Enhancing fungal production of galactaric acid

Galactaric (mucic) acid is a symmetrical six carbon diacid which can be produced by oxidation of galactose with nitric acid, electrolytic oxidation of d-galacturonate or microbial conversion of d-galacturonate. Both salts and the free acid of galactarate have relatively low solubility, which may create challenges for a microbial host. Galactaric acid was most soluble at pH values around 4.7 in the presence of ammonium or sodium ions and less soluble in the presence of potassium ions. Solubility increased with increasing temperature. Production of galactaric acid by Trichoderma reesei D-161646 was dependent on temperature, pH and medium composition, being best at pH 4 and 35 °C. Up to 20 g L^?1 galactaric acid were produced from d-galacturonate using a fed-batch strategy with lactose as co-substrate and both ammonium and yeast extract as nitrogen sources. Crystals of galactaric acid were observed to form in the broth of some fermentations.

     

Production of optically pure d-lactic acid from brown rice using metabolically engineered Lactobacillus plantarum

Simultaneous saccharification and fermentation (SSF) of d-lactic acid was performed using brown rice as both a substrate and a nutrient source. An engineered Lactobacillus plantarum NCIMB 8826 strain, in which the ʟ-lactate dehydrogenase gene was disrupted, produced 97.7 g/L d-lactic acid from 20% (w/v) brown rice without any nutrient supplementation. However, a significant amount of glucose remained unconsumed and the yield of lactic acid was as low as 0.75 (g/g-glucose contained in brown rice). Interestingly, the glucose consumption was significantly improved by adapting L. plantarum cells to the low-pH condition during the early stage of SSF (8–17 h). As a result, 117.1 g/L d-lactic acid was produced with a high yield of 0.93 and an optical purity of 99.6% after 144 h of fermentation. SSF experiments were repeatedly performed for ten times and d-lactic acid was stably produced using recycled cells (118.4–129.8 g/L). On average, d-lactic acid was produced with a volumetric productivity of 2.18 g/L/h over 48 h.

     

Enhancing l-malate production of Aspergillus oryzae FMME218-37 by improving inorganic nitrogen utilization

Microbial l-malate production from renewable feedstock is a promising alternative to petroleum-based chemical synthesis. However, high l-malate production of Aspergillus oryzae was achieved to date using organic nitrogen, with inorganic nitrogen still unable to meet industrial applications. In the current study, we constructed a screening system and nitrogen supply strategy to improve l-malate production with ammonium sulphate [(NH₄)₂SO₄] as the sole nitrogen source. First, we generated and identified a high-producing mutant FMME218-37, which stably boosted l-malate production from 30.73 to 78.12 g/L, using a combined screening system with morphological characteristics. Then, by analyzing the fermentation parameters and physiological characteristics, we further speculated the key factor was the unbalance of carbon and nitrogen absorption. Finally, the titer and productivity of l-malate was increased to 95.2 g/L and 0.57 g/(L h) by regulating the nitrogen supply module to balance carbon and nitrogen absorption, which represented the highest level in A. oryzae with (NH₄)₂SO₄ as nitrogen source achieved to date. Moreover, our findings using a low-cost substrate may lead to building an economical cell factory of A. oryzae for l-malate production.

     

l-3-n-Butylphthalide Activates Akt/mTOR Signaling,Inhibits Neuronal Apoptosis and Autophagy and Improves Cognitive Impairment in Mice with Repeated Cerebral Ischemia–Reperfusion Injury

