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Li, Mi

Publications and source records attributed to Li, Mi.

87 records · Page 5
All sourcesNASA NTRSDOE OSTI
DOE OSTI2017

31 P NMR Characterization of Tricin and Its Structurally Similar Flavonoids

Tricin, a flavonoid metabolite, has been recently identified as a component of lignin in select monocot plants. This finding has initiated consideration on updating the lignin biosynthesis pathway. Here, we report a rapid method of determination of tricin in corn stover lignin, based on 31 P nuclear magnetic resonance (NMR) spectroscopy by phosphitylating with 2-chloro-4,4,5,5-tetramethyl-1,3,2-dioxaphospholane (TMDP). Nine other flavonoids, with similar structure to tricin, have also been examined using the current method. The application of 31 P NMR enables rapid identification of tricin-like flavonoids in the heterogeneous lignin polymer. The well resolved spectroscopic peaks from these derivatized flavonoids and lignin functional groups provide important information for the determination of flavonoids individually or their association with lignin.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2017

The DnaK Chaperone Uses Different Mechanisms To Promote and Inhibit Replication of Vibrio cholerae Chromosome 2

Replication of Vibrio cholerae chromosome 2 (Chr2) depends on molecular chaperone DnaK to facilitate binding of the initiator (RctB) to the replication origin. The binding occurs at two kinds of site, 12-mers and 39-mers, which promote and inhibit replication, respectively. Here we show that DnaK employs different mechanisms to enhance the two kinds of binding. We found that mutations in rctB that reduce DnaK binding also reduce 12-mer binding and initiation. The initiation defect is suppressed by second-site mutations that increase 12-mer binding only marginally. Instead, they reduce replication inhibitory mechanisms: RctB dimerization and 39-mer binding. One suppressing change was in a dimerization domain which is folded similarly to the initiator of an iteron plasmid—the presumed progenitor of Chr2. In plasmids, DnaK promotes initiation by reducing dimerization. A different mutation was in the 39-mer binding domain of RctB and inactivated it, indicating an alternative suppression mechanism. Paradoxically, although DnaK increases 39-mer binding, the increase was also achieved by inactivating the DnaK binding site of RctB. This result suggests that the site inhibits the 39-mer binding domain (via autoinhibition) when prevented from binding DnaK. Taken together, our results reveal an important feature of the transition from plasmid to chromosome: the Chr2 initiator retains the plasmid-like dimerization domain and its control by chaperones but uses the chaperones in an unprecedented way to control the inhibitory 39-mer binding.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2017

Effects of organosolv and ammonia pretreatments on lignin properties and its inhibition for enzymatic hydrolysis

Lignin offers structural support and protection for plant cell walls; but, it also contributes to biomass recalcitrance and the costs of biofuel production via the biological pathway. Organosolv and ammonia pretreatments have been developed to reduce biomass recalcitrance and improve sugar release performance during enzymatic hydrolysis. It is believed that lignin properties are related to its inhibition on enzymatic hydrolysis; therefore, understanding the characteristics of lignin is a key for effective biomass conversion to biofuels. In this study, an organosolv pretreatment using 60% ethanol with 1.25% H 2 SO 4 significantly deconstructed poplar lignin and reduced its molecular weights due to the cleavage of lignin inter-unit linkages. The organosolv pretreatment increased the contents of phenolic OH units and the lignin residue showed a high cellulase maximum adsorption capacity. Ammonia pretreatment with 5% ammonium hydroxide was not as effective as organosolv pretreatment on lignin deconstruction. Organosolv lignin residue had lower lignin S/G ratio than the untreated one. Furthermore, when compared to the organosolv lignin residue and untreated lignin, ammonia lignin residue had a higher cellulase adsorption affinity. In addition, the effects of lignin on cellulose hydrolysis was investigated and the results suggested that the presence of lignin with cellulose substrates reduced cellulose hydrolysis, and its inhibitory effect was primarily determined by the lignin properties after each pretreatment. The organosolv pretreatment resulted in a slightly lower cellulase binding strength (249.7 mL g -1 ) on poplar lignin than that on untreated samples (261.1 mL g -1 ), while ammonia lignin residue showed a higher cellulase binding strength (402.8 mL g -1 ) and had more significant inhibition effect on cellulose hydrolysis. Our results demonstrated that the binding strength significantly affected the lignin-derived inhibition on enzymatic hydrolysis of cellulose in the cellulose-lignin mixtures.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2016

