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Methods for biosynthesizing 1,3butadiene

A technology of biosynthesis and butadiene, applied in biochemical equipment and methods, waste fuels, enzymes, etc., can solve the problem of less catalytic terminal vinyl groups

Inactive Publication Date: 2015-01-28
INVISTA TEXTILES (U K) LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, these enzymatic activities rarely catalyze the formation of terminal vinyl groups

Method used

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  • Methods for biosynthesizing 1,3butadiene
  • Methods for biosynthesizing 1,3butadiene
  • Methods for biosynthesizing 1,3butadiene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0232] Enzyme activity of mevalonate diphosphate decarboxylase accepting 3-hydroxypent-4-enoate as substrate

[0233] His-tagged MDD genes from S. cerevisiae, S. epidermidis and S. pneumoniae were cloned and expressed in E. coli in shake flask cultures containing Luria Broth medium.

[0234] The pellet from each induced shake flask culture was collected by centrifugation, and the pellet was resuspended and dissolved. Cell debris was separated from the supernatant by centrifugation and filtered using a 0.2 μm filter. The MDD enzyme was purified from the supernatant using Ni-affinity chromatography, concentrated and ultrafiltered by using a 10 kDa polyethersulfone membrane into 50 mM tromethamine buffer (pH=7.5), 100 mM NaCl and 5% (v / v) Buffer exchange with glycerol.

[0235] Native enzyme activity in the presence of 50mM Tris-HCl (pH = 7.5), 100mM NaCl, 5% (v / v) glycerol, 10mM MgCl 2 , 15 mM ATP and 5 mM the natural substrate mevalonate diphosphate (from Sigma Aldrich) buff...

Embodiment 2

[0241] Amino acid residues that enhance mevalonate diphosphate decarboxylase activity in accepting 3-hydroxypent-4-enoate as a substrate

[0242] Figure 13 Amino acid sequences of MDD enzymes from S. cerevisiae, S. epidermidis and S. pneumoniae are provided, with conserved residues within the enzyme's catalytic cleft in bold.

[0243] Using total protein concentration and purity from densitometry, the enzyme concentration was 385 μg / mL for purified MDD from S. cerevisiae and 88 μg / mL for purified MDD from S. pneumoniae.

[0244] Assuming incomplete conversion of 3-hydroxypent-4-enoic acid as an unnatural substrate, the specific conversion of MDD from S. cerevisiae was 809 [(peak area of ​​m / z 54 ion) / (μg MDD)] and The MDD from S. pneumoniae was 3200 [(peak area of ​​m / z 54 ion) / (μg MDD)]. Thus, the specific conversion rate of MDD from S. pneumoniae was about four times that of MDD from S. cerevisiae. The specific conversion of MDD from S. epidermidis was between that of MD...

Embodiment 3

[0248] Enzymatic activity of isoprene synthase accepting trans-2-butenylpyrophosphate as substrate

[0249] The his-tagged isoprene synthase (ISPS) gene from Populus alba was cloned and expressed in E. coli in shake flask cultures containing Luria Broth medium.

[0250] The pellet from each induced shake flask culture was collected by centrifugation, and the pellet was resuspended and dissolved. Cell debris and supernatant were centrifuged and filtered through a 0.2 μm filter. ISPS enzyme variants were purified from supernatants using Ni-affinity chromatography, concentrated and buffer exchanged into 50 mM tromethamine buffer (pH=7.5), 100 mM NaCl and 5% (v / v ) in glycerol.

[0251] The natural enzyme activity was in the presence of 50mM Tris·HCl (pH=7.5), 100mM NaCl, 5% (v / v) glycerol, 20mM MgCl 2 and 5 mM of the natural substrate dimethylallyl diphosphate (from Sigma-Aldrich) at 30°C. Native activity assays were performed in 2 mL septum-sealed vials, thereby allowing acc...

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Abstract

This document describes biochemical pathways for producing butadiene by forming two vinyl groups in a butadiene synthesis substrate. These pathways described herein rely on enzymes such as mevalonate diphosphate decarboxylase, isoprene synthase, and dehydratases for the final enzymatic step.

Description

[0001] Cross References to Related Applications [0002] This application claims priority to US Application 61 / 566,085, filed December 2, 2011, and US Application 61 / 714,883, filed October 17, 2012, the entire disclosures of which are incorporated herein by reference. technical field [0003] The present invention relates to methods for the biosynthesis of 1,3-butadiene, and more particularly, to the use of one or more isolated enzymes such as dehydrogenases, monooxygenases, desaturases, dehydratases, and decarboxylases, Alternatively, 1,3-butadiene is synthesized using recombinant host cells expressing one or more of these enzymes. Background technique [0004] 1,3-butadiene (hereinafter referred to as butadiene) is used in the production of synthetic rubber (including styrene-butadiene-rubber (SBR), polybutadiene (PB), styrene-butadiene latex (SBL), acrylonitrile-butadiene-styrene resin (ABS), nitrile rubber, and adiponitrile), which are important monomers in the manufact...

Claims

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Application Information

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IPC IPC(8): C12P5/02C12N9/00C07C11/167
CPCY02E50/343C12N9/88C12N9/0004C12Y401/01033C12Y402/03027C12P5/026C12N15/52Y02E50/30
Inventor P.S.珀尔曼C.陈A.波特斯A.V.E.康拉蒂
Owner INVISTA TEXTILES (U K) LTD
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