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Chauhan, Sushma,Hou, Chen Yuan,Jung, Sang Taek,Kang, Taek Jin Academic Press 2017 Analytical biochemistry Vol.532 No.-
<P><B>Abstract</B></P> <P>A myc-tag and of which recognition by an antibody 9E10 has long been used for the detection and purification of recombinant proteins. We have previously expanded the application of the tag to the specific detection and purification of backbone-cyclized proteins. Here we sought a more practical way to using the 9E10 antibody by expressing its single chain antibody (scAb) form in <I>Escherichia coli</I>. The combined use of a strong T7 promoter and auto-induction strategy rather than early to mid-log induction of a Lac promoter resulted in the soluble over-expression of 9E10 scAb. However, the co-expression of a chaperone, Skp, was absolutely necessary for the activity even when the protein was expressed in a soluble manner. We could purify about 4 mg of 9E10 scAb from 1 l of culture, and the resulting scAb could be used to detect and purify the backbone-cyclized protein as the parental full-length 9E10. Moreover, the immunoaffinity resin prepared using 9E10 scAb could be regenerated several times after the elution of bound proteins using an acid, which added more value to the ready preparation of the active antibody in bacteria.</P>
Soluble expression of horseradish peroxidase in <i>Escherichia coli</i> and its facile activation
Chauhan, Sushma,Kang, Taek Jin Elsevier 2018 Journal of bioscience and bioengineering Vol.126 No.4
<P>Horseradish peroxidase (HRP) is widely used as a marker enzyme in immunoassays and biosensors, and can possibly be used in industries such as waste water treatments or fine chemical synthesis. Cost-effective production of active HRP is thus very important in the related fields. Also, engineering of HRP for its better performance in the designated application is expected to make the enzyme even more important in several areas of research and industry. One of obstacles to this end and to the large scale production of the enzyme has been its facile expression in a bacterial host. Here we show that HRP could be overexpressed as a soluble form by fusing the enzyme with <I>Escherichia coli</I> phosphoglycerate kinase (PGK). After simple incubation with calcium ion, hemin, and oxidized glutathione, PGK-HRP could be fully activated showing a higher molar specific activity than plant-derived HRP. Our established procedure did not use tedious and inefficient refolding steps that have been used to activate HRP produced as inclusion bodies and thus is superior in its overall yield (>72 mg purified HRP conjugate per L culture) to existing methods. By co-expressing PGK-HRP with ferrochelatase in a special host that permitted the formation of disulfide bonds in the cytoplasm, the activation steps could be simplified even though the resulting specific activity was low.</P>
Solubility enhancement of indigo dye through biochemical reduction and structural modification
박현아,김하얀,Sushma Chauhan,Pamidimarri DVN Sudheer,최권영 한국화학공학회 2022 Korean Journal of Chemical Engineering Vol.39 No.8
Indigo is one of the most widely used dyes for textiles and is thus produced in large quantities. Owing toits low solubility, a chemical-dependent continuous reduction-oxidation process is often used for indigo dyeing. Unfortunately,the indigo dyeing process has adopted a hazardous reducing agent such as Na2S2O4, to reduce indigo into leucoindigo,thus causing serious water pollution in the process. To avoid this, the use of chemical reducing reagents wasbanned, creating a need to identify alternative reducing agents or to develop more eco-friendly dyeing processes. In thisreview, alternative reducing reagents such as biochemicals, plant fruits, biocatalysts, and microbes, which are less harmfulto the environment than chemical reducing reagents, are summarized with their reducing reactions and performance. In addition, alternative modifications of indigo that bypass the use of reducing reagents have also been brieflyintroduced. The reducing chemicals and processes summarized have their respective merits and drawbacks; however,further research is required to obtain profitable dyeing performance that meets economic goals.
Mohit Mishra,Sushma Chauhan,Balasubramanian Velramar,Rakesh Kumar Soni,Sudheer Deva Venkata Narayana Pamidimarri 한국화학공학회 2021 Korean Journal of Chemical Engineering Vol.38 No.4
The production of various organic acids from vegetable waste via a facile and cost-effective method utilizing characterized synthetic microbial consortia is described in this study. Five bacterial species with the ability to produce organic acids from vegetable waste biomass were isolated and identified as Lactobacillus casei, Lactobacillus acidophilus, Bacillus megaterium, Pseudomonas florescence and Escherichia coli. Using these cultures, mixed acid fermentation was developed and was efficient in producing various organic acids. The total organic acids accumulated using optimized fermentation conditions was found to be 72.44±3.43 g L1. The acetic acid was produced as major acid accumulated up to 25.27±1.26 g L1, followed by lactic acid 19.11±1.73 g L1. Efforts were also put forth to check the ability to produce methane by the anaerobic digestion process. Up to 14.97mL g1 biomass methane was produced during the anaerobic digestion process. The technology developed in this study is a carbon-neutral process for managing vegetable food waste with economic benefit. The developed technology will have great economic potential and add value to vegetable food waste management.
