High cell density fed-batch fermentation for the production of recombinant <Emphasis Type="Italic">E. coli</Emphasis> K-12 ghost vaccine against streptococcal disease

The bacterial ghost system is a novel vaccine delivery method which provides versatile carrier functions for foreign antigens with excellent natural intrinsic adjuvant properties. In this study, ghost bacteria of E. coli K-12/pHCE-InaN-GAPDH-ghost 27 SDM were created for mass production of a Streptococcus iniae ghost vaccine. The optimal fed-batch process for high cell density culture of E. coli K-12/pHCE-InaN-GAPDH-ghost 27 SDM was developed using the nutrient feeding strategy with Riesenberg defined medium. Fermentation was conducted in four phases as follow: (1) initial batch phase, (2) fed-batch phase for high cell density culture, (3) thermal induction phase for the formation of ghost by the expression of lysis gene E, and (4) high temperature holding phase to increase ghost formation efficiency. The maximum ghost bacteria vaccine (GBV) was obtained from the fed-batch fermentation of 34.9 g dry cell weight (dcw)/L. The expression of antigen glyceraldehyde 3-phosphate dehydrogenase (GAPDH) on the ghost cell with a high temperature holding phase was confirmed with outer-membrane protein fractionation using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting. Results indicate no damage to the expressed antigen on the ghost cell surfaces even after the temperature was increased to 47°C for high efficiency ghost cell formation. Efficacy of the GBV was evaluated by the challenge test in which vaccinated Olive flounder were infected with live S. iniae. The E. coli K-12 host strain, E. coli K-12/pHCE vector control, and formalin-killed cell (FKC) -treated vaccine groups showed 100, 100, and 65% cumulative mortality, respectively. The GBV-treated groups showed 50% cumulative mortality with increased survival ratios. Hence, the immunoprotective efficacy of GBV against S. iniae was better than that of the FKC vaccine. Therefore, the GBV is proposed as an effective vaccine in aquaculture for the prevention of streptococcal disease.     

Effect of heating rate on pDNA production by E. coli

The effects of heating rate (HR) on the performance of two-phase (batch followed by fed-batch) high cell-density cultivations (HCDC) of E. coli DH5α for the production of plasmid DNA (pDNA) were investigated. Optimal temperatures for the HCDC, as selected from shake flask experiments at constant temperatures between 30 and 45 °C, were 35 °C for biomass accumulation in the batch phase and 42 °C for inducing pDNA replication during the fed-batch. In HCDC the temperature was increased at HR of 0.025, 0.05, 0.10 and 0.25 °C/min and the performance of the cultivations were compared to a HCDC run at constant temperature (35 °C). Compared to constant 35 °C, heat-induced HCDC accumulated up to 50% less biomass within the same cultivation time and acetate and glucose accumulated to high concentrations. The overall specific productivity (Q_P) and average pDNA yield (Y_p/x) in HCDC at 35 °C were 0.22 ± 0.02 mg/g h and 5.3 ± 0.00 mg/g, respectively. Such parameters were maximum at a HR of 0.05 °C/min, reaching 0.56 ± 0.06 mg/g h and 9.3 ± 0.6 mg/g, respectively. At HR above 0.5 °C/min, Y_p/x remained relatively constant, whereas Q_P tended to decrease. The supercoiled pDNA fraction remained around 80% at all HR. Bioreactors were equipped with a capacitance/conductivity probe. In all cases biomass concentration correlated closely with the capacitance signal and acetate and glucose accumulation was accompanied by an increase in the conductivity signal. Thus, it was possible to calculate acetate and biomass concentrations, as well as μ, from online capacitance and conductivity signals using estimators. Altogether, in this study it was shown that it is possible to maximize pDNA productivity by choosing an appropriate HR and that relevant parameters can be estimated by capacitance/conductivity signals, which are useful for better process control and development.     

