tag:blogger.com,1999:blog-18414946506137088622024-03-21T08:55:13.193-07:00Nzomics a blog about nitrile-active enzymesUnknownnoreply@blogger.comBlogger170125tag:blogger.com,1999:blog-1841494650613708862.post-62958679077756894212016-09-27T05:31:00.000-07:002016-09-27T05:31:40.629-07:00Preparation of CLEAs of a recombinant NHase ES-NHT-118<div class="WordSection1">
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<span style="background-color: black;"><span style="color: white;"><span style="font-family: Arial, Helvetica, sans-serif;"><b>Preparation of Cross-linked Enzyme Aggregates of Nitrile Hydratase ES-NHT-118 from<i> E. coli</i> by Macromolecular Cross-linking Agent</b></span><o:p></o:p></span></span></div>
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<span style="background-color: black;"><span style="color: white;"><i>Liya Zhou, Haixia Mou, Jing Gao, Li Ma, Ying He and Yanjun Jiang</i><o:p></o:p></span></span></div>
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<span style="background-color: black;"><span style="color: white;"><a href="http://www.sciencedirect.com/science/article/pii/S100495411630458X">Chinese Journal of Chemical Engineering (in press)</a><o:p></o:p></span></span></div>
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<span style="background-color: black;"><span style="color: white;"><a href="http://dx.doi.org/10.1016/j.cjche.2016.08.027" target="doilink"><span style="background-attachment: initial; background-clip: initial; background-image: initial; background-origin: initial; background-position: initial; background-repeat: initial; background-size: initial; border: 1pt none; font-family: "arial" , sans-serif; font-size: 10pt; padding: 0cm; text-decoration: none;">http://dx.doi.org/10.1016/j.cjche.2016.08.027</span></a><o:p></o:p></span></span></div>
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<span style="background-color: black; color: white; font-family: Arial, Helvetica, sans-serif;"><span style="background-attachment: initial; background-clip: initial; background-image: initial; background-origin: initial; background-position: initial; background-repeat: initial; background-size: initial;">Cross-linked enzyme aggregates (CLEAs) of nitrile hydratase (NHase) ES-NHT-118 from<span class="apple-converted-space"> </span><em><span style="border: none 1.0pt; padding: 0cm;">E. coli</span></em><span class="apple-converted-space"> </span>were prepared by using ammonium sulfate as precipitating agent followed by cross-linking with dextran polyaldehyde for the first time. In this process, egg white was added as an amine source to aid formation of CLEAs. The optimal conditions of the immobilization process were determined. Michaelis constants (<em><span style="border: none 1.0pt; padding: 0cm;">K</span></em></span><sub><span style="background-attachment: initial; background-clip: initial; background-image: initial; background-origin: initial; background-position: initial; background-repeat: initial; background-size: initial; border: 1pt none; font-size: 9pt; padding: 0cm;">m</span></sub></span><span style="background-attachment: initial; background-clip: initial; background-image: initial; background-origin: initial; background-position: initial; background-repeat: initial; background-size: initial;"><span style="background-color: black; color: white; font-family: Arial, Helvetica, sans-serif;">) of free NHase and NHase CLEAs were also determined. The NHase CLEAs exhibited increased stability at varied pH and temperature conditions compared to its free counterpart. When exposed to high concentrations of acrylamide, NHase CLEAs also exhibited effective catalytic activity.</span><span style="background-color: white; color: white; font-family: arial, sans-serif;"><o:p></o:p></span></span></div>
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Unknownnoreply@blogger.com0tag:blogger.com,1999:blog-1841494650613708862.post-60836204776804576572016-09-01T04:12:00.002-07:002016-09-01T04:12:52.173-07:00ChromSoc nitrilase flow chemistry project 5We have looked at the conversion of 4-cyanopyridine to isonicotinic acid using our <a href="http://nzomics.blogspot.co.uk/2016/08/chromsoc-nitrilase-flow-chemistry_3.html">mesoscale flow chemistry apparatus</a> containing a nitrilase enzyme immobilized in alginate. Here is an example of our results.<br />
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<a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEhx5sL1nq4s_Fm-77wNqBhgLnQlF_zZk6cm3a3kI2HRQREgJ_i50_mZ53kRKaT8UwhqgAwLE0xr21ZYoubgwniedUtrVC0J2IpdsZTrq6lf_L2FMFVRFMwXttYG_d9pxG8FKY9HyqUkvTI/s1600/NIT21+alginate+beads+flow+on+4CNpyridine.png" imageanchor="1" style="margin-left: 1em; margin-right: 1em;"><img border="0" height="216" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEhx5sL1nq4s_Fm-77wNqBhgLnQlF_zZk6cm3a3kI2HRQREgJ_i50_mZ53kRKaT8UwhqgAwLE0xr21ZYoubgwniedUtrVC0J2IpdsZTrq6lf_L2FMFVRFMwXttYG_d9pxG8FKY9HyqUkvTI/s320/NIT21+alginate+beads+flow+on+4CNpyridine.png" width="320" /></a></div>
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Rob flowed the reaction medium through the device, and took a measurement of the amount of ammonia (using <a href="http://pubs.rsc.org/en/Content/ArticleLanding/2015/CC/c4cc06021k#!divAbstract">our colorimetric nitrilase assay</a>) available after each cycle. Each cycle took about 20 minutes. It has not reached completion but seems to be a fairly robust system as far as it goes.Unknownnoreply@blogger.com0tag:blogger.com,1999:blog-1841494650613708862.post-64339555961830970052016-08-05T05:53:00.000-07:002016-08-05T05:53:14.117-07:00The cysteinesulfenic Acid in NHase as catalytic nucleophile<div class="WordSection1">
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<b>Time-Resolved Crystallography of the Reaction Intermediate of Nitrile Hydratase: Revealing a Role for the Cysteinesulfenic Acid Ligand as a Catalytic Nucleophile.</b></div>
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<i>Yamanaka, Y., Kato, Y., Hashimoto, K., Iida, K., Nagasawa, K., Nakayama, H., Dohmae, N., Noguchi, K., Noguchi, T., Yohda, M. and Odaka, M.</i></div>
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<i>Angew. Chem. Int. Ed., </i><b>(2015)</b><i>, </i>54: 10763–10767.</div>
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The reaction mechanism of nitrile hydratase (NHase) was investigated using time-resolved crystallography of the mutant NHase, in which βArg56, strictly conserved and hydrogen bonded to the two post-translationally oxidized cysteine ligands, was replaced by lysine, and pivalonitrile was the substrate. The crystal structures of the reaction intermediates were determined at high resolution (1.2–1.3 Å). In combination with FTIR analyses of NHase following hydration in H218O, we propose that the metal-coordinated substrate is nucleophilically attacked by the O(SO−) atom of αCys114-SO−, followed by nucleophilic attack of the S(SO−) atom by a βArg56-activated water molecule to release the product amide and regenerate αCys114-SO−.<o:p></o:p></div>
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<span style="mso-fareast-language: EN-GB;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEhhGEEstD-l7ejQKTSJ36B4vezpFO27lMaGjxmCtH5vG9NPcEVt-GE5bnIhXPr1lChZt_KPosJdW3eAEhOj6ux_KXgW7FPFgkooj8jkpxkRAkU9A4sVED57heoeT-g_9FokTow4zKiuXHQ/s1600/image001-729988.gif"><img alt="" border="0" id="BLOGGER_PHOTO_ID_6315325195881179522" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEhhGEEstD-l7ejQKTSJ36B4vezpFO27lMaGjxmCtH5vG9NPcEVt-GE5bnIhXPr1lChZt_KPosJdW3eAEhOj6ux_KXgW7FPFgkooj8jkpxkRAkU9A4sVED57heoeT-g_9FokTow4zKiuXHQ/s320/image001-729988.gif" /></a></span><o:p></o:p></div>
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Unknownnoreply@blogger.com0tag:blogger.com,1999:blog-1841494650613708862.post-63139947046142438342016-08-05T05:38:00.000-07:002016-08-05T05:38:40.997-07:00High concentration synthesis of 3-hydroxypropionic acid <div class="WordSection1">
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<b>Enzymatic synthesis of 3-hydroxypropionic acid at high productivity by using free or immobilized cells of recombinant Escherichia coli</b><o:p></o:p></div>
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<o:p> </o:p><i>Shanshan Yua, Peiyuan Yao, Jianjiong Li, Jie Ren, Jing Yuan, Jinhui Feng, Min Wang, Qiaqing Wu, Dunming Zhu,</i></div>
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<o:p>Journal of Molecular Catalysis B: Enzymatic, Volume 129, July 2016, Pages 37-42</o:p></div>
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<o:p><a href="http://www.sciencedirect.com/science/article/pii/S1381117716300509">doi:10.1016/j.molcatb.2016.03.011</a></o:p></div>
