Purification and characterization of a novel extracellular halophilic and organic solvent-tolerant amylopullulanase from the haloarchaeon, Halorubrum sp. strain Ha25

A halophilic archaeon, Halorubrum sp. strain Ha25, produced extracellular halophilic organic solvent-tolerant amylopullulanase. The maximum enzyme production was at high salt concentration, 3–4 M NaCl. Optimum pH and temperature for enzyme production were 7.0 and 40 °C, respectively. Molecular mass of purified enzyme was estimated to be about 140 kDa by SDS–PAGE. This enzyme was active on pullulan and starch as substrates. The apparent K _m for the enzyme activity on pullulan was 4 mg/ml and for soluble starch was 1.8 mg/ml. Optimum temperature for amylolytic and pullulytic activities was 50 °C. Optimum pH for amylolytic activity was 7 and for pullulytic activity was 7.5. This enzyme was active over a wide range of concentrations (0–4.5 M) of NaCl. The effect of organic solvents on the enzyme activities showed that this enzyme was more stable in the presence of non-polar organic solvents than polar solvents. This study is the first report on amylopullulanase production in halophilic bacteria and archaea.     

Characteristic of immobilized cephalosporin C acylase and its application in one-step enzymatic conversion of cephalosporin C to 7-aminocephalosporanic acid

Cephalosporin C (CPC) acylase is an enzyme which hydrolyzes CPC to 7-aminocephalosporanic acid (7-ACA) directly, and therefore has great potential in industrial application. In this study, the CPC acylase from a recombinant Escherichia coli was purified to high purity by immobilized metal affinity chromatography, and the CPC acylase was covalently attached to three kinds of epoxy supports, BB-2, ES-V-1 and LX-1000EP. The immobilized CPC acylase with LX-1000EP as the support shows the highest activity (81 U g⁻¹) suggesting its potential in industrial 7-ACA production. The activity of immobilized enzyme was found to be optimal at pH between 8.5 and 9.5 and to increase with temperature elevation until 55 °C. Immobilized CPC acylase showed good stability at pH between 8.0 and 9.5 and at temperature up to 40 °C. To avoid product degradation, the production of 7-ACA utilizing immobilized enzyme was carried out at 25 °C, pH 8.5 in a designed reactor. Under optimal reaction conditions, a very high 7-ACA yield of 96.7% was obtained within 60 min. In the results of repeated batch production of 7-ACA, 50% activity of the initial cycle was maintained after being recycled 24 times and the average conversion rate of CPC reached 98%.     

Comparative biochemical characterization of soluble and chitosan immobilized β-galactosidase from Kluyveromyces lactis NRRL Y1564

An investigation was conducted on the production of β-galactosidase (β-gal) by different strains of Kluyveromyces, using lactose as a carbon source. The maximum enzymatic activity of 3.8 ± 0.2 U/mL was achieved by using Kluyveromyces lactis strain NRRL Y1564 after 28 h of fermentation at 180 rpm and 30 °C. β-gal was then immobilized onto chitosan and characterized based on its optimal operation pH and temperature, its thermal stability and its kinetic parameters (K_m and V_max) using o-nitrophenyl β-d-galactopyranoside as substrate. The optimal pH for soluble β-gal activity was found to be 6.5 while the optimal pH for immobilized β-gal activity was found to be 7.0, while the optimal operating temperatures were 50 °C and 37 °C, respectively. At 50 °C, the immobilized enzyme showed an increased thermal stability, being 8 times more stable than the soluble enzyme. The immobilized enzyme was reused for 10 cycles, showing stability since it retained more than 70% of its initial activity. The immobilized enzyme retained 100% of its initial activity when it was stored at 4 °C and pH 7.0 for 93 days. The soluble β-gal lost 9.4% of its initial activity when it was stored at the same conditions.     

