Optimization of culture medium and conditions for penicillin acylase production by streptomyces lavendulae ATCC 13664

The culture medium for Streptomyces lavendulae ATCC 13664 was optimized on a shake-flask scale by using a statistical factorial design for enhanced production of penicillin acylalse. This extracellularenzyme recently has been reported to bea penicillin Kacylase, presenting also high hydrolytic activity against penicillin V and other natural aliphatic penicillins such as penicillin K, penicillin F, and penicillin dihydroF,. The factorial design indicated that the main factors that positively affect penicillin acylase production by S. lavendulae were the concentration of yeast extract and the presence of oligoelements in the fermentation medium, whereas the presence of olive oil in the medium had no effect on enzyme production. An initial concentration of 2.5% (w/v) yeast extract and 3 μg/mL of CuSO₄·5H₂O was found to be best for acylase production. In such optimized culture medium, fermentation, of the microorganism yielded 289 IU/L of enzyme in 72 h when employing a volume medium/volume flask ratio of 0.4 and a 300-rpm shaking speed. The presence of copper, alone and in combination with other metals, stimulated biomass as well as penicillin acylase production. The time course of penicillin acylase production was also studied in the optimized medium and conditions. Enzyme production showed catabolite repression by different carbon sources such as glucose, lactose, citrate, glycerol, and glycine.     

A Process to Produce Penicillin G Acylase by Surface-Adhesion Fermentation Using <Emphasis Type="Italic">Mucor griseocyanus</Emphasis> to Obtain 6-Aminopenicillanic Acid by Penicillin G Hydrolysis

The production of extracellular and mycelia-associated penicillin G acylase (maPGA) with Mucor griseocyanus H/55.1.1 by surface-adhesion fermentation using Opuntia imbricata, a cactus, as a natural immobilization support was studied. Enzyme activity to form 6-aminopencillanic acid (6-APA) from penicillin G was assayed spectrophotometrically. The penicillin G hydrolysis to 6-APA was evaluated at six different times using PGA samples recovered from the skim milk medium at five different incubation times. Additionally, the effect of varying the penicillin G substrate concentration level on the PGA enzyme activity was also studied. The maximum reaction rate, V _max, and the Michaelis constant, K _M, were determined using the Michaelis–Menten model. The maximum levels for maPGA and extracellular activity were found to be 2,126.50 international unit per liter (IU/l; equal to 997.83 IU/g of support) at 48 h and 755.33 IU/l at 60 h, respectively. Kinetics of biomass production for total biomass showed a maximum growth at 60 h of 3.36 and 2.55 g/l (equal to 0.012 g of biomass per gram of support) for the immobilized M. griseocyanus biomass. The maPGA was employed for the hydrolysis of penicillin G to obtain 6-APA in a batch reactor. The highest quantity of 6-APA obtained was 226.16 mg/l after 40-min reaction. The effect of substrate concentration on maPGA activity was evaluated at different concentrations of penicillin G (0–10 mM). K _M and V _max were determined to be 3.0 × 10⁻³ M and 4.4 × 10⁻³ mM/min, respectively.     

Production of Raw Starch-Saccharifying Thermostable and Neutral Glucoamylase by the Thermophilic Mold <Emphasis Type="Italic">Thermomucor indicae-seudaticae</Emphasis> in Submerged Fermentation

Among physical and nutritional parameters optimized by “one variable at a time” approach, four cultural variables (sucrose, MgSO₄ ^.7H₂O, inoculum size, and incubation period) significantly affected glucoamylase production. These variables were, therefore, selected for optimization using response surface methodology. The p-values of the coefficients for linear effect of sucrose and inoculum size were less than 0.0001, suggesting them to be the key experimental variables in glucoamylase production. The enzyme production (34 U/ml) attained under optimized conditions (sucrose, 2%; MgSO₄ ^.7H₂O, 0.13%; yeast extract, 0.1%; inoculum size, 5 × 10⁶ spores per 50 ml production medium; incubation time, 48 h; temperature, 40°C; and pH 7.0) was comparable with the value predicted by polynomial model (34.2 U/ml). An over all 3.1-fold higher enzyme titers were attained due to response surface optimization. The experimental model was validated by carrying out glucoamylase production in shake flasks of increasing capacity (0.25–2.0 l) and 22-l laboratory bioreactors (stirred tank and airlift), where the enzyme production was sustainable. Furthermore, the fermentation time was reduced from 48 h in shake flasks to 32 h in bioreactors.     