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3-Hydroxypropionic acid (3-HP) is an important platform chemical for organic synthesis and high performance polymers. This paper describes an effective enzymatic method for the synthesis of 3-HP was achieved by using free or immobilized recombinant <i>Escherichia coli</i> BL21(DE3) cells harboring a nitrilase gene from environmental sample (NIT190). Under the optimal conditions (100 mmol/L Tris-HCl buffer, pH 8.0, 30 °C), the maximum substrate concentration which led to 100% hydrolysis by using free cells within 24 h was 4.5 mol/L (319.5 g/L). Furthermore, immobilization of the whole cells enhanced their substrate tolerance (up to 7.0 mol/L), stability, and reusability. The immobilized cells could be reused for up to 30 batches, and 70% of enzyme activity was retained after 74 batches in distilled water. A productivity (36.9 g/(L h)) was obtained after isolation and purification of 3-HP from the first 30 batches.</div>
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<a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEjZQdx8vEAtHdvh1OYhNc7Opz4o02639VxIhlrtAs-9ZlOUaEbnZ-ZTM10SP0licBqomWwMmZAtF2LIYKp8ZfNfcIIWkob-jrPep6opowiCQ2NDmKIN134dSY2Jbjp9C46EJZwp6WkEdCI/s1600/3HP+nitrilase+free+cell+and+immobilized.jpg" imageanchor="1" style="margin-left: 1em; margin-right: 1em;"><img border="0" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEjZQdx8vEAtHdvh1OYhNc7Opz4o02639VxIhlrtAs-9ZlOUaEbnZ-ZTM10SP0licBqomWwMmZAtF2LIYKp8ZfNfcIIWkob-jrPep6opowiCQ2NDmKIN134dSY2Jbjp9C46EJZwp6WkEdCI/s1600/3HP+nitrilase+free+cell+and+immobilized.jpg" /></a></div>
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Figure shows free cell substrate tolerance (a) compared to three immobilized cell methods (b-d).<br /><div class="MsoNormal">
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Unknownnoreply@blogger.com0tag:blogger.com,1999:blog-1841494650613708862.post-47389865311956299312016-08-05T05:27:00.000-07:002016-08-05T05:27:25.676-07:00Simultaneous KRED and NHase/amidase activity<div class="WordSection1">
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<b>Developing a Biocascade Process: Concurrent Ketone Reduction-Nitrile Hydrolysis of 2-Oxocycloalkanecarbonitriles</b><o:p></o:p></div>
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<i>Elisa Liardo, Nicolás Ríos-Lombardía, Francisco Morís, Javier González-Sabín, and Francisca Rebolledo</i><o:p></o:p></div>
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<i>Org. Lett., <b>2016</b>, 18 (14), pp 3366–3369</i></div>
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<i><a href="http://pubs.acs.org/doi/abs/10.1021/acs.orglett.6b01510">DOI: 10.1021/acs.orglett.6b01510</a></i></div>
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<o:p> </o:p><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEhAOjMTyRtrLVENbgrCGIX5O214jrPjyVM9p3a-lq2XuQ-y1oJAm33iEFmzuSHVzyLAUZ5zJeu2kR_dBzOTqf2JncjxzEPMtDyttTjZ92fgHtNL8z5Qfa8-CmJhj08u7fxevMpP7MYsEMo/s1600/image001-799900.gif"><img alt="" border="0" id="BLOGGER_PHOTO_ID_6314627134550957010" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEhAOjMTyRtrLVENbgrCGIX5O214jrPjyVM9p3a-lq2XuQ-y1oJAm33iEFmzuSHVzyLAUZ5zJeu2kR_dBzOTqf2JncjxzEPMtDyttTjZ92fgHtNL8z5Qfa8-CmJhj08u7fxevMpP7MYsEMo/s320/image001-799900.gif" /></a></div>
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<o:p> A stereoselective bioreduction of 2-oxocycloalkanecarbonitriles was concurrently coupled to a whole cell-catalyzed nitrile hydrolysis in one-pot. The first step, mediated by ketoreductases, involved a dynamic reductive kinetic resolution, which led to 2-hydroxycycloalkanenitriles in very high enantio- and diastereomeric ratios. Then, the simultaneous exposure to nitrile hydratase and amidase from whole cells of <i>Rhodococcus rhodochrous</i> provided the corresponding 2-hydroxycycloalkanecarboxylic acids with excellent overall yield and optical purity for the all-enzymatic cascade.</o:p></div>
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Unknownnoreply@blogger.com0tag:blogger.com,1999:blog-1841494650613708862.post-52742117057309380312016-08-03T05:54:00.000-07:002016-08-03T05:54:23.343-07:00ChromSoc nitrilase flow chemistry project 4Once you have the track filled with immobilized enzyme and the two halves stuck together, you just need to condition it and check there are no leaks.<br />
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<a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEgiwqwkjrbuwYIK5fDUnifDx13OYLXnwGhoa-jgGULaThyphenhyphenIIaLnuXIhTA22lGWYFdmunsRedz12qOeEAshiJyc7-6ekmp5pua_5j3n9jaesnPPqBov4SGJdaM48iDhuwZxbq6YAw11gAVM/s1600/13621358_625613934286399_1673697843_o.jpg" imageanchor="1" style="margin-left: 1em; margin-right: 1em;"><img border="0" height="181" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEgiwqwkjrbuwYIK5fDUnifDx13OYLXnwGhoa-jgGULaThyphenhyphenIIaLnuXIhTA22lGWYFdmunsRedz12qOeEAshiJyc7-6ekmp5pua_5j3n9jaesnPPqBov4SGJdaM48iDhuwZxbq6YAw11gAVM/s320/13621358_625613934286399_1673697843_o.jpg" width="320" /></a></div>
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Then it is just a case of getting the reaction going using a water bath to get the appropriate temperature. We tend to set it up so that we have a separate starting and receiving flask so that we can track aliquots through the enzyme bed, but you can just the two pipe operating out of /into the same flask obviously.</div>
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<a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEgFMjKT1WY0f2ZeLhFdwO9EqoMBt3x28wsphi18FDMJUOZzq7LlLbyID8qv2uCHeozUWt57SvHiSHfN-x9Oe156TMRzlAtYnzG37BhDELC5-utjfpBMMW9dC496zjzQcEPoVzJJ0lDAjWc/s1600/13699377_631511273696665_1919783869_o.jpg" imageanchor="1" style="margin-left: 1em; margin-right: 1em;"><img border="0" height="181" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEgFMjKT1WY0f2ZeLhFdwO9EqoMBt3x28wsphi18FDMJUOZzq7LlLbyID8qv2uCHeozUWt57SvHiSHfN-x9Oe156TMRzlAtYnzG37BhDELC5-utjfpBMMW9dC496zjzQcEPoVzJJ0lDAjWc/s320/13699377_631511273696665_1919783869_o.jpg" width="320" /></a></div>
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Unknownnoreply@blogger.com0tag:blogger.com,1999:blog-1841494650613708862.post-17468022418263127902016-08-03T05:46:00.001-07:002016-08-03T05:54:54.894-07:00ChromSoc nitrilase flow chemistry project 3Rob has shown made a supply of the plates that go together to make a flow cell for flow biocatalysis. The 3D printed master copy (the one with the wall around the shape) has provided another silicone mould which has then be used to make polyurethane casts.<br />
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<a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEionexo_8DrCFfQlJER9JcdNU3cGQRexASSwctzTngqprEEYv0WGHQHhs8Ei2f4s55jh8PSx6Fz-jgLkcn_9v-aT7TqHke5It_nOM3b6lwuv22iBmDgYysm2r3w9ELq2B7peN8lyh4D3Gc/s1600/13632885_625613970953062_1816306551_o.jpg" imageanchor="1" style="margin-left: 1em; margin-right: 1em;"><img border="0" height="320" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEionexo_8DrCFfQlJER9JcdNU3cGQRexASSwctzTngqprEEYv0WGHQHhs8Ei2f4s55jh8PSx6Fz-jgLkcn_9v-aT7TqHke5It_nOM3b6lwuv22iBmDgYysm2r3w9ELq2B7peN8lyh4D3Gc/s320/13632885_625613970953062_1816306551_o.jpg" width="181" /></a></div>
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<a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEiBE7DEF9IwirWzEfOWhq_aXcWgAo3ch2OeLXnZCPlbhOG7BGSI97G5CnmUP3Uvr3lHDDD0rWyCRhbn4Bp5A0j89miVxLKL0XvSpGyAt2MIw70fdQ68dpr6koyAl8lhAslRF5HWyYjD7wo/s1600/13632839_625613957619730_1849849328_o.jpg" imageanchor="1" style="margin-left: 1em; margin-right: 1em;"><img border="0" height="181" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEiBE7DEF9IwirWzEfOWhq_aXcWgAo3ch2OeLXnZCPlbhOG7BGSI97G5CnmUP3Uvr3lHDDD0rWyCRhbn4Bp5A0j89miVxLKL0XvSpGyAt2MIw70fdQ68dpr6koyAl8lhAslRF5HWyYjD7wo/s320/13632839_625613957619730_1849849328_o.jpg" width="320" /></a></div>
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They can then be stuck together with the track filled with immobilized enzymes. Alongside these reactions we are also running comparable batch reactions in glassware to see how they compare.</div>
Unknownnoreply@blogger.com0tag:blogger.com,1999:blog-1841494650613708862.post-38549078706495944062016-06-29T09:19:00.000-07:002016-06-29T09:19:05.049-07:00ChromSoc nitrilase flow chemistry project 2We have a range of nitrilases which we use as starting points for all our projects. Most of them are listed in this <a href="http://pubs.rsc.org/en/Content/ArticleHtml/2015/CC/c4cc06021k">ChemComm</a>. We've used them both as cell free extract and with many different types of immobilization. A good place to start we have found is in simple alginate beads which are easy to make, and give a consistent performance under our standard reaction conditions. Their synthesis using a powered syringe dropping into a stirred beaker has a somewhat hypnotic quality.<br />