Factors influencing β-glucosidase production, activity and stability inNectria catalinensis

The influence of different cultivation conditions on β-glucosidase production and of some parameters on the activity and stability of this enzyme were studied inNectria catalinensis. Maximal β-glucosidase production was achieved with ammonium nitrate (0.5 g N/L) as nitrogen source. Tween 80, Tween 20 and Triton X-100 increased β-glucosidase yields, Tween 80 was the most effective for enzyme release and growth at a concentration of 3.4 mmol/L. On the other hand, Tween 20 and Triton X-100 had an inhibitory effect onN. catalinensis growth. A temperature of 23°C and an initial pH of cultures of 6.5 were optimal for biomass and β-glucosidase production. Under optimal cultural conditions (ammonium nitrate, 0.5 g N/L; Tween 80, 3.4 mmol/L; 23°C; initial pH 6.5) the β-glucosidase yield was increased more than five fold respect to the initial state. Optimal temperature for β-glucosidase activity was 45°C, the initial activity dropped 60 % after 6 h of incubation at this temperature. Optimal pH for enzyme activity was 5.3. At this pH the β-glucosidase was completely stable after 3 d of incubation. TheV andK _m values calculated from Lineweaver-Burk and Eadie-Hofstee plots were 0.23 μmol 4-nitrophenol per min per mg of protein and 0.25 mmol 4-nitrophenol β-d-glucopyranoside per L, respectively. The activation energy according to Arrhenius plot was 49.6 KJ/mol.     

Molecular cloning and expression of an extracellular α-amylase gene from an Antarctic deep sea psychrotolerant <Emphasis Type="Italic">Pseudomonas stutzeri</Emphasis> strain 7193

Psychrotolerant Pseudomonas stutzeri strain 7193 capable of producing an extracellular α-amylase was isolated from deep sea sediments of Prydz Bay, Antarctic. The 59678-Da protein (AmyP) was encoded by 1665-bp gene (amyP). The deduced amino acid sequence was identified with four regions, which are conserved in amylolytic enzymes and form a catalytic domain, and was predicted to be maltotetraose forming extracellular amylase by using the I-TASSER online server. Purification of AmyP amylases from both the recombinant of Escherichia coli Top 10 F′ and strain 7193 was conducted. Biochemical characterization revealed that the optimal amylase activity was observed at pH 9.0 and temperature 40°C. The enzymes were unstable at temperatures above 30°C, and only retain half of their highest activity after incubation at 60°C for 5 min. Thin-layer chromatography analysis of the products of the amylolytic reaction showed the presence of maltotetraose, maltotriose, maltose and glucose in the starch hydrolysate.     

Pestalotiopsis mangiferae isolated from cocoa leaves and concomitant tannase and gallic acid production

The enzyme tannase is of great industrial and biotechnological importance for the hydrolysis of vegetable tannins, reducing their undesirable effects and generating products for a wide range of processes. Thus, the search for new microorganisms that permit more stable tannase production is of considerable importance. A strain of P. mangiferae isolated from cocoa leaves was selected and investigated for its capacity to produce tannase enzymes and gallic acid through submerged fermentation. The assessment of the variables affecting tannase production by P. mangiferae showed that tannic acid, ammonium nitrate and temperature were the most significant (8.4 U/mL). The variables were analyzed using Response Surface Methodology - RSM (Box-Behnken design), with the best conditions for tannase production being: 1.9% carbon source, 1% nitrogen source and temperature of 23 °C. Tannase activity doubled (16.9 U/mL) after the optimization process when compared to the initial fermentation. A pH of 7.0 was optimal for the tannase and it presented stability above 80% with pH between 4.0 and 7.0 after 2h of incubation. The optimal temperature was 30 °C and activity remained at above 80% at 40–60 °C after 1 h. Production of gallic acid was achieved with 1% tannic acid (0.9 mg/mL) and P. mangiferae had not used up the gallic acid produced by tannic acid hydrolysis after 144 h of fermentation. A 5% tannic acid concentration was the best for gallic acid production (1.6 mg/mL). These results demonstrate P. mangiferae’s potential for tannase and gallic acid production for biotechnological applications.     