l-3-n-Butylphthalide (l-NBP) exerts neuroprotective effects in animal models of cerebral ischemia, but its potential benefits in repeated cerebral ischemia–reperfusion (RCIR) injury remain unknown. We investigated the effect of l-NBP on cognitive impairment induced by RCIR in mice. Male C57Bl/6 mice received sham surgery or bilateral common carotid artery occlusion (3 times, 20 min each) and were orally administered preoperative l-NBP (30 mg/kg/day, 7 days), postoperative l-NBP (30 or 60 mg/kg/day, 28 days) or postoperative vehicle (28 days). Learning and memory were assessed by the Morris water maze task and step-down passive avoidance test. Nissl staining was used to identify pathologic changes in the hippocampal CA1 region. The expressions of proteins associated with signaling, apoptosis and autophagy were assessed by quantitative PCR and western blot. RCIR induced deficits in learning and memory that were alleviated by preoperative or postoperative l-NBP administration. Pathologic lesions in the hippocampal CA1 region induced by RCIR were less severe in mice treated with l-NBP. Preoperative or postoperative l-NBP administration in mice receiving RCIR promoted hippocampal expression of phospho-Akt and phospho-mTOR (suggesting activation of Akt/mTOR signaling), increased the Bcl-2/Bax ratio (indicating suppression of apoptosis) and reduced the LC3-II/LC3-I ratio (implying inhibition of autophagy). Preoperative or postoperative l-NBP administration also depressed hippocampal levels of beclin-1 mRNA (indicating suppression of autophagy). These findings suggest that the effect of l-NBP to alleviate learning and memory deficits in mice following RCIR may involve activation of Akt/mTOR signaling and regulation of the expressions of proteins related to apoptosis and autophagy.

     

Purification,characterization, gene cloning and nucleotide sequencing of D-stereospecific amino acid amidase from soil bacterium: Delftia acidovorans

The D-amino acid amidase-producing bacterium was isolated from soil samples using an enrichment culture technique in medium broth containing D-phenylalanine amide as a sole source of nitrogen. The strain exhibiting the strongest activity was identified as Delftia acidovorans strain 16. This strain produced intracellular D-amino acid amidase constitutively. The enzyme was purified about 380-fold to homogeneity and its molecular mass was estimated to be about 50 kDa, on sodium dodecyl sulfate polyacrylamide gel electrophoresis. The enzyme was active preferentially toward D-amino acid amides rather than their L-counterparts. It exhibited strong amino acid amidase activity toward aromatic amino acid amides including D-phenylalanine amide, D-tryptophan amide and D-tyrosine amide, yet it was not specifically active toward low-molecular-weight D-amino acid amides such as D-alanine amide, L-alanine amide and L-serine amide. Moreover, it was not specifically active toward oligopeptides. The enzyme showed maximum activity at 40°C and pH 8.5 and appeared to be very stable, with 92.5% remaining activity after the reaction was performed at 45°C for 30 min. However, it was mostly inactivated in the presence of phenylmethanesulfonyl fluoride or Cd²⁺, Ag⁺, Zn²⁺, Hg²⁺ and As³⁺ . The NH₂ terminal and internal amino acid sequences of the enzyme were determined; and the gene was cloned and sequenced. The enzyme gene damA encodes a 466-amino-acid protein (molecular mass 49,860.46 Da); and the deduced amino acid sequence exhibits homology to the D-amino acid amidase from Variovorax paradoxus (67.9% identity), the amidotransferase A subunit from Burkholderia fungorum (50% identity) and other enantioselective amidases.

     

Production of d-lactic acid by Lactobacillus delbrueckii ssp. delbrueckii from orange peel waste: techno-economical assessment of nitrogen sources

In this study, the effect of several organic nitrogen sources (namely peptone, meat extract—ME, yeast extract—YE, and corn steep liquor—CSL) on d-lactic acid production by Lactobacillus delbrueckii ssp. delbrueckii has been studied. While lactic acid bacteria (LAB) are well-known for their complex nutritional requirements, organic nitrogen source-related cost can be as high as 38% of total operational costs (OPEX), being its nature and concentration critical factors in the growth and productivity of the selected strain. Corn steep liquor (CSL) has been chosen for its adequacy, on the grounds of the d-lactic acid yield, productivity, and its cost per kilogram of product. Finally, orange peel waste hydrolysate supplemented with 37 g/l CSL has been employed for d-lactic acid production, reaching a final yield of 88% and a productivity of 2.35 g/l h. CSL cost has been estimated at 90.78$/ton of d-lactate.

