Continuous production of high-purity fructooligosaccharides and ethanol by immobilized Aspergillus japonicus and Pichia heimii

High-purity fructooligosaccharides (FOS) were produced from sucrose by an innovative process incorporating immobilized Aspergillus japonicus and Pichia heimii cells. Intracellular FTase of A. japonicus converted sucrose into FOS and glucose, and P. heimii fermented glucose mainly into ethanol. The continuous production of FOS was carried out using a tanks-in-series bioreactor consisting of three stirred tanks. When a solution composed of 1 g L^?1 yeast extract and 300 g L^?1 sucrose was fed continuously to the bioreactor at a dilution rate of 0.1 h^?1, FOS at a purity of up to 98.2 % could be achieved and the value-added byproduct ethanol at 79.6 g L^?1 was also obtained. One gram of sucrose yielded 0.62 g FOS and 0.27 g ethanol. This immobilized dual-cell system was effective for continuous production of high-purity FOS and ethanol for as long as 10 days.     

Reaction Kinetics of the Invertase from Yeast (S. Cerevisiae)

Invertase converts sucrose to glucose and fructose. The reaction mechanism for the formation of glucose and fructose was studied by stopped flow spectrophotometer and circular dichroism. The reaction mechanism follows biphasic mode with rate constants of k₁0.0053 s^?1?±?0.001 s^?1 and k₂ 0.030 s^?1?±?0.01 s^?1 for 25 mM concentration of sucrose. Far UV circular dichroic spectrum of invertase in presence of sucrose shows 18 % increase in β conformation as a function of time. Taken together, the invertase hydrolysis follows biphasic mode where it undergoes conformational changes followed by hydrolysis of the sucrose.     

Highly selective hydrolysis for the outer glucose at the C-20 position in ginsenosides by β-glucosidase from Thermus thermophilus and its application to the production of ginsenoside F2 from gypenoside XVII

β-Glucosidase from Thermus thermophilus has specific hydrolytic activity for the outer glucose at the C-20 position in protopanaxadiol-type ginsenosides without hydrolysis of the inner glucose. The hydrolytic activity of the enzyme for gypenoside XVII was optimal at pH 6.5 and 90 °C, with a half-life of 1 h with 3 g enzyme l^?1 and 4 g gypenoside XVII l^?1. Under the optimized conditions, the enzyme converted the substrate gypenoside XVII to ginsenoside F₂ with a molar yield of 100 % and a productivity of 4 g l^?1 h^?1. The conversion yield and productivity of ginsenoside F₂ are the highest reported thus far among enzymatic transformations.     

Characterization of a recombinant l-rhamnose isomerase from Dictyoglomus turgidum and its application for l-rhamnulose production

A putative recombinant enzyme from Dictyoglomus turgidum was characterized and immobilized on Duolite A568 beads. The native enzyme was a 46 kDa tetramer. Its activity was highest for l-rhamnose, indicating that it is an l-rhamnose isomerase. The maximum activities of both the free and immobilized enzymes for l-rhamnose isomerization were at pH 8.0 and 75 °C in the presence of Mn²⁺. Under these conditions, the half-lives of the free and immobilized enzymes were 28 and 112 h, respectively. In a packed-bed bioreactor, the immobilized enzyme produced an average of 130 g l-rhamnulose l^?1 from 300 g l-rhamnose l^?1 after 240 h at pH 8.0, 70 °C, and 0.6 h^?1, with a productivity of 78 g l^?1 h^?1 and a conversion yield of 43 %. To the best of our knowledge, this is the first report describing the enzymatic production of l-rhamnulose.     

Purification and evaluation of the enzymatic properties of a novel fructosyltransferase from Aspergillus oryzae: a potential biocatalyst for the synthesis of sucrose 6-acetate

A novel fructosyltransferase (AoFT) capable of synthesizing sucrose 6-acetate (S6A) from sucrose and glucose 6-acetate has been purified to homogeneity from Aspergillus oryzae ZZ-01. Its molecular mass was ~50 kDa by SDS-PAGE; optimal activity was at 45 °C and it was stable from pH 4.5 to 7.5 with an optimum pH of 6. Mg²⁺, K⁺ (5 mM), propanol, toluene (50 %, v/v), Tween 20 or Triton X-100 (1 %, w/v) increased the transfructosylation activity by 20, 17, 17, 10, 25 and 20 %, respectively. An overall conversion of 32 % was achieved under optimal conditions over 24 h. This is the first report that the purified and characterized the fructosyltransferase from Aspergillus capable of synthesis of S6A from sucrose and glucose 6-acetate.     

