Separation of bio-based glucaric acid via antisolvent crystallization and azeotropic drying
A downstream process was developed for producing pure monopotassium glucarate and crystalline glucaric acid from a fermentation broth.
Choi, Hoon↗
SEARCH · Engineering Papers
Results for “Lactones”
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The quorum-quenching lactonase from Alicyclobacter acidoterrestris : purification, kinetic characterization, crystallization and crystallographic analysis
Lactonases comprise a class of enzymes that hydrolyze lactones, including acyl-homoserine lactones (AHLs); the latter are used as chemical signaling molecules by numerous Gram-negative bacteria. Lactonases have therefore been demonstrated to quench AHL-based bacterial communication. In particular, lactonases are capable of inhibiting bacterial behaviors that depend on these chemicals, such as the formation of biofilms or the expression of virulence factors. A novel representative from the metallo-β-lactamase superfamily, named AaL, was isolated from the thermoacidophilic bacterium Alicyclobacter acidoterrestris . Kinetic characterization proves AaL to be a proficient lactonase, with catalytic efficiencies ( k cat / K m ) against AHLs in the region of 10 5 M -1 s -1 . AaL exhibits a very broad substrate specificity. Its structure is expected to reveal the molecular determinants for its substrate binding and specificity, as well as to provide grounds for future protein-engineering projects. Here, the expression, purification, characterization, crystallization and X-ray diffraction data collection of AaL at 1.65Å resolution are reported.
Alicyclobacter acidoterrestris↗
Structural and Mechanistic Roles of Novel Chemical Ligands on the SdiA Quorum-Sensing Transcription Regulator
Bacteria engage in chemical signaling, termed quorum sensing (QS), to mediate intercellular communication, mimicking multicellular organisms. The LuxR family of QS transcription factors regulates gene expression, coordinating population behavior by sensing endogenous acyl homoserine lactones (AHLs). However, some bacteria (such as Escherichia coli) do not produce AHLs. These LuxR orphans sense exogenous AHLs but also regulate transcription in the absence of AHLs. Importantly, this AHL-independent regulatory mechanism is still largely unknown. Here we present several structures of one such orphan LuxR-type protein, SdiA, from enterohemorrhagic E. coli (EHEC), in the presence and absence of AHL. SdiA is actually not in an apo state without AHL but is regulated by a previously unknown endogenous ligand, 1-octanoyl-rac-glycerol (OCL), which is ubiquitously found throughout the tree of life and serves as an energy source, signaling molecule, and substrate for membrane biogenesis. While exogenous AHL renders to SdiA higher stability and DNA binding affinity, OCL may function as a chemical chaperone placeholder that stabilizes SdiA, allowing for basal activity. Structural comparison between SdiA-AHL and SdiA-OCL complexes provides crucial mechanistic insights into the ligand regulation of AHL-dependent and -independent function of LuxR-type proteins. Importantly, in addition to its contribution to basic science, this work has implications for public health, inasmuch as the SdiA signaling system aids the deadly human pathogen EHEC to adapt to a commensal lifestyle in the gastrointestinal (GI) tract of cattle, its main reservoir. These studies open exciting and novel avenues to control shedding of this human pathogen in the environment. IMPORTANCE Quorum sensing refers to bacterial chemical signaling. The QS acyl homoserine lactone (AHL) signals are recognized by LuxR-type receptors that regulate gene transcription. However, some bacteria have orphan LuxR-type receptors and do not produce AHLs, sensing them from other bacteria. We solved three structures of the E. coli SdiA orphan, in the presence and absence of AHL. SdiA with no AHL is not in an apo state but is regulated by a previously unknown endogenous ligand, 1-octanoyl-rac-glycerol (OCL). OCL is ubiquitously found in prokaryotes and eukaryotes and is a phospholipid precursor for membrane biogenesis and a signaling molecule. While AHL renders to SdiA higher stability and DNA-binding affinity, OCL functions as a chemical chaperone placeholder, stabilizing SdiA and allowing for basal activity. Our studies provide crucial mechanistic insights into the ligand regulation of SdiA activity.