Microbial surface displaying formate dehydrogenase and its application in optical detection of formate

In the present work, NAD⁺-dependent formate dehydrogenase (FDH), encoded by fdh gene from Candida boidinii was successfully displayed on Escherichia coli cell surface using ice nucleation protein (INP) from Pseudomonas borealis DL7 as an anchoring protein. Localization of matlose binding protein (MBP)-INP-FDH fusion protein on the E. coli cell surface was characterized by SDS-PAGE and enzymatic activity assay. FDH activity was monitored through the oxidation of formate catalyzed by cell-surface-displayed FDH with its cofactor NAD⁺, and the production of NADH can be detected spectrometrically at 340 nm. After induction for 24 h in Luria-Bertani medium containing isopropyl-β-d-thiogalactopyranoside, over 80% of MBP-INP-FDH fusion protein present on the surface of E. coli cells. The cell-surface-displayed FDH showed optimal temperature of 50 °C and optimal pH of 9.0. Additionally, the cell-surface-displayed FDH retained its original enzymatic activity after incubation at 4 °C for one month with the half-life of 17 days at 40 °C and 38 h at 50 °C. The FDH activity could be inhibited to different extents by some transition metal ions and anions. Moreover, the E. coli cells expressing FDH showed different tolerance to solvents. The recombinant whole cell exhibited high formate specificity. Finally, the E. coli cell expressing FDH was used to assay formate with a wide linear range of 5–700 μM and a low limit of detection of 2 μM. It is anticipated that the genetically engineered cells may have a broad application in biosensors, biofuels and cofactor regeneration system.     

Operation of a fixed-bed bioreactor in batch and fed-batch modes for production of inulinase by solid-state fermentation

This work is focused on the inulinase production by solid-state fermentation (SSF) in a fixed-bed reactor (34 cm diameter and 50 cm height) with working capacity of 2-kg of dry substrate operated in batch and fed-batch modes. It was investigated different strategies for feeding the inlet air in the bioreactor (saturated and unsaturated air) as alternative to remove the metabolic heat generated during the microbial growth by evaporative cooling. The kinetic evaluation of the process carried out in batch mode using unsaturated air showed that the evaporative cooling decreasing the mean temperature of the solid-bed, although the enzyme production was lower than that obtained using saturated air. Results showed that maximum enzyme activity (586 ± 63 U gds⁻¹) was obtained in the fed-batch mode using saturated air after 24 h of fermentation. The enzymatic extract obtained by fed-batch mode was characterized and presented optimum temperature and pH in the range of 52–57 °C and 4.8–5.2, respectively. For a temperature range from 40 to 70 °C the enzyme presented decimal reduction time, D-value, ranging from 5748 to 47 h, respectively. For a pH range from 3.5 to 5.5 the enzyme showed good stability, presenting D-values higher than 2622 h. In terms of Michaelis–Mentem parameters were demonstrated that the crude inulinase activity presented higher affinity for substrate sucrose compared to inulin.     

Heterologous production of a temperature and pH-stable laccase from Bacillus vallismortis fmb-103 in Escherichia coli and its application

To enhance laccase yield, the laccase gene from Bacillus vallismortis fmb-103 was cloned and heterologously expressed in Escherichia coli BL21 (DE3) cells. The auto-induction strategy was applied during fermentation, and the process was controlled, as follows: Cu²⁺ was added when the optical density at 600 nm (OD₆₀₀) was 0.3, the fermentation temperature was adjusted to 16 °C when the OD₆₀₀ was 0.9, and fermentation was stopped after 50 h. The yield of recombinant laccase was up to 3420 U/L, as assayed by 2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid). Recombinant laccase was purified 4.47-fold by heating for 10 min at 70 °C and dialyzing against 50–60% ammonium sulfate, retained more than 50% activity after 10 h at 70 °C, and demonstrated broad pH stability. Malachite green was efficiently degraded by recombinant laccase, especially in combination with mediators. These results provided a basis for the future application of recombinant laccase to malachite green degradation.     

