A one-pot two-enzyme system on the production of high value-added D-allulose from Jerusalem artichoke tubers

A one-pot two-enzyme reaction system was developed to produce high-fructose syrup (HFS) containing healthy rare sugar d-allulose from Jerusalem artichoke (JA). Inulin in JA was converted through the cascade reaction of a novel exo-inulinase from Bacillus velezensis (BvInu) and D-allulose 3-epimerase from Ruminococcus sp. (RDAE). BvInu and RDAE were expressed in Bacillus subtilis and successfully secreted into the supernatant, which decrease the production cost and avoid enzyme purification. The optimal temperature and stability of extracellular BvInu significantly increased, thereby promoting the catalytic activity of the two enzymes for the cascade reaction in one pot. Inulin in JA powder was almost completely converted into monosaccharides in the treatment with the optimal ratio of BvInu : RDAE (80 : 40 U/g inulin) at 50 °C for 2 h. The ratio of D-glucose, D-fructose, and D-allulose in the product was approximately 1:3:1, and the yield on JA powder was 67%. The system exhibits potential high-valued application of non-grain crops on producing HFS with D-allulose.     

Expression of exoinulinase genes in Saccharomyces cerevisiae to improve ethanol production from inulin sources

To improve inulin utilization and ethanol fermentation, exoinulinase genes from the yeast Kluyveromyces marxianus and the recently identified yeast, Candida kutaonensis, were expressed in Saccharomyces cerevisiae. S. cerevisiae harboring the exoinulinase gene from C. kutaonensis gave higher ethanol yield and productivity from both inulin (0.38 vs. 0.34 g/g and 1.35 vs. 1.22 g l^?1 h^?1) and Jerusalem artichoke tuber flour (0.47 vs. 0.46 g/g and 1.62 vs. 1.54 g l^?1 h^?1) compared with the strain expressing the exoinulinase gene from K. marxianus. Thus, the exoinulinase gene from C. kutaonensis is advantageous for engineering S. cerevisiae to improve ethanol fermentation from inulin sources.     

Cloning of exoinulinase gene from Penicillium janthinellum strain B01 and its high-level expression in Pichia pastoris

Aims: The aim of this study is to improve exoinulinase production by expression of a cloned exoinulinase gene inuA1 (GenBank accession no. JF961344 ) from Penicillium janthinellum strain B01 in Pichia pastoris. Methods and Results: A full‐length cDNA of exoinulinase gene (inuA1) was cloned from P. janthinellum strain B01 using RACE PCR. An open reading frame (ORF) of 2115 bp is interrupted by a single intron of 67 bp. The fragment encodes a signal peptide with 20 amino acids and a mature protein with 684 amino acids. The inuA1 was subcloned to the pPICZαC expression vector and succesfully over‐expressed in Pichia pastoris X‐33. The highest activity of exoinlinase reached 272·8 U ml^?1 in the fermentation liquid. It was c. 11‐fold of that produced by wild‐strain B01. A large amount of fructose was identified after the hydrolysis of inulin with the crude recombinant exoinulinase. The recombinant exoinulinase was purified and characterized. The molecular weight of the purified recombinant exoinulianse was 100 kDa. The mass spectrometry result indicated that the purified protein was indeed recombinant exoinulinase. The optimal pH and temperature of the purified recombinant exoinulianse were 4·5 and 50°C, respectively. Conclusions: An exoinulinase gene of P. janthinellum strain B01 was cloned, sequenced and over‐expressed successfully in P. pastoris. Significance and Impact of the Study: Only a few genes have been cloned from P. janthinellum because its molecular biology is poorly understood. In this study, we cloned and over‐expressed inuA1 gene of P. janthinellum in P. pastoris. This recombinant exoinulinase can be used to hydrolyse inulin to produce fructose and facilitate the biofuel production from inulin resources.     