59 BASIC BIOLOGICAL SCIENCES↗
Quorum sensing modulates microbial community structure through regulation of secondary metabolites
Bacteria are recognized for their diverse metabolic capabilities, yet the impact of microbe-microbe interactions on multispecies community structure and dynamics is poorly understood. Cell-to-cell signaling in the form of quorum sensing (QS) often regulates secondary metabolite production and microbial interactions. Here, we examine how acylhomoserine lactone (AHL)-mediated QS impacts microbial community structure in a 10-member synthetic community of isolates from Populus deltoides . To explore the role of QS in microbial community structure and dynamics, we disrupted AHL signaling by exogenous addition of AiiA-lactonase, an enzyme that cleaves the lactone ring. Microbial community structure resulting from signal inactivation, as measured by 16S rRNA amplicon sequencing and secondary metabolite production, was assessed after successive passaging of the community. Further, we investigated the impact of quorum quenching on specific microbe-microbe interactions using pairwise inhibition assays. Our results indicate that AHL inactivation alters the relative abundance of dominant community members at later passages but does not impact the overall membership in the community. Quorum quenching significantly alters the metabolic profile in lactonase-treated communities. This metabolic alteration impacts microbe-microbe interactions through decreased inhibition of other community members. Together, these results indicate that QS impacts microbial community structure through the regulation of secondary metabolites in dominant members and that the membership of microbial communities can be relatively stable despite changes in metabolic profiles.
59 BASIC BIOLOGICAL SCIENCES↗
Enabling Catalytic Strategies for Biomass Conversion
This research program employed a mix of fundamental investigations of catalytic reactivity with targeted approaches for the catalytic synthesis of monomers and renewable polymers. We investigated the mechanisms of selective aerobic oxidation of polyols and carbohydrates with Pd catalysts with a special focus on the role of hydrogen peroxide and peroxy intermediates in an effort to increase catalyst lifetime. We also extended our studies on the selective oxidation of sugars to ketoses and the oxidative lactonization of 1,5-diols to generate new families of lactone monomers.
36 MATERIALS SCIENCE↗
A possible prebiotic synthesis of pantetheine, a precursor to coenzyme A
The involvement of coenzyme A in many enzyme reactions suggests that it acted in this capacity very early in the development of life on Earth. Particularly relevant in this regard is its role in the activation of amino acids and hydroxy acids in the biosynthesis of some peptide antibiotics--a mechanism of peptide synthesis that forms the basis for the proposal that a thioester world could have preceded the RNA world. The components of coenzyme A have been shown to be probable prebiotic compounds: beta-alanine, pantoyl lactone and cysteamine and possibly adenosine. We show here that the pantetheine moiety of coenzyme A (which also occurs in a number of enzymes) can be synthesized in yields of several per cent by heating pantoyl lactone, beta-alanine and cysteamine at temperatures as low as 40 degrees C. These components are extremely soluble and so would have been preferentially concentrated in evaporating bodies of water, for example on beaches and at lagoon margins. Our results show that amide bonds can be formed at temperatures as low as 40 degrees C, and provide circumstantial support for the suggestion that pantetheine and coenzyme A were important in the earliest metabolic systems.
NASA Discipline Number 52-20↗
Biomass pre-treatment for co-production of high-concentration C5- and C6-carbohydrates and their derivatives
Described is a method of processing biomass to separate it into a liquid fraction enriched in solubilized C5-sugar-containing oligomers and C-5 sugar monomers and a solid fraction enriched in substantially insoluble cellulose and C6-sugar-containing oligomers. The method includes the steps of reacting biomass with a solvent system comprising water, at least one lactone, or at least one furan, or at least one cyclic ether, and at least one acid, for a time and at a temperature to yield the liquid and solid fractions. The liquid and solid fractions may then be separated. Gamma-valeroloactone is a preferred lactone for use in the solvent system. Tetrahydrofuran is a preferred furan species for use in the solvent system.
