Characterization of isolated starch from Isatis indigotica Fort. root and anhydro-sugars preparation using its decoction residues.

Isatis indigotica Fort. root (Ban-lan-gen, IIR), a traditional Chinese medicine (TCM), has an ancient and well-documented history for its medicinal properties. Aside from epigoitrin, indole alkaloids, and their corresponding derivatives as medicinal ingredients, it also contains lots of biomass such as starch. Herein, a new starch was isolated from IIR and the physicochemical properties such as amylose content, moisture content, ash content, morphology, thermal properties, and crystallography were characterized systematically. The amylose content of IIR starch was 19.84 ± 0.85%, and the size and shape of starch granules is ellipsoidal shape with sizes from 2 to 10 µm. IIR starch exhibited a C-type pattern and had 25.92% crystallinity (higher than that of corn starch). The gelatinization temperature of IIR starch was 58.68-75.41 °C, and its gelatinization enthalpy was ΔH gel = 4.33 J/g. After decocting, the IIR's residues can be used to prepare anhydro-sugars in a polar aprotic solvent. The total carbon yield of levoglucosan (LG), levoglucosenone (LGO), 5-hydroxymethylfurfural (HMF), and furfural (FF) could reach 69.81% from IIR's decoction residues in 1,4-dioxane with 15 mM H2SO4 as the catalyst. Further, the residues after dehydration were prepared into biochar by thermochemical conversion and the BET surface area of biochar was 1749.46 m2/g which has good application prospect in soil improvement and alleviates obstacles of IIR continuous cropping.

D-Lactic acid production by Sporolactobacillus inulinus YBS1-5 with simultaneous utilization of cottonseed meal and corncob residue.

d-Lactic acid, is an important organic acid produced from agro-industrial wastes by Sporolactobacillus inulinus YBS1-5 was investigated to reduce the raw material cost of fermentation. The YBS1-5 strain could produce d-lactic acid by using cottonseed meal as the sole nitrogen source. For efficient utilization, the cottonseed meal was enzymatically hydrolyzed and simultaneously utilized during d-lactic acid fermentation. Corncob residues are rich in cellulose and can be enzymatically hydrolyzed without pretreatment. The hydrolysate of this lignocellulosic waste could be utilized by strain YBS1-5 as a carbon source for d-lactic acid production. Under optimal conditions, a high d-lactic acid concentration (107.2g/L) was obtained in 7-L fed-batch fermenter, with an average productivity of 1.19g/L/h and a yield of 0.85g/g glucose. The optical purity of d-lactic acid in the broth was 99.2%. This study presented a new approach for low-cost production of d-lactic acid for an industrial application.

Effect of lignocellulose-derived inhibitors on the growth and D-lactic acid production of Sporolactobacillus inulinus YBS1-5.

The impact of lignocellulose-derived inhibitors on the cell growth and D-lactic production of Sporolactobacillus inulinus YBS1-5 was investigated. At high concentrations, both furans and phenolics, such as furfural, HMF, syringaldehyde and vanillin, affected cell growth and D-lactic acid production and syringaldehyde exhibited the highest. Further experiments showed that only vanillin caused cellular membrane damage. Based on the Biolog approach, in vivo studies on intact S. inulinus cells indicated that phenolics had a stronger inhibitory effect than furan derivatives on the metabolic activity of the concerned substrates related with the key enzymes of D-lactic acid fermentation. The direct in vitro inhibitory effect of the model compounds on the four key enzymes displayed similar patterns. Syringaldehyde was the strongest inhibitor. In general, comparison with published results for other microorganisms indicated that strain YBS1-5 was a robust microorganism against inhibitors of lignocellulose hydrolysate. Notably, in concentrated corn stover hydrolysate, S. inulinus YBS1-5 produced 70.7 g/L D-lactic acid, which was 87.7 % of the yield from the control experiment. However, the fermentation time was prolonged 36 h. In order to improve fermentation rate, a detoxification technology or more robust mutant to phenolics especially syringaldehyde should be developed.