Continuous production of fructose-syrups from inulin by immobilized inulinase from recombinantSaccharomyces cerevisiae

Recombinant exoinulinase was partially purified from the culture supernatant ofS. cerevisiae by (NH₄)₂SO₄ precipitation and PEG treatment. The purified inulinase was immobilized onto Amino-cellulofine with glutaraldehyde as a cross-linking agent. Immobilization yield based on the enzyme activity was about 15%. Optimal pH and temperature of immobilized enzyme were found to be 5.0 and 60°C, respectively. The enzyme activity was stably maintained in the pH ranges of 4.5 to 6.0 at 60°C. 100% of enzyme activity was observed even after incubation for 24 hr at 60°C. In the operation of a packed-bed reactor containing 412 U inulinase, dahalia inulin of 7.5%(w/v) concentration was completely hydrolyzed at flow rate of 2.0 mL/min at 60°C, resulting in a volumetric productivity of 693 g-reducing sugars/L/h. Under the reaction conditions of 1.0 mL/min flow rate with 2.5% inulin at 60°C, the reactor was successfully operated over 30 days without loss of inulinase activity.     

Study on survival,growth, haematology and body composition of Cyprinus carpio under different acute and chronic salinity regimes

Pakistan’s most of the land is less productive or no productivity at all due to erosion and salinity of the soil, which can be utilized to develop fisheries. The project, “Survival, growth and body composition of Cyprinus carpio under different salinity regimes” was undertaken in two phases. In the first phase susceptibility of Cyprinus carpio at four salinity levels in triplicate within 0–10 g L⁻¹NaCl for 96 h in each aquarium was checked after one week acclamation at 0 g L⁻¹, 2 g L⁻¹ and 4 g L⁻¹ NaCl. LC₅₀ values varied from 7.67 to 10.65 g L⁻¹ after 96 h for C. carpio. Percentage mortality of the fish and important water quality parameters after every 12 h were observed for a period of 96-h. Probit analysis showed that 96-h LC₅₀ values ranged from 7.67 to 10.65 g L⁻¹. During experimental period aquaria water temperature ranged from 29.6 to 33.7 °C, pH values fluctuated between 7.8 and 9.7, Electrical conductivity values ranged from 2.40 to 20.13 dSm⁻¹ and Dissolved oxygen ranged between 2.23 and 10 mg L⁻¹. Sub-lethal salt concentration i.e. 0 g L⁻¹ to 3 g L⁻¹ NaCl upto 40 days showed that growth of C. carpio decreased with the increase of water salinity levels and ceased at 4 g L⁻¹ salinity and increase in salinity have negatively affected hematological parameters.     

Efficient chemoenzymatic synthesis of (S)-α-amino-4-fluorobenzeneacetic acid using immobilized penicillin amidase

An efficient chemoenzymatic route was developed for synthesis of (S)-α-amino-4-fluorobenzeneacetic acid, a valuable chiral intermediate of Aprepitant, using immobilized penicillin amidase catalyzed kinetic resolution of racemic N-phenylacetyl-4-fluorophenylglycine. The optimum temperature, pH and agitation rate of the reaction were determined to be 40 °C, 9.5 and 300 rpm, respectively. Kinetic resolution of 80 g L⁻¹ N-phenylacetyl-4-fluorophenylglycine by immobilized amidase 20 g L⁻¹ resulted in 49.9% conversion and >99.9% e.e. within 3 h. The unreacted N-phenylacetyl-4-fluorophenylglycine can be easily racemized and then recycled as substrate. The production of (S)-α-amino-4-fluorobenzeneacetic acid was further amplified in 1 L reaction system, affording excellent conversion (49.9%) and enantioselectivity (99.9%). This chemoenzymatic approach was demonstrated to be promising for industrial production of (S)-α-amino-4-fluorobenzeneacetic acid.     