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<br /><br /><iframe allowfullscreen='allowfullscreen' webkitallowfullscreen='webkitallowfullscreen' mozallowfullscreen='mozallowfullscreen' width='320' height='266' src='https://www.blogger.com/video.g?token=AD6v5dxt11Tan_x0EQpoIbZBYiFpOczFEkI4WOweEWi5gCD4_yO0YV17nT6gqihVXWuDJ5GtnL9nScZY8AObKLO2WA' class='b-hbp-video b-uploaded' frameborder='0'></iframe></div>
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<br />Unknownnoreply@blogger.com0tag:blogger.com,1999:blog-1841494650613708862.post-79173294789024882622016-06-29T08:48:00.000-07:002016-06-29T08:48:10.944-07:00ChromSoc nitrilase flow chemistry project 1I have an undergraduate student, Rob, working with us this summer on nitrilase reactions. He is kindly sponsored by the <a href="http://www.chromsoc.com/">Chromatographic Society</a> to work on a project using HPLC and GC to compare batch processes run on immobilized enzyme in a flask with those run on the same enzyme preparation using our in-house mesoscale flow reactor system. The flow reactor system is something we have been working on for a while and its genesis was part of a project based around using 3D printing to make bespoke laboratory equipment which is described in the Tumblr blog <a href="http://nzomics.tumblr.com/">here</a>, with a video of the system in operation doing a nitro reduction <a href="http://nzomics.tumblr.com/post/100689426055/flow-biocatalysis-using-a-3d-printed-device-nitro">here</a>. My intention is to relate here a real time log of progress with this project.<br />
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<a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEhkIi-1iZ3Z5cuuwoCnEHljAnbO3kwFwdPT3KdYC_9e66uYtNsXHNLmPevLPDVRbWlSCCkTvqsaZmyHwglIFLovyjado3vUVY-mxieExQPf_aW5DHoHmKSApNj8-mdLI6BNnb7T4SPS57c/s1600/flow+track+overlay.png" imageanchor="1" style="margin-left: 1em; margin-right: 1em;"><img border="0" height="320" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEhkIi-1iZ3Z5cuuwoCnEHljAnbO3kwFwdPT3KdYC_9e66uYtNsXHNLmPevLPDVRbWlSCCkTvqsaZmyHwglIFLovyjado3vUVY-mxieExQPf_aW5DHoHmKSApNj8-mdLI6BNnb7T4SPS57c/s320/flow+track+overlay.png" width="237" /></a></div>
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Two copies of the track fit together, and the solution</div>
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is flowed through. The track contains enzyme immobilized</div>
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on beads.</div>
<br />Unknownnoreply@blogger.com0tag:blogger.com,1999:blog-1841494650613708862.post-27128578812774261592016-04-22T05:35:00.001-07:002016-04-22T05:35:25.977-07:00Active Site investigations on the Fe-centred NHase from Comamonas testosteroni Ni1<div class="WordSection1">
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<b>Analyzing the catalytic role of active site residues in the Fe-type nitrile hydratase from <i>Comamonas testosteroni</i> Ni1</b><o:p></o:p></div>
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<b><i>Salette Martinez, Rui Wu, Karoline Krzywda, Veronika Opalka, Hei Chan, Dali Liu , Richard C. Holz</i></b><o:p></o:p></div>
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<a href="http://link.springer.com/article/10.1007/s00775-015-1273-3">JBIC Journal of Biological Inorganic Chemistry (2015), 20/5, 885-894</a><o:p></o:p></div>
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<span style="background-color: black;"><span style="color: white;"><span style="background-attachment: initial; background-clip: initial; background-image: initial; background-origin: initial; background-position: initial; background-repeat: initial; background-size: initial; font-family: "arial" , "sans-serif"; font-size: 10pt;">A strictly conserved active site arginine residue (αR157) and two histidine residues (αH80 and αH81) located near the active site of the Fe-type nitrile hydratase from<span class="apple-converted-space"> </span><em><span style="font-family: "arial" , "sans-serif";">Comamonas testosteroni</span></em><span class="apple-converted-space"> </span>Ni1 (<em><span style="font-family: "arial" , "sans-serif";">Ct</span></em>NHase), were mutated. These mutant enzymes were examined for their ability to bind iron and hydrate acrylonitrile. For the αR157A mutant, the residual activity (<em><span style="font-family: "arial" , "sans-serif";">k</span></em><span class="apple-converted-space"> </span></span><sub><span style="background-attachment: initial; background-clip: initial; background-image: initial; background-origin: initial; background-position: initial; background-repeat: initial; background-size: initial; font-family: "arial" , "sans-serif";">cat</span></sub><span style="background-attachment: initial; background-clip: initial; background-image: initial; background-origin: initial; background-position: initial; background-repeat: initial; background-size: initial; font-family: "arial" , "sans-serif"; font-size: 10pt;"> = 10 ± 2 s</span><sup><span style="background-attachment: initial; background-clip: initial; background-image: initial; background-origin: initial; background-position: initial; background-repeat: initial; background-size: initial; font-family: "arial" , "sans-serif";">−1</span></sup><span style="background-attachment: initial; background-clip: initial; background-image: initial; background-origin: initial; background-position: initial; background-repeat: initial; background-size: initial; font-family: "arial" , "sans-serif"; font-size: 10pt;">) accounts for less than 1 % of the wild-type activity (<em><span style="font-family: "arial" , "sans-serif";">k</span></em><span class="apple-converted-space"> </span></span><sub><span style="background-attachment: initial; background-clip: initial; background-image: initial; background-origin: initial; background-position: initial; background-repeat: initial; background-size: initial; font-family: "arial" , "sans-serif";">cat</span></sub><span style="background-attachment: initial; background-clip: initial; background-image: initial; background-origin: initial; background-position: initial; background-repeat: initial; background-size: initial; font-family: "arial" , "sans-serif"; font-size: 10pt;"> = 1100 ± 30 s</span><sup><span style="background-attachment: initial; background-clip: initial; background-image: initial; background-origin: initial; background-position: initial; background-repeat: initial; background-size: initial; font-family: "arial" , "sans-serif";">−1</span></sup><span style="background-attachment: initial; background-clip: initial; background-image: initial; background-origin: initial; background-position: initial; background-repeat: initial; background-size: initial; font-family: "arial" , "sans-serif"; font-size: 10pt;">) while the<span class="apple-converted-space"> </span><em><span style="font-family: "arial" , "sans-serif";">K</span></em><span class="apple-converted-space"> </span></span><em><sub><span style="background-attachment: initial; background-clip: initial; background-image: initial; background-origin: initial; background-position: initial; background-repeat: initial; background-size: initial; font-family: "arial" , "sans-serif";">m</span></sub></em><span class="apple-converted-space"><sub><span style="background-attachment: initial; background-clip: initial; background-image: initial; background-origin: initial; background-position: initial; background-repeat: initial; background-size: initial; font-family: "arial" , "sans-serif";"> </span></sub></span><span style="background-attachment: initial; background-clip: initial; background-image: initial; background-origin: initial; background-position: initial; background-repeat: initial; background-size: initial; font-family: "arial" , "sans-serif"; font-size: 10pt;">value is nearly unchanged at 205 ± 10 mM. On the other hand, mutation of the active site pocket αH80 and αH81 residues to alanine resulted in enzymes with<span class="apple-converted-space"> </span><em><span style="font-family: "arial" , "sans-serif";">k</span></em><span class="apple-converted-space"> </span></span><sub><span style="background-attachment: initial; background-clip: initial; background-image: initial; background-origin: initial; background-position: initial; background-repeat: initial; background-size: initial; font-family: "arial" , "sans-serif";">cat</span></sub><span