Physicochemical structural changes of cellulosic substrates during enzymatic saccharification

Enzymatic hydrolysis represents one of the major steps and barriers in the commercialization process of converting cellulosic substrates into biofuels and other value added products. It is usually achieved by a synergistic action of enzyme mixture typically consisting of multiple enzymes such as glucanase, cellobiohydrolase and β-glucosidase with different mode of actions. Due to the innate biomass recalcitrance, enzymatic hydrolysis normally starts with an initial fast rate of hydrolysis followed by a rapid decrease of rate toward the end of hydrolysis. With majority of literature studies focusing on the effect of key substrate characteristics on the initial rate or final yield of enzymatic hydrolysis, information about physicochemical structural changes of cellulosic substrates during enzymatic hydrolysis is still quite limited. Consequently, what slows down the reaction rate toward the end of hydrolysis is not well understood. Lastly, this review highlights recent advances in understanding the structural changes of cellulosic substrates during the hydrolysis process, to better understand the fundamental mechanisms of enzymatic hydrolysis.

09 BIOMASS FUELS↗
DOE OSTI2016

An in-depth understanding of biomass recalcitrance using natural poplar variants as the feedstock

Here, in an effort to better understand the biomass recalcitrance, six natural poplar variants were selected as feedstocks based on previous sugar release analysis. Compositional analysis and physicochemical characterizations of these poplars were performed and the correlations between these physicochemical properties and enzymatic hydrolysis yield were investigated. Gel permeation chromatography (GPC) and 13 C solid state NMR were used to determine the degree of polymerization (DP) and crystallinity index (CrI) of cellulose, and the results along with the sugar release study indicated that cellulose DP likely played a more important role in enzymatic hydrolysis. Simons’ stain revealed that the accessible surface area of substrate significantly varied among these variants from 17.3 to 33.2 mg g$–1\atop{biomass}$ as reflected by dye adsorption, and cellulose accessibility was shown as one of the major factors governing substrates digestibility. HSQC and 31 P NMR analysis detailed the structural features of poplar lignin variants. Overall, cellulose relevant factors appeared to have a stronger correlation with glucose release, if any, than lignin structural features. Lignin structural features, such as a phenolic hydroxyl group and the ratio of syringyl and guaiacyl (S/G), were found to have a more convincing impact on xylose release. Low lignin content, low cellulose DP, and high cellulose accessibility generally favor enzymatic hydrolysis; however, recalcitrance cannot be simply judged on any single substrate factor.

09 BIOMASS FUELS↗
DOE OSTI2016

Current understanding of the correlation of lignin structure with biomass recalcitrance

Lignin, a complex aromatic polymer in terrestrial plants, contributes significantly to biomass recalcitrance to microbial and/or enzymatic deconstruction. To reduce biomass recalcitrance, substantial endeavors have been exerted on pretreatment and lignin engineering in the past few decades. Lignin removal and/or alteration of lignin structure have been shown to result in reduced biomass recalcitrance with improved cell wall digestibility. While high lignin content is usually a barrier to a cost-efficient application of bioresources to biofuels, the direct correlation of lignin structure and its concomitant properties with biomass remains unclear due to the complexity of cell wall and lignin structure. Advancement in application of biorefinery to production of biofuels, chemicals, and bio-derived materials necessitates a fundamental understanding of the relationship of lignin structure and biomass recalcitrance. In this mini-review, we focus on recent investigations on the influence of lignin chemical properties on bioprocessability—pretreatment and enzymatic hydrolysis of biomass. Furthermore, lignin-enzyme interactions and the effects of lignin compositional units, hydroxycinnamates, and lignin functional groups on biomass recalcitrance have been highlighted, which will be useful not only in addressing biomass recalcitrance but also in deploying renewable lignocelluloses efficiently.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2016

Effect of torrefaction temperature on lignin macromolecule and product distribution from HZSM-5 catalytic pyrolysis