09 BIOMASS FUELS↗
Synthesis of polyketides from low cost substrates by the thermotolerant yeast Kluyveromyces marxianus
Kluyveromyces marxianus is a promising nonconventional yeast for biobased chemical production due to its rapid growth rate, high TCA cycle flux, and tolerance to low pH and high temperature. Unlike Saccharomyces cerevisiae, K. marxianus grows on low-cost substrates to cell densities that equal or surpass densities in glucose, which can be beneficial for utilization of lignocellulosic biomass (xylose), biofuel production waste (glycerol), and whey (lactose). Additionally, we have evaluated K. marxianus for the synthesis of polyketides, using triacetic acid lactone (TAL) as the product. The 2-pyrone synthase (2-PS) was expressed on a CEN/ARS plasmid in three different strains, and the effects of temperature, carbon source, and cultivation strategy on TAL levels were determined. The highest titer was obtained in defined 1% xylose medium at 37°C, with substantial titers at 41 and 43°C. The introduction of a high-stability 2-PS mutant and a promoter substitution increased titer four-fold. 2-PS expression from a multi-copy pKD1-based plasmid improved TAL titers a further five-fold. Combining the best plasmid, promoter, and strain resulted in a TAL titer of 1.24 g/L and a yield of 0.0295 mol TAL/mol carbon for this otherwise unengineered strain in 3 ml tube culture. This is an excellent titer and yield (on xylose) before metabolic engineering or fed-batch culture relative to other hosts (on glucose), and demonstrates the promise of this rapidly growing and thermotolerant yeast species for polyketide production.
59 BASIC BIOLOGICAL SCIENCES↗
Xenicanes attenuate pro-inflammatory 5-lipoxygenase: Prospective natural anti-inflammatory leads from intertidal brown seaweed Padina tetrastromatica
Two previously unreported xenicane class of novaxenicin-type xenicin diterpenoids (1–2) bearing cyclonona[d]furo [2,3-b]pyrandiol and three xeniolide-type diterpenoids with unprecedented octahydrocyclonona[c]pyran-3(1H)-one backbones (3–5) were separated from the organic extract of the intertidal seaweed Padina tetrastomatica (family Dictyotaceae), collected from southern India. The compounds were deduced to bear a xenicane moiety with a 2-oxabicyclo[7.4.0]tridecane cyclic system. The structures of these specialized metabolites were attributed based on the extensive nuclear magnetic resonance spectral analyses, and comparison of related compounds. Xeniolide-type diterpenes (3–5) registered significantly greater attenuation potential against pro-inflammatory 5-lipoxygenase (IC{sub 50} ~ 2.04 mM) than that exhibited by non-steroidal anti-inflammatory drug ibuprofen (IC{sub 50} 4.50 mM, P< 0.05). The xeniolide derivative octahydro-1,7-dihydroxy-4-(4{sup 1}-hydroxy-4{sup 2}-methylpropyl)-6-(6{sup 1}-hydroxy-6{sup 2}-propenyl)-10-methyl-cyclonona[c]pyran-3(1H)-one (5) exhibited comparable antioxidant activity (DPPH IC{sub 50} 1.73 mM) along with standard antioxidative agent α-tocopherol (IC{sub 50} < 2 mM). In silico molecular modelling studies were performed to designate the 5-lipoxygenase inhibitory mechanism of the xenicanes, and the comparison of docking parameters suggested that the xeniolide derivative 5 exhibited least binding energy of −11.56 kcal mol{sup −1}, and that was corroborated with its greater inhibition potential against the pro-inflammatory enzyme. These results demonstrated that the xeniolide-type diterpenoids with previously unreported δ-lactone cyclononane framework might constitute promising anti-inflammatory leads with pro-inflammatory 5-lipoxygenase enzyme inhibitory activities.
60 APPLIED LIFE SCIENCES↗
Candida antarctica Lipase B as Catalyst for Cyclic Esters Synthesis, Their Polymerization and Degradation of Aliphatic Polyesters
Candida antarctica: lipase B (CALB) is the most widely studied enzyme, due to its high selectivity and catalytic activity in organic and polymer synthesis. To avoid organometallic catalysts in the synthesis of biodegradable polyesters, the enzymatic ring-opening polymerization of cyclic esters can be carried out with CALB as a biocatalyst. This paper reviews selected examples of the application of lipases in polymer chemistry covering the synthesis of cyclic ester monomers, linear and star oligomers, as well as well-defined high molar mass polymers with improved mechanical properties. It is shown that by the selection of appropriate conditions of temperature and solvent assortment, CALB effectively catalyzes the polymerization of large size lactones obtaining polyesters with M{sub n} up to 80 kg mol{sup −1}. The chemical structure and main applications of described materials are also discussed. As the polymer recycling is an important topic from environmental point of view, we also described the potential applications of CALB in degradation process of aliphatic polyesters.