Characterization of recombinant L-ribose isomerase acquired from Cryobacterium sp. N21 with potential application in L-ribulose production

l-Ribose isomerase (lRI) is an enzyme that can catalyze the reversible isomerization between l-ribose and l-ribulose. It can also perform the conversion between many aldoses into their corresponding ketoses. l-RI was produced from Cryobacterium sp. N21 (CrL-RIse), and l-ribose was utilized as a substrate. The recombinant l-RI gene was cloned and overexpressed from Cryobacterium sp. N21. The purification of CrL-RIse was performed by metal-affinity chromatography. The enzyme displayed a corresponding band with an approximate size of 35 kDa on the SDS-PAGE analysis. The protein for this gene contains 266 amino acids with an expected molecular weight (M_w) of 29.6 kDa. The measured M_w of CrL-RIse calculated by HPLC was 125 kDa. CrL-RIse was extremely active in glycine buffer at 35 °C, pH 9.0, showing a specific activity of 54.96 U mg⁻¹. CrL-RIse displayed no major increase in activity with metal ions, excluding Mn²⁺. The estimated Km, K_cat, K_cat/Km and V_max values of CrL-RIse were 37.8 mM, 10,416 min⁻¹, 275.43 min⁻¹ mM⁻¹, and 250 U mg⁻¹, respectively. The rate of l-ribulose production was 31 % (6.24, 12.11, and 20.89 g L⁻¹) at equilibrium by utilizing 20, 40, and 70 g L⁻¹ of the substrate, respectively. The results indicated that CrL-RIse has the capability to manufacture l-ribulose from l-ribose.     

Effective biodegradation of 2,4,6-trinitrotoluene using a novel bacterial strain isolated from TNT-contaminated soil

In this environmental-sample based study, rapid microbial-mediated degradation of 2,4,6-trinitrotoluene (TNT) contaminated soils is demonstrated by a novel strain, Achromobacter spanius STE 11. Complete removal of 100 mg L⁻¹ TNT is achieved within only 20 h under aerobic conditions by the isolate. In this bio-conversion process, TNT is transformed to 2,4-dinitrotoluene (7 mg L⁻¹), 2,6-dinitrotoluene (3 mg L⁻¹), 4-aminodinitrotoluene (49 mg L⁻¹) and 2-aminodinitrotoluene (16 mg L⁻¹) as the key metabolites. A. spanius STE 11 has the ability to denitrate TNT in aerobic conditions as suggested by the dinitrotoluene and NO₃ productions during the growth period. Elemental analysis results indicate that 24.77 mg L⁻¹ nitrogen from TNT was accumulated in the cell biomass, showing that STE 11 can use TNT as its sole nitrogen source. TNT degradation was observed between pH 4.0–8.0 and 4–43 °C; however, the most efficient degradation was at pH 6.0–7.0 and 30 °C.     

Enhanced expression of endoinulinase from Aspergillus niger by codon optimization in Pichia pastoris and its application in inulooligosaccharide production

In the present study, the endoinulinase gene (EnInu) from Aspergillus niger CICIM F0620 was optimized according to the codon usage of Pichia pastoris and both the native and the optimized gene were expressed in P. pastoris. Use of the optimized gene resulted in the secretion of recombinant endoinulinase activity that reached 1,349 U ml^?1, 4.18 times that observed using the native gene. This is the highest endoinulinase activity reported to date. The recombinant enzyme was optimally active at pH 6.0 and 60 °C. Moreover, inulooligosaccharides production from inulin was studied using the recombinant enzyme produced from the optimized gene. After 8 h under optimal conditions, which included 400 g l^?1 inulin, an enzyme concentration of 40 U g^?1 substrate, 50 °C and pH 6.0, the inulooligosaccharide yield was 91 %. The high substrate concentration and short reaction time described here should reduce production costs distinctly, compared with the conditions used in previous studies. Thus, this study may provide the basis for the industrial use of this recombinant endoinulinase for the production of inulooligosaccharides.     