Torrefaction is a low-temperature process considered as an effective pretreatment technique to improve the grindability of biomass as well as enhance the production of aromatic hydrocarbons from Catalytic Fast Pyrolysis (CFP). For this paper, this study was performed to understand the effect of torrefaction temperature on structural changes in the lignin macromolecule and its subsequent influence on in-situ CFP process. Lignin extracted from southern pine and switchgrass (via organosolv treatment) was torrefied at four different temperatures (150, 175, 200 and 225 °C) in a tubular reactor. Between the two biomass types studied, lignin from pine appeared to have greater thermal stability during torrefaction when compared with switchgrass lignin. The structural changes in lignin as a result of torrefaction were followed by using FTIR spectroscopy, solid state CP/MAS 13 C NMR, 31 P NMR spectroscopy and it was found that higher torrefaction temperature (200 and 225 °C) caused polycondensation and de-methoxylation of the aromatic units of lignin. Gel permeation chromatography analysis revealed that polycondensation during torrefaction resulted in an increase in the molecular weight and polydispersity of lignin. The torrefied lignin was subsequently used in CFP experiments using H + ZSM-5 catalyst in a micro-reactor (Py-GC/MS) to understand the effect of torrefaction on the product distribution from pyrolysis. It was observed that although the selectivity of benzene-toluene-xylene compounds from CFP of pine improved from 58.3% (torrefaction temp at 150 °C) to 69.0% (torrefaction temp at 225 °C), the severity of torrefaction resulted in a loss of overall aromatic hydrocarbon yield from 11.6% to 4.9% under same conditions. Torrefaction at higher temperatures also increased the yield of carbonaceous residues from 63.9% to 72.8%. Finally, overall, torrefying lignin caused structural transformations in both type of lignins (switchgrass and pine), which is ultimately detrimental to achieving a higher aromatic hydrocarbon yield from CFP.

09 BIOMASS FUELS↗
DOE OSTI2016

Elucidating structural characteristics of biomass using solution-state 2 D NMR with a mixture of deuterated dimethylsulfoxide and hexamethylphosphoramide

In recent developments of NMR methods for characterization of lignocellulosic biomass allow improved understanding of plant cell-wall structures with minimal deconstruction and modification of biomass. This study introduces a new NMR solvent system composed of dimethylsulfoxide (DMSO- d 6 ) and hexamethylphosphoramide (HMPA- d 18 ). HMPA as a co-solvent enhanced swelling and mobility of the biomass samples; thereby it allowed enhancing signals of NMR spectra. Moreover, the structural information of biomass was successfully analyzed by the proposed NMR solvent system (DMSO- d 6 /HMPA-d 18 ; 4:1, v/v ) with different biomass. The proposed bi-solvent system does not require derivatization or isolation of biomass, facilitating a facile sample preparation and involving with no signals overlapping with biomass peaks. Furthermore, it also allows analyzing biomass with a room-temperature NMR probe instead of cryo-probes, which are traditionally used for enhancing signal intensities.

09 BIOMASS FUELS↗
DOE OSTI2016

Structure and Functional Properties of the Active Form of the Proteolytic Complex, ClpP1P2, from Mycobacterium tuberculosis

The ClpP protease complex and its regulatory ATPases, ClpC1 and ClpX, in Mycobacterium tuberculosis (Mtb) are essential and, therefore, promising drug targets. The Mtb ClpP protease consists of two heptameric rings, one composed of ClpP1 and the other of ClpP2 subunits. Formation of the enzymatically active ClpP1P2 complex requires binding of N-blocked dipeptide activators. We have found a new potent activator, benzoyl-leucine-leucine (Bz-LL), that binds with higher affinity and promotes 3–4-fold higher peptidase activity than previous activators. Bz-LL-activated ClpP1P2 specifically stimulates the ATPase activity of Mtb ClpC1 and ClpX. The ClpC1P1P2 and ClpXP1P2 complexes exhibit 2–3-fold enhanced ATPase activity, peptide cleavage, and ATP-dependent protein degradation. The crystal structure of ClpP1P2 with bound Bz-LL was determined at a resolution of 3.07 Å and with benzyloxycarbonyl-Leu-Leu (Z-LL) bound at 2.9 Å. Bz-LL was present in all 14 active sites, whereas Z-LL density was not resolved. Surprisingly, Bz-LL adopts opposite orientations in ClpP1 and ClpP2. In ClpP1, Bz-LL binds with the C-terminal leucine side chain in the S1 pocket. One C-terminal oxygen is close to the catalytic serine, whereas the other contacts backbone amides in the oxyanion hole. In ClpP2, Bz-LL binds with the benzoyl group in the S1 pocket, and the peptide hydrogen bonded between parallel β-strands. The ClpP2 axial loops are extended, forming an open axial channel as has been observed with bound ADEP antibiotics. Thus occupancy of the active sites of ClpP allosterically alters sites on the surfaces thereby affecting the association of ClpP1 and ClpP2 rings, interactions with regulatory ATPases, and entry of protein substrates.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2016