Pamidimarri D. V. N Sudheer,윤주현,Sushma Chauhan,강택진,최권영 한국생물공학회 2017 Biotechnology and Bioprocess Engineering Vol.22 No.6
In this study, the production of 9-(nonanoyloxy) nonanoic acid from oleic acid was investigated. The whole cell biotransformation of oleic acid includes OhyA (hydratase), ADH (alcohol dehydrdogenase), and BVMO (Baeyer-Villiger Monooxygenase) enzymes consecutively. BVMOs are known to catalyze oxidative cleavage of long chain aliphatic ketones (e.g., 2-decanone, 10-ketooctadecanoic acid). However, the enzymes are difficult to overexpress in a soluble form in microorganisms. Thereby, this study has focused on screening and functional expression of the BVMOs in Escherichia coli. Initially BVMOs were selected by protein sequence analysis and were examined for their ability to express in soluble and active form to generate 9-(nonanoyloxy)nonanoic acid from oleic acid. Secondly various optimization strategies of inducer concentrations, co-expression with molecular chaperones, and different media conditions were investigated. Among the 9 BVMOs screened, three BVMOs were found to produce the target product and among these, Di_BVMO3 isolated from Dietzia sp. D5 was found to be best. Further, the soluble expression of Di_BVMO3 was enhanced by adding phosphoglycerate kinase as N-terminal fusion tag. The whole cell biotransformation with fusion enzyme resulted in 3 ~ 5-fold enhancement in product formation compared with the non-fusion counterpart. Final productivity up to 105.3 mg/L was achieved. Besides Di-BVMO3, other two new BVMOs of Rh_BVMO4 from Rhodococcus sp. RHA1 and AFL838 from Aspergillus flavus NRRL3357 were screened for production of 9-(nonanoyloxy)nonanoic acid and could be used for whole cell biotransformation reaction of other long chain ketones.
Sudheer, Pamidimarri D.V.N,Seo, Dahee,Kim, Eun-Joo,Chauhan, Sushma,Chunawala, J.R.,Choi, Kwon-Young Elsevier 2018 Enzyme and microbial technology Vol.119 No.-
<P><B>Abstract</B></P> <P>Production of (Z)-11-(heptanoyloxy)undec-9-enoic acid from recinoleic acid was achieved by whole-cell biotransformation by <I>Escherichia coli,</I> utilizing crude glycerol as the sole carbon source. Whole-cell biotransformation resulted in ∼93% conversion of the substrate ricinoleic acid to (Z)-11-(heptanoyloxy)undec-9-enoic acid. We replaced the inducer-dependent promoter system (T7 and Rhm promotors) with a constitutive promoter system. This resulted in successful expression of ADH, FadL, and E6-BVMO, without costly inducer addition. Efficacy evaluation of the whole-cell biotransformation by inducer-free system by five different <I>E. coli</I> strains revealed that the highest product titer was accumulated in <I>E. coli</I> BW25113 strain. The engineered inducer-free system using crude glycerol as the sole carbon source showed competitive performance with induction systems. Optimized conditions resulted in the accumulation of 7.38 ± 0.42 mM (Z)-11-(heptanoyloxy)undec-9-enoic acid, and when 10 mM substrate was used as feed concentration, the product titer reached 2.35 g/L. The inducer-free construct with constitutive promoter system that this study established, which utilizes the waste by-product crude glycerol, will pave the way for the economic synthesis of many industrially important chemicals, like (Z)-11-(heptanoyloxy)undec-9-enoic acid.</P> <P><B>Highlights</B></P> <P> <UL> <LI> Conversion of recinoleic acid into (Z)-11-(heptanoyloxy)undec-9-enoic acid. </LI> <LI> BVMO-ADH-FadL expressing <I>E. coli</I> biotransformation system. </LI> <LI> Induction-free biotransformation system development. </LI> <LI> Waste by-product crude glycerol utilization as sole carbon source. </LI> </UL> </P>