Modulation of guanosine 5′-diphosphate-d-mannose metabolism in recombinant Escherichia coli for production of guanosine 5′-diphosphate-l-fucose

Guanosine 5’-diphosphate (GDP)-l-fucose, an activated form of a nucleotide sugar, plays an important role in a wide range of biological functions. In this study, the enhancement of GDP-l-fucose production was attempted by supplementation of mannose, which is a potentially better carbon source to be converted into GDP-l-fucose than glucose, and combinatorial overexpression of the genes involved in the biosynthesis of GDP-d-mannose, a precursor of GDP-l-fucose. Supply of a mannose and glucose led to a 1.3-fold-increase in GDP-l-fucose concentration (52.5 ± 0.8 mg l^?1) in a fed-batch fermentation of recombinant E. coli BL21star(DE3) overexpressing the gmd and wcaG genes, compared with the case using glucose as a sole carbon source. A maximum GDP-l-fucose concentration of 170.3 ± 2.3 mg l^?1, corresponding to a 4.4-fold enhancement compared with the control strain overexpressing gmd and wcaG genes only, was achieved in a glucose-limited fed-batch fermentation of a recombinant E. coli BL21star(DE3) strain overexpressing manB, manC, gmd and wcaG genes. Further improvement of GDP-l-fucose production was not obtained by additional overexpression of the manA gene.     

Simultaneous saccharification and fermentation of kraft pulp by recombinant Escherichia coli for phenyllactic acid production

Simultaneous saccharification and fermentation (SSF) of renewable cellulose for the production of 3-phenyllactic acid (PhLA) by recombinant Escherichia coli was investigated. Kraft pulp recovered from biomass fractionation processes was used as a model cellulosic feedstock and was hydrolyzed using 10–50 filter paper unit (FPU) g⁻¹ kraft pulp of a commercial cellulase mixture, which increased the glucose yield from 21% to 72% in an enzyme dose-dependent manner. PhLA fermentation of the hydrolyzed kraft pulp by a recombinant E. coli strain expressing phenylpyruvate reductase from Wickerhamia fluorescens TK1 produced 1.9 mM PhLA. The PhLA yield obtained using separate hydrolysis and fermentation was enhanced from 5.8% to 42% by process integration into SSF of kraft pulp (20 g L⁻¹) in a complex medium (pH 7.0) at 37 °C. The PhLA yield was negatively correlated with the initial glucose concentration, with a five-fold higher PhLA yield observed in culture medium containing 10 g L⁻¹ glucose compared to 100 g L⁻¹. Taken together, these results suggest that the PhLA yield from cellulose in kraft pulp can be improved by SSF under glucose-limited conditions.     

Biochemical characteristics of phytases from fungi and the transformed microorganism

Five sources of phytases were used to study their biochemical characteristics. Phytase E was from an original Escherichia coli (E. coli), phytase PI and PG from the transformed Pichia pastoris (P. pastoris) with phytase gene of E. coli, phytase B and R from Aspergillus niger (A. niger). The results showed that the relative phytase activities had no significant changes when temperature was below 60 °C (P>0.05), and then decreased significantly with temperature increasing (P<0.01). The fungal phytase with the phytase gene from A. niger had the higher thermostability than the bacterial phytase with the phytase gene from E. coli; i.e. at 70 °C, 27–58% of phytase activity (compared with 30 °C) was retained for the bacterial phytase, and 73–96% for the fungal phytase; at 90 °C, 20–47% was retained for the bacterial phytase, and 41–52% for the fungal phytase, especially for the most thermostable phytase R (P<0.01). The optimum pH ranges were 3.0–4.5 for the bacterial phytases and 5.0–5.5 for the fungal phytases (P<0.01). When pH levels were 1, 7 and 8, only 3–7% of phytase activity (compared with the maximum phytase activity at a pH point) was retained for both bacterial and fungal phytases. The amount of inorganic P released from soybean meal was significantly increased when the levels of phytase activity in the soybean meal increased from 0 to 1.0 U/g soybean meal (P<0.01), except for phytase PI. The maximum P released was obtained at 1 U/g soybean meal for all five kinds of phytases (P<0.01). The most economical phytase concentration for P released was 0.25 U/g for phytase PI and B, and 0.50–1.0 U/g for phytase PG, E and R. In addition, the linear and non-linear regression models were established to estimate phytase activity and its characteristics very easily and economically.     