Characterization of hyperthermostable α-amylase from Geobacillus sp. IIPTN

A newly isolated Geobacillus sp. IIPTN (MTCC 5319) from the hot spring of Uttarakhand's Himalayan region produced a hyperthermostable α-amylase. The microorganism was characterized by biochemical tests and 16S rRNA gene sequencing. The optimal temperature and pH were 60°C and 6.5, respectively, for growth and enzyme production. Although it was able to grow in temperature ranges from 50 to 80°C and pH 5.5–8.5. Maximum enzyme production was in exponential phase with activity 135 U ml⁻¹ at 60°C. Assayed with cassava as substrate, the enzyme displayed optimal activity 192 U ml⁻¹ at pH 5.0 and 80°C. The enzyme was purified to homogeneity with purification fold 82 and specific activity 1,200 U mg⁻¹ protein. The molecular mass of the purified enzyme was 97 KDa. The values of K _m and V _max were 36 mg ml⁻¹ and 222 μmol mg⁻¹ protein min⁻¹, respectively. The amylase was stable over a broad range of temperature from 40°C to 120°C and pH ranges from 5 to 10. The enzyme was stimulated with Mn²⁺, whereas it was inhibited by Hg²⁺, Cu²⁺, Zn²⁺, Mg²⁺, and EDTA, suggesting that it is a metalloenzyme. Besides hyperthermostability, the novelty of this enzyme is resistance against protease.     

Biochemical characterization of a novel thermostable xyloglucanase from an alkalothermophilic Thermomonospora sp.

Xyloglucanase from an extracellular culture filtrate of alkalothermophilic Thermomonospora sp. was purified to homogeneity with a molecular weight of 144 kDa as determined by SDS-PAGE and exhibited specificity towards xyloglucan with apparent K _m of 1.67 mg/ml. The enzyme was active at a broad range of pH (5–8) and temperatures (40–80°C). The optimum pH and temperature were 7 and 70°C, respectively. The enzyme retained 100% activity at 50°C for 60 h with half-lives of 14 h, 6 h and 7 min at 60, 70 and 80°C, respectively. The kinetics of thermal denaturation revealed that the inactivation at 80°C is due to unfolding of the enzyme as evidenced by the distinct red shift in the wavelength maximum of the fluorescence profile. Xyloglucanase activity was positively modulated in the presence of Zn²⁺, K⁺, cysteine, β-mercaptoethanol and polyols. Thermostability was enhanced in the presence of additives (polyols and glycine) at 80°C. A hydrolysis of 55% for galactoxyloglucan (GXG) from tamarind kernel powder (TKP) was obtained in 12 h at 60°C and 6 h at 70°C using thermostable xyloglucanases, favouring a reduction in process time and enzyme dosage. The enzyme was stable in the presence of commercial detergents (Ariel), indicating its potential as an additive to laundry detergents.     

C-Terminal proline-rich sequence broadens the optimal temperature and pH ranges of recombinant xylanase from Geobacillus thermodenitrificans C5

Efficient utilization of hemicellulose entails high catalytic capacity containing xylanases. In this study, proline rich sequence was fused together with a C-terminal of xylanase gene from Geobacillus thermodenitrificans C5 and designated as GthC5ProXyl. Both GthC5Xyl and GthC5ProXyl were expressed in Escherichia coli BL21 host in order to determine effect of this modification. The C-terminal oligopeptide had noteworthy effects and instantaneously extended the optimal temperature and pH ranges and progressed the specific activity of GthC5Xyl. Compared with GthC5Xyl, GthC5ProXyl revealed improved specific activity, a higher temperature (70 °C versus 60 °C) and pH (8 versus 6) optimum, with broad ranges of temperature and pH (60–80 °C and 6.0–9.0 versus 40–60 °C and 5.0–8.0, respectively). The modified enzyme retained more than 80% activity after incubating in xylan for 3 h at 80 °C as compared to wild −type with only 45% residual activity. Our study demonstrated that proper introduction of proline residues on C-terminal surface of xylanase family might be very effective in improvement of enzyme thermostability. Moreover, this study reveals an engineering strategy to improve the catalytic performance of enzymes.     

Production and optimization of cellulase-free, alkali-stable xylanase by Bacillus pumilus SV-85S in submerged fermentation