Strategies for Enhancing the Production of Penicillin G Acylase from Bacillus badius: Influence of Phenyl Acetic Acid Dosage

Bacillus badius isolated from soil has been identified as potential producer of penicillin G acylase (PGA). In the present study, batch experiments performed at optimized inoculum size, temperature, pH, and agitation yielded a maximum PGA of 9.5 U/ml in shake flask. The experiments conducted in bioreactor with different oxygen flow rates revealed that 0.66 vvm oxygen flow rate could be sufficient for the maximum PGA activity of 12.7 U/ml. From a detailed investigation on the strategies of the addition of phenyl acetic acid (PAA) for increasing the production of PGA, it was found that the controlled addition of 10 ml of 0.1 % (w/v) PAA once in every 2 h from 6th hour of growth showed the maximum PGA activity of 32 U/ml. Thus, our studies for the first time showed that at concentration above 0.1 % (w/v) PAA, the PGA production decreased. This selective condition paves the way for less costly bioprocess for the production of PGA.     

Immobilization of Penicillin G Acylase on Calcined Layered Double Hydroxides

A hydrotalcite-like Mg2 /Al3 layered double hydroxide (LDH) material was prepared by means of amodified coprecipitation method involving a rapid mixing step followed by a separate aging process. LDH calcined at 500℃ , denoted as CLDH, was characterized by XRD, IR and BET surface area measurements.CLDH has a poor crystalline MgO-like structure with a high surface area and porosity. CLDH was used as asupport for the immobilization of penicillin G acylase(PGA). The effect of varying the immobilization conditions, such as pH, contact time and the ratio of enzyme to support, on the activity of the immobilized enzymein the hydrolysis of penicillin G has been studied. It was found that the activity of the immobilized enzyme decreased slightly with decreasing pH and reached a maximum after a contact time of 24 h. The activity of theimmobilized enzyme increased with increasing the ratio of enzyme to support. It was found that the adsorption of PGA inhibited the expected reaction of CLDH with an aqueous medium to regenerate a LDH phase. Itsoriginal activity(36%) after 15 cycles of reuse of the immobilized enzyme was retained, but no further loss in the activity was observed.     

Process optimisation for the biosynthesis of cellulase by Bacillus PC-BC6 and its mutant derivative Bacillus N3 using submerged fermentation

This study deals with optimisation of cultural conditions for enhanced production of cellulase by Bacillus PC-BC6 and its mutant derivative Bacillus N3. Influence of different variables including incubation time, temperature, inoculum size, pH, nitrogen sources and metal ions has been studied. The optimum conditions for cellulase production were incubation period of 72 h, inoculum size 4% incubation temperature 37°C, pH 7, 0.25% ammonium sulphate, 0.2% peptone as inorganic and organic nitrogen source in case of Bacillus PC-BC6. In case of mutant Bacillus N3, optimal conditions were incubation period of 48 h, incubation temperature 37°C, inoculum size 3%, pH 7, 0.2% ammonium chloride and 0.15% yeast extract. Presence of MnSO₄ and CaCl₂ enhances the enzyme production by Bacillus PC-BC6 and mutant Bacillus N3, respectively. This study was innovative and successful in producing cellulase economically by using cheap indigenous substrate Saccharum spontaneum.     