The occurrence of tricin and its derivatives in plants

Our understanding of the structure and biosynthetic pathway of lignin, a phenylpropanoid heteropolymer, continues to evolve, especially with the discovery of new lignin monomers/structural moieties such as monolignol acetate, hydroxycinnamyl aldehyde/alcohol, and p-hydroxybenzoate in the past decades. Recently, tricin has been reported as a component incorporated into monocot lignin. As a flavonoid compound widely distributed in herbaceous plants, tricin has been extensively studied due to its biological significance in plant growth as well as its potential for pharmaceutical importance. Tricin is biosynthesized as a constituent of plant secondary metabolites through a combination of phenylpropanoid and polyketide pathways. Tricin occurs in plants in either free or conjugated forms such as tricin-glycosides, tricin-lignans, and tricin-lignan-glycosides.The emergence of tricin covalently incorporated with lignin biopolymer implies the possible association of lignification and tricin biosynthesis. This review summarizes the occurrence of tricin and its derivatives in plants. Additionally, synthesis, potential application, and characterization of tricin are discussed.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2016

Analytical Methods for Biomass Characterization during Pretreatment and Bioconversion

Lignocellulosic biomass has been introduced as a promising resource for alternative fuels and chemicals because of its abundance and complement for petroleum resources. Biomass is a complex biopolymer and its compositional and structural characteristics largely vary depending on its species as well as growth environments. Because of complexity and variety of biomass, understanding its physicochemical characteristics is a key for effective biomass utilization. Characterization of biomass does not only provide critical information of biomass during pretreatment and bioconversion, but also give valuable insights on how to utilize the biomass. For better understanding biomass characteristics, good grasp and proper selection of analytical methods are necessary. This chapter introduces existing analytical approaches that are widely employed for biomass characterization during biomass pretreatment and conversion process. Diverse analytical methods using Fourier transform infrared (FTIR) spectroscopy, gel permeation chromatography (GPC), and nuclear magnetic resonance (NMR) spectroscopy for biomass characterization are reviewed. In addition, biomass accessibility methods by analyzing surface properties of biomass are also summarized in this chapter.

accessibility↗
DOE OSTI2015

Antigenic Determinants of the Bilobal Cockroach Allergen Bla g 2

Bla g 2 is a major indoor cockroach allergen associated with the development of asthma. Antigenic determinants on Bla g 2 were analyzed by mutagenesis based on the structure of the allergen alone and in complex with monoclonal antibodies that interfere with IgE antibody binding. The structural analysis revealed mechanisms of allergen-antibody recognition through cation-π interactions. Single and multiple Bla g 2 mutants were expressed in Pichia pastoris and purified. The triple mutant K132A/K251A/F162Y showed an ~100-fold reduced capacity to bind IgE, while preserving the native molecular fold, as proven by x-ray crystallography. This mutant was still able to induce mast cell release. T-cell responses were assessed by analyzing Th1/Th2 cytokine production and the CD4+ T-cell phenotype in peripheral blood mononuclear cell cultures. Although T-cell activating capacity was similar for the KKF mutant and Bla g 2 based on CD25 expression, the KKF mutant was a weaker inducer of the Th2 cytokine IL-13. Furthermore, this mutant induced IL-10 from a non-T-cell source at higher levels that those induced by Bla g 2. Lastly, our findings demonstrate that a rational design of site-directed mutagenesis was effective in producing a mutant with only 3 amino acid substitutions that maintained the same fold as wild type Bla g 2. These residues, which were involved in IgE antibody binding, endowed Bla g 2 with a T-cell modulatory capacity. The antigenic analysis of Bla g 2 will be useful for the subsequent development of recombinant allergen vaccines.

59 BASIC BIOLOGICAL SCIENCES↗