class="apple-converted-space"><span style="background-attachment: initial; background-clip: initial; background-image: initial; background-origin: initial; background-position: initial; background-repeat: initial; background-size: initial; font-family: "arial" , "sans-serif"; font-size: 10pt;"> </span></span><span style="background-attachment: initial; background-clip: initial; background-image: initial; background-origin: initial; background-position: initial; background-repeat: initial; background-size: initial; font-family: "arial" , "sans-serif"; font-size: 10pt;">values of 220 ± 40 and 77 ± 13 s</span><sup><span style="background-attachment: initial; background-clip: initial; background-image: initial; background-origin: initial; background-position: initial; background-repeat: initial; background-size: initial; font-family: "arial" , "sans-serif";">−1</span></sup><span style="background-attachment: initial; background-clip: initial; background-image: initial; background-origin: initial; background-position: initial; background-repeat: initial; background-size: initial; font-family: "arial" , "sans-serif"; font-size: 10pt;">, respectively, and<span class="apple-converted-space"> </span><em><span style="font-family: "arial" , "sans-serif";">K</span></em><span class="apple-converted-space"> </span></span><em><sub><span style="background-attachment: initial; background-clip: initial; background-image: initial; background-origin: initial; background-position: initial; background-repeat: initial; background-size: initial; font-family: "arial" , "sans-serif";">m</span></sub></em><span class="apple-converted-space"><sub><span style="background-attachment: initial; background-clip: initial; background-image: initial; background-origin: initial; background-position: initial; background-repeat: initial; background-size: initial; font-family: "arial" , "sans-serif";"> </span></sub></span><span style="background-attachment: initial; background-clip: initial; background-image: initial; background-origin: initial; background-position: initial; background-repeat: initial; background-size: initial; font-family: "arial" , "sans-serif"; font-size: 10pt;">values of 187 ± 11 and 179 ± 18 mM. The double mutant (αH80A/αH81A) was also prepared and provided an enzyme with a<span class="apple-converted-space"> </span><em><span style="font-family: "arial" , "sans-serif";">k</span></em><span class="apple-converted-space"> </span></span><sub><span style="background-attachment: initial; background-clip: initial; background-image: initial; background-origin: initial; background-position: initial; background-repeat: initial; background-size: initial; font-family: "arial" , "sans-serif";">cat</span></sub><span class="apple-converted-space"><span style="background-attachment: initial; background-clip: initial; background-image: initial; background-origin: initial; background-position: initial; background-repeat: initial; background-size: initial; font-family: "arial" , "sans-serif"; font-size: 10pt;"> </span></span><span style="background-attachment: initial; background-clip: initial; background-image: initial; background-origin: initial; background-position: initial; background-repeat: initial; background-size: initial; font-family: "arial" , "sans-serif"; font-size: 10pt;">value of 132 ± 3 s</span><sup><span style="background-attachment: initial; background-clip: initial; background-image: initial; background-origin: initial; background-position: initial; background-repeat: initial; background-size: initial; font-family: "arial" , "sans-serif";">−1</span></sup><span class="apple-converted-space"><span style="background-attachment: initial; background-clip: initial; background-image: initial; background-origin: initial; background-position: initial; background-repeat: initial; background-size: initial; font-family: "arial" , "sans-serif"; font-size: 10pt;"> </span></span><span style="background-attachment: initial; background-clip: initial; background-image: initial; background-origin: initial; background-position: initial; background-repeat: initial; background-size: initial; font-family: "arial" , "sans-serif"; font-size: 10pt;">and a<span class="apple-converted-space"> </span><em><span style="font-family: "arial" , "sans-serif";">K</span></em><span class="apple-converted-space"> </span></span><em><sub><span style="background-attachment: initial; background-clip: initial; background-image: initial; background-origin: initial; background-position: initial; background-repeat: initial; background-size: initial; font-family: "arial" , "sans-serif";">m</span></sub></em><span class="apple-converted-space"><sub><span style="background-attachment: initial; background-clip: initial; background-image: initial; background-origin: initial; background-position: initial; background-repeat: initial; background-size: initial; font-family: "arial" , "sans-serif";"> </span></sub></span><span style="background-attachment: initial; background-clip: initial; background-image: initial; background-origin: initial; background-position: initial; background-repeat: initial; background-size: initial; font-family: "arial" , "sans-serif"; font-size: 10pt;">value of 213 ± 61 mM. These data indicate that all three residues are catalytically important, but not essential. X-ray crystal structures of the αH80A/αH81A, αH80W/αH81W, and αR157A mutant<span class="apple-converted-space"> </span><em><span style="font-family: "arial" , "sans-serif";">Ct</span></em>NHase enzymes were solved to 2.0, 2.8, and 2.5 Å resolutions, respectively. In each mutant enzyme, hydrogen-bonding interactions crucial for the catalytic function of the αCys</span><sup><span style="background-attachment: initial; background-clip: initial; background-image: initial; background-origin: initial; background-position: initial; background-repeat: initial; background-size: initial; font-family: "arial" , "sans-serif";">104</span></sup><span style="background-attachment: initial; background-clip: initial; background-image: initial; background-origin: initial; background-position: initial; background-repeat: initial; background-size: initial; font-family: "arial" , "sans-serif"; font-size: 10pt;">-SOH ligand are disrupted. Disruption of these hydrogen bonding interactions likely alters the nucleophilicity of the sulfenic acid oxygen and the Lewis acidity of the active site Fe(III) ion.<o:p></o:p></span></span></span></div>
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<span style="mso-fareast-language: EN-GB;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEiEhFbUp3CzPSxWj-8I52gT77lsB07bcdiAfvCgM0ANkq4Zo7mU6_kpobj_5T03ks-ceiGX5gjcXoSy6Hci_8FgYn3ByPqOiDgTtI0H7RSYjCdlTqpGHQgjYSyOwprUYW7bNQTIRuIGsvQ/s1600/image001-733262.jpg"><img alt="" border="0" id="BLOGGER_PHOTO_ID_6276356971343219922" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEiEhFbUp3CzPSxWj-8I52gT77lsB07bcdiAfvCgM0ANkq4Zo7mU6_kpobj_5T03ks-ceiGX5gjcXoSy6Hci_8FgYn3ByPqOiDgTtI0H7RSYjCdlTqpGHQgjYSyOwprUYW7bNQTIRuIGsvQ/s320/image001-733262.jpg" /></a></span><o:p></o:p></div>
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<span style="background-attachment: initial; background-clip: initial; background-color: black; background-image: initial; background-origin: initial; background-position: initial; background-repeat: initial; background-size: initial; color: white; font-family: "arial" , "sans-serif"; font-size: 10pt;">3D content at </span><a href="http://proteopedia.org/w/Journal:JBIC:32">http://proteopedia.org/w/Journal:JBIC:32</a> <o:p></o:p></div>
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Unknownnoreply@blogger.com0tag:blogger.com,1999:blog-1841494650613708862.post-78732672715901452392016-04-22T05:05:00.002-07:002016-04-22T05:05:43.006-07:00BASF opens new bio-acrylamide plant in Bradford, UK<div class="MsoNormal">
From <o:p></o:p></div>
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<a href="http://www.worldofchemicals.com/media/basf-opens-new-bio-acrylamide-plant-in-bradford-uk/9710.html">http://www.worldofchemicals.com/media/basf-opens-new-bio-acrylamide-plant-in-bradford-uk/9710.html</a><o:p></o:p></div>
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“BRADFORD, UK: BASF said it has opened a new world-scale
bio-acrylamide (BioACM) production facility at its site in Bradford, a major
investment that will help ensure long-term future of one of the UK’s largest
chemical manufacturing facilities, which employs around 600 people.”<o:p></o:p></div>
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“The development of a biocatalytic manufacturing process for
acrylamide started in Bradford with collaboration with Huddersfield University.