Influence of carbon and nitrogen sources and temperature on hyperproduction of a thermotolerant β-glucosidase from synthetic medium by Kluyveromyces marxianus

The effect of carbon source and its concentration, inoculum size, yeast extract concentration, nitrogen source, pH of the fermentation medium, and fermentation temperature on β-glucosidase production by Kluyveromyces marxianus in shake-flask culture was investigated. These were the independent variables that directly regulated the specific growth and β-glucosidase production rate. The highest product yield, specific product yield, and productivity of β-glucosidase occurred in the medium (pH 5.5) inoculated with 10% (v/v) inoculum of the culture. Cellobiose (20 g/L) significantly improved β-glucosidase production measured as product yield (Y _P/S ) and volumetric productivity (Q _P ) followed by sucrose, lactose, and xylose. The highest levels of productivity (144 IU/[L·h]) of β-glucosidase occurred on cellobiose in the presence of CSL at 35°C and are significantly higher than the values reported by other researchers on almost all other organisms. The thermodynamics and kinetics of β-glucosidase production and its deactivation are also reported. The enzyme was substantially stable at 60°C and may find application in some industrial processes.     

Di-functional magnetic nanoflowers: A highly efficient support for immobilizing penicillin G acylase

The novel di-functional magnetic nanoflowers (DMNF) which had both epoxy groups and hydrophilic catechol as well as phthaloquinone groups capable of covalently coupling of penicillin G acylase (PGA) were characterized by scanning electron microscopy, transmission electron microscope (TEM), vibrating sample magnetometer, N₂ adsorption, and so on. The studies showed that DMNF possessed “hierarchical petal” structure of nanosheets had specific saturation magnetization of 39.7 emu/g and average pore diameter of 25.4 nm as well as specific surface area of 17.28 m²/g. For hydrolysis of penicillin G potassium catalyzed by the PGA immobilized on DMNF with enzyme loading of 106 mg/g-support, its apparent activity reached 2,667 U/g, which benefited from the “hierarchical petal” and large pore structure of the magnetic DMNF leading to high enzyme loading and fast diffusion of substrate molecules to the immobilized PGA to reaction. The apparent activity of the immobilized PGA could keep 2,408 U/g (above 90% of its initial activity) after repeating use for 10 cycles. The magnetic immobilized PGA exhibited excellent operational stability due to covalently coupling of the enzyme molecules between the support by covalent interaction of the amino groups of PGA and the reactive groups of epoxy, catechol, and phthaloquinone groups on DMNF. Furthermore, the PGA displayed good acid and alkaline resistance as well as thermal stability by immobilization using DMNF.     

Xylanase production by the thermophilic mold Humicola lanuginosa in solid-state fermentation

Among the lignocellulosic substrates tested, wheat bran supported a high xylanase (EC 3.2.1.8) secretion by Humicola lanuginosa in solid-state fermentation (SSF). Enzyme production reached a peak in 72 h followed by a decline thereafter. Enzyme production was very high (7832 U/g of dry moldy bran) when wheat bran was moistened with tap water at a substrate-to-moistening agent ratio of 1:2.5 (w/v) and an inoculum level of 3 × 10⁶ spores/10 g of wheat bran at a water activity (a _w ) of 0.95. Cultivation of the mold in large enamel trays yielded a xylanase titer comparable with that in flasks. Parametric optimization resulted in a 31% increase in enzyme production in SSF. Xylanase production was approx 23-fold higher in SSF than in submerged fermentation (SmF). A threshold constitutive level of xylanase was secreted by H. lanuginosa in a medium containing glucose as the sole carbon source. The enzyme was induced by xylose and xylan. Enzyme synthesis was repressed beyond 1.0% (w/v) xylose in SmF, whereas it was unaffected up to 3.0% (w/w) in SSF, suggesting a minimization of catabolite repression in SSF.     

Xylanase Production by Bacillus circulans D1 Using Maltose as Carbon Source

Bacillus circulans D1 is a good producer of extracellular thermostable xylanase. Xylanase production in different carbon sources was evaluated and the enzyme synthesis was induced by various carbon sources. It was found that d-maltose is the best inducer of the enzyme synthesis (7.05 U/mg dry biomass at 48 h), while d-glucose and d-arabinose lead to the production of basal levels of xylanase. The crude enzyme solution is free of cellulases, even when the microorganism was cultivated in a medium with d-cellobiose. When oat spelt xylan was supplemented with d-glucose, the repressive effect of this sugar on xylanase production was observed at 24 h, only when used at 5.0 g/L, leading to a reduction of 60% on the enzyme production. On the other hand, when the xylan medium was supplemented with d-xylose (3.0 or 5.0 g/L), this effect was more evident (80 and 90% of reduction on the enzyme production, respectively). Unlike that observed in the xylan medium, glucose repressed xylanase production in the maltose medium, leading to a reduction of 55% on the enzyme production at 24 h of cultivation. Xylose, at 1.0 g/L, induced xylanase production on the maltose medium. On this medium, the repressive effect of xylose, at 3.0 or 5.0 g/L, was less expressive when compared to its effect on the xylan medium.     

