Stability and stabilisation of penicillin acylase

The stability of an oligomeric enzyme, penicillin acylase, was studied in aqueous media. The enzyme was produced by mutant cells of Escherichia coli ATCC 9637, extracted from the periplasmic space by osmotic shock and further purified using a pseudo‐affinity adsorption process. Enzyme stabilisation attempts were performed with salts, alcohols and sugars. The highest levels of retained activity were obtained in the presence of 15% (w/v) ammonium or sodium sulfate. A kinetic model was proposed to describe the inactivation of penicillin acylase, taking into account results obtained in stability assays performed at different temperatures and with different enzyme concentrations. According to this model, the inactivation of penicillin acylase involves an intermediary active precursor of the enzyme, formed prior to dissociation into sub‐units. © 1999 Society of Chemical Industry     

Penicillin Acylase‐Catalyzed Solid‐State Ampicillin Synthesis

The ability of immobilized penicillin acylase from E. coli to retain a remarkable catalytic activity in solid‐state systems has been demonstrated. Stabilization of immobilized penicillin acylase by inorganic salt hydrates allowed us to exploit nearly the whole catalytic activity of the enzyme at a very low water content. Using this technique, enzymatic synthesis of ampicillin in solid‐state systems was performed with high yields (up to 70% starting from equimolar mixture of reagents) and rates comparable to the corresponding values in homogeneous solutions and heterogeneous systems, “aqueous solution‐precipitate”. Peculiarities of the enzymatic solid‐state acyl transfer process, such as absence of the clear‐cut maximum on the ampicillin accumulation curves and dependence of the synthetic efficiency on the enzyme loading, have been observed. The space‐time yield of solid‐state enzymatic ampicillin synthesis was shown to be up to ten times higher compared to the homogeneous solutions and heterogeneous “aqueous solution‐precipitate” systems.     

Penicillin acylase release from Escherichia coli cells by mechanical cell disruption and permeabilization

The release of Penicillin acylase from Escherichia coli cells through mechanical cell disruption using high‐pressure homogenization and sonication was studied. From these cell disruption processes, the enzyme activity was totally released although with low specific activities, 0.1–0.3 IU(mg prot)^?1. Intracellular total soluble protein release was quantified and modelled by a first order kinetic model. The effect of the driving force for each mechanical method, namely acoustic power input and homogenization pressure, on the respective kinetic disruption constants was also analysed. The release of Penicillin acylase by cell permeabilization using osmotic shock was also evaluated. The effects of cell concentration, penicillin acylase activity in E coli cells, type of buffer, pH, hypertonic solution composition, temperature and time used for osmotic shock were evaluated. Using cold osmotic shock, highly selective penicillin acylase release was attained with specific enzyme activities of about 4 IU(mg prot)^?1 and enzyme activity release yields higher than 90%. The high purity of the penicillin acylase was a consequence of the optimized differential enzyme release method which was validated by SDS gel electrophoresis. © 2002 Society of Chemical Industry     

A polyacrylic acid as a carrier for immobilization of penicillin acylase

A copolymer of acrylic acid with divinylbenzene was synthesized by suspension polymerization. This polymer is an effective carrier. Penicillin acylase was immobilized on this carrier to convert benzylpenicillin to 6‐aminopenicillanic acid, which may be employed in the manufacture of semisynthetic penicillins. Factors that affect the activity of immobilized penicillin acrylase, such as temperature, pH, and amount of native enzyme, were studied. Under suitable conditions, the activity and activity recovery of the immobilized enzyme were 3100 U/g (dry carrier, p‐dimethylaminobenzaldehyde method) and 59.7%, respectively. The immobilized penicillin acylase shows a remarkable increase in stability. At 40°C and pH 8.0 the value of the kinetic Michaelis–Menten constant (K_m) of the immobilized enzyme is 2.8 × 10^?3 mol/L, and the value of activation energy of enzyme catalysis is 71.5 kJ/mol. © 2002 Wiley Periodicals, Inc. J Appl Polym Sci 86: 2067–2069, 2002     

Stabilization of penicillin V acylase from Streptomyces lavendulae by covalent immobilization