Strategies for high titre plasmid DNA production in Escherichia coli DH5α

The production of plasmid pEGFP-N1 in Escherichia coli DH5α was optimised. A strategy evaluating different media components separately was not successful (OD < 2.5, low plasmid titres), a statistical approach via a Plackett Burman design (11 parameters) allowed some improvement (7 mg/L plasmid, OD₆₀₀ 8.5). Generally, high biomass did not correlate with high plasmid titres. When conditions were transferred to the bioreactor (batch operation) little improvement in plasmid titres (10 mg/L plasmid, OD₆₀₀ 20) was observed. By switching to a fed-batch procedure with linear feeding these values increased to 20 mg/L plasmid (OD₆₀₀ 50). By using an adaptive feeding strategy, plasmid titres could be increased to 50 mg/L. Finally, by combining a growth controlled (reduced temperature (35 °C), low dO₂) initial batch phase with an adaptive feeding strategy in the fed-batch phase (37 °C, glucose-/dO₂-limitation) we were reproducibly able to produce up to 250 mg/L of plasmid DNA in cultures that reached a final OD₆₀₀ of 80.     

Molecular characterization and molasses fermentation performance of a wild yeast strain operating in an extremely wide temperature range

Molasses fermentation performance by both a cryotolerant and a thermophilic yeast (strain AXAZ-1) isolated from grapes in Greece was evaluated in an extremely wide temperature range (3–40 °C). Sequence analysis of the 5.8S internal transcribed spacer and the D1/D2 ribosomal DNA (rDNA) regions assigned isolate to Saccharomyces cerevisiae. Restriction fragment length polymorphism of the mitochondrial DNA showed that strain AXAZ-1 is genetically divergent compared to other wild strains of Greek origin or commercial yeast starters. Yeast cells growing planktonically were capable of fermentation in a wide temperature spectrum, ranging from 3 °C to 38 °C. Immobilization of yeast on brewer’s spent grains (BSG) improved the thermo-tolerance of the strain and enabled fermentation at 40 °C. Time to complete fermentation with the immobilized yeast ranged from 20 days at 3 to 38 h at 40 °C. The daily ethanol productivity reached maximum (58.1 g/L) and minimum (2.5 g/L) levels at 30 and 3 °C, respectively. The aroma-related compounds’ profiles of immobilized cells at different fermentation temperatures were evaluated by using solid phase microextraction (SPME) gas chromatography–mass spectrometry (GC–MS). Molasses fermentation resulted in a high quality fermentation product due to the low concentrations of higher and amyl alcohols at all temperatures tested. Strain AXAZ-1 is very promising for the production of ethanol from low cost raw materials, as it was capable to perform fermentations of high ethanol concentration and productivities in both low and high temperatures.     

Estudios de la viabilidad y la vitalidad frente al congelado de la levadura probiótica Saccharomyces boulardii: efecto del preacondicionamiento fisiológico