Biological Real-Time Reaction Calorimeter Studies for the Production of Penicillin G Acylase from Bacillus badius

Penicillin G acylase (PGA) is a commercially important enzyme that cleaves penicillin G to 6-amino penicillanic acid (6-APA) and phenyl acetic acid (PAA). The strain Bacillus badius has been identified as potential producer of PGA. A detailed calorimetric investigation on PGA production was carried out to enable generation of thermokinetic data possible for commercial application. Reaction calorimetric studies coupled with respirometric studies suggested that enzyme activity of the species B. badius was calorimetrically traceable. Three phases of growth were distinctly noticeable in the metabolic heat-time curve. Increase in enzymatic activity with restricted growth confirmed intracellular nature of the production process. The estimated heat yields due to biomass growth, 10.026 kJ/g, substrate consumption 22.761 kJ/g, and oxygen uptake 383?±?10 kJ/mol helped to understand the energetic of the organism under study. Low oxycalorific coefficient confirmed the existence of fermentation-coupled metabolism of B. badius.     

Purification and Characterization of Thermostable Chitinase from <Emphasis Type="Italic">Bacillus licheniformis</Emphasis> SK-1

Chitinase was purified from the culture medium of Bacillus licheniformis SK-1 by colloidal chitin affinity adsorption followed by diethylamino ethanol-cellulose column chromatography. The purified enzyme showed a single band on sodium dodecyl sulfate polyacrylamide gel electrophoresis. The molecular size and pI of chitinase 72 (Chi72) were 72 kDa and 4.62 (Chi72) kDa, respectively. The purified chitinase revealed two activity optima at pH 6 and 8 when colloidal chitin was used as substrate. The enzyme exhibited activity in broad temperature range, from 40 to 70°C, with optimum at 55°C. It was stable for 2 h at temperatures below 60°C and stable over a broad pH range of 4.0–9.0 for 24 h. The apparent K _m and V _max of Chi72 for colloidal chitin were 0.23 mg ml⁻¹ and 7.03 U/mg, respectively. The chitinase activity was high on colloidal chitin, regenerated chitin, partially N-acetylated chitin, and chitosan. N-bromosuccinamide completely inhibited the enzyme activity. This enzyme should be a good candidate for applications in the recycling of chitin waste.     

Optimization in the immobilization of penicillin G acylase by entrapment in xerogel particles with magnetic properties

Biocatalysis presents a sound alternative to chemical synthesis in the field of drug production, given the highly selective nature of biological catalysts. Penicillin G Acylase (PGA) from E. coli is currently used to hydrolyze penicillin G (PG) and catalyzes the synthesis of β-lactam antibiotics. In this work, particular emphasis is given to recent developments in penicillin G acylase immobilization, by entrapment simultaneously with nano-magnetic particles in a silica matrix. The sol–gel biocatalytic particles were prepared either by a conventional method (crushed powder) or by a more recent approach, based in an emulsion system using 150 mM AOT/isooctane, which allowed for the formation of spherical micro- and nanobeads. The effects on PGA activity of different sol–gel precursors, additives, enzyme concentration, aging, drying conditions and mechanical stability were evaluated. After these optimization studies, a mechanically stable carrier based on porous xerogels silica matrixes, starting from tetramethoxysilane (TMOS) with 65–67% PGA activity yield in these carriers allowed an immobilization yield of 74 mg protein g_{dry sol–gel}⁻¹ and 930 Ug_{dry sol–gel}⁻¹ for specific activity were obtained.     

