•This work is the first time reported in research of structure and bioactivity of an alkali-soluble single-component polysaccharide (named CPTC-2) isolated and purified from Taxus chinensis var. mairei by ion-exchange and gel-permeation chromatography in series.•The authors used a variety of methods to analyze the structure properties of polysaccharide and get a fully understanding of the structure of CPTC-2. Moreover, the combination of MTS assay and flow cytometry method was adopted to study the antitumor function of CPTC-2.•Finally, the partial relationship between structure of the polysaccharide and its antitumor activity was obtained.An alkali-soluble single-component polysaccharide, named CPTC-2, was isolated and purified from the leaves of Taxus chinensis var. mairei. by ion-exchange and gel-permeation chromatography in series. The weight-average molecular mass (Mw) of CPTC-2 was about 73.53 kDa determined by gel permeation chromatography (GPC). The structural characteristics of CPTC-2 were analyzed by gas chromatography (GC), Infrared (IR) spectrum, nuclear magnetic resonance (NMR) spectroscopy, periodate oxidation and Smith degradation studies, as well as methylation analysis. The results showed that CPTC-2 consisted of glucose, mannose, xylose, arabinose, rhamnose, and galactose with a molar ratio of 1.00:0.32:0.27:3.34:1.22:1.84. CPTC-2 was mainly composed of one type of sugar, α-glycosidic linkage. It had a backbone composed of α-(1 → 3) Araf, α-(1 → 5) Araf and α-(1 → 4) Galp with branches composed of α-(1 → 3,5) Araf and β-(1 → 3,6) Manp. In vitro anti-tumor experiments with SGC-7901 cells were performed and assessed by MTS (MPEG-2 Transport Stream) method and flow cytometry. The results showed CPTC-2 could inhibit the growth of SGC-7901 cells in a concentration-dependent manner via increased apoptosis. The relationship between the structure of CPTC-2 and its anti-tumor activity indicated that the α-configuration glycosidic bond residues may be essential for the anti-tumor activity of this polysaccharide.

The Rhodobacter capsulatushemA gene, which encodes 5-aminolevulinic acid synthase (ALAS), was expressed in Escherichia coli Rosetta (DE3) and the enzymatic properties of the purified recombinant ALAS (RC-ALAS) were studied. Compared with ALASs encoded by hemA genes from Agrobacterium radiobacter (AR-ALAS) and Rhodobacter sphaeroides (RS-ALAS), the specific activity of RC-ALAS reached 198.2 U/mg, which was about 31.2% and 69.5% higher than those of AR-ALAS (151.1 U/mg) and RS-ALAS (116.9 U/mg), respectively. The optimum pH values and temperatures of the three above mentioned enzymes were all pH 7.5 and 37 °C, respectively. Moreover, RC-ALAS was more sensitive to pH, while the other two were sensitive to temperature. The effects of metals, ethylene diamine tetraacetic acid (EDTA), and sodium dodecyl sulfate (SDS) on the three ALASs were also investigated. The results indicate that they had the same effects on the activities of the three ALASs. SDS and metal ions such as Co2+, Zn2+, and Cu2+ strongly inhibited the activities of the ALASs, while Mn2+ exerted slight inhibition, and K+, Ca2+, Ba2+, Mg2+, or EDTA had no significant effect. The specificity constant of succinyl coenzyme A [(kcat/Km)S-CoA] of RC-ALAS was 1.4989, which was higher than those of AR-ALAS (0.7456) and RS-ALAS (1.1699), showing its high catalytic efficiency. The fed-batch fermentation was conducted using the recombinant strain containing the R. capsulatushemA gene, and the yield of 5-aminolevulinic acid (ALA) achieved was 8.8 g/L (67 mmol/L) under the appropriate conditions.

Xylitol is a five-carbon sugar alcohol with potential for use as a sweetener. Industrially, xylitol is currently produced by chemical hydrogenation of d-xylose using Raney nickel catalysts and this requires expensive separation and purification steps as well as high pressure and temperature that lead to environmental pollution. Highly efficient biotechnological production of xylitol using microorganisms is gaining more attention and has been proposed as an alternative process. Although the biotechnological method has not yet surpassed the advantages of chemical reduction in terms of yield and cost, various strategies offer promise for the biotechnological production of xylitol. In this review, the focus is on the most recent developments of the main metabolic engineering strategies for improving the production of xylitol.

A 24-membered ring macrolide compound, macrolactin A has potential applications in pharmaceuticals for its anti-infectious and antiviral activity. In this study, macrolactin A was produced by a marine bacterium, which was identified as Bacillus subtilis by 16S ribosomal RNA (rRNA) sequence analysis. Electrospray ionization mass spectrometry (ESI/MS) and nuclear magnetic resonance (NMR) spectroscopy analyses were used to characterize this compound. To improve the production, response surface methodology (RSM) involving Box-Behnken design (BBD) was employed. Faeces bombycis, the main by-product in sericulture, was used as a nitrogen source in fermentation. The interactions between three significant factors, F. bombycis, soluble starch, and (NH4)2SO4 were investigated. A quadratic model was constructed to fit the production and the factors. Optimum medium composition was obtained by analysis of the model. When cultivated in the optimum medium, the production of macrolactin A was increased to 851 mg/L, 2.7 times as compared to the original. This study is also useful to find another way in utilizing F. bombycis.