Effect of tannins on growth and amylase production by Calvatia gigantea

The effect of tannins was investigated on growth and α-amylase (α-1,4-glucan 4-glucanohydrolase, EC 3.2.1.1) production by the edible fungal species Calvatia gigantea, grown in a laboratory-scale fermenter on acorn starch media containing up to 2 g tannins l⁻¹. No inhibition of both growth and amylase excretion was observed when the fungus was cultivated on media containing 40 to 100 times higher tannin concentration than that reported to inhibit microbial growth. Amylase excretion was enhanced when starch was dry sterilized but specific growth rate was higher when starch was wet sterilized. Biomass and amylase production increased with increasing substrate concentration and specific growth rate reached its maximum value at 20 g l⁻¹ starch concentration. The optimum pH of biomass and amylase productionwas 5.0–5.5 and 6.0−6.5 respectively and that of temperature was 29–32 and 29–30°C respectively. Maximum yields of 68 250 U amylase and 0.58–0.60 g biomass g⁻¹ acorn were obtained at optimum growth conditions. A plot of reciprocal growth rate vs. reciprocal starch concentration made it possible to calculate K_s = 0.84 g acorn starch l⁻¹ and μ_max = 0.249 h⁻¹.     

Purification and characteristics of two exoinulinases from Chrysosporium pannorum

Two types of exoinulinase (2,1-β-d-fructan fructanohydrolase EC 3.2.1.7) were purified from Chrysosporium pannorum AHU 9700. The enzymes, F2 and F3, were glyco-proteins having isoelectric points around pH 4.6 and 4.45. The molecular weights were estimated by SDS-polyacrylamide gel electrophoresis to be 84,000 and 70,000, respectively. The enzymes were active on inulin, sucrose, raffinose, stachyose, and fructo-oligosaccharides, but not on melezitose. Levan could be hydrolyzed by exoinulinase F3, but not by exoinulinase F2. The hydrolysis products of inulin by the two enzymes were fructose and small amounts of glucose.     

Inulinase production by a marine yeast <Emphasis Type="Italic">Pichia guilliermondii</Emphasis> and inulin hydrolysis by the crude inulinase

Marine yeast strain 1, isolated from the surface of a marine alga, was found to secrete a large amount of inulinase into the medium. This marine yeast was identified as a strain of Pichia guilliermondii according to the results of routine yeast identification and molecular methods. The crude inulinase produced by this marine yeast worked optimally at pH 6.0 and 60°C. The optimal medium for inulinase production was seawater containing 4.0% (w/v) inulin and 0.5% (w/v) yeast extract, while the optimal cultivation conditions for inulinase production were pH 8.0, 28°C and 170 rpm. Under the optimal conditions, over 60 U ml⁻¹ of inulinase activity was produced within 48 h of fermentation in shake flasks. A large amount of monosaccharides and a trace amount of oligosaccharides were detected after the hydrolysis, indicating that the crude inulinase had a high exoinulinase activity.     

Biosynthesis of lipids by Rhodosporidium toruloides ATCC 10788

The limiting amount of nitrogen required to trigger lipid accumulation in the oleaginous yeast Rhodosporidium toruloides ATCC 10788 was studied, batchwise, by subjecting washed mid-exponentially grown cells to nitrogen at levels of 10⁻² M down to 10⁻⁴ M per g l⁻¹ of lean cells (2–5% fat content) in a mineral medium where glucose was present at 35 g l⁻¹. The results showed that lipid accumulation always started sometime after nitrogen reached a level of 3 × 10⁻⁵ M and the specific initial lipid productivity was constant. Furthermore, the cells were subjected to nine combinations of temperature and pH, from (25° C, pH 4.5) to (35° C, pH 7.5) in the mineral medium supplemented with 0.5 g l⁻¹ of yeast extract and 1 g l⁻¹ (NH₄)₂SO₄. As was expected, lipid content in the cells was higher at 25° C, but pH around 6.0–7.5 slightly enhanced the effect of lower temperature. The effect of pH was also noticed to affect the size of changes in the temporal profiles of the oil's fatty acid distribution prior to nitrogen depletion, whereas no significant difference in the fatty acid composition of the oil was shown after exhaustion of nitrogen from the medium for all combinations of temperature and pH.     