Continuous production of extracellular l-glutaminase by ca-alginate-immobilized marine Beauveria bassiana BTMF S-10 in packed-bed reactor

l-Glutamine amidohydrolase (l-glutaminase, EC 3.5.1.2) is a therapeutically and industrially important enzyme. Because it is a potent antileukemic agent and a flavor-enhancing agent used in the food industry, many researchers have focused their attention on l-glutaminase. In this article, we report the continuous production of extracellular l-glutaminase by the marine fungus Beauveria bassiana BTMF S-10 in a packed-bed reactor. Parameters influencing bead production and performance under batch mode were optimized in the order-support (Na-alginate) concentration, concentration of CaCl₂ for bead preparation, curing time of beads, spore inoculum concentration, activation time, initial pH of enzyme production medium, temperature of incubation, and retention time. Parameters optimized under batch mode for l-glutaminase production were incorporated into the continuous production studies. Beads with 12×10⁸ spores/g of beads were activated in a solution of 1% glutamine in seawater for 15 h, and the activated beads were packed into a packed-bed reactor. Enzyme production medium (pH 9.0) was pumped through the bed, and the effluent was collected from the top of the column. The effect of flow rate of the medium, substrate concentration, aeration, and bed height on continuous production of l-glutaminase was studied. Production was monitored for 5 h in each case, and the volumetric productivity was calculated. Under the optimized conditions for continuous production, the reactor gave a volumetric productivity of 4.048 U/(mL·h), which indicates that continuous production of the enzyme by Ca-alginate-immobilized spores is well suited for B. bassiana and results in a higher yield of enzyme within a shorter time. The results indicate the scope of utilizing immobilized B. bassiana for continuous commercial production of l-glutaminase.     

乙烯基Ia3d介孔硅分子筛的环氧化及其固定化青霉素酰化酶(英文)

采用直接共聚法合成表面含有乙烯基的具有立方相Ia3d结构的介孔硅分子筛(V-ClMS),然后对乙烯基团进行环氧化制备得到表面环氧基功能化的介孔硅分子筛(E-CIMS),采用X射线衍射、N2吸附-脱附、透射电镜、热重分析和13C固体核磁共振对制备的介孔硅分子筛进行了表征.结果表明,表面含有乙烯基的V-ClMS介孔硅分子筛能被一步成功合成,并易于发生环氧化而获得表面环氧基功能化的E-CIMS介孔硅分子筛.将E-CIMS介孔硅分子筛作为载体用于固定化青霉素G酰化酶(PGA),研究了表面环氧基团对固定化PGA初活性和操作稳定性的影响.结果表明,随着表面环氧基团数量的增加,介孔硅分子筛孔径减小,表面疏水性增加,导致载酶量和初活性减小.但介孔硅分子筛表面适量的环氧基团能增强E-CIMS介孔硅分子筛与PGA之间的相互作用,从而提高固定化PGA的操作稳定性.     

High-yield bacillus subtilis protease production by solid-state fermentation

A Bacillus subtilis isolate was shown to be able to produce extracellular protease in solid-state fermentations (SSF) using soy cake as culture medium. A significant effect of inoculum concentration and physiological age on protease production was observed. Maximum activities were obtained for inocula consisting of exponentially growing cells at inoculum concentrations in the range of 0.7–2.0 mg g⁻¹. A comparative study on the influence of cultivation temperature and initial medium pH on protease production in SSF and in submerged fermentation (SF) revealed that in SSF a broader pH range (5–10), but the same optimum temperature (37°C), is obtained when compared to SF. A kinetic study showed that enzyme production is associated with bacterial growth and that enzyme inactivation begins before biomass reaches a maximum level for both SF and SSF. Maximum protease activity and productivity were 960 U g⁻¹ and 15.4 U g⁻¹ h⁻¹ for SSF, and 12 U mL⁻¹ and 1.3 U mL⁻¹ h⁻¹ for SF. When SSF protease activity was expressed by volume of enzyme extract, the enzyme level was 10-fold higher and the enzyme productivity 45% higher than in SF. These results indicate that this bacterial strain shows a high biotechnological potential for protease production in solid-state fermentation.     