The aim of this study was to evaluate the vitality and viability of the probiotic yeast Saccharomyces boulardii after freezing/thawing and the physiological preconditioning effect on these properties. The results indicate that the specific growth rate (0.3/h^?1) and biomass (2-3 × 10⁸ cells/ml) of S. boulardii obtained in flasks shaken at 28 °C and at 37 °C were similar. Batch cultures of the yeast in bioreactors using glucose or sugar-cane molasses as carbon sources, reached yields of 0.28 g biomass/g sugar consumed, after 10 h incubation at 28 °C; the same results were obtained in fed batch fermentations. On the other hand, in batch cultures, the vitality of cells recovered during the exponential growth phase was greater than the vitality of cells from the stationary phase of growth. Vitality of cells from fed-batch fermentations was similar to that of stationary growing cells from batch fermentations. Survival to freezing at –20 °C and subsequent thawing of cells from batch cultures was 0.31% for cells in exponential phase of growth and 11.5% for cells in stationary phase. Pre-treatment of this yeast in media with water activity (a_w) 0.98 increased the survival to freezing of S. boulardii cells stored at –20 °C for 2 months by 10 fold. Exposure of the yeast to media of reduced a_w and/or freezing/thawing process negatively affected cell vitality. It was concluded that stress conditions studied herein decrease vitality of S. boulardii. Besides, the yeast strain studied presented good tolerance to bile salts even at low pH values.     

Production of an anti-MUC1 C595 dbFv antibody fragment in recombinant Escherichia coli

The production of C595 diabody fragment (dbFv) in Escherichia coli (E. coli) HB2151 clone has been explored. The comparison of fermentation processes mode demonstrated that a higher biomass inoculum operation enhanced C595 dbFv production. It was demonstrated that a concentration of 12.1 mg l⁻¹ broth of dbFv and a cell concentration of 23.6 g l⁻¹ broth were achieved at the end of 75 l fermentation.     

Cloning,expression, purification and characterization of an oligomeric His-tagged thermophilic esterase from Thermus thermophilus HB27

The esterase E34Tt (YP_004875.1) from Thermus thermophilus HB27 was cloned, expressed in Escherichia coli as a His-tagged protein, purified and characterized. The gene sequence was subcloned into a T-vector, released with the restriction enzymes BamHI and HindIII, ligated to a pET-21d(+) vector, and transferred to E. coli BL21 (DE3) cells. Inducer concentration (isopropyl β-d-1-thiogalactopyranoside, IPTG) and cultivation time before and after induction were optimized. Best results were obtained by adding 0.25 mM IPTG after 8 h of cultivation and maintaining the induction during 4 extra hours. Most of the enzyme (94%) remained membrane-associated and had to be extracted with a detergent. From the membrane crude extract, the His-tagged E34Tt was purified as a dimer (71.8 kDa) in a single purification step by using metal affinity chromatography. The Rosso's model was used to optimize the reaction conditions. E34Tt-His₆ was active in a wide temperature (19.7–79.4 °C) and pH range (4.0–9.3), and maximal activity was determined at pH 6.3 and 58.2 °C, which is 10–18 °C higher than the optimal reaction temperature of the previously reported variants expressed in mesophilic yeasts. E34Tt-His₆ preferentially hydrolyzed esters with ten carbon atoms, and was highly thermostable (half-life of 107.9 min at 85 °C), suggesting that E34Tt-His₆ has potential for industrial applications.     

Mixed culture of Saccharomyces cerevisiae and Acetobacter pasteurianus for acetic acid production

Mixed culture of Saccharomyces cerevisiae and Acetobacter pasteurianus was carried out for high yield of acetic acid. Acetic acid production process was divided into three stages. The first stage was the growth of S. cerevisiae and ethanol production, fermentation temperature and aeration rate were controlled at 32 °C and 0.2 vvm, respectively. The second stage was the co-culture of S. cerevisiae and A. pasteurianus, fermentation temperature and aeration rate were maintained at 34 °C and 0.4 vvm, respectively. The third stage was the growth of A. pasteurianus and production of acetic acid, fermentation temperature and aeration rate were controlled at 32 °C and 0.2 vvm, respectively. Inoculation volume of A. pasteurianus and S. cerevisiae was 16% and 0.06%, respectively. The average acetic acid concentration was 52.51 g/L under these optimum conditions. To enhance acetic acid production, a glucose feeding strategy was subsequently employed. When initial glucose concentration was 90 g/L and 120 g/L glucose was fed twice during fermentation, acetic acid concentration reached 66.0 g/L.     