     

Comprehensive assessment of the l-lysine production process from fermentation of sugarcane molasses

l-Lysine is an essential amino acid that can be produced by chemical processes from fossil raw materials, as well as by microbial fermentation, the latter being a more efficient and environmentally friendly procedure. In this work, the production process of l-lysine-HCl is studied using a systematic approach based on modeling and simulation, which supports decision making in the early stage of process design. The study considers two analysis stages: first, the dynamic analysis of the fermentation reactor, where the conversion of sugars from sugarcane molasses to l-lysine with a strain of Corynebacterium glutamicum is carried out. In this stage, the operation mode (either batch or fed batch) and operating conditions of the fermentation reactor are defined to reach the maximum technical criteria. Afterwards, the second analysis stage relates to the industrial production process of l-lysine-HCl, where the fermentation reactor, upstream processing, and downstream processing are included. In this stage, the influence of key parameters on the overall process performance is scrutinized through the evaluation of several technical, economic, and environmental criteria, to determine a profitable and sustainable design of the l-lysine production process. The main results show how the operating conditions, process design, and selection of evaluation criteria can influence in the conceptual design. The best plant design shows maximum product yield (0.31 g l-lysine/g glucose) and productivity (1.99 g/L/h), achieving 26.5% return on investment (ROI) with a payback period (PBP) of 3.8 years, decreasing water and energy consumption, and with a low potential environmental impact (PEI) index.

     

Sodium ions activated phosphofructokinase leading to enhanced d-lactic acid production by Sporolactobacillus inulinus using sodium hydroxide as a neutralizing agent

Sporolactobacillus inulinus is a superior d-lactic acid-producing bacterium and proposed species for industrial production. The major pathway for d-lactic acid biosynthesis, glycolysis, is mainly regulated via the two irreversible steps catalyzed by the allosteric enzymes, phosphofructokinase (PFK) and pyruvate kinase. The activity level of PFK was significantly consistent with the cell growth and d-lactic acid production, indicating its vital role in control and regulation of glycolysis. In this study, the ATP-dependent PFK from S. inulinus was expressed in Escherichia coli and purified to homogeneity. The PFK was allosterically activated by both GDP and ADP and inhibited by phosphoenolpyruvate; the addition of activators could partly relieve the inhibition by phosphoenolpyruvate. Furthermore, monovalent cations could enhance the activity, and Na⁺ was the most efficient one. Considering this kind activation, NaOH was investigated as the neutralizer instead of the traditional neutralizer CaCO₃. In the early growth stage, the significant accelerated glucose consumption was achieved in the NaOH case probably for the enhanced activity of Na⁺-activated PFK. Using NaOH as the neutralizer at pH 6.5, the fermentation time was greatly shortened about 22 h; simultaneously, the glucose consumption rate and the d-lactic acid productivity were increased by 34 and 17%, respectively. This probably contributed to the increased pH and Na⁺-promoted activity of PFK. Thus, fermentations by S. inulinus using the NaOH neutralizer provide a green and highly efficient d-lactic acid production with easy subsequent purification.

     

An aldonolactonase AltA from Penicillium oxalicum mitigates the inhibition of β-glucosidase during lignocellulose biodegradation

Efficient deconstruction of lignocellulose is achieved by the synergistic action of various hydrolytic and oxidative enzymes. However, the aldonolactones generated by oxidative enzymes have inhibitory effects on some cellulolytic enzymes. In this work, d-glucono-1,5-lactone was shown to have a much stronger inhibitory effect than d-glucose and d-gluconate on β-glucosidase, a vital enzyme during cellulose degradation. AltA, a secreted enzyme from Penicillium oxalicum, was identified as an aldonolactonase which can catalyze the hydrolysis of d-glucono-1,5-lactone to d-gluconic acid. In the course of lignocellulose saccharification conducted by cellulases from P. oxalicum or Trichoderma reesei, supplementation of AltA was able to relieve the decrease of β-glucosidase activity obviously with a stimulation of glucose yield. This boosting effect disappeared when sodium azide and ethylenediaminetetraacetic acid (EDTA) were added to the saccharification system to inhibit the activities of oxidative enzymes. In summary, we describe the first heterologous expression of a fungal secreted aldonolactonase and its application as an efficient supplement of cellulolytic enzyme system for lignocellulose biodegradation.