Subsequent work by scientists from Britain, Germany, South Africa and US, made
significant improvements to the performance of the biocatalyzed conversion
technology.”<o:p></o:p></div>
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<img src="https://www.basf.com/images/uk/Bradford_3564lo.jpg-renditions/cq5dam.web.4-3.6.jpg" /></div>
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Image from <a href="https://www.basf.com/gb/en/company/about-us/Locations/Bradford.html">BASF.com</a></div>
Unknownnoreply@blogger.com0tag:blogger.com,1999:blog-1841494650613708862.post-61480725760407448812016-04-19T06:09:00.001-07:002016-04-19T06:09:57.654-07:00Creating bioactive molecules from online data<div class="MsoNormal">
From a genome sequence to that protein in your hand is a
well –established process<o:p></o:p></div>
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Until I started collaborating closely with biologists ten
years ago, my impression of how easy it was to get a sample of a complex
biomolecule like a protein was based on popular science stories describing
days/months of tedious purification of buckets of biomass (sea sponges, exotic fungi,
cow bile, etc) to end up with a single vial containing so little it was
invisible to the naked eye.<o:p></o:p></div>
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Follow <a href="https://medium.com/@jjbperry/creating-bioactive-molecules-from-online-data-ad2537a76ef1#.u29hxlpu7" target="_blank">the link</a> to see the schematic explained.</div>
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<a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEgDyr4XWB7BCdZ3kn2scCLkoEo1NF-xX_z0x1tOz7XBOoTfvtyeL9MD4mNAmcSSgU9SGFla706tgJjuNh5EpfmNWPlGv2m42JLwiB7kA27lpnh4bKL9QZZMeHfZ-nUWACmkjLtL4yBBQoM/s1600/expression+schematic.jpg" imageanchor="1" style="margin-left: 1em; margin-right: 1em;"><img border="0" height="177" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEgDyr4XWB7BCdZ3kn2scCLkoEo1NF-xX_z0x1tOz7XBOoTfvtyeL9MD4mNAmcSSgU9SGFla706tgJjuNh5EpfmNWPlGv2m42JLwiB7kA27lpnh4bKL9QZZMeHfZ-nUWACmkjLtL4yBBQoM/s320/expression+schematic.jpg" width="320" /></a></div>
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Unknownnoreply@blogger.com1tag:blogger.com,1999:blog-1841494650613708862.post-79811266710474389942016-04-18T04:43:00.000-07:002016-04-18T04:43:31.186-07:00A poster on Production of 2,6-difluorobenzamide using the NHase from Aurantimonas manganoxydans<div class="WordSection1">
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<b>Production of <a href="http://www.chemspider.com/ImagesHandler.ashx?id=78873&w=250&h=250"> 2, 6- difluorobenzamide</a> via whole-cell biocatalysis by nitrile hydratase from <i>Aurantimonas manganoxydans</i><o:p></o:p></b></div>
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Lirong Yang<o:p></o:p></div>
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<a href="https://sim.confex.com/sim/2015/webprogram/Paper30317.html">AGM Society for Industrial Microbiology & Biotechnology</a><o:p></o:p></div>
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Nitrile hydratases (NHases) are enzymes which catalyze the hydration of nitriles, converting them into their corresponding amides. Amides are an important intermediates for pharmaceutical and pesticide industry. For example, 2, 6 – difluorobenzamide is used for the synthesis of fluorinated benzoyl urea pesticide.<o:p></o:p></div>
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Four NHase genes from <i>Aurantimonas manganoxydans</i> ATCC BAA-1229, <i>Klebsiella oxytoca</i> KCTC 1686, <i>Pseudomonas putida</i> NRRL-18668, <i>Comamonas testosteroni</i> 5-MGAM-4D were cloned and functionally expressed in <i>Escherichia coli</i> BL21 (DE3). All of the recombinant NHases can catalyze the hydration of 2, 6-difluorobenzonitrile to produce 2, 6-difluorobenzamide. Among them, the NHase from <i>Aurantimonas manganoxydans</i> ATCC BAA-1229 showed the highest activity. <o:p> </o:p></div>
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<span style="mso-fareast-language: EN-GB;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEhuKO9vwM5xeAeUbS9fAuibSOE5wi9M-306w1YIhfrE8x4SLu9J4yd7-v7_Cmqggve1A-rTz_pRlK6ey_-B6PAMH4CLbB8FRKspynZbPMfQod1uVJaf_feiNlP9V4S5TinTe4XXue9VAqw/s1600/image001-705869.png"><img alt="" border="0" id="BLOGGER_PHOTO_ID_6274860058685360002" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEhuKO9vwM5xeAeUbS9fAuibSOE5wi9M-306w1YIhfrE8x4SLu9J4yd7-v7_Cmqggve1A-rTz_pRlK6ey_-B6PAMH4CLbB8FRKspynZbPMfQod1uVJaf_feiNlP9V4S5TinTe4XXue9VAqw/s320/image001-705869.png" /></a></span><o:p></o:p></div>
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Unknownnoreply@blogger.com0tag:blogger.com,1999:blog-1841494650613708862.post-72709953043613704922016-04-18T04:32:00.000-07:002016-04-18T04:32:08.630-07:00Assisting soluble expression of recombinant and active Co-centred NHase<div class="WordSection1">
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<span style="color: #2e2e2e; font-family: arial, sans-serif;"><span style="font-size: 22px;">Chaperones-assisted soluble expression and maturation of recombinant Co-type nitrile hydratase in Escherichia coli to avoid the need for a low induction temperature</span></span></div>
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Xiaolin Pei, Qiuyan Wang, Lijun Meng, Jing Li, Zhengfen Yang, Xiaopu Yin, Lirong Yang, Shaoyun Chen and Jianping Wu<o:p></o:p></div>
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Journal of Biotechnology, Volume 203, 10 June 2015, Pages 9–16<o:p></o:p></div>
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<a href="http://dx.doi.org/10.1016/j.jbiotec.2015.03.004" target="doilink"><span style="background: white; border: none 1.0pt; color: #316c9d; font-family: "arial" , "sans-serif"; font-size: 10.0pt; padding: 0cm;">doi:10.1016/j.jbiotec.2015.03.004</span></a><o:p></o:p></div>
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Nitrile hydratase (NHase) is an important industrial enzyme that biosynthesizes high-value amides. However, most of NHases expressed in Escherichia coli easily aggregate to inactive inclusion bodies unless the induction temperature is reduced to approximately 20 °C. The NHase from <i>Aurantimonas manganoxydans</i> has been functionally expressed in E. coli, and exhibits considerable potential for the production of nicotinamide in industrial application. In this study, the effects of chaperones including GroEL/ES, Dnak/J-GrpE and trigger factor on the expression of the recombinant Co-type NHase were investigated. The results indicate that three chaperones can significantly promote the active expression of the recombinant NHase at 30 °C. The total NHase activities reached to 263 and 155 U/ml in shake flasks when the NHase was co-expressed with GroEL/ES and DnaK/J-GrpE, which were 52- and 31-fold higher than the observed activities without chaperones, respectively. This increase is possibly due to the soluble expression of the recombinant NHase assisted by molecular chaperones. Furthermore, GroEL/ES and DnaK/J-GrpE were determined to promote the maturation of the Co-type NHase in E. coli under the absence of the parental activator gene. These knowledge regarding the chaperones effect on the NHase expression are useful for understanding the biosynthesis of Co-type NHase.<o:p></o:p></div>
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Unknownnoreply@blogger.com0tag:blogger.com,1999:blog-1841494650613708862.post-17096229107409905002016-04-18T03:56:00.000-07:002016-04-18T03:56:17.745-07:00NHase hydration of alicyclic 𝜶,𝝎-dinitrile 1-cyanocyclohexaneacetonitrile to 1-cyanocyclohexaneacetamide<div class="WordSection1">
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<b>Highly regioselective and efficient production of 1-cyanocyclohexaneacetamide by <i>Rhodococcus aetherivorans </i>ZJB1208 nitrile hydratase<o:p></o:p></b></div>
