328-50-7Relevant articles and documents
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Clutterbuck
, p. 519 (1927)
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Barreto,Barreto
, (1961)
Microbial formation of α ketoglutaric acid from D xylonic acid
Ohsugi,Takahashi
, p. 1257 - 1258 (1976)
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Integrating error-prone PCR and DNA shuffling as an effective molecular evolution strategy for the production of α-ketoglutaric acid by l-amino acid deaminase
Hossain, Gazi Sakir,Shin, Hyun-Dong,Li, Jianghua,Wang, Miao,Du, Guocheng,Liu, Long,Chen, Jian
, p. 46149 - 46158 (2016)
l-Amino acid deaminases (LAADs; EC 1.4.3.2) belong to a family of amino acid dehydrogenases that catalyze the formation of α-keto acids from l-amino acids. In a previous study, a whole cell biocatalyst with the l-amino acid deaminase (pm1) from Proteus mirabilis was developed for the one-step production of α-ketoglutarate (α-KG) from l-glutamic acid, and the α-KG titer reached 12.79 g L-1 in a 3 L batch bioreactor. However, the product α-KG strongly inhibited pm1 activity, and the titer of α-KG was comparatively lower than expected. Therefore, in this study, multiple rounds of error-prone polymerase chain reaction (PCR) and gene shuffling were integrated for the molecular engineering of pm1 to further improve the catalytic performance and α-KG titer. A variant (pm1338g4), which contained mutations in 34 amino acid residues, was found to have enhanced catalytic efficiency. In a batch system, the α-KG titer reached 53.74 g L-1 when 100 g of monosodium glutamate was used as a substrate. Additionally, in a fed-batch biotransformation system, the maximum α-KG titer reached 89.11 g L-1 when monosodium glutamate was continuously fed at a constant rate of 6 g L-1 h-1 (from 4 to 23 h) with an initial concentration of 50 g L-1. Analysis of the kinetics of the mutant variant showed that these improvements were achieved due to enhancement of the reaction velocity (from 56.7 μM min-1 to 241.8 μM min-1) and substrate affinity (the Km for glutamate decreased from 23.58 to 6.56 mM). A possible mechanism for the enhanced substrate affinity was also evaluated by structural modeling of the mutant. Our findings showed that the integration of error-prone PCR and gene shuffling was an effective method for improvement of the catalytic performance of industrial enzymes.
From a dimer to a monomer: Construction of a chimeric monomeric isocitrate dehydrogenase
Tian, Changqing,Wen, Bin,Bian, Mingjie,Jin, Mingming,Wang, Peng,Xu, Lei,Zhu, Guoping
, p. 2396 - 2407 (2021/10/29)
Many isocitrate dehydrogenases (IDHs) are dimeric enzymes whose catalytic sites are located at the intersubunit interface, whereas monomeric IDHs form catalytic sites with single polypeptide chains. It was proposed that monomeric IDHs were evolved from dimeric ones by partial gene duplication and fusion, but the evolutionary process had not been reproduced in laboratory. To construct a chimeric monomeric IDH from homo-dimeric one, it is necessary to reconstitute an active center by a duplicated region; to properly link the duplicated region to the rest part; and to optimize the newly formed protein surface. In this study, a chimeric monomeric IDH was successfully constructed by using homo-dimeric Escherichia coli IDH as a start point by rational design and site-saturation mutagenesis. The ~67 kDa chimeric enzyme behaved as a monomer in solution, with a Km of 61 μM and a kcat of 15 s?1 for isocitrate in the presence of NADP+ and Mn2+. Our result demonstrated that dimeric IDHs have a potential to evolve monomeric ones. The evolution of the IDH family was also discussed.
METHODS FOR IMPROVING YIELDS OF L-GLUFOSINATE
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Page/Page column 32, (2020/03/29)
Compositions and methods for the production of L-glufosinate are provided. The method involves converting racemic glufosinate to the L-glufosinate enantiomer or converting PRO to L-glufosinate in an efficient manner. In particular, the method involves the specific amination of PRO to L-glufosinate, using L-glutamate, racemic glutamate, or another amine source as an amine donor. PRO can be obtained by the oxidative deamination of D-glufosinate to PRO (2- oxo-4-(hydroxy(methyl)phosphinoyl)butyric acid) or generated via chemical synthesis. PRO is then converted to L-glufosinate using a transaminase in the presence of an amine donor. When the amine donor donates an amine to PRO, L-glufosinate and a reaction by product are formed. Because the PRO remaining represents a yield loss of L-glufosinate, it is desirable to minimize the amount of PRO remaining in the reaction mixture. Degradation, other chemical modification, extraction, sequestration, binding, or other methods to reduce the effective concentration of the by-product, i.e., the corresponding alpha ketoacid or ketone to the chosen amine donor will shift the reaction equilibrium toward L-glufosinate, thereby reducing the amount of PRO and increasing the yield of L-glufosinate. Therefore, the methods described herein involve the conversion or elimination of the alpha ketoacid or ketone by-product to another product to shift the equilibrium towards L-glufosinate.