This paper reports the production of a cellulase-free and alkali-stable xylanase in high titre from a newly isolated Bacillus pumilus SV-85S using cheap and easily available agro-residue wheat bran. Optimization of fermentation conditions enhanced the enzyme production to 2995.20 ± 200.00 IU/ml, which was 9.91-fold higher than the activity under unoptimized basal medium (302.2 IU/ml). Statistical optimization using response-surface methodology was employed to obtain a cumulative effect of peptone, yeast extract, and potassium nitrate (KNO₃) on enzyme production. A 2³ central composite design best optimized the nitrogen source at the 0 level for peptone and yeast extract and at the −α level for KNO₃, along with 5.38-fold increase in xylanase activity. Addition of 0.1% tween 80 to the medium increased production by 1.5-fold. Optimum pH for xylanase was 6.0. The enzyme was 100% stable over the pH range from 5 to 11 for 1 h at 37°C and it lost no activity, even after 3 h of incubation at pH 7, 8, and 9. Optimum temperature for the enzyme was 50°C, but the enzyme displayed 78% residual activity even at 65°C. The enzyme retained 50% activity after an incubation of 1 h at 60°C. Characteristics of B. pumilus SV-85S xylanase, including its cellulase-free nature, stability in alkali over a long duration, along with high-level production, are particularly suited to the paper and pulp industry.     

Optimization of production of C-phycocyanin and extracellular polymeric substances by Arthrospira sp.

The key factors influencing the production of C-phycocyanin (C-PC) and extracellular polymeric substances (EPS) by photoautotrophic culture of Arthrospira sp. were optimized using Taguchi method. Six factors were varied at either three or two levels as follows: light intensity at three levels; three initial culture pHs; two species of Arthrospira; three concentrations of Zarrouk’s medium; three rates of aeration of the culture with air mixed with 2% v/v carbon dioxide; and two incubation temperatures. All cultures ran for 14 days. The optimal conditions for the production of C-PC and EPS were different. For both products, the best cyanobacterium proved to be Arthrospira maxima IFRPD1183. The production of C-PC was maximized with the following conditions: a light intensity of 68 µmol photons m⁻² s⁻¹ (a diurnal cycle of 16-h photoperiod and 8-h dark period), an initial pH of 10, the full strength (100%) Zarrouk’s culture medium, an aeration rate of 0.6 vvm (air mixed with 2% v/v CO₂) and a culture temperature of 30 °C. The concentration of Zarrouk’s medium was the most important factor influencing the final concentration of C-PC. The optimal conditions for maximal production of EPS were as follows: a light intensity of 203 µmol photons m⁻² s⁻¹ with the earlier specified light–dark cycle; an initial pH of 9.5; a 50% strength of Zarrouk’s medium; an aeration rate of 0.2 vvm (air mixed with 2% v/v CO₂); and a temperature of 35 °C. Production of C-PC and EPS in raceway ponds is discussed.

     

Characterization of a thermostable lipase showing loss of secondary structure at ambient temperature

A gene encoding extracellular lipase was cloned and characterized from metagenomic DNA extracted from hot spring soil. The recombinant gene was expressed in E. coli and expressed protein was purified to homogeneity using hydrophobic interactions chromatography. The mature polypeptide consists of 388 amino acids with apparent molecular weight of 43 kDa. The enzyme displayed maximum activity at 50°C and pH 9.0. It showed thermal stability up to 40°C without any loss of enzyme activity. Nearly 80% enzyme activity was retained at 50°C even after incubation for 75 min. However above 50°C the enzyme displayed thermal instability. The half life of the enzyme was determined to be 5 min at 60°C. Interestingly the CD spectroscopic study carried out in the temperature range of 25–95°C revealed distortion in solution structure above 35°C. However the intrinsic tryptophan fluorescence spectroscopic study revealed that even with the loss of secondary structure at 35°C and above the tertiary structure was retained. With p-nitrophenyl laurate as a substrate, the enzyme exhibited a K _m , V _max and K _cat of 0.73 ± 0.18 μM, 239 ± 16 μmol/ml/min and 569 s⁻¹ respectively. Enzyme activity was strongly inhibited by CuCl₂, HgCl₂ and DEPC but not by PMSF, eserine and SDS. The protein retained significant activity (~70%) with Triton X-100. The enzyme displayed 100% activity in presence of 30% n-Hexane and acetone.     