Increased production of γ-lactones from hydroxy fatty acids by whole Waltomyces lipofer cells induced with oleic acid

Among several fatty acids tested, oleic acid was selected as the most efficient inducer for the production of 4-hydroxydodecanoic acid, a metabolite of β-oxidation, by Waltomyces lipofer. Cells were induced by incubation for 12 h in a medium containing 10 g l^?1 yeast extract, 10 g l^?1 peptone, 5 g l^?1 oleic acid, 1 g l^?1 glucose, and 0.05 % (w/v) Tween 80. The optimal reaction conditions for the production of γ-lactones by induced cells were pH 6.5, 35 °C, 200 rpm, 0.71 M Tris, 60 g l^?1 hydroxy fatty acid, and 20 g l^?1 cells. Non-induced cells produced 38 g l^?1 γ-dodecalactone from 60 g l^?1 10-hydroxystearic acid after 30 h, with a conversion yield of 63 % (w/w) and a productivity of 1.3 g l^?1 h^?1 under the optimized conditions, whereas induced cells produced 51 g l^?1 γ-dodecalactone from 60 g l^?1 10-hydroxystearic acid after 30 h, with a conversion yield of 85 % (w/w) and a productivity of 1.7 g l^?1 h^?1. The conversion yield and productivity of induced cells were 22 % and 1.3-fold higher, respectively, than those of non-induced cells. Induced cells also produced 28 g l^?1 γ-decalactone and 12 g l^?1 γ-butyrolactone from 60 g l^?1 12-hydroxystearic acid and 60 g l^?1 10-hydroxydecanoic acid, respectively, after 30 h. The concentration, conversion yield, and productivity of γ-dodecalactone and γ-decalactone are the highest reported thus far. This is the first study on the biotechnological production of γ-butyrolactone.     

Oxalic acid production by Aspergillus niger

Aspergillus niger is able to produce a quite high concentration of oxalic acid using sucrose as carbon and energy source. Operating at pH higher than 6 and an enriched N and P medium is necessary in order to conduct the fermentation towards oxalic acid production. A pH?shift technique, operating at acid pH?in the first two days and then setting pH?to 6, allowed the productivity to slightly increase in shaking flasks cultures up to 3.0?kg/m³?·?d, with a final oxalic acid concentration of 29?kg/m³. When operating at more controlled conditions, in a stirred tank, both productivity and oxalic acid concentration were improved (4.1?kg/m³?·?d and 33.8?kg/m³, respectively). However the main drawback of this fermentation is the low yield attained (about 0.3?kg oxalic acid/kg sucrose) because most of glucose, resulting from the hydrolysis of sucrose by the extracellular enzymes secreted at the beginning of the fermentation, is very quickly oxidised to gluconic acid, a process which is favoured at a pH?close to 6. Milk whey was proved to be a very good substrate as it allows oxalic acid to be produced with a similar productivity (2.5?kg/m³?·?d in shaking flasks) giving excellent yields of almost 0.6?kg oxalic acid/kg lactose.     

Production of ganoderic acid by Ganoderma lucidum RCKB-2010 and its therapeutic potential

The newly isolated basidiomycetous fungus, identified as Ganoderma lucidum RCKB-2010 was tested for production of ganoderic acid (GA) under submerged fermentation conditions. Production of GA under liquid static cultivation condition was found to be 2,755.88 mg L^?1 on the 25th day of incubation, whereas under shaking cultivation conditions the maximum production of GA was observed to be 373.75 mg L^?1. ¹H NMR analysis revealed clearly that the fungal extracts possessed a lanostane skeleton, confirming the presence of GA. Interestingly, GA was found to have potential to inhibit the proliferation of HeLa cells and U87 human glioma cells in a dose dependent manner. In addition, GA was also found to possess antibacterial activity, exhibiting a minimal inhibitory concentration of 0.25 mg mL^?1 against standard strains of Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus and Staphylococcus epidermidis. GA produced in the present study holds potential as a potent anticancer agent.     