36 MATERIALS SCIENCE↗
Artesunate inhibits adipogeneis in 3T3-L1 preadipocytes by reducing the expression and/or phosphorylation levels of C/EBP-α, PPAR-γ, FAS, perilipin A, and STAT-3
Differentiation of preadipocyte, also called adipogenesis, leads to the phenotype of mature adipocyte. However, excessive adipogenesis is closely linked to the development of obesity. Artesunate, one of artemisinin-type sesquiterpene lactones from Artemisia annua L., is known for anti-malarial and anti-cancerous activities. In this study, we investigated the effect of artesunate on adipogenesis in 3T3-L1 preadipocytes. Artesunate strongly inhibited lipid accumulation and triglyceride (TG) synthesis during the differentiation of 3T3-L1 preadipocytes into adipocytes at 5 μM concentration. Artesunate at 5 μM also reduced not only the expressions of CCAAT/enhancer-binding protein-α (C/EBP-α), peroxisome proliferator-activated receptor-γ (PPAR-γ), fatty acid synthase (FAS), and perilipin A but also the phosphorylation levels of signal transducer and activator of transcription-3 (STAT-3) during adipocyte differentiation. Moreover, artesunate at 5 μM reduced leptin, but not adiponectin, mRNA expression during adipocyte differentiation. Taken together, these findings demonstrate that artesunate inhibits adipogenesis in 3T3-L1 preadipoytes through the reduced expression and/or phosphorylation levels of C/EBP-α, PPAR-γ, FAS, perilipin A, and STAT-3. -- Highlights: •Artesunate, an artemisinin derivative, inhibits adipogenesis. •Artesunate inhibits C/EBP-α, PPAR-γ, FAS, perilipin A, and STAT-3 in 3T3-L1 adipocytes. •Artesunate reduces leptin, but not adiponectin, expression in 3T3-L1 adipocytes. •Artesunate thus may have therapeutic potential against obesity.
60 APPLIED LIFE SCIENCES↗
Withaferin A induces apoptosis by ROS-dependent mitochondrial dysfunction in human colorectal cancer cells
Withaferin A (WA) is an active steroidal lactone derived from the herbal plant Withania somnifera, which exhibits antitumor activity with reactive oxygen species (ROS) modulating in a variety of cancer models, such as breast cancer, lung cancer and pancreatic cancer. However, to the best of our knowledge, the direct effect and mechanism of WA on CRC cells has not been previously determined. The present study investigated the anti-tumor effects of WA on CRC cells in vitro, and explored the mechanisms of action. The flow cytometry was applied for detecting the accumulation of ROS with the treatment of withaferin A. We performed the flow cytometry and western blot to evaluate the withaferin A induced apoptosis and cell cycle arrest in human colon cancer cells. And to verify the ROS accumulation induced mitochondrial dysfunction after the treatment of withaferin A, fluorescence microscope and western blot were applied. WA exerted a dose-dependent cytotoxic effect on HCT-116 and RKO cells. The effect was associated with ROS-mediated cell cycle arrest and the expression of apoptotic proteins. In addition, WA promoted ROS production and decreased mitochondrial membrane potential accompanying with mitochondrial dysfunction. Taken together, these results strongly indicated that WA directly inhibits cell growth and induces apoptosis in CRC cells through ROS-mediated mitochondrial dysfunction and JNKs pathway, and WA may be a promising potential candidate for therapeutic application of CRC.
60 APPLIED LIFE SCIENCES↗
Gulo regulates the proliferation, apoptosis and mesenchymal-to-epithelial transformation of metanephric mesenchyme cells via inhibiting Six2
During kidney development, the balance between self-renewal and differentiation of metanephric mesenchyme (MM) cells, mainly regulated by Sine oculis-related homeobox 2 (Six2), is critical for forming mature kidney. L-gulono-γ-lactone oxidase (Gulo), a crucial enzyme for vitamin C synthesis, reveals a different expression at various stages during kidney development, but its function in the early renal development remains unknown. In this work, we aim to study the role of Gulo in MM cells at two differentiation stages. We found that Gulo expression in undifferentiated MM (mK3) cells was lower than in differentiated MM (mK4) cells. Over-expression of Gulo can promote mesenchymal-to-epithelial transformation (MET) and apoptosis and inhibit the proliferation in mK3 cells. Knock-down of Gulo in mK4 cells made its epithelial character cells unstabilized, facilitated the proliferation and restrained the apoptosis. Furthermore, we found that Six2 was negatively regulated by Gulo, and over-expression or knock-down of Six2 was able to rescue partially the MET, proliferation and apoptosis of MM cells caused by Gulo. In conclusion, these findings reveal that Gulo promotes the MET and apoptosis, and inhibits proliferation in MM cells by down-regulating Six2.
