","volume":"134 ","pages":"Pages 51-60"},"PeriodicalIF":0.0,"publicationDate":"2016-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1016/j.molcatb.2016.09.010","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"81834826","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Immobilization of lipase on mesoporous silica nanoparticles with hierarchical fibrous pore","authors":"Zafar Ali,&nbsp;Lei Tian,&nbsp;Panpan Zhao,&nbsp;Baoliang Zhang,&nbsp;Nisar Ali,&nbsp;Muhammad Khan,&nbsp;Qiuyu Zhang","doi":"10.1016/j.molcatb.2016.10.011","DOIUrl":"10.1016/j.molcatb.2016.10.011","url":null,"abstract":"<div><p><span><span>Lipase from Candida Ragusa (CRL) was successfully covalently immobilized on fibrous silica nanoparticles KCC-1, and the properties of </span>immobilized enzyme were investigated. Mesoporous fibrous silica nanoparticles (MSNPs) were synthesized with particles size 200</span> <!-->nm pore size 15–30<!--> <span><span>nm; followed by amino-functionalization. Scanning Electron Microscopy (SEM), Transmittance Electronic microscopy (TEM), </span>Fourier Transform Infrared Spectroscopy (FT-IR) and N</span>₂ adsorption were used for the characterization of nanoparticles. Further SiO₂@NH₂ nanoparticles were activated by glutaraldehyde as a bifunctional cross linker, and were used for lipase immobilization. The applied approach for support preparation, activation, and optimization of immobilization conditions, led to better resistance to temperature and pH inactivation in comparison to the free lipase, and hence widened the reaction pH and temperature regions, with the optimum pH and temperature of 7.5 and 40<!--> <!-->°C, respectively. The immobilized Lipase Candida Ragusa (ICRL) maintained above 81% of the initial activity after 28<!--> <!-->days and 80% activity after 8 repeated cycles. Thus ICRL showed improved storage stability reusability and 700<!--> <!-->U/g of protein as immobilization efficiency.</p></div>","PeriodicalId":16416,"journal":{"name":"Journal of Molecular Catalysis B-enzymatic","volume":"134 ","pages":"Pages 129-135"},"PeriodicalIF":0.0,"publicationDate":"2016-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1016/j.molcatb.2016.10.011","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"90789266","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Studies of carbon monoxide dehydrogenase from Oligotropha carboxidovorans","authors":"Stephanie Dingwall,&nbsp;Jarett Wilcoxen ,&nbsp;Dimitri Niks,&nbsp;Russ Hille","doi":"10.1016/j.molcatb.2016.10.007","DOIUrl":"10.1016/j.molcatb.2016.10.007","url":null,"abstract":"<div><p>We have undertaken physicochemical studies of the CO dehydrogenase from the aerobe <em>Oligotropha carboxidovorans</em>, probing both the binuclear Mo- and Cu-containing active site where CO is oxidized to CO₂ and the enzyme’s FAD, where the reducing equivalents obtained from CO are transferred to the quinone pool. Regarding the FAD site, we have characterized the semiquinone oxidation state by EPR and identified it to be of the blue neutral form with a linewidth of 20<!--> <!-->G. The signature long-wavelength absorbance of FADH<img> is also observed in the absorption spectrum of partially reduced enzyme at low pH. The enzyme exhibits a pH-dependent absorption spectrum in the oxidized state that is lost upon covalent modification of the enzyme by the flavin-specific agent diphenyliodonium cation. The pH dependence is attributed to Tyr 193 of the FAD-containing CoxM subunit, which sits atop the <em>re</em> face of the isoalloxazine ring in van der Waals contact with it. Electron equilibration among the enzyme’s four redox-active centers (including two [2Fe-2S] clusters in addition to the binuclear center and FAD) is found to be pH-dependent, but too fast to be followed using a stopped-flow pH jump protocol. Electron transfer from the iron-sulfur clusters to the FAD is thus much faster than in other members of the xanthine oxidase family of molybdenum-containing enzymes to which CO dehydrogenase belongs. Finally, a complex of the binuclear center with bicarbonate has been characterized by EPR, where the absence of observed hyperfine coupling using ¹³C-labeled bicarbonate suggests strongly that the bicarbonate is not directly coordinated to the Mo(V) of the partially reduced binuclear center.