Engineering the intracellular metabolism of <Emphasis Type="Italic">Escherichia coli</Emphasis> to produce gamma-aminobutyric acid by co-localization of GABA shunt enzymes

Objectives

To direct the carbon flux from Krebs cycle into the gamma-aminobutyric acid (GABA) shunt pathway for the production of GABA by protein scaffold introduction in Escherichia coli.

Results

Escherichia coli was engineered to produce GABA from glucose by the co-localization of enzymes succinate semialdehyde dehydrogenase (GadD), GABA aminotransferase (PuuE) and GABA transporter (GadC) by protein scaffold. 0.7 g GABA l^?1 was produced from 10 g glucose l^?1 while no GABA was produced in wild type E. coli. pH 6 and 30 °C were optimum for GABA production, and GABA concentration increased to 1.12 g GABA l^?1 when 20 g glucose l^?1 was used. When competing metabolic networks were inactivated, GABA increased by 24 % (0.87 g GABA l^?1).

Conclusions

The novel GABA production system was constructed by co-localization of GABA shunt enzymes.

     

Expression and evaluation of enzymes required for the hydrolysis of galactomannan

The cost-effective production of bioethanol from lignocellulose requires the complete conversion of plant biomass, which contains up to 30 % mannan. To ensure utilisation of galactomannan during consolidated bioprocessing, heterologous production of mannan-degrading enzymes in fungal hosts was explored. The Aspergillus aculeatus endo-β-mannanase (Man1) and Talaromyces emersonii α-galactosidase (Agal) genes were expressed in Saccharomyces cerevisiae Y294, and the Aspergillus niger β-mannosidase (cMndA) and synthetic Cellvibrio mixtus β-mannosidase (Man5A) genes in A. niger. Maximum enzyme activity for Man1 (374 nkat ml^?1, pH 5.47), Agal (135 nkat ml^?1, pH 2.37), cMndA (12 nkat ml^?1, pH 3.40) and Man5A (8 nkat ml^?1, pH 3.40) was observed between 60 and 70 °C. Co-expression of the Man1 and Agal genes in S. cerevisiae Y294[Agal-Man1] reduced the extracellular activity relative to individual expression of the respective genes. However, the combined action of crude Man1, Agal and Man5A enzyme preparations significantly decreased the viscosity of galactomannan in locust bean gum, confirming hydrolysis thereof. Furthermore, when complemented with exogenous Man5A, S. cerevisiae Y294[Agal-Man1] produced 56 % of the theoretical ethanol yield, corresponding to a 66 % carbohydrate conversion, on 5 g l^?1 mannose and 10 g l^?1 locust bean gum.     

Production of aglycone protopanaxatriol from ginseng root extract using Dictyoglomus turgidum β-glycosidase that specifically hydrolyzes the xylose at the C-6 position and the glucose in protopanaxatriol-type ginsenosides

The hydrolytic activity of a recombinant β-glycosidase from Dictyoglomus turgidum that specifically hydrolyzed the xylose at the C-6 position and the glucose in protopanaxatriol (PPT)-type ginsenosides followed the order Rf > Rg₁ > Re > R₁ > Rh₁ > R₂. The production of aglycone protopanaxatriol (APPT) from ginsenoside Rf was optimal at pH 6.0, 80 °C, 1 mg ml^?1 Rf, and 10.6 U ml^?1 enzyme. Under these conditions, D. turgidum β-glycosidase converted ginsenoside R₁ to APPT with a molar conversion yield of 75.6 % and a productivity of 15 mg l^?1 h^?1 after 24 h by the transformation pathway of R₁ → R₂ → Rh₁ → APPT, whereas the complete conversion of ginsenosides Rf and Rg₁ to APPT was achieved with a productivity of 1,515 mg l^?1 h^?1 after 6.6 h by the pathways of Rf → Rh₁ → APPT and Rg₁ → Rh₁ → APPT, respectively. In addition, D. turgidum β-glycosidase produced 0.54 mg ml^?1 APPT from 2.29 mg ml^?1 PPT-type ginsenosides of Panax ginseng root extract after 24 h, with a molar conversion yield of 43.2 % and a productivity of 23 mg l^?1 h^?1, and 0.62 mg ml^?1 APPT from 1.35 mg ml^?1 PPT-type ginsenosides of Panax notoginseng root extract after 20 h, with a molar conversion yield of 81.2 % and a productivity of 31 mg l^?1 h^?1. This is the first report on the APPT production from ginseng root extract. Moreover, the concentrations, yields, and productivities of APPT achieved in the present study are the highest reported to date.     