Enhanced production of d-lactic acid by Sporolactobacillus sp.Y2-8 mutant generated by atmospheric and room temperature plasma.

To improve the production of d-lactic acid, atmospheric and room temperature plasma (ARTP) was used to generate mutations in Sporolactobacillus sp. Y2-8. An efficient mutant YBS1-5 was rapidly isolated by implanting ARTP twice with a 100 W radio-frequency power input, 10 standard liters per minute of the helium flow, and a 2 mm treatment distance. Significant improvement of d-lactic acid productivity (1.39 g L(-1) H(-1) ) by YBS1-5 was achieved, and it was 41.84% higher than the productivity (0.98 g L(-1) H(-1) ) of Y2-8. Moreover, the dry cell weight of YBS1-5 was 16.7% higher than that of Y2-8. Metabolic activities of concerned substrates related with key enzymes of d-lactic acid fermentation were analyzed by Biolog approach. Results showed that the activities of the key enzymes glucokinase and d-lactate dehydrogenase in mutant YBS1-5 were increased by approximately 45% and 66%, respectively, in comparison with those of the strain Y2-8. Fed-batch fermentation further improved the productivity; 127 g L(-1) d-lactic acid in 74 H by YBS1-5 with higher productivity (1.72 g L(-1) H(-1) ) was achieved. The subculture experiments indicated that YBS1-5 was genetically stable after eight generations.

Mn²âº/Mg ²âº-dependent pyruvate kinase from a D-lactic acid-producing bacterium Sporolactobacillus inulinus: characterization of a novel Mn²âº-mediated allosterically regulated enzyme.

Sporolactobacillus inulinus has attracted scientific and commercial interest due to its high efficiency in D-lactic acid production. Pyruvate kinase (PYK) is one of the key regulatory points in glycolysis, and well-activated PYK can improve D-lactic acid production. A novel Mn(2+)/Mg(2+)-dependent PYK from S. inulinus was expressed in Escherichia coli and purified to homogeneity. Kinetic characterization demonstrated that the S. inulinus PYK had drastically higher activity and affinity toward substrates in the presence of Mn(2+) compared to those of the common PYK cofactor Mg(2+), and the circular dichroism spectra of the S. inulinus PYK suggested a Mn(2+)-mediated allosteric activation. The S. inulinus PYK was also allosterically regulated by ribose-5-phosphate or AMP activation and inorganic phosphate or ATP inhibition. The inhibition could be marked reduced or fully eliminated in the presence of activators. The result of fermentations by S. inulinus Y2-8 showed that the extracellular-added MnSO4 and KH2PO4 significantly affected glycolysis flux and D-lactic acid production, which is consistent with the allosteric regulation of Mn(2+) and inorganic phosphate on PYK. The sophisticated regulatory role of PYK would establish the foundation of substantial disturbance or restructuring of cellular metabolism for improving the S. inulinus D-lactic acid production.

Highly efficient synthesis of endomorphin-2 under thermodynamic control catalyzed by organic solvent stable proteases with in situ product removal.

An efficient enzymatic synthesis of endomorphin-2 (EM-2) was achieved using organic solvent stable proteases in nonaqeous media, based on thermodynamic control and an in situ product removal methodology. The high stability of biocatalysts in organic solvents enabled the aleatoric modulation of the nonaqueous reaction media to shift thermodynamic equilibrium toward synthesis. Peptide Boc-Phe-Phe-NH2 was synthesized with a high yield of 96% by the solvent stable protease WQ9-2 in monophase medium with an economical molar ratio of the substrate of 1:1. The tetrapeptide Boc-Tyr-Pro-Phe-Phe-NH2 was synthesized with a yield of 88% by another organic solvent tolerant protease PT121 from Boc-Tyr-Pro-OH and Phe-Phe-NH2 in an organic-aqueous biphasic system. The reaction-separation coupling in both enzymatic processes provides "driving forces" for the synthetic reactions and gives a high yield and high productivity without purification of the intermediate, thereby making the synthesis more amenable to scale-up.