60 APPLIED LIFE SCIENCES↗
Sustainable, degradable and malleable low-dielectric-constant thermosets derived from biomass for recyclable green electronics
In view of their excellent thermal and chemical resistance, favorable adhesion properties, high tensile strength, and other advantages, epoxy thermosets have numerous industrial applications such as coating, adhesive, and composite material production. However, bisphenol A (BPA)-based resins, which account for a significant fraction of the above thermosets, adversely affect human health by interfering with the normal functioning of the endocrine system and cannot be easily decomposed and recycled. Herein, degradable and sustainable bio-based epoxy thermosets with crosslinked network structures are prepared by the epoxidation of isosorbide with epichlorohydrin and the curing of the produced isosorbide diglycidyl ether (ISDGE) with cyclic lactones through a cationic ring-opening reaction. The ISDGE thermosets fully decompose into soluble and recyclable products under mildly basic conditions within three days, which is ascribed to the presence of ester moieties within the polymer network structure. Compared to a representative BPA-based epoxy, ISDGE-based thermosets exhibit lower dielectric constants and are more flexible. Moreover, the glass fibers in ISDGE-based prepregs can be fully recovered after on-demand degradation. Furthermore, our work provides promising eco-friendly alternatives to conventional epoxy thermosets and paves the way for the reduction of plastic waste generation and the development of recyclable green electronics.
37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
Goniothalamin prevents the development of chemically induced and spontaneous colitis in rodents and induces apoptosis in the HT-29 human colon tumor cell line
Colon cancer is the third most incident type of cancer worldwide. One of the most important risk factors for colon cancer development are inflammatory bowel diseases (IBD), thus therapies focusing on IBD treatment have great potential to be used in cancer prevention. Nature has been a source of new therapeutic and preventive agents and the racemic form of the styryl-lactone goniothalamin (GTN) has been shown to be a promising antiproliferative agent, with gastroprotective, antinociceptive and anti-inflammatory effects. As inflammation is a well-known tumor promoter, the major goal of this study was to evaluate the therapeutic and preventive potentials of GTN on chemically induced and spontaneous colitis, as well as the cytotoxic effects of GTN on a human colon tumor cell line (HT-29). GTN treatments inhibited TNBS-induced acute and chronic colitis development in Wistar rats, reducing myeloperoxidase levels and inflammatory cells infiltration in the mucosa. In spontaneous-colitis using IL-10 deficient mice (C57BL/6 background), GTN prevented colitis development through downregulation of TNF-α, upregulation of SIRT-1 and inhibition of proliferation (PCNA index), without signs of toxicity after three months of treatment. In HT-29 cells, treatment with 10 μM of GTN induced apoptosis by increasing BAX/BCL2, p-JNK1/JNK1, p-P38/P38 ratios as well as through ROS generation. Caspase 8, 9 and 3 activation also occurred, suggesting caspase-dependent apoptotic pathway, culminating in PARP-1 cleavage. Together with previous data, these results show the importance of GTN as a pro-apoptotic, preventive and therapeutic agent for IBD and highlight its potential as a chemopreventive agent for colon cancer. - Highlights: • Goniothalamin (GTN) inhibits the development of TNBS-induced colitis in rats. • Moreover, GTN prevents the development of spontaneous colitis in IL-10 deficient mice. • This activity relies on downregulation of TNF-α and upregulation of SIRT-1 expression. • GTN induces intrinsic apoptosis in HT-29 cells, involving ROS, MAPK and caspases. • These results highlight GTN as a potential chemopreventive agent for colon cancer.