Cloning,deletion, and overexpression of a glucose oxidase gene in <Emphasis Type="Italic">Aureobasidium</Emphasis> sp. P6 for Ca<Superscript>2+</Superscript>-gluconic acid overproduction

This study aimed to overexpress a glucose oxidase gene (GOD1) in Aureobasidium sp. P6 to achieve Ca²⁺-gluconic acid (GA) overproduction. The GOD1 gene was cloned, deleted, and overexpressed. A protein deduced from the GOD1 gene of Aureobasidium sp. P6 strain had 1824 bp that encoded a protein with 606 amino acids, with a conserved NADB-ROSSMAN domain and a GMC-oxred domain. Deleting the GOD1 gene made the disruptant GOK1 completely lose the ability to produce GA and GOD1 activity, whereas overexpressing the GOD1 gene rendered the transformant GOEX8 to produce considerably more Ca²⁺-GA (160.5?±?5.6 g/L) and higher GOD1 activity (1438.6?±?73.2 U/mg of protein) than its parent P6 strain (118.7?±?4.3 g/L of Ca²⁺-GA and 1100.0?±?23.6 U/mg of GOD1 protein). During a 10-L fermentation, the transformant GOEX8 grown in the medium containing 160.0 g/L of glucose produced 186.8?±?6.0 g/L of Ca²⁺-GA, the yield was 1.2 g/g of glucose, and the volumetric productivity was 1.7 g/L/h. Most of the produced GOD1 were located in the yeast cell wall. The purified product was identified to be a GA. The transformant GOEX8 overexpressing the GOD1 gene could produce considerably more Ca²⁺-GA (186.8?±?6.0 g/L) than its wild-type strain P6.     

Production of a novel compound, 10,12-dihydroxystearic acid from ricinoleic acid by an oleate hydratase from Lysinibacillus fusiformis

A recombinant oleate hydratase from Lysinibacillus fusiformis converted ricinoleic acid to a product, whose chemical structure was identified as the novel compound 10,12-dihydroxystearic acid by gas chromatograph/mass spectrometry, Fourier transform infrared, and nuclear magnetic resonance analysis. The reaction conditions for the production of 10,12-dihydroxystearic acid were optimized as follows: pH?6.5, 30 °C, 15 g?l^?1 ricinoleic acid, 9 mg?ml^?1 of enzyme, and 4 % (v/v) methanol. Under the optimized conditions, the enzyme produced 13.5 g?l^?1 10,12-dihydroxystearic acid without detectable byproducts in 3 h, with a conversion of substrate to product of 90 % (w/w) and a productivity of 4.5 g?l^?1?h^?1. The emulsifying activity of 10,12-dihydroxystearic acid was higher than that of oleic acid, ricinoleic acid, stearic acid, and 10-hydroxystearic acid, indicating that 10,12-dihydroxystearic acid can be used as a biosurfactant.     

Micropropagation of cocoyam (Xanthosoma sagittifolium L. Schott) in temporary immersion bioreactor

This study aims at establishing a temporary immersion technique for large-scale propagation of cocoyam (Xanthosoma sagittifolium). Sucrose was experimented with as a determinant factor for shoot multiplication in this culture system. The highest proliferation rate (68 ± 7) occurred with 20 g l^?1 sucrose in the culture medium. This concentration appeared to be the optimal amount due to its promoting effect on plantlet development. The acclimatized plantlets showed a continuous effect of sucrose treatment during ex vitro growth, especially in low sucrose concentration. This fact is evidenced by the low survival rate (0.13 ± 0.12) and the poor chlorophyll content (1.180 ± 0.076 mg g^?1) recorded on plantlets derived from 15 g l^?1 of sucrose. The treatment with 60 g l^?1 of sucrose prior to acclimatization was efficient for roots induction and elongation. The analysis of pH revealed a fluctuation from one subculture to another, with an overall pH decrease under all treatments tested and, thus, indicates that plants release proton during growth. This feature had an impact on in vitro plantlets growth. The potentiality of the temporary immersion technique to foster the production of Xanthosoma sagittifolium is discussed.     