Glucokinase contributes to glucose phosphorylation in D-lactic acid production by Sporolactobacillus inulinus Y2-8.

Sporolactobacillus inulinus, a homofermentative lactic acid bacterium, is a species capable of efficient industrial D-lactic acid production from glucose. Glucose phosphorylation is the key step of glucose metabolism, and fine-tuned expression of which can improve D-lactic acid production. During growth on high-concentration glucose, a fast induction of high glucokinase (GLK) activity was observed, and paralleled the patterns of glucose consumption and D-lactic acid accumulation, while phosphoenolpyruvate phosphotransferase system (PTS) activity was completely repressed. The transmembrane proton gradient of 1.3-1.5 units was expected to generate a large proton motive force to the uptake of glucose. This suggests that the GLK pathway is the major route for glucose utilization, with the uptake of glucose through PTS-independent transport systems and phosphorylation of glucose by GLK in S. inulinus D-lactic acid production. The gene encoding GLK was cloned from S. inulinus and expressed in Escherichia coli. The amino acid sequence revealed significant similarity to GLK sequences from Bacillaceae. The recombinant GLK was purified and shown to be a homodimer with a subunit molecular mass of 34.5 kDa. Strikingly, it demonstrated an unusual broad substrate specificity, catalyzing phosphorylation of 2-deoxyglucose, mannitol, maltose, galactose and glucosamine, in addition to glucose. This report documented the key step concerning glucose phosphorylation of S. inulinus, which will help to understand the regulation of glucose metabolism and D-lactic acid production.

Efficient production of D-glucosaminic acid from D-glucosamine by Pseudomonas putida GNA5.

D-glucosaminic acid was produced efficiently from glucosamine by oxidative fermentation using a newly isolated strain, Pseudomonas putida GNA5. After optimization of the fermentation process, 51.5 g L(-1) D-glucosaminic acid was produced from an initial concentration of 60 g L(-1) D-glucosamine-HCl after 72 h of oxidative fermentation, which corresponded to a molar yield of 95.4%. This production process is potentially of considerable economic significance because very few by-products were detected. Furthermore, D-glucosaminic acid was accumulated stably during the oxidative fermentation process without the addition of an inhibitor of D-glucosaminic acid breakdown, even though D-glucosamine was exhausted. These results suggest that the mechanisms of D-glucosaminic acid-related metabolism differ between Pseudomonas putida GNA5 and the strain Pseudomonas genera, which was previously reported to produce D-glucosaminic acid.

Breeding of D(-)-lactic acid high producing strain by low-energy ion implantation and preliminary analysis of related metabolism.

The low-energy nitrogen ion beam implantation technique was used in the breeding of mutant D(-)-lactic-acid-producing strains. The wild strain Sporolactobacillus sp. DX12 was mutated by an N(+) ion beam with energy of 10keV and doses ranging from 0.4 x 10(15) to 6.60 x 10(15) ions/cm(2). Combined with an efficient screening method, an efficient mutant Y2-8 was selected after two times N(+) ion beam implantation. By using the mutant Y2-8, 121.6g/l of D-lactic acid was produced with the molar yields of 162.1% to the glucose. The yield of D-lactic acid by strain Y2-8 was 198.8% higher than the wild strain. Determination of anaerobic metabolism by Biolog MT2 was used to analyze the activities of the concerned enzymes in the lactic acid metabolic pathway. The results showed that the activities of the key enzymes responded on the substrates such as 6-phosphofructokinase, pyruvate kinase, and D-lactate dehydrogenase were considerably higher in the mutants than the wild strain. These might be affected by ion beam implantation.