Cloning,purification and characterization of a thermostable β-galactosidase from Thermotoga naphthophila RUK-10

A novel β-galactosidase gene (Tnap1577) from the hyperthermophilic bacterium Thermotoga naphthophila RUK-10 was cloned and expressed in Escherichia coli BL21 (DE3) cells to produce β-galactosidase. The recombinant β-galactosidase was purified in three steps: heat treatment to deactivate E. coli proteins, Ni-NTA affinity chromatography and Q-sepharose chromatography. The optimum temperatures for the hydrolysis of o-nitrophenyl-β-d-galactoside (o-NPG) and lactose with the recombinant β-galactosidase were found to be 90 °C and 70 °C, respectively. The corresponding optimum pH values were 6.8 and 5.8, respectively. The molecular mass of the enzyme was estimated to be 70 kDa by SDS-PAGE analysis. Thermostability studies showed that the half-lives of the recombinant enzyme at 75 °C, 80 °C, 85 °C and 90 °C were 10.5, 4, 1, and 0.3 h, respectively. Kinetic studies on the recombinant β-galactosidase revealed K_m values for the hydrolysis of o-NPG and lactose of 1.31 mM and 1.43 mM, respectively. These values are considerably lower than those reported for other hyperthermophilic β-galactosidases, indicating high intrinsic affinity for these substrates. The recombinant β-galactosidase from Thermotoga naphthophila RUK-10 also showed transglycosylation activity in the synthesis of alkyl galactopyranoside. This additional activity suggests the enzyme has potential for broader biotechnological applications beyond the degradation of lactose.     

Characterization and overproduction of a thermo-alkaline pectate lyase from alkaliphilic Bacillus licheniformis with potential in ramie degumming

A thermo-alkaline pectate lyase (BliPelA) gene from an alkaliphilic Bacillus licheniformis strain was cloned and overexpressed in Escherichia coli. Mature BliPelA exhibited maximum activity at pH 11 and 70 °C, and demonstrated cleavage capability on a broad range of substrates such as polygalacturonic acid, pectins, and methylated pectins. The highest specific activity, of 320 U mg⁻¹, was towards polygalacturonic acid. Significant ramie (Boehmeria nivea) fiber weight loss (21.5%) was obtained following enzyme treatment and combined enzyme-chemical treatment (29.3%), indicating a high ramie degumming efficiency of BliPelA. The total activity of recombinant BliPelA reached 1450.1 U ml⁻¹ with a productivity of 48.3 U ml⁻¹ h⁻¹ under high-cell-density cultivation with a glycerol exponential feeding strategy for 30 h in 1-l fed-batch fermenter, and 1380.1 U ml⁻¹ with a productivity of 57.5 U ml⁻¹ h⁻¹ after 24 h under constant glucose feeding in a 20-l fermenter using E. coli as the host. The enzyme yields reached 4.5 and 4.3 g l⁻¹ in 1-l and 20-l fed-batch fermenters, respectively, which are higher than those of most reported alkaline Pels. Based on these promising properties and high-level production, BliPelA shows great potential for application in ramie degumming in textile industry.     

Heterologous expression of a newly screened β-agarase from Alteromonas sp. GNUM1 in Escherichia coli and its application for agarose degradation

The gene of agaG1 from Alteromonas sp. GNUM1 encoding a β-agarase (AgaG1) was heterologously expressed in E. coli BL21 (DE3). The recombinant strain was cultured at 37 °C and then AgaG1 was expressed at 25 °C and 0.5 mM IPTG. The optimum conditions for AgaG1 to hydrolyze agarose were pH 7.0 and 40 °C. The main products of agarose hydrolysis by AgaG1 were confirmed to be neoagarobiose and neoagarotetraose. A new agarose hydrolysis process using AgaG1 was developed, in which the reaction temperature was adjusted stepwise to avoid gelation problem with no chemical pretreatment step. The enzyme AgaG1 was found to be very effective and highly selective. When 10.0 g/L agarose was hydrolyzed, 98% of the agarose added was converted to 3.8 and 6.4 g/L of neoagarobiose and neoagarotetraose, respectively.     