Cloning and characterization of an exoinulinase from Bacillus polymyxa

A gene encoding an exoinulinase (inu) from Bacillus polymyxa MGL21 was cloned and sequenced. It is composed of 1455 nucleotides, encoding a protein (485 amino acids) with a molecular mass of 55522 Da. Inu was expressed in Escherichia coli and the His-tagged exoinulinase was purified. The purified enzyme hydrolyzed sucrose, levan and raffinose, in addition to inulin, with a sucrose/inulin ratio of 2. Inulinase activity was optimal at 35°C and pH 7, was completely inactivated by 1 mM Ag⁺ or Hg²⁺. The K _m and V _max values for inulin hydrolysis were 0.7 mM and 2500 M min^–1 mg^–1 protein. The enzyme acted on inulin via an exo-attack to produce fructose mainly.     

Effects of oxygen volumetric mass transfer coefficient and pH on lipase production by Staphylococcus warneri EX17

The principal objectives of this study were to evaluate the kinetics of lipase production by Staphylococcus warneri EX17 under different oxygen volumetric mass transfer coefficients (k_La) and pH conditions in submerged bioreactors, using glycerol (a biodiesel by-product) as a carbon source. Cultivations were conducted at different k_La (26, 38, 50, and 83 h⁻¹) and pH values (6.0, 7.0, and 8.0). The optimal k_La and pH were 38 h⁻¹ and 7.0, respectively. Under these conditions, the maximal cell production obtained was 8.0 g/L, and the volumetric and specific lipase production reached high levels of activity, approximately 800 U/L and 150 U/g cell, respectively, after 12 h of cultivation. This result was approximately five times higher than that obtained in the shake flask cultures. The relationship between cell growth and lipase production was found to be associated with growth by the Luedeking-Piret model.     

Characterization and properties of an inulinase from a thermophilic bacteria

The inulinase of the thermophilic bacterial strain LCB41 (Bacillus sp.) was produced in fermentor using a mineral medium containing inulin as carbon source. The enzyme content was as high as the known inulinase producers and most of the activity was found in the culture medium. The enzyme was stable at high temperature and active at neutral and slightly basic pH. Fructose is liberated as the sole reaction product of inulin hydrolysis, classifying the enzyme as an exoinulinase. Inulin and sucrose were both hydrolyzed at appreciable rates with an (I/S) ratio of 0·40 and (V_m/K_m)₁/(V_m/K_m)_S = 9·9. The enzyme was less inhibited than yeast invertase or Kluyveromyces fragilis inulinase at high sucrose concentrations. The inulinase of strain LCB41 is a good candidate for industrial hydrolysis of inulin or sucrose.     

Expression,characterization and structural profile of a heterodimeric β-galactosidase from the novel strain Lactobacillus curieae M2011381

A heterodimeric β-galactosidase was discovered in the novel strain Lactobacillus curieae M2011381. The gene encoding the enzyme was expressed in Escherichia coli BL21 (DE3). The specific enzyme activities of the recombinant holoenzyme (LacLM) and large subunit (LacL) measured 11.4 U/mg and 3.8 U/mg, respectively. The k_cat/K_m values of LacLM and LacL were 740 mM⁻¹ s⁻¹ and 1.40 mM⁻¹ s⁻¹, respectively. LacLM showed maximum activity at pH 8.0 and 55 °C, and it could maintain its activity at a neutral pH and below 45 °C. LacLM displayed both hydrolysis and transgalactosylation activity on 200 g/L lactose. When LacLM was added to milk, the lactose was hydrolyzed after 6 h without galactooligosaccharide generation. The sequence alignment and homology modeling of the structures of the holoenzyme and subunits revealed that LacL has a catalytic domain with a catalytic dyad, Glu470 and Glu538, and small subunit LacM is a β-sheet domain with a conserved Trp294. The molecular docking of LacLM helped to illustrate the roles of both subunits in the reaction with lactose.     