     

A type D ferulic acid esterase from Streptomyces werraensis affects the volume of wheat dough pastries

A type D ferulic acid esterase (FAE) was identified in the culture supernatant of Streptomyces werraensis, purified, sequenced, and heterologously produced in E. coli BL21(DE3)Star by co-expressing chaperones groES-groEL (69 U L⁻¹). The unique enzyme with a mass of about 48 kDa showed no similarity to other FAEs, and only moderate homology (78.5%) to a Streptomycete β-xylosidase. The purified reSwFAED exhibited a temperature optimum of 40 °C, a pH optimum in the range from pH seven to eight and a clear preference for bulky natural substrates, such as 5-O-trans-feruloyl-l-arabinofuranose (FA) and β-d-xylopyranosyl-(1→2)-5-O-trans-feruloyl-l-arabinofuranose (FAX), compared to the synthetic standard substrate methyl ferulate. Treatment of wheat dough with as little as 0.03 U or 0.3 U kg⁻¹ reSwFAED activity resulted in a significant increase of the bun volume (8.0 or 9.7%, resp.) after baking when combined with polysaccharide-degrading enzymes from Aspergillus. For the first time, the long-standing, but rarely proven positive effect of a FAE in baking was confirmed.

     

Thermostable and highly specific l-aspartate oxidase from Thermococcus litoralis DSM 5473: cloning,overexpression, and enzymological properties

We successfully expressed the l-aspartate oxidase homolog gene (accession no: OCC_06611) of Thermococcus litoralis DSM 5473 in the soluble fraction of Escherichia coli BL21 (DE3) using a pET21b vector with 6X His tag at its C-terminus. The gene product (Tl-LASPO) showed l-aspartate oxidase activity in the presence of FAD in vitro, and this report is the first that details an l-aspartate oxidase derived from a Thermococcus species. The homologs of Tl-LASPO existed mainly in archaea, especially in the genus of Thermococcus, Pyrococcus, Sulfolobus, and Halobacteria. The quaternary structure of Tl-LASPO was homotrimeric with a subunit molecular mass of 52 kDa. The enzyme activity of Tl-LASPO increased with temperature up to 70 °C. Tl-LASPO was active from pH 6.0 to 9.0, and its highest activity was at pH 8.0. Tl-LASPO was stable at 80 °C for 1 h. The highest k _cat/K _m value was observed in assays at 70 °C. Tl-LASPO was highly specific for l-aspartic acid. Tl-LASPO utilized fumaric acid, 2,6-dichlorophenolindophenol, and ferricyanide in addition to FAD as a cofactor under anaerobic conditions. The absorption spectrum of holo-Tl-LASPO exhibited maxima at 380 and 450 nm. The FAD dissociation constant, K _d, of the FAD-Tl-LASPO complex was determined to be 5.9 × 10⁻⁹ M.

     

An integrative expression vector for <Emphasis Type="Italic">Actinosynnema pretiosum</Emphasis>

Background  

The Actinomycete Actinosynnema pretiosum ssp. auranticum has commercial importance due to its production of ansamitocin P-3 (AP-3), a potent antitumor agent. One way to increase AP-3 production would be to constitutively express selected genes so as to relieve bottlenecks in the biosynthetic pathway; however, an integrative expression vector for A. pretiosum is lacking. The aim of this study was to construct a vector for heterologous gene expression in A. pretiosum.     

An Update on Design and Pharmacology of Dendritic Poly(l-lysine)

Dendritic nanomaterials are unique due to their flexible architectures. So far, many structural analogues of dendritic poly(l-lysine) have been developed. Since its monomer unit is a biodegradable amino acid, poly(l-lysine) derived nanocarriers are biocompatible and safe. In this overview, structural diversity of dendritic poly(l-lysine) scaffold and patents filed on them so far are described. Furthermore, biopharmaceutical properties and therapeutic activity modulations observed from their drug delivery applications are highlighted. Poly(l-lysine) based dendriplexes, dendrosomes and dendrisomes remain novel and nearly unexplored. Since structural modifications can control the biopharmaceutical properties of aforementioned scaffold, achieving programmed drug delivery is possible. Many such structures have demonstrated not only excellent carrier characteristics but few intrinsic therapeutic activities also. A poly(l-lysine) dendrimer product VivaGel is currently under consideration in a new drug application category of various regulatory bodies. As dendritic poly(l-lysine) scaffold is biocompatible unlike many other nanocarriers, its clinical utilization would prove considerably beneficial.