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<span style="font-family: "iskoola pota" , "sans-serif"; mso-fareast-language: EN-GB;">Ren-Chao Zheng, Xin-Jian Yina and Yu-Guo Zheng<o:p></o:p></span></div>
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<span style="font-family: "iskoola pota" , "sans-serif"; mso-fareast-language: EN-GB;"><a href="http://onlinelibrary.wiley.com/doi/10.1002/jctb.4724/epdf" target="_blank">J Chem Technol Biotechnol 2016; 91: 1314–1319</a><o:p></o:p></span></div>
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<span style="font-family: "iskoola pota" , "sans-serif"; mso-fareast-language: EN-GB;">DOI 10.1002/jctb.4724<o:p></o:p></span></div>
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A newly isolated NHase producing strain<i>, Rhodococcus aetherivorans</i> ZJB1208, was successfully used for hydration of1-CCHAN. Some key parameters of the biocatalytic process, including reaction temperature, pH, catalyst loading and substrate loading, were optimized. The fed-batch biotransformation was performed in non-buffered water system with the continuous precipitation of 1-cyanocyclohexaneacetamide. The substrate loading was increased up to 864 g L<sup>−1</sup> (6.0 mol L<sup>−1</sup>), giving a product concentration of 966.7 g L<sup>−1</sup> and biocatalyst yield (g product/g cat) of 204.2.<o:p></o:p></div>
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Unknownnoreply@blogger.com0tag:blogger.com,1999:blog-1841494650613708862.post-24700528625840882632016-04-18T02:17:00.002-07:002016-04-18T02:51:45.863-07:00Clues towards the role of amide carbonyl groups in the active site of a cobalt centred NHase<div class="WordSection1">
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<b><span style="font-family: "advtt2cba4af3.b" , "serif"; font-size: 12.0pt;">Role of the Amide Carbonyl Groups in the Nitrile Hydratase Active Site for Nitrile Coordination Using Co(III) Complex with N2S3-type Ligand<o:p></o:p></span></b></div>
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<span style="font-family: "advtt5843c571" , "serif"; font-size: 12.0pt;">Takuma Yano, Tomohiro Ikeda, Tomonori Shibayama, Tomohiko Inomata, Yasuhiro Funahashi,2 Tomohiro Ozawa,</span><span style="font-family: "advoteebdf440" , "sans-serif"; font-size: 12.0pt;">*</span><span style="font-family: "advtt5843c571" , "serif"; font-size: 12.0pt;"> and Hideki Masuda</span><span style="font-family: "advoteebdf440" , "sans-serif"; font-size: 12.0pt;">*<o:p></o:p></span></div>
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<span style="font-family: "arial black" , "sans-serif"; font-size: 10.0pt;"><a href="https://www.jstage.jst.go.jp/article/cl/44/6/44_150084/_article">Chemistry Letters Vol. 44 (2015) No. 6 P 761-763</a><o:p></o:p></span></div>
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<span style="font-family: "advtt5843c571" , "serif"; font-size: 12.0pt;">The role of the amide carbonyl oxygens in the nitrile hydratase (NHase) active site for the nitrile coordination to the metal center was studied using a distorted square-pyramidal N2S3-type Co(III) complex at room temperature in the presence of a folded-sheet mesoporous material (FSM) or sodium cation.<o:p></o:p></span></div>
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Unknownnoreply@blogger.com0tag:blogger.com,1999:blog-1841494650613708862.post-23838066562306418372016-04-04T04:57:00.000-07:002016-04-04T04:57:53.264-07:00A proposed catalytic mechanism for NHase via a cyclic intermediate assessed by QM/MM <div class="WordSection1">
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<b><span style="background-color: black; color: white; font-size: 13.5pt;">Catalytic Mechanism of Nitrile Hydratase Subsequent to Cyclic Intermediate Formation: A QM/MM Study<o:p></o:p></span></b></div>
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<span style="background-color: black; color: white; font-size: 13.5pt;">Megumi Kayanuma, Mitsuo Shoji, Masafumi Yohda, Masafumi Odaka, and Yasuteru Shigeta<o:p></o:p></span></div>
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<span style="background-color: black; color: white; font-size: 13.5pt;">J. Phys. Chem. B, Article ASAP<o:p></o:p></span></div>
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<b><span style="background-color: black; color: white; font-size: 13.5pt;">DOI</span></b><span style="font-size: 13.5pt;"><span style="background-color: black; color: white;">: 10.1021/acs.jpcb.5b11363</span><o:p></o:p></span></div>
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<span style="background-color: black;"><span style="color: white;"><span style="font-family: "times new roman" , "serif"; font-size: 10.5pt; line-height: 115%;">The catalytic mechanism of an Fe-containing nitrile hydratase (NHase) subsequent to the formation of a cyclic intermediate was investigated using a hybrid quantum mechanics/molecular mechanics (QM/MM) method. We identified the following mechanism: (i) proton transfer from βTyr72 to the substrate via αSer113, and cleavage of the S–O bond of αCys114–SO</span><sup><span style="font-family: "times new roman" , "serif"; font-size: 9.5pt; line-height: 115%;">–</span></sup><span class="apple-converted-space"><span style="font-family: "times new roman" , "serif"; font-size: 10.5pt; line-height: 115%;"> and formation of a disulfide bond between αCys109 and αCys114; (ii) direct attack of a water molecule on the sulfur atom of αCys114, which resulted in the generation of both an imidic acid and a renewed sulfenic cysteine; and (iii) isomerization of the imidic acid to the amide. In addition, to clarify the role of βArg56K, which is one of the essential amino residues in the enzyme, we analyzed a βR56K mutant in which βArg56 was replaced by Lys. The results suggest that βArg56 is necessary for the formation of disulfide intermediate by stabilizing the cleavage of the S–O bond via a hydrogen bond with the oxygen atom of αCys114–SO</span></span><sup><span style="font-family: "times new roman" , "serif"; font-size: 9.5pt; line-height: 115%;">–</span></sup><span style="font-family: "times new roman" , "serif"; font-size: 10.5pt; line-height: 115%;">.<o:p></o:p></span></span></span></div>
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<span style="font-family: "times new roman" , "serif"; mso-fareast-language: EN-GB;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEjFfG5xtJBLb8CvqvA8uoWTqi5rv8q8TgzOZssQz3_dRqs9AEtCUzaC7YqxnTIx2vX3n90gGowV58XcYvTfc1KF_GEjt7Wm2wjV0yo1hB-7hP8ZZxOQtsbEdF2GBQKwHooMj1Na2q2KuC8/s1600/image001-713012.png"><img alt="" border="0" id="BLOGGER_PHOTO_ID_6269667905074392050" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEjFfG5xtJBLb8CvqvA8uoWTqi5rv8q8TgzOZssQz3_dRqs9AEtCUzaC7YqxnTIx2vX3n90gGowV58XcYvTfc1KF_GEjt7Wm2wjV0yo1hB-7hP8ZZxOQtsbEdF2GBQKwHooMj1Na2q2KuC8/s320/image001-713012.png" /></a></span><span style="font-family: "times new roman" , "serif";"><o:p></o:p></span></div>
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Unknownnoreply@blogger.com0tag:blogger.com,1999:blog-1841494650613708862.post-78512534505777599452016-03-07T08:46:00.000-08:002016-03-07T08:46:56.775-08:00Formation of Nitrile Hydratase CLEAs in Mesoporous Silica<div class="WordSection1">
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<b>Formation of Nitrile Hydratase Cross-Linked Enzyme Aggregates in Mesoporous Onion-like Silica: Preparation and Catalytic Properties<o:p></o:p></b></div>
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Jing Gao, Qi Wang, Yanjun Jiang*, Junkai Gao, Zhihua Liu, Liya Zhou, and Yufei Zhang,<o:p></o:p></div>
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<cite><span style="background: white; color: black; font-family: "calibri" , "sans-serif";">Ind. Eng. Chem. Res.</span></cite><span style="background: white; color: black;">,<span class="apple-converted-space"> </span><span class="citationyear"><b>2015</b></span>,<span class="apple-converted-space"> </span><span class="citationvolume"><i>54</i></span><span class="apple-converted-space"> </span>(1), pp 83–90<o:p></o:p></span></div>