A novel nitrilase from Ralstonia eutropha H16 and its application to nicotinic acid production

A novel aliphatic nitrilase, REH16, was found in Ralstonia eutropha H16 and overexpressed in Escherichia coli BL21(DE3), and its enzymatic properties were studied. The temperature and pH optima were 37 °C and 6.6, respectively, and the best thermostability of the nitrilase was observed at 25 °C, which preserved 95% of activity after 120 h of incubation. REH16 has a broad hydrolytic activity toward aliphatic and heterocyclic nitriles and showed high tolerance of 3-cyanopyridine; this enzyme could hydrolyze as high as 100 mM 3-cyanopyridine completely. To improve the 3-cyanopyridine conversion efficiency in an aqueous reaction system, water-miscible organic solvents were tested, and ethanol (10% v/v) was chosen as the optimal co-solvent. Finally, under optimized conditions, using the fed-batch reaction mode, total of 1050 mM 3-cyanopyridine was hydrolyzed completely in 20.8 h with eight substrate feedings, yielding 129.2 g/L production of nicotinic acid and thus showing a potential for industrial application.

     

Production and characterization of a new cyclodextrin glycosyltransferase from Bacillus firmus isolated from Brazilian soil

A new CGTase was obtained from Bacillus firmus, strain 7B, isolated from oat soil culture, using a high alkaline pH medium containing 1% Na₂CO₃. The enzyme was characterized in soluble form, for pH 5–11, temperature from 30 to 85 °C, using a 1% maltodextrin substrate solution and appropriate buffers. It produced mainly β-CD and the cell-free supernatant had a precipitating activity measured by the trichloroethylene method that is a 100-fold greater than that of the enzyme of Bacillus firmus, strain 37, previously studied by our group. The molecular weight of the pure protein was measured as 56,230 Da with SDS-PAGE. The optimum temperature for the enzyme activity was 50 °C and it was most active at pH 6.0. Thermal deactivation was noticeable above 65 °C and the enzyme was highly stable below 60 °C. The influence of substrate or product concentration on the initial rate of CD production was studied and the kinetic parameters were determined. The enzyme showed cyclization activity on different raw and hydrolyzed starches and hydrolyzed cornstarch gave the highest activity.     

Screening of yeast strains capable of hyperproducing amylolytic enzymes

Summary Fifteen strains of yeast, which produced an extracellular amylolytic enzymes, were isolated from nature. One of them produced more than 100 times the enzyme activity in comparison with the 14 strains and the extremely hyperproducing strain of yeast was identified asCandida sp. 347. Paper chromatograms of the amylolytic enzyme demonstrated activity of amyloglucosidase. The optimum pH for activity of the enzyme was 5.5–6.0 and optimum temperature was 60°C.     

Characterization of a recombinant amylolytic enzyme of hyperthermophilic archaeon Thermofilum pendens with extremely thermostable maltogenic amylase activity

A gene (Tpen_1458) encoding a putative alpha amylase from hyperthermophilic archaeon Thermofilum pendens (TfMA) was cloned and expressed in Escherichia coli. The recombinant amylolytic enzyme was purified by Ni-NTA affinity chromatography and its catalytic properties were examined. Purified TfMA was extremely thermostable with a half-life of 60 min at an optimal temperature of 95°C. TfMA activity increased to 136% in the presence of 5 mM CaCl₂. Maximal activity was measured toward γ-cyclodextrin with a specific activity of 56 U/mg using copper bicinchoninate method. TfMA catalyzed the ring-opening reaction by cleaving one α-1,4-glycosidic linkage of cyclodextrin to produce corresponding single maltooligosaccharide at the initial time. The final products from cyclodextrins, linear maltooligosaccharides, and starch were glucose and maltose, and TfMA could also degrade pullulan and amylase inhibitor acarbose to panose and acarviosine-glucose, respectively. These results revealed that TfMA is a novel maltogenic amylase.     

Directed immobilization of CGTase: The effect of the enzyme orientation on the enzyme activity and its use in packed-bed reactor for continuous production of cyclodextrins

In this study, two different approaches were assessed in order to direct the immobilization of a cyclodextrin glycosyltransferase on functionalized silica support, one by amino groups using glutaraldehyde activation (Si-NH-G-CGTase) and other by disulfide bond through the Cys on the enzyme surface (Si-SH-CGTase). The efficiency of the immobilization of the enzyme by the Cys in Si-SH was four times higher than with the amino group linkage in Si-NH-G (2.86% and 11.91%, respectively). After immobilization, the optimum pH remained at 5.5 for the two derivatives and the optimum temperature was 70 °C for the free enzyme, 80 °C for Si-SH-CGTase and 90 °C for Si-NH-G-CGTase. Both preparations were used for continuous production of cyclodextrins, and Si-NH-G-CGTase presented higher total productivity, retaining 100% of its initial activity for at least 200 h, while the Si-SH-CGTase presented only 40% at the same time. The Si-SH-CGTase could be reloaded with new enzymes linked by disulfide bonds and was able to be used for more than 200 h.     