     

Fusion of agarase and neoagarobiose hydrolase for mono-sugar production from agar

In enzymatic saccharification of agar, endo- and exo-agarases together with neoagarobiose hydrolase (NABH) are important key enzymes for the sequential hydrolysis reactions. In this study, a bifunctional endo/exo-agarase was fused with NABH for production of mono-sugars (d-galactose and 3,6-anhydro-l-galactose) from agar using only one fusion enzyme. Two fusion enzymes with either bifunctional agarase (Sco3476) or NABH (Zg4663) at the N-terminus, Sco3476–Zg4663 (SZ) and Zg4663–Sco3476 (ZS), were constructed. Both fusion enzymes exhibited their optimal agarase and NABH activities at 40 and 35 °C, respectively. Fusions SZ and ZS enhanced the thermostability of the NABH activity, while only fusion SZ showed a slight enhancement in the NABH catalytic efficiency (K _cat/K _M) from 14.8 (mg/mL)⁻¹ s⁻¹ to 15.8 (mg/mL)⁻¹ s⁻¹. Saccharification of agar using fusion SZ resulted in 2-fold higher mono-sugar production and 3-fold lower neoagarobiose accumulation when compared to the physical mixture of Sco3476 and Zg4663. Therefore, this fusion has the potential to reduce enzyme production cost, decrease intermediate accumulation, and increase mono-sugar yield in agar saccharification.

     

Stabilization of Angiotensin-(1-7) in Cardioprotective Solutions

Angiotensin-(1-7) (Ang 1-7) has been previously studied in combination with an antioxidant containing preparation as a cardioprotective reperfusion solution. In this study a stability improvement of aqueous Ang 1-7 solutions was observed. However, no data was provided on the responsibilities and causes of the noticed stability enhancement. Therefore, the influence of pH and pharmaceutical additives as well as the effect of the single specific agents present in the antioxidant preparation such as α-ketoglutaric acid (α-KG), 5-hydroxymethylfurfural (5-HMF), N-acetyl-seleno-l-methionine (NASeLM) and N-acetyl-l-methionine (NALM) on the stability was evaluated. Analyses were performed by an HPLC method with fluorescence detection. Crucial instability was found in a pH range of 5.0–7.5 without addition of the antioxidative mixture. Zetasizing confirmed the presence of microparticles and MS studies showed no degradation products within 25 days. 5-HMF was identified as main component for stability enhancement of Ang 1-7 solution. By adding this substance the stability of the cardioprotective peptide solution can be prolonged and appears as a promising approach for transplant purposes.

     

Enzymatic liquefaction of agarose above the sol–gel transition temperature using a thermostable endo-type β-agarase,Aga16B

The main carbohydrate of red macroalgae is agarose, a heterogeneous polysaccharide composed of d-galactose and 3,6-anhydro-l-galactose. When saccharifying agarose by enzymes, the unique physical properties of agarose, namely the sol–gel transition and the near-insolubility of agarose in water, limit the accessibility of agarose to the enzymes. Due to the lower accessibility of agarose to enzymes in the gel state than to the sol state, it is important to prevent the sol–gel transition by performing the enzymatic liquefaction of agarose at a temperature higher than the sol–gel transition temperature of agarose. In this study, a thermostable endo-type β-agarase, Aga16B, originating from Saccharophagus degradans 2-40^T, was characterized and introduced in the liquefaction process. Aga16B was thermostable up to 50 °C and depolymerized agarose mainly into neoagarooligosaccharides with degrees of polymerization 4 and 6. Aga16B was applied to enzymatic liquefaction of agarose at 45 °C, which was above the sol–gel transition temperature of 1 % (w/v) agarose (∼35 °C) when cooling agarose. This is the first systematic demonstration of enzymatic liquefaction of agarose, enabled by determining the sol–gel temperature of agarose under specific conditions and by characterizing the thermostability of an endo-type β-agarase.