</p></div>","PeriodicalId":16416,"journal":{"name":"Journal of Molecular Catalysis B-enzymatic","volume":"134 ","pages":"Pages 317-322"},"PeriodicalIF":0.0,"publicationDate":"2016-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1016/j.molcatb.2016.10.007","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"88399037","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"A highly efficient immobilized MAS1 lipase for the glycerolysis reaction of n-3 PUFA-rich ethyl esters","authors":"Xiumei Wang ,&nbsp;Daoming Li ,&nbsp;Weifei Wang ,&nbsp;Bo Yang ,&nbsp;Yonghua Wang","doi":"10.1016/j.molcatb.2016.09.012","DOIUrl":"10.1016/j.molcatb.2016.09.012","url":null,"abstract":"<div><p>This study reported that immobilized MAS1 lipase showed high catalytic efficiency in the production of triacylglycerols (TAG) highly enriched with n-3 polyunsaturated fatty acids (PUFA) by glycerolysis of ethyl esters (EE). Immobilized MAS1 lipase was found to have no regiospecificity and be a more suitable catalyst for the glycerolysis of n-3 PUFA-rich EE compared with other enzymes. Higher TAG content (73.9%) and EE conversion (82%) were obtained by immobilized MAS1 lipase than those by Novozym 435 (29.6% and 54.8%, respectively) and Lipozyme RM IM (10% and 49%, respectively). Besides, the effects of temperature, enzyme loading and n-3 PUFA-rich EE/glycerol molar ratio on TAG content were evaluated using response surface methodology. The results showed that temperature, enzyme loading and n-3 PUFA-rich EE/glycerol molar ratio had significant effects on TAG content. The maximum TAG content (76.5%) was achieved under the optimal conditions (enzyme loading of 163.8<!--> <!-->U/g substrate, n-3 PUFA-rich EE/glycerol molar ratio of 4.13:1 at 65<!--> <!-->°C). Subsequently, the glycerolysis reaction mixtures were further purified by molecular distillation and highly pure n-3 PUFA-rich TAG (96.2%) with similar fatty acids composition to the substrate (EE) was obtained in the final products. In addition, the obtained final products had low acid value and peroxide value (0.03<!--> <!-->mg KOH/g and 3.2<!--> <!-->meq/kg, respectively). These results indicated that immobilized MAS1 lipase is a promising catalyst for the synthesis of TAG in industrial application.</p></div>","PeriodicalId":16416,"journal":{"name":"Journal of Molecular Catalysis B-enzymatic","volume":"134 ","pages":"Pages 25-31"},"PeriodicalIF":0.0,"publicationDate":"2016-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1016/j.molcatb.2016.09.012","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"72463489","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Differential effect of a chemical denaturant on activity and stability of a serine protease in nonaqueous media","authors":"Shivcharan Prasad ,&nbsp;Villendra S. Negi ,&nbsp;Joydev K. Laha ,&nbsp;Ipsita Roy","doi":"10.1016/j.molcatb.2016.09.011","DOIUrl":"10.1016/j.molcatb.2016.09.011","url":null,"abstract":"<div><p>With reference to industrial application, reusability of the biocatalyst is an important criterion which determines the cost of the final product. Urea-induced structural perturbation of proteases has led to higher enzymatic activity, especially in nonaqueous media. The mechanism behind this phenomenon has not been investigated in detail. Using the transesterification activity of subtilisin Carlsberg in nonaqueous media as an illustration, we report that the higher activity is due to simultaneous decrease in Michaelis constant and increase in turnover number of the enzyme. However, we show that this perturbed architecture is unable to retain the high activity-conformation for further rounds of catalysis. Thus, we conclude that the use of an enzyme for commercial applications is dependent upon a compromise between activity and (operational) stability.</p></div>","PeriodicalId":16416,"journal":{"name":"Journal of Molecular Catalysis B-enzymatic","volume":"134 ","pages":"Pages 32-36"},"PeriodicalIF":0.0,"publicationDate":"2016-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1016/j.molcatb.2016.09.011","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"78823187","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Thermostability enhancement of xylanase Aspergillus fumigatus RT-1","authors":"Mohd Khairul Hakimi bin Abdul Wahab ,&nbsp;Mohd Anuar bin Jonet ,&nbsp;Rosli Md Illias","doi":"10.1016/j.molcatb.2016.09.020","DOIUrl":"10.1016/j.molcatb.2016.09.020","url":null,"abstract":"<div><p>This study aimed to improve the thermostability of <em>endo</em>-1,4-β-xylanase (afxynG1) from <em>Aspergillus fumigatus</em> RT-1 using error-prone PCR. Since the wild type enzyme has an optimum temperature stability at 50<!--> <!-->°C, the improvement of its stability will widen its application in industries with operating processes at higher temperatures. A library containing approximately 5000 afxynG1 mutants was generated and thermally screened at 60°<!--> <!-->C for 30<!--> <!-->min. Four mutants (T16A/T39I/L176Q, S68R, A60D and Q47P/S159R) were selected for enzymatic characterization because of their higher catalytic activity compared to the wild type. Among these mutants, the mutant T16A/T39I/L176Q showed highest stability at 70<!--> <!