The 5-aminolevulinate (ALA) synthase gene (hemA) from Agrobacterium radiobacter zju-0121, which was cloned previously in our laboratory, contains several rare codons. To enhance the expression of this gene, Escherichia coli Rosetta(DE3), which is a rare codon optimizer strain, was picked out as the host to construct an efficient recombinant strain. Cell extracts of the recombinant E. coli were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under the appropriate conditions. The results indicated that the activity of ALA synthase expressed in Rosetta(DE3)/pET-28a(+)-hemA was about 20% higher than that in E. coli BL21(DE3). Then the effects of precursors (glycine and succinate) and glucose, which is an inhibitor for ALA dehydratase as well as the carbon sources for cell growth, on the production of 5-aminolevulinate were investigated. Based on an optimal fed-batch culture system described in our previous work, up to 6.5 g/l (50 mM) ALA was produced in a 15-l fermenter.

The 5-aminolevulinate (ALA) synthase gene (hemA) containing several codons rarely used by Escherichia coli was cloned from the genome of Rhodobacter sphaeroides and optimized in two strains of Escherichia coli: BL21(DE3) and Rosetta(DE3), which is a rare codon optimizer strain. The effects of initial isopropyl-β-d-thiogalactopyranoside (IPTG) concentration, induction time, and temperature on enzyme activity were studied and compared for two strains. The results indicated that the ALA synthase expressed by Rosetta(DE3)/pET-28a(+)-hemA was higher than that by BL21(DE3)/pET-28a(+)-hemA. The initial precursors, glycine and succinate, and initial glucose, which is an inhibitor for both ALA synthase and dehydratase, were observed to be the key factors affecting ALA production. ALA synthase activity was generally higher with Rosetta(DE3) than with BL21(DE3), so was ALA biosynthesis. Based on the optimal culture system using Rosetta(DE3), the yield of ALA achieved 3.8 g/l (29 mM) under the appropriate conditions in fermenter.

•Stabilizing cis-epoxysuccinate hydrolase by using protein engineering.•Five substitutions was isolated and integrated together.•The half-life and T5015 of integrated mutant 5X-1increased by 33.5-fold and 20 °C.•The range of optimum pHs of mutant 5X-1 extends from 8.0–9.0 to 5.0–10.0.cis-Epoxysuccinate hydrolase (CESH) is efficient in catalyzing conversion of cis-epoxysuccinate into l-(+)-tartaric acid, and the enzyme has been successfully applied in industry. However, low thermostability limits its use in large-scale applications. To improve the stability of CESH, we conducted directed evolution and used strategies belonging to semi-rational redesign. Mutant 1X-1 (Q122R) was harvested from directed evolution. Its half-life at 50 °C (t1/2, 50 °C) increased from 8.5 min to 31.6 min, and the T5015 (temperature at which the activity of enzyme decreased by 50% in 15 min) increased from 44.0 °C to 49.5 °C. Substitutions F26V and I83R predicted by multiple sequence alignments were introduced into mutant 1X-1, and the t1/2, 50 °C and T5015 of mutant 3X (Q122R, F26V and I83R) increased to 170.8 min and 55.4 °C. Simulated mutation based on in silico structural modelling was used in constructing mutant 5X (Q122R, F26V, I83R, D8K and S90R), for which the t1/2, 50 °C and T5015 increased to 237.1 min and 62.4 °C, respectively. Site-saturated mutagenesis was employed on amino acid residues Asp-8, Phe-26, Ile-83, Ser-90, and Gln-122 to maximize the thermostability of mutant 5X. Mutant 5X-1 (Q122R, F26W, I83R, D8K and S90R) was isolated; its t1/2,50 °C increased to 293.2 min, 34.5-fold that of the wild-type enzyme and the T5015 increased to 64.8 °C. Moreover, the effective working ranges of pH of mutant 5X-1 extended to 5.0–10.0 from 8.0–9.0 for the wild-type enzyme.Download full-size image

5-Aminolevulinic acid (ALA) is a common precursor for tetrapyrrole compounds in all kinds of organisms and has wide applications in agriculture and medicines. In this study, a new strategy, i.e. short-term dissolved oxygen (DO) shock during aerobic fermentation, was introduced to produce 5-aminolevulinic acid with a recombinant E. coli. Effects of duration time of DO shock operation on plasmid concentration, intracellular ALA synthase (ALAS) activity and ALA production were investigated in Erlenmeyer shake flasks. The results indicated that both ALAS activity and ALA yield were enhanced in an anaerobic operation of 45 min in the early exponential phase during fermentation, while they decreased when the anaerobic operation time was further increased to 60 min. The DO shock protocol was confirmed with the fed-batch fermentation in a 15 L fermenter and the ALA production achieved 9.4 g·L−1 (72 mmol·L−1), which is the highest yield in the fermentation broth reported up to now.