60 APPLIED LIFE SCIENCES↗
Identification of a compound isolated from German chamomile (Matricaria chamomilla) with dermal sensitization potential
German chamomile is one of the most popular herbal ingredients used in cosmetics and personal care products. Allergic skin reactions following topical application of German chamomile have been occasionally reported, although it is not fully understood which of the chemical constituents is responsible for this adverse effect. In the present work, three candidate sensitizers were isolated from German chamomile based on activity-guided fractionation of chamomile extracts tested using the in vitro KeratinoSens™ assay. The compounds were identified as the polyacetylene tonghaosu (1), and both trans- and cis-glucomethoxycinnamic acids (2 and 3). These three compounds were classified as non- to weakly reactive using in chemico methods; however, aged tonghaosu was found to be more reactive when compared to freshly isolated tonghaosu. The polyacetylene (1) constituent was determined to be chemically unstable, generating a small electrophilic spirolactone, 1,6-dioxaspiro[4.4]non-3-en-2-one (4), upon aging. This small lactone (4) was strongly reactive in both in chemico HTS- and NMR-DCYA methods and further confirmed as a potential skin sensitizer by Local Lymph Node Assay (LLNA). - Highlights: • Fractions of German chamomile tested positive in the KeratinoSens™ assay. • Three compounds containing structural alerts were isolated and tested with in chemico methods. • The polyacetylene tonghaosu was found to be unstable and categorized as potential pre-hapten. • A degradation product of tonghaosu tested as positive dermal sensitizer in animal studies.
60 APPLIED LIFE SCIENCES↗
Engineering quorum-sensing circuits in Synechococcus elongatus PCC 7942 towards self-inducible systems
Despite significant potential for cyanobacteria as sustainable bioproduction chases, there are limited examples of scaled cyanobacterial bioproduction. In part, this is because most cyanobacterial species are poorly adapted to bioreactor cultivation conditions and lack features that facilitate biomass growth and harvesting at scale. We explored quorum sensing (QS) pathways derived from heterotrophic microbes as a method for autoinduction of gene expression circuits coordinated to population density in cyanobacteria. Here, we integrated genetic modules designed to produce and detect the diffusible QS signal, acyl-homoserine lactones (AHLs), in the cyanobacterial model, Synechococcus elongatus PCC 7942 (S. elongatus). We demonstrate that S. elongatus heterologously produces sufficient AHL signals to activate gene expression in a dose-dependent and population density-responsive manner. A hybrid combination of AHL synthesis enzyme from Vibrio fischeri (Lux system) with the transcription factor receiver from Pseudomonas aeruginosa (Las system) provides an ideal activation ratio and mitigates toxicity observed with some AHL systems. As a proof of concept, we coupled the QS pathway to the expression of a cell division inhibitory gene, cdv3, facilitating late-phase cell elongation, cell sedimentation, and improved biomass recovery. Our findings provide a foundation for the development of auto-induction systems leverageable to improve cyanobacterial biotechnology applications.
Acyl homoserine lactones (AHL)↗
Production of a δ-Lactam from Glucose through Integrating Biological and Chemical Catalysis
We present a new strategy for the production of a δ-lactam from glucose that integrates biological production of triacetic acid lactone (TAL, 4-hydroxy-6-methyl-2H-2-one) with catalytic transformation of TAL into 6-methylpiperidin-2-one (MPO) through metabolic engineering, isomerization, amination, and catalytic hydrogenation/hydrogenolysis. We developed a sustainable and antibiotic-free fed-batch fermentation using genetically modified Rhodotorula toruloides IFO0880. This process achieved a yield of 2-hydroxy-6-methyl-4H-pyran-4-one (2H4P) at 0.05 g/g of glucose, corresponding to a 9.9 g/L titer. By adjusting the pH of the fermentation broth to 2, 2H4P was quantitatively converted into TAL. The TAL in the fermentation broth was directly converted by aminolysis into 4-hydroxy-6-methylpyridin-2(1H)-one (HMPO), which achieved an 18.5% yield with 94.3% purity. The HMPO yield was lower in the fermentation broth than in a clean feedstock (32.2%), suggesting that the biological impurities are inhibitors in this reaction. Further investigation revealed that lower pH levels and reduced TAL concentrations in the fermentation broth significantly decreased HMPO yields. Subsequently, the precipitated HMPO was filtered and dried and then subjected to the final catalytic conversion in H2O solvent, achieving a MPO yield of 91.8%. Furthermore, this integrated approach demonstrated the direct use of TAL in the filtered aqueous fermentation broth without the need to isolate TAL.
37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