Trans-glycosylation capacity of a highly glycosylated multi-specific β-glucosidase from Fusarium solani

An extracellular β-glucosidase from Fusaruim solani cultivated on wheat bran was purified by only two chromatographic steps. The purified enzyme exhibited optimal temperature and pH at 60 °C and pH 5, respectively. The purified β-glucosidase behaves as a very large protein due to its high degree of glycosylation. More interestingly, the endoglycosidase H (Endo H) treatment led to 97.55% loss of its initial activity after 24 h of treatment. Besides, the addition of Tunicamycin (nucleoside antibiotic blocking the N-glycosylation first step) during the culture of the fungus affected seriously the glycosylation of the enzyme. Both treatments (endo H and Tunicamycin) strengthened the idea that the hyperglycosylation is involved in the β-glucosidase activity and thermostability. This enzyme was also shown to belong to class III of β-glucosidases (multi-specific) since it was able to act on either cellobiose, gentiobiose or sophorose which are disaccharide composed of two units of d-glucose connected by β1–4, β1–6 and β1–2 linkage, respectively. The β-glucosidase activity was strongly enhanced by ferrous ion (Fe²⁺) and high ionic strength (1 M KCl). The purified enzyme exhibited an efficient transglycosylation capacity allowing the synthesis of cellotriose and cellotetraose using cellobiose as donor.

     

Biochemical characterization of an alkaline surfactant-stable keratinase from a new keratinase producer,Bacillus zhangzhouensis

A new keratinase producer, Bacillus sp. BK111, isolated from a poultry feather was identified as Bacillus zhangzhouensis, which is the first report for its keratinolytic activity. The keratinase production was optimized, followed by the enzyme purification and characterization using biochemical assays. A 2.34-fold increase was observed in the enzyme production under optimized conditions. The enzyme was characterized as a serine protease with 42 kDa molecular weight, stable in a wide range of temperature and pH with maximum keratinolytic activity at 60 °C and pH 9.5. The enzyme had a wide range of different substrates with the best performance on the feather meal substrate. Metal ions of Ca²⁺, K⁺, Na⁺ and Mn²⁺ enhanced the enzyme activity. The enzyme showed a great deal of stability in the presence of ethanol, methanol, acetone, 2-propanol, dimethyl sulfoxide, Tween-80 and Triton X-100. Dithiothreitol (DTT), as a reducing agent, caused a twofold increase in keratinolytic activity. The half-life of the enzyme at optimum temperature was calculated to be 125 min and the ratio of keratinolytic:caseinolytic for the enzyme was 0.8. Our results showed the remarkable features of the enzyme that make it suitable for biotechnological usages.

     

Simultaneous liquefaction,saccharification, and fermentation of l-lactic acid using aging paddy rice with hull by an isolated thermotolerant Enterococcus faecalis DUT1805

Simultaneous liquefaction, saccharification, and fermentation (SLSF) has attracted much attention for the production of bio-based chemicals, including l-lactic acid, due to its high efficiency and low cost. In this study, a lactic acid-producing bacterium with high tolerance of temperature up to 55 °C was isolated and characterized as Enterococcus faecalis DUT1805. Various strategies of stepwise controlled temperature were proposed and investigated for glucose utilization. The results indicated that E. faecalis DUT 1805 exhibited an optimal temperature at 50 °C, which could achieve temperature compatibility of enzyme, saccharification, and fermentation, and decrease the possibility of contamination by the other microorganisms during the large-scale fermentation. To reduce the cost of raw material and operation for lactic acid production, aging paddy rice with hull (APRH) was used in l-lactic acid production by simultaneous liquefaction, saccharification, and fermentation (SLSF). An open SLSF operation at 50 °C and pH 6.5, and 17% (w/v) solid loading in 5 L bioreactors was demonstrated with the lactic acid titer, yield, and productivity of 73.75 g/L, 87% to initial starch, and 2.17 g/(L h), respectively.