Hydrophobic charge-induction chromatography, a novel chromatographic technique for bioseparation, was developed to isolate and purify bovine IgG with high purity. In this work, the raw IgG solution, a precipitate from bovine colostrum powder solution with 40% (wt/vol) ammonium sulfate, was dissolved in 50 mM phosphate buffer and used as loading solution for investigating chromatographic conditions on a mercapto-ethyl-pyridine (MEP) HyperCel (Pall Corp., Port Washington, NY) sorbent. The initial IgG concentration had no effect on the dynamic binding capacity of MEP HyperCel resin, but the linear velocity of loading solution had an obvious effect on the dynamic IgG binding capacity and IgG recovery. The maximum linear velocity was optimized as 0.4 cm/min of loading solution, and 90% recovery of IgG was achieved. Under these optimized binding conditions, the pH and ionic strength for the elution process were selected as pH 4.5 and 0.5 M NaCl, respectively. Subsequently, hydrophobic charge-induction chromatography was performed on a MEP HyperCel sorbent to isolate IgG using bovine colostrum whey as the loading solution. Under the optimized operation conditions, a remarkable process improvement in IgG purification was received, which includes a yield of 91.5%, a purity of 93.9% (wt/wt), and a purification factor of 6.8. The results indicated that MEP HyperCel chromatography offers an efficient means to purify IgG from bovine colostrums.

The rapid and incomplete oxidation of sugars, alcohols, and polyols by the gram-negative bacterium Gluconobacter oxydans facilitates a wide variety of biological applications. For the conversion of glucose to 5-keto-d-gluconate (5-KGA), a promising precursor of the industrial substance L-(+)-tartaric acid, G. oxydans DSM2343 was genetically engineered to strain ZJU2, in which the GOX1231 and GOX1081 genes were knocked out in a markerless fashion. Then, a secondary alcohol dehydrogenase (GCD) from Xanthomonas campestris DSM3586 was heterologously expressed in G. oxydans ZJU2. The 5-KGA production and cell yield were increased by 10% and 24.5%, respectively. The specific activity of GCD towards gluconate was 1.75 ± 0.02 U/mg protein, which was 7-fold higher than that of the sldAB in G. oxydans. Based on the analysis of kinetic parameters including specific cell growth rate (μ), specific glucose consumption rate (qs) and specific 5-KGA production rate (qp), a dissolved oxygen (DO) control strategy was proposed. Finally, batch fermentation was carried out in a 15-L bioreactor using an initial agitation speed of 600 rpm to obtain a high μ for cell growth. Subsequently, DO was continuously maintained above 20% to achieve a high qp to ensure a high accumulation of 5-KGA. Under these conditions, the maximum concentration of 5-KGA reached 117.75 g/L with a productivity of 2.10 g/(L·h).

d-Glucose, l-arabinose, d-mannose, d-xylose, and cellobiose are saccharification products of lignocellulose and important carbon sources for industrial fermentation. The fermentation efficiency with each of the five sugars and the mixture of the two most dominant sugars, d-glucose and d-xylose, was evaluated for acetone–butanol–ethanol (ABE) fermentation by Clostridium acetobutylicum ATCC 824. The utilization efficacy of the five reducing sugars was in the order of d-glucose, l-arabinose, d-mannose, d-xylose and cellobiose. d-Xylose, the second most abundant component in lignocellulosic hydrolysate, was used in the fermentation either as sole carbon source or mixed with glucose. The results indicated that maintaining pH at 4.8, the optimal pH value for solventogenesis, could increase d-xylose consumption when it was the sole carbon source. Different media containing d-glucose and d-xylose at different ratios (1:2, 1:5, 1.5:1, 2:1) were then attempted for the ABE fermentation. When pH was at 4.8 and xylose concentration was five times that of glucose, a 256.9% increase in xylose utilization and 263.7% increase in solvent production were obtained compared to those without pH control. These results demonstrate a possible approach combining optimized pH control and d-glucose and d-xylose ratio to increase the fermentation efficiency of lignocellulosic hydrolysate.

The production of acrylates from biomass-originated lactic acid is of extraordinary importance, to overcome the increasing worldwide shortage of petroleum. In this study, the catalytic dehydration of methyl lactate over a calcium sulfate catalyst, with various promoters, has been carried out to identify potential catalyst/promoter combinations for acrylate production. The best catalyst for methyl acrylate formation in this study has been calcium sulfate, with cupric sulfate and phosphates as promoters. The optimal mass ratio of m(CaSO4):m(CuSO4):m(Na2HPO4):m(KH2PO4) is 150.0:13.8:2.5:1.2. Effects of carrier gas, reaction temperature, feed concentration as well as contact time on the dehydration of methyl lactate have been investigated. With nitrogen as a carrier gas, a combined yield of acrylic acid and methyl acrylate is 63.9% from 60% (by mass) methyl lactate at 400°C with 7.7 seconds contact time.