Recombinant Fab expression and secretion in Escherichia coli continuous culture at medium cell densities: Influence of temperature

Production of recombinant antibody fragments (Fabs) in Escherichia coli has gained interest because of the recognised advantages of this expression system and because Fabs do not require glycosylation. However, more comprehensive studies on the factors that influence expression conditions and product yield are still required for full process development. In this work, the effect of growth temperature on the periplasmatic expression of the 3H6 Fab in E. coli was studied in carbon-limited continuous cultures operated at medium cell densities. Three different temperatures were assayed, namely 37, 33 and 30 °C. Results showed that biomass yield was not affected within this temperature range whilst product yield increased as temperature decreased. Periplasmic Fab secretion corresponded to 30% of the produced Fab protein and its efficiency was irrespective of the process temperature. Moreover, considerable product leakage to the culture supernatant was detected in all cases, ranging from about 40% at 37 °C to almost 70% at 30 °C. Besides, plasmid loss was observed along process time indicating a selective pressure against plasmid-bearing cells. This study supports the potential of continuous cultivations of E. coli at medium cell densities under well controlled conditions as a tool for characterising the impact of environmental parameters and cell physiology under protein production conditions.     

A novel low-temperature-active exo-inulinase identified based on Molecular-Activity strategy from Sphingobacterium sp. GN25 isolated from feces of Grus nigricollis

A novel glycosyl hydrolase family 32 exo-inulinase (InuAGN25) gene was cloned from Sphingobacterium sp. GN25 isolated from feces of Grus nigricollis. InuAGN25 showed the highest identity of 54.3% with a putative levanase recorded in GenBank. Molecular-Activity strategy was proposed to predict InuAGN25 to be a low-temperature-active exo-inulinase before experiments performance. Molecular analyses included progressive sequential, phylogenetic and structural analyses. InuAGN25 was effectively expressed in Escherichia coli. The purified recombinant InuAGN25 showed characteristics of low-temperature-active enzymes: (1) the enzyme retained 55.8% of the maximum activity at 20 °C, 35.8% at 10 °C, and even 8.2% at 0 °C; (2) the enzyme exhibited 75.8, 30.5 and 10.8% of the initial activity after preincubation for 60 min at 45, 50 and 55 °C, respectively; (3) K_m values of the enzyme toward inulin were 2.8, 3.0, 3.2 and 5.8 mg ml⁻¹ at 0, 10, 20 and 40 °C, respectively. Fructose was the main product of inulin and Jerusalem artichoke tubers hydrolyzed by the purified recombinant InuAGN25 at room temperature, 10 °C and 0 °C. These results suggested the Molecular-Activity strategy worked efficiently and made InuAGN25 promising for the production of fructose at low temperatures.     

Ethanol and furfural production from corn stover using a hybrid fractionation process with zinc chloride and simultaneous saccharification and fermentation (SSF)

A two-stage hybrid fractionation process was investigated to produce cellulosic ethanol and furfural from corn stover. In the first stage, zinc chloride (ZnCl₂) was used to selectively solubilize hemicellulose. During the second stage, the remaining treated solids were converted into ethanol using commercial cellulase and Saccharomyces cerevisiae or recombinant Escherichia coli, KO11. This hybrid fractionation process recovered 93.8% of glucan, 89.7% of xylan, 71.1% of arabinan, and 74.9% of lignin under optimal reaction conditions (1st stage: 5% acidified ZnCl₂, 7.5 ml/min, 150 °C (10 min) and 170 °C (10 min); 2nd stage: simultaneous saccharification and fermentation (SSF) using S. cerevisiae). The furfural yield from the hemicellulose hydrolysates was 58%. The SSF of the treated solids resulted in 69–98% of the theoretical maximum ethanol yields based on the glucan content in the treated solids. After fermentation, the solid residues contained primarily lignin. Based on the total lignin in untreated corn stover, the lignin recovery yield was 74.9%.