Application of factorial design to the study of xylitol production from eucalyptus hemicellulosic hydrolysate

This study deals with the bioconversion of xylose into xylitol by Candida guilliermondii FTI 20037 using eucalyptus hemicellulosic hydrolysate obtained by acid hydrolysis. The influence of various parameters (ammonium sulfate, rice bran, pH, and xylose concentration) on the production of xylitol was evaluated. The experiments were based on multivariate statistical concepts, with the application of factorial design techniques to identify the most important variables in the process. The levels of these variables were quantified by the response surface methodology, which permitted the establishment of a significant mathematical model with a coefficient determination of R ²=0.92. The best results (xylitol=10.0 g/L, yield factor=0.2 g/g, and productivity=0.1 g/[L·h]) were attained with hydrolysate containing ammonium sulfate (1.1 g/L), rice bran (5.0 g/L), and xylose (initial concentration of 60.0 g/L), after 72 h of fermentation. The pH of fermentation was adjusted to 8.0 and the inoculum level utilized was 3 g/L.     

Recent progress in the development of immobilized penicillin G acylase for chemical and industrial applications: A mini‐review

Immobilized penicillin G acylase (PGA) as an important industrial catalyst can catalyze penicillin G potassium (PG) to 6‐aminopenicillanic acid (6‐APA). 6‐APA is an important intermediate for semisynthetic penicillin drugs, which occupies a huge market space in the anti‐inflammatory field; as a result, immobilized PGA occupies a huge market space in the pharmaceutical field. However, at present, there are different degrees of defects in the preparation and production process of immobilized PGAs on the market because of the huge demand; therefore, the performance of immobilized PGA and its productivity will bring huge economic benefits to enterprises. Therefore, research on immobilized PGA has always been a focus. This review first introduces the source, classification, structure, and catalytic mechanism of PGA and then studies the development of immobilization methods, immobilized carriers, reaction media, enzyme activity regeneration, and reactors of immobilized PGA in recent years.     

The engineering and immobilization of penicillin G acylase onto thermo‐sensitive tri‐block copolymer system

In this work, the relationships between catalytic performances of penicillin G acylase (PGA) and the molar ratio of carrier, thermo‐sensitive tri‐block polymer, poly (N,N‐diethylacrylamide‐b‐ β‐hydroxyethyl methacrylate‐b‐glycidyl methacrylate) (PDEA‐b‐PHEMA‐b‐PGMA) were studied firstly, and result documented the optimal molar ratio was n_DEA:n_HEMA:n_GMA = 100:47:24, which presented a suitable lower critical solution temperature (LCST) of 35°C and the activity retention ratio of 80.62% (±0.50%). Based on the suitable carrier, immobilization conditions were investigated and optimized. When pH of solution, concentration of PGA, immobilized time, and immobilization temperature were 8.0, 1/10 (m/v), 16 hours, and 36°C, respectively, enzyme loading capacity (L), enzyme activity (Ea), and activity retention ratio (Ar) of PGA arrived at the highest value of 21 223 U, 16 199 U/g, and 93.50% (±0.50%), respectively. Besides, the response rate (Rr) of immobilized PGA was the same as free PGA, the reusable stability (Rs) was 77.00% (±1.00%) after using for 11 times, which indicated that the carrier has better compatibility with L, Ar, Rs, and Rr.     

Comparative study of amidase production by free and immobilized Escherichia coli cells

Escherichia coli NCIM 2569 was evaluated for its potential for amidase production under submerged fermentation. Among the various amide compounds screened, maximum substrate specificity and enzyme yield (8.1 U/mL) were obtained by using 1% acetamide. Fermentation was carried out at 30°C in shake-flask culture under optimized process conditions. A maximum of 0.52 U/mL of intracellular amidase activity was also obtained from cells incubated for 24 h. Studies were also performed to elucidate the optimal conditions (gel concentration, initial biomass, curing period of beads, and calcium ion concentration in the production medium) for immobilization of whole cells. By using E. coli cells entrapped in alginate, a maximum of 6.2 U/mL of enzyme activity was obtained after 12 h of incubation under optimized conditions. Using the immobilized cells, three repeated batches were carried out successfully, and 85% of the initial enzyme activity was retained in the second and third batches. The study indicated that the immobilized E. coli cells offered certain advantages such as less time for maximum enzyme production, more stability in the enzyme production rate, and repeated use of the biocatalyst.