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Nitrile hydratase CLEAs) were formed in mesoporous onion-like silica (NHase-CLEAs@MOS) by using macromolecular dextran polyaldehyde as a cross-linker through the carrier-bound CLEAs method. Effect of pH, thermal and storage stability, and kinetic parameters of NHase-CLEAs@MOS were also studied. The maximum amount of NHase absorbed in MOS was 535 mg/g. Under optimized conditions, the maximum activity recovery of NHase-CLEAs@MOS was 48.2%. The stabilities of NHase-CLEAs@MOS were improved significantly compared to the NHase@MOS prepared by physical adsorption and free NHase. <o:p></o:p></div>
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<span style="mso-fareast-language: EN-GB;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEh5xSaPB4LtRKKr1QICbGvi0lo-crIphizOSy6vO8ikwbdyh6yK5ZWn25LQNjPXSeO3gM4LAdY2fQOHMmMYLK8h9xSQ0gYgqKPIbBipXsBe6ggahYSWdX4jBhi0_cgJ-e5-dt_CARgEa24/s1600/image001-769079.gif"><img alt="" border="0" id="BLOGGER_PHOTO_ID_6259316845015936898" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEh5xSaPB4LtRKKr1QICbGvi0lo-crIphizOSy6vO8ikwbdyh6yK5ZWn25LQNjPXSeO3gM4LAdY2fQOHMmMYLK8h9xSQ0gYgqKPIbBipXsBe6ggahYSWdX4jBhi0_cgJ-e5-dt_CARgEa24/s320/image001-769079.gif" /></a></span><o:p></o:p></div>
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Unknownnoreply@blogger.com0tag:blogger.com,1999:blog-1841494650613708862.post-26634267073721871072016-03-07T06:18:00.000-08:002016-03-07T06:18:30.807-08:00Enzymatic cascade synthesis of (S)-2-hydroxycarboxylic amides and acids using a hydroxynitrile lyase, nitrile-active enzymes and an amidase<div class="WordSection1">
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This <a href="http://www.sciencedirect.com/science/article/pii/S1381117714002343"> review</a> (<i>Journal of Molecular Catalysis B: Enzymatic</i>, 114, <b>2015</b>, 25–30) by van Rantwijk and Stolz covers the bienzymatic conversion of aldehydes into enantiomerically pure hydroxycarboxylic acids and amides via an enzymatic cascade of hydrocyanation and nitrile hydration/hydrolysis. It compares results obtained via cross-linked enzyme aggregates (CLEAs) as well as whole-cell <i>Escherichia coli</i> expressing two enzymes. It highlights these methods’ potential for yielding near-quantitative yield and ee at synthetically relevant concentrations.<o:p></o:p></div>
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<span style="mso-fareast-language: EN-GB;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEhyRuxtIv-Cx-caj25aRKtstwj1sM9iSDG7Fy40qu6YtdzmkYlW4tn7cRoyoJOKC2nZ2l18tCcJWyHPK6ncFP25cBguIv4phsAiwIE_M4NQEnIbDLM8kPAJ7bbXzU9DViFE6X-gjmZPCjM/s1600/image001-718471.jpg"><img alt="" border="0" id="BLOGGER_PHOTO_ID_6259314477546440626" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEhyRuxtIv-Cx-caj25aRKtstwj1sM9iSDG7Fy40qu6YtdzmkYlW4tn7cRoyoJOKC2nZ2l18tCcJWyHPK6ncFP25cBguIv4phsAiwIE_M4NQEnIbDLM8kPAJ7bbXzU9DViFE6X-gjmZPCjM/s320/image001-718471.jpg" /></a></span><o:p></o:p></div>
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Unknownnoreply@blogger.com0tag:blogger.com,1999:blog-1841494650613708862.post-8311282553786671162016-02-29T04:59:00.000-08:002016-02-29T04:59:24.135-08:00Sequence alignment of Nit6803 from Syechocystis sp. PCC6803 with other nitrilases<div class="WordSection1">
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<span style="mso-fareast-language: EN-GB;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEirRZnw4KElcz88gkR1p7CHcKG2-eBm6baaIaD1AzVEQQ7E5-AadFihig_QEEe7_WXrOirvQjHIJj0l_TFGufFw92k4MirHKrEM4hyphenhyphenUkj-T0VlTd0LRmiWU7kYBIG7K0xMhQQQmjQ_UqbE/s1600/image001-775074.png"><img alt="" border="0" id="BLOGGER_PHOTO_ID_6256695648785548658" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEirRZnw4KElcz88gkR1p7CHcKG2-eBm6baaIaD1AzVEQQ7E5-AadFihig_QEEe7_WXrOirvQjHIJj0l_TFGufFw92k4MirHKrEM4hyphenhyphenUkj-T0VlTd0LRmiWU7kYBIG7K0xMhQQQmjQ_UqbE/s320/image001-775074.png" /></a></span><o:p></o:p></div>
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Secondary structure elements are drawn on the basis of structures of Nit6803 and shown at the top of the aligned sequences. <span style="font-family: "symbol";">b</span>-Sheets are shown as arrows in yellow, whereas <span style="font-family: "symbol";">a</span>-helices are shown as bars in red. Residues involved in enzymatic catalysis are indicated are highlighted in red rectangle, whereas the proposed key residue involved in substrate preference is highlighted in blue rectangle. Nit6803, PaNit, PH0642, DNCAase and RrNit indicate <i>Syechocystis</i> sp. PCC6803 nitrilase (GI: 16331918), hyperthermophilic nitrilase (GI: 14521598), <i>Pyrococcus horikoshii</i> hypothetical protein (GI: 14590532), N-carbamoyl-D-amino-acid amidohydrolase (GI: 34921541) and <i>Rhodococcus rhodochrous</i> ATCC 33278 nitrilase (GI: 417384). <o:p></o:p></div>
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<o:p> from <a href="http://www.sciencedirect.com/science/article/pii/S1047847714002123" target="_blank">this paper</a> by Yuan, Wei and co-workers.</o:p></div>
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Unknownnoreply@blogger.com0tag:blogger.com,1999:blog-1841494650613708862.post-60992330430879487902016-02-27T08:11:00.000-08:002016-02-27T08:11:58.384-08:00NCBI Sequence numbers for nitrile hydratase and nitrilase to 27/2.16<div class="MsoNormal" style="margin: 0cm 0cm 10pt;">
<span style="font-family: "calibri";">Looking at the bare search term "nitrile hydratase" amongst protein sequences (and remember, most aren’t but it’s a rough measure), today gives me 10224 hits, of which 4117 were RefSeq data. </span></div>
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<span style="font-family: "calibri";">There are 80688 sequences labelled as "nitrilase" (not sure how robust that is currently), of which 20872 are pegged as RefSeq data.</span></div>
Unknownnoreply@blogger.com1tag:blogger.com,1999:blog-1841494650613708862.post-57505616614292645052016-02-27T08:02:00.001-08:002016-02-29T05:00:36.419-08:00A crystal structure of nitrilase Nit6803 from Syechocystis sp. PCC6803<a href="http://www.rcsb.org/pdb/explore/explore.do?structureId=3WUY" target="_blank"><img src="http://www.rcsb.org/pdb/images/3WUY_bio_r_500.jpg?bioNum=1" height="400" width="400" /></a><br />
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<span style="color: #444444; font-size: 1.1em; line-height: 17.9998px;">PDB:</span><span style="color: #444444; font-size: 1.1em; line-height: 17.9998px;"> </span><span class="highlight" style="color: #444444; font-size: 1.1em; line-height: 17.9998px;">3WUY</span><span style="color: #444444; font-size: 1.1em; line-height: 17.9998px;">_A</span></h1>
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<div class="seq gbff" id="viewercontent1" sequencesize="10592" style="line-height: 1.3em; margin: 0.5em 0px 0px; padding: 0px;" val="742261201" virtualsequence="true">
<pre style="font-family: monospace, serif; overflow: visible; white-space: pre-wrap; width: 50em; word-wrap: break-word; zoom: 1;">>gi|742261201|pdb|3WUY|A Chain A, Crystal Structure Of Nit6803
<span class="ff_line" id="gi_742261201_1">GSHMLGKIMLNYTKNIRAAAAQISPVLFSQQGTMEKVLDAIANAAKKGVELIVFPETFVPYYPYFSFVEP</span>
<span class="ff_line" id="gi_742261201_71">PVLMGKSHLKLYQEAVTVPGKVTQAIAQAAKTHGMVVVLGVNEREEGSLYNTQLIFDADGALVLKRRKIT</span>
<span class="ff_line" id="gi_742261201_141">PTYHERMVWGQGDGAGLRTVDTTVGRLGALACWEHYNPLARYALMAQHEQIHCGQFPGSMVGQIFADQME</span>
<span class="ff_line" id="gi_742261201_211">VTMRHHALESGCFVINATGWLTAEQKLQITTDEKMHQALSGGCYTAIISPEGKHLCEPIAEGEGLAIADL</span>
<span class="ff_line" id="gi_742261201_281">DFSLIAKRKRMMDSVGHYARPDLLQLTLNNQPWSALEANPVTPNAIPAVSDPELTETIEALPNNPIFSH</span></pre>