Cloning,Heterologous Expression,Purification and Characterization of M12 Mutant of Aspergillus niger Glucose Oxidase in Yeast Pichia pastoris KM71H

Aspergillus niger glucose oxidase (GOx) genes for wild-type (GenBank accession no. X16061, swiss-Prot; P13006) and M12 mutant (N2Y, K13E, T30 V, I94 V, K152R) were cloned into pPICZαA vector for expression in Pichia pastoris KM71H strain. The highest expression level of 17.5 U/mL of fermentation media was obtained in 0.5 % (v/v) methanol after 9 days of fermentation. The recombinant GOx was purified by cross-flow ultrafiltration using membranes of 30 kDa molecular cutoff and DEAE ion-exchange chromatography at pH 6.0. Purified wt GOx had k _cat of 189.4 s^?1 and K _m of 28.26 mM while M12 GOx had k _cat of 352.0 s^?1 and K _m of 13.33 mM for glucose at pH 5.5. Specificity constants k _cat/K _m of wt (6.70 mM^?1 s^?1) and M12 GOx (26.7 mM^?1 s^?1) expressed in P. pastoris KM71H were around three times higher than for the same enzymes previously expressed in Saccharomyces cerevisiae InvSc1 strain. The pH optimum and sugar specificity of M12 mutant of GOx remained similar to the wild-type form of the enzyme, while thermostability was slightly decreased. M12 GOx expressed in P. pastoris showed three times higher activity compared to the wt GOx toward redox mediators like N,N-dimethyl-nitroso-aniline used for glucose strips manufacturing. M12 mutant of GOx produced in P. pastoris KM71H could be useful for manufacturing of glucose biosensors and biofuel cells.     

Microbial transformation of ginsenoside Rb1 to compound K by Lactobacillus paralimentarius

In this study, the major ginsenoside Rb1 was transformed into the more pharmacologically active minor compound K by food grade Lactobacillus paralimentarius LH4, which was isolated from kimchi, a traditional Korean fermented food. The enzymatic reaction was analyzed by TLC, HPLC, and NMR. Using the cell-free enzyme of Lactobacillus paralimentarius LH4 at optimal conditions for 30 °C at pH 6.0, 1.0 mg ml^?1 ginsenoside Rb1 was transformed into 0.52 mg ml^?1 compound K within 72 h, with a corresponding molar conversion yield of 88 %. The cell-free enzyme hydrolyzed the two glucose moieties attached to the C-3 position and the outer glucose moiety attached to the C-20 position of the ginsenoside Rb1. The cell-free enzyme hydrolyzed the ginsenoside Rb1 along the following pathway: ginsenoside Rb1 → gypenoside XVII and ginsenoside Rd → ginsenoside F2 → compound K. Our results indicate that Lactobacillus paralimentarius LH4 has the potential to be applied for the preparation of compound K in the food industry.     

Two-stage fermentation process for enhanced mannitol production using Candida magnoliae mutant R9

Mutants of Candida magnoliae NCIM 3470 were generated by treatment of ultra-violet radiations, ethyl methyl sulphonate and N-methyl-N′-nitro-N-nitrosoguanidine. Mutants with higher reductase activity were screened by means of 2,3,5-triphenyl tetrazolium chloride agar plate assay. Among the screened mutants, the mutant R9 produced maximum mannitol (i.e. 46 g l^?1) in liquid fermentation medium containing 250 g l^?1 glucose and hence was selected for further experiments. Preliminary optimization studies were carried out on shake-flask level which increased the mannitol production to 60 g l^?1 in liquid fermentation medium containing 300 g l^?1 glucose. A two-stage fermentation process comprising of growth phase and production phase was employed. During the growth phase, glucose was supplemented and aerobic conditions were maintained. Thereafter, the production phase was initiated by supplementing fructose and switching to anaerobic conditions by discontinuing aeration and decreasing the speed of agitation. The strategy of two-stage fermentation significantly enhanced the production of mannitol up to 240 g l^?1, which is the highest among all fermentative production processes and corresponds to 81 % yield and 4 g l^?1 h^?1 productivity without formation of any by-product.     