Fed-batch fermentation of recombinant Citrobacter freundii with expression of a violacein-synthesizing gene cluster for efficient violacein production from glycerol

The extensive prospects of violacein in the pharmaceutical industry have attracted increasing interest. However, the fermentation levels of violacein are currently inadequate to meet the demands of industrial production. This study was undertaken to develop an efficient process for the production of violacein by recombinant Citrobacter freundii. The effects of dissolved oxygen (DO) and pH on cell growth and violacein production in batch cultures were investigated first. When the DO and pH of the medium were controlled at around 25% and 7.0, respectively, the biomass and concentration of violacein were maximized. Based on the consumption of nutrients in the medium observed during batch culture, a fed-batch fermentation strategy with controlled DO and pH was implemented. By continuously feeding glycerol, NH₄Cl, and l-tryptophan at a constant feeding rate of 16 mL h⁻¹, the final concentration of violacein reached 4.13 g L⁻¹, which was 4.09-fold higher than the corresponding batch culture, and the maximal dry cell weight (DCW) and average violacein productivity obtained for the fed-batch culture were 3.34 g DCW L⁻¹ and 82.6 mg L⁻¹ h⁻¹, respectively. To date, this is the first report on the efficient production of violacein by genetically engineered strains in a fermentor.     

Considerazioni su Gelsi Ingialliti per Fame

Jerusalem artichoke (Helianthus tuberosus L.), an important crop, containing over 50% inulin in its tubers on a dry weight basis is an agricultural and industrial crop with a great potential for production of ethanol and industrial products. Inulin is a good substrate for bioethanol production. Saccharomyces cerevisiae 6525 can produce high concentrations of ethanol, but it cannot synthesize inulinase. In this study, a new integration vector carrying inuA1 gene encoding exoinulinase was constructed and transformed into 18SrDNA site of industrial strain S. cerevisiae 6525. The obtained transformant, BR8, produced 1.1 U mL^? 1 inulinase activity within 72 h and the dry cell weight reached 12.3 g L^? 1 within 48 h. In a small-scale fermentation, BR8 produced 9.5% (v/v) ethanol, with a productivity rate of 0.385 g ethanol per gram inulin, while wild-type S. cerevisiae 6525 produced only 3.3% (v/v) ethanol in the same conditions. In a 5-L fermentation, BR8 produced 14.0% (v/v) ethanol in fermentation medium containing inulin and 1% (w/v) (NH4)₂SO₄. The engineered S. cerevisiae 6525 carrying inuA1 converted pure nonhydrolyzed inulin directly into high concentrations of ethanol.     

Sulfate reduction and microbial community of autotrophic biocathode in response to acidity

Microbial electrolysis cells (MECs) with autotrophic biocathode are a promising technology for removal of pollutants in wastewater. The aim of this study was to investigate the effect of initial acidity of wastewater on performance of sulfate-reducing biocathodes. MECs with biocathodes were operated with initial pH values of catholyte ranged from 3.0 to 7.0. The optimum initial pH value was 6.0 with a maximum sulfate reductive rate and biomass of 57 mg L⁻¹ d⁻¹ and 2.1 ± 0.4 mg g⁻¹, respectively. With initial pH 7.0, the pH value of catholyte increased to 9.8 ± 0.2 after an operation cycle, which resulted in low performance of the biocathode. A considerable sulfate reductive rate of 31 ± 0.85 mg L⁻¹ d⁻¹ was achieved with initial pH 3.0. Desulfovibrio sp. grew dominantly with abundance of 46%–66% in the cathode biofilm with initial pH values from 3.0 to 6.0 and contributed to the sulfate reduction. Clostridium and Parapedobacter also had high abundance in pH 6.0 cathode, indicated that interspecies electron transfer between electrochemical active and sulfate-reducing bacteria could play an important role in sulfate removal. The results suggest that acidity of catholyte is an important factor to be considered to utilize autotrophic biocathode MECs for wastewater treatment.