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<span style="color: #444444; font-size: 1.1em; line-height: 17.9998px;">PDB:</span><span style="color: #444444; font-size: 1.1em; line-height: 17.9998px;"> </span><span class="highlight" style="color: #444444; font-size: 1.1em; line-height: 17.9998px;">3WUY</span><span style="color: #444444; font-size: 1.1em; line-height: 17.9998px;">_B</span></h1>
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<div class="seq gbff" id="viewercontent1" sequencesize="10592" style="line-height: 1.3em; margin: 0.5em 0px 0px; padding: 0px;" val="742261202" virtualsequence="true">
<pre style="font-family: monospace, serif; overflow: visible; white-space: pre-wrap; width: 50em; word-wrap: break-word; zoom: 1;">>gi|742261202|pdb|3WUY|B Chain B, Crystal Structure Of Nit6803
<span class="ff_line" id="gi_742261202_1">GSHMLGKIMLNYTKNIRAAAAQISPVLFSQQGTMEKVLDAIANAAKKGVELIVFPETFVPYYPYFSFVEP</span>
<span class="ff_line" id="gi_742261202_71">PVLMGKSHLKLYQEAVTVPGKVTQAIAQAAKTHGMVVVLGVNEREEGSLYNTQLIFDADGALVLKRRKIT</span>
<span class="ff_line" id="gi_742261202_141">PTYHERMVWGQGDGAGLRTVDTTVGRLGALACWEHYNPLARYALMAQHEQIHCGQFPGSMVGQIFADQME</span>
<span class="ff_line" id="gi_742261202_211">VTMRHHALESGCFVINATGWLTAEQKLQITTDEKMHQALSGGCYTAIISPEGKHLCEPIAEGEGLAIADL</span>
<span class="ff_line" id="gi_742261202_281">DFSLIAKRKRMMDSVGHYARPDLLQLTLNNQPWSALEANPVTPNAIPAVSDPELTETIEALPNNPIFSH</span></pre>
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Unknownnoreply@blogger.com0tag:blogger.com,1999:blog-1841494650613708862.post-35463295979383532202016-02-27T07:34:00.000-08:002016-02-27T07:34:09.049-08:00A new thermophilic nitrilase from Pyrococcus sp. M24D13 <div class="MsoNormal">
<b>A new thermophilic
nitrilase from an Antarctic hyperthermophilic microorganism</b> by <i>Geraldine V. Dennett and Jenny M. Blamey</i><o:p></o:p></div>
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<a href="http://journal.frontiersin.org/article/10.3389/fbioe.2016.00005/abstract" target="_blank"><b><i>Front. Bioeng. Biotechnol</i>.</b> doi: 10.3389/fbioe.2016.00005</a><o:p></o:p></div>
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<span style="font-family: "Calibri","sans-serif"; font-size: 11.0pt; line-height: 115%; mso-ansi-language: EN-GB; mso-ascii-theme-font: minor-latin; mso-bidi-font-family: "Times New Roman"; mso-bidi-language: AR-SA; mso-bidi-theme-font: minor-bidi; mso-fareast-font-family: Calibri; mso-fareast-language: EN-US; mso-fareast-theme-font: minor-latin; mso-hansi-theme-font: minor-latin;">Several environmental samples from Antarctica
were collected and enriched to search for microorganisms with nitrilase
activity. A new thermostable nitrilase from a novel hyperthermophilic archaea <i>Pyrococcus</i> sp. M24D13 was purified and
characterized. The activity of this enzyme increased as the temperatures rise
from 70 up to 85 °C. Its optimal activity occurred at 85 °C and pH 7.5. This
new enzyme shows a remarkable resistance to thermal inactivation retaining more
than 50% of its activity even after 8 h of incubation at 85 °C. In addition,
this nitrilase is highly versatile demonstrating activity towards different
substrates such as benzonitrile (60 mM, aromatic nitrile) </span><span style="font-family: Calibri, sans-serif; font-size: 11pt; line-height: 115%;">and
butyronitrile (60 mM, aliphatic nitrile). Moreover the enzyme NitM24D13 also
presents cyanidase activity.</span>Unknownnoreply@blogger.com0tag:blogger.com,1999:blog-1841494650613708862.post-17953911635479152712016-02-27T07:09:00.001-08:002016-02-27T07:09:10.172-08:00Mutagenesis of a fungal nitrilase from Gibberella intermedia for improved rate and different acid/amide<div class="MsoNormal">
<b>Engineering of a
fungal nitrilase for improving catalytic activity and reducing by-product
formation in</b> <b>the absence of
structural information</b> from <i>Jin-Song
Gong, Heng Li, Zhen-Ming Lu, Xiao-Juan Zhang, Qiang Zhang, Jiang-Hong Yu, Zhe-Min Zhou, Jin-Song Shi and Zheng-Hong Xu</i><o:p></o:p></div>
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<b><i>Catal. Sci. Technol</i></b>., 2016, <a href="http://pubs.rsc.org/en/content/articlelanding/2016/cy/c5cy01535a#!divAbstract" target="_blank">DOI: 10.1039/C5CY01535A</a><o:p></o:p></div>
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This study employs sequence analysis and saturation
mutagenesis to improve the catalytic activity and reduce the by-product
formation of fungal nitrilase in the absence of structural information.
Site-saturation mutagenesis of isoleucine 128 and asparagine 161 in the fungal
nitrilase from <i>Gibberella intermedia</i>
was performed and mutants I128L and N161Q showed higher catalytic activity
toward 3-cyanopyridine and weaker amide forming ability than the wild-type.
Moreover, the activity of double mutant I128L–N161Q was improved by 100% and
the amount of amide formed was reduced to only one third of that of the
wild-type. The stability of the mutants was significantly enhanced at 30 and 40
°C. The catalytic efficiency of the mutant enzymes was substantially improved.
In this study, we successfully applied a novel approach that required no
structural information and minimal workload of mutant screening for engineering
of fungal nitrilase.<o:p></o:p></div>
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<a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEjuy-UVAPXhwOPakegUSl6HZyWYHklRDSFbRZa51pM4ZgadQ_LGj3OYW8PHJoGO4FikNHy02N7CJS1ZrjicljR4Y6maGaAriXH1XGWsVtzis5vdpM_qnRhfaRP9VT8mk_cPIA6IrczSo2I/s1600/3cyanopyridine.png" imageanchor="1" style="margin-left: 1em; margin-right: 1em;"><img border="0" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEjuy-UVAPXhwOPakegUSl6HZyWYHklRDSFbRZa51pM4ZgadQ_LGj3OYW8PHJoGO4FikNHy02N7CJS1ZrjicljR4Y6maGaAriXH1XGWsVtzis5vdpM_qnRhfaRP9VT8mk_cPIA6IrczSo2I/s1600/3cyanopyridine.png" /></a></div>
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Unknownnoreply@blogger.com0tag:blogger.com,1999:blog-1841494650613708862.post-73163277196017161002016-02-27T06:49:00.000-08:002016-02-27T06:49:18.092-08:00Immobilization of nitrilase for synthesis of 2-hydroxy-4-(methylthio) butanoic acid<b>Immobilization of
nitrilase on bioinspired silica for efficient synthesis of
2-hydroxy-4-(methylthio) butanoic acid from 2-hydroxy-4-(methylthio)
butanenitrile </b> from <i>Li-Qun Jin, Dong-Jing Guo, Zong-Tong Li,
Zhi-Qiang Liu, Yu-Guo Zheng</i><br />
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<b><i>Journal of Industrial Microbiology & Biotechnology</i></b>, <a href="http://link.springer.com/article/10.1007/s10295-016-1747-5" target="_blank">DOI 10.1007/s10295-016-1747-5</a><o:p></o:p></div>
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This paper describes a simple and effective method to
immobilize recombinant nitrilase, for efficient production of
2-hydroxy-4-(methylthio) butanoic acid from 2-hydroxy-4-(methylthio)
butanenitrile. The immobilized enzyme displayed better thermal stability, pH
stability and shelf life compared to free nitrilase. Moreover, it showed
excellent reusability and could be recycled up to 16 batches without
significant loss in activity. 200 mM 2-hydroxy-4-(methylthio) butanenitrile was
completely converted by the immobilized enzyme within 30 min, and the
accumulation amount of 2-hydroxy-4-(methylthio) butanoic acid reached 130
mmol/g of immobilized beads after 16 batches.<o:p></o:p></div>
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<a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEg9c55KIRtbXJ9jk4AhjLbDYujxmj-9b9HvBd8FUiham5_kXeHnYBr-ce_Nj1YR_QNP9yGMt1bIiWq7lZ_JpaK35BOMuGJH_6HyDX8vXngeRmYDQMxSGIjDC7pfim9kwKbNb0R8O4opfKQ/s1600/2H4MSBnitrile.png" imageanchor="1" style="margin-left: 1em; margin-right: 1em;"><img border="0" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEg9c55KIRtbXJ9jk4AhjLbDYujxmj-9b9HvBd8FUiham5_kXeHnYBr-ce_Nj1YR_QNP9yGMt1bIiWq7lZ_JpaK35BOMuGJH_6HyDX8vXngeRmYDQMxSGIjDC7pfim9kwKbNb0R8O4opfKQ/s1600/2H4MSBnitrile.png" /></a></div>
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