A xylanase from <Emphasis Type="Italic">Streptomyces sp.</Emphasis> FA1: heterologous expression,characterization, and its application in Chinese steamed bread

Xylanases (EC 3.2.1.8) are hydrolytic enzymes that have found widespread application in the food, feed, and paper-pulp industries. Streptomyces sp. FA1 xynA was expressed as a secreted protein in Pichia pastoris, and the xylanase was applied to the production of Chinese steamed bread for the first time. The optimal pH and the optimal temperature of XynA were 5.5 and 60 °C, respectively. Using beechwood as substrate, the K _m and V _max were 2.408 mg mL^?1 and 299.3 µmol min^?1 mg^?1, respectively. Under optimal conditions, a 3.6-L bioreactor produced 1374 U mL^?1 of XynA activity at a protein concentration of 6.3 g L^?1 after 132 h of fermentation. Use of recombinant XynA led to a greater increase in the specific volume of the CSB than could be achieved using commercial xylanase under optimal conditions. This study provides the basis for the application of the enzyme in the baking industry.     

Kinetics of growth and ethanol formation from a mix of glucose/xylose substrate by Kluyveromyces marxianus UFV-3

The fermentation of both glucose and xylose is important to maximize ethanol yield from renewable biomass feedstocks. In this article, we analyze growth, sugar consumption, and ethanol formation by the yeast Kluyveromyces marxianus UFV-3 using various glucose and xylose concentrations and also under conditions of reduced respiratory activity. In almost all the conditions analyzed, glucose repressed xylose assimilation and xylose consumption began after glucose had been exhausted. A remarkable difference was observed when mixtures of 5 g L^?1 glucose/20 g L^?1 xylose and 20 g L^?1 glucose/20 g L^?1 xylose were used. In the former, the xylose consumption began immediately after the glucose depletion. Indeed, there was no striking diauxic phase, as observed in the latter condition, in which there was an interval of 30 h between glucose depletion and the beginning of xylose consumption. Ethanol production was always higher in a mixture of glucose and xylose than in glucose alone. The highest ethanol concentration (8.65 g L^?1) and cell mass concentration (4.42 g L^?1) were achieved after 8 and 74 h, respectively, in a mixture of 20 g L^?1 glucose/20 g L^?1 xylose. When inhibitors of respiration were added to the medium, glucose repression of xylose consumption was alleviated completely and K. marxianus was able to consume xylose and glucose simultaneously.     

Culture medium optimization of a new bacterial extracellular polysaccharide with excellent moisture retention activity

A new kind of extracellular polysaccharide (EPS) from Pseudomonas fluorescens PGM37 was obtained and culture media was optimized using the statistical methods single factor experiments and response surface methodology (RSM) design. As a result, the optimum cultivation conditions initial pH value, medium volume, inoculum size, temperature, and rotation speed were 7.5, 100 mL/250 mL, 5 %, 28 °C, and 180 rpm, respectively. The optimized media: sucrose 36.23 g?L^?1, yeast extract 3.32 g?L^?1, sodium chloride 1.13 g?L^?1, and calcium chloride 0.20 g?L^?1. The maximum predicted yield of EPS was 10.1163 g?L^?1 under these conditions. The validation data was 10.012 g?L^?1, which could strongly confirm the correlation between the experimental and theoretical values. Gas Chromatography analysis revealed that the polymer was made up of mannose and glucose in the ratio of 1:1. Infrared spectroscopy showed that the polysaccharide had β-D-pyranoid configuration and contained no other substituent. Graded by different multiples of alcohol after specific degradation by enzyme and then detected by LC-ESI-MS, the EPS structure was β-D-Glcp-(1, 4)-β-D-Manp-(1, 4)-β-D-Glcp-(1, 4)-β-D-Manp. The moisture retention ability of the EPS was found to be superior to glycerol and only a little inferior to hyaluronic acid (HA), which presented potential application value in cosmetics and clinical medicine fields.