-->°C and retained 45.9% of its activity after 60<!--> <!-->min of incubation while the wild type had lost its activity completely after 50<!--> <!-->min of incubation. The other mutants, A60D, S68R and Q47P/S159R also showed improvement in thermostability by retaining 33.2%, 25.8% and 23.8% of their activity respectively. The optimum temperature for mutants also significantly increased. The optimum temperature for T16A/T39I/L176Q increased up to 70<!--> <!-->°C, followed by A60D increased up to 60<!--> <!-->°C while the rest remained the same, similar to the wild type enzyme. The mutant T16A/T39I/L176Q had the highest half-life time (t_1/2) of 42<!--> <!-->min at 70°<!--> <!-->C, which is a 3.5-fold increase compared to the wild type enzyme which only showed a t_1/2 of 12<!--> <!-->min at 70<!--> <!-->°C. This is followed by mutant A60D, t_1/2 of 31<!--> <!-->min (2.7-fold), S68R, t_1/2 of 29<!--> <!-->min (2.4-fold) and Q47P/S159R, t_1/2 of 27<!--> <!-->min (2.25-fold). Based on homology modelling conducted to analyze the <em>mutants’ structures</em>, it showed that hydrophobicity and hydrogen bonds were the driving forces that lead to the improvement of the thermal stability of these xylanase mutants.</p></div>","PeriodicalId":16416,"journal":{"name":"Journal of Molecular Catalysis B-enzymatic","volume":"134 ","pages":"Pages 154-163"},"PeriodicalIF":0.0,"publicationDate":"2016-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1016/j.molcatb.2016.09.020","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"73953362","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Special issue OxiZymes 2016","authors":"Willem van Berkel,&nbsp;Marco Fraaije,&nbsp;Frank Hollmann","doi":"10.1016/j.molcatb.2016.11.009","DOIUrl":"10.1016/j.molcatb.2016.11.009","url":null,"abstract":"","PeriodicalId":16416,"journal":{"name":"Journal of Molecular Catalysis B-enzymatic","volume":"134 ","pages":"Page 273"},"PeriodicalIF":0.0,"publicationDate":"2016-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1016/j.molcatb.2016.11.009","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"73291630","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Purification and characterization of a fibrinolytic enzyme from the food-grade fungus, Neurospora sitophila","authors":"Xiao lan Liu,&nbsp;Narasimha kumar Kopparapu,&nbsp;Hong chen Zheng,&nbsp;Priti Katrolia,&nbsp;Yong ping Deng,&nbsp;Xi qun Zheng","doi":"10.1016/j.molcatb.2016.10.006","DOIUrl":"10.1016/j.molcatb.2016.10.006","url":null,"abstract":"<div><p>A fibrinolytic protease was purified from the culture supernatant of a GRAS fungus, <em>Neurospora sitophila</em>. The enzyme displayed a molecular mass of 34<!--> <!-->kDa, as estimated by SDS-PAGE and gel filtration chromatography. The isoelectric point (pI) of the enzyme was 9.3<!--> <!-->±<!--> <!-->0.2 as determined by iso-electric focusing (IEF). It was maximally active at pH 7.6 and 41<!--> <!-->°C and displayed remarkable stability in a wide pH range (4–11) and up to 52<!--> <!-->°C. The enzyme activity was inhibited by phenylmethane sulfonyl fluoride (PMSF) and ethylenediamine tetracetic acid (EDTA), indicating that it is a metal-dependent serine protease. It was found to be a direct acting plasmin like protein which efficiently cleaved the α-chain of fibrin(ogen), followed by β-chain and γ-chain. Three internal peptide sequences LASTANSGVLSGLLAGTVGGK; AYTSKSSVPSSVGLAR; LLDTGLNTAHSDFNR were determined by Q-TOF2. These results indicate no sequence similarities with other fibrinolytic enzymes suggesting it to be a novel enzyme. This fibrinolytic enzyme may be developed as a safe potential candidate for oral administration as a functional food additive or as a drug for prevention and/or treatment of thrombolytic diseases.</p></div>","PeriodicalId":16416,"journal":{"name":"Journal of Molecular Catalysis B-enzymatic","volume":"134 ","pages":"Pages 98-104"},"PeriodicalIF":0.0,"publicationDate":"2016-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1016/j.molcatb.2016.10.006","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"73787123","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"A Green approach towards the synthesis of chiral alcohols using functionalized alginate immobilized Saccharomyces cerevisiae cells","authors":"Narmada Muthineni, M. S. Arnipalli, Sridhar Bojja, H. Meshram, A. Srivastava, Bhaskar Rao Adari","doi":"10.1016/J.MOLCATB.2016.10.016","DOIUrl":"https://doi.org/10.1016/J.MOLCATB.2016.10.016","url":null,"abstract":"","PeriodicalId":16416,"journal":{"name":"Journal of Molecular Catalysis B-enzymatic","volume":"19 1","pages":"233-237"},"PeriodicalIF":0.0,"publicationDate":"2016-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"79286726","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}