Penicillin V acylase from the actinomycete Streptomyces lavendulae ATCC 13664 has been immobilized to epoxy‐activated acrylic beads (Eupergit C®) by covalent binding. Further linkage of bovine serum albumin after enzyme immobilization was carried out in order to remove the remaining oxirane groups of the support. The obtained immobilized biocatalyst displayed double exponential deactivation kinetics at temperatures below 55 °C, while the native enzyme followed single exponential decay at the same temperatures. We concluded that soluble penicillin acylase was deactivated in one step, whereas the immobilized enzyme showed an enzymatic intermediate state which is highly thermostable. As a consequence of the immobilization process, the enzyme displayed a 10‐fold increase in its half‐life at 40 °C. At this temperature, the enzymatic intermediate state was progressively destabilized as the pH of the medium was increased. Thus, the optimum pH range for the immobilized enzyme preparation was established as being from 7.0 to 8.0. Higher pH values led to quicker enzyme deactivation. © 2001 Society of Chemical Industry     

Selective reverse micellar extraction of penicillin acylase from E coli

The recovery of penicillin acylase from E coli by a new reverse micellar treatment is described. The results are compared with the cell disruption by ultrasound followed by reverse micellar extraction. The process gave selective extraction of penicillin acylase directly from the periplasmic space of E coli without disrupting the cells. Unlike ultrasonication which breaks open the cells entirely, making subsequent processing difficult and expensive, reverse micellar treatment of cells gave a moderate recovery of 60% of enzyme activity in a highly pure form. © 2001 Society of Chemical Industry     

Optimization of transglutaminase extraction produced by Bacillus circulans BL32 on solid‐state cultivation

BACKGROUND: This paper reports investigations of the extraction of transglutaminase (TGase) produced by Bacillus circulans BL32 on solid‐state cultivation in order to obtain a crude extract with the highest possible specific activity. The optimization of downstream processing parameters for the effective recovery of the enzyme was carried out using response surface methodology based on the central composite rotatable design (CCRD) to reduce losses in the cultivated solids and to obtain a crude extract as concentrated as possible. Several solvents and temperatures were tested, followed by a 2³ factorial design performed to optimize conditions extraction time, mechanical agitation, and solid:liquid ratio. RESULTS: The mathematical model showed that solid:liquid ratio has a significant negative effect on transglutaminase recovery. The optimal conditions for extraction were: water as solvent at 7 °C; 5 min extraction time; agitation speed 250 rpm; and 1:6 solid:liquid ratio. Under these conditions the model predicts a maximum response of 0.291 U mg^?1 of protein of transglutaminase activity recovery, very closely matching experimental activity of 0.285 U mg^?1 of protein. TGase recovery achieved under the optimized extraction conditions, according to the CCRD, was 2.5‐fold higher than that obtained under non‐optimized conditions previously employed. CONCLUSION: Results show that TGase can be produced in cheap solid state cultivations and the optimization of its downstream processing parameters can improve enzyme recovery in crude extracts and may have important impacts on enzyme costs. Copyright © 2008 Society of Chemical Industry     

Effect of dextran polymers on the stability of soluble Escherichia coli penicillin G acylase

The stabilisation of Escherichia coli penicillin G acylase (PGA) by dextran polymers (of molecular weight 11.5, 37.7 and 71 kDa) was studied. The inactivation of both the native and dextran‐containing enzyme preparations obeyed first‐order kinetics at the temperature and pH values studied. The optimal concentrations of dextran polymers of molecular weight 11.5, 37.7 and 71 kDa stabilising PGA against inactivation were 50, 20 and 7.5 mmol dm⁻³ respectively. Dextran 11500 (11.5 kDa) gave 100‐fold protection of PGA against thermal inactivation of enzyme above 50 °C. The kinetic constants of the enzyme were slightly altered, but temperature and pH profiles were not altered by the dextrans. © 1999 Society of Chemical Industry     

Stabilization of native penicillin G acylase by ionic liquids

Five different ionic liquids, based on dialkylimidazolium cations associated with perfluorinated and bis{(trifluoromethyl)sulfonyl}imide anions, were used to investigate the scope and limitations of these new solvents as media for penicillin G acylase‐catalyzed reactions. Deactivation of the native enzyme in ionic liquids (ILs) and in organic solvents (toluene, dichloromethane and 2‐propanol) at low water content and 40 °C was investigated using the hydrolysis of penicillin G as activity test. Native penicillin G acylase shows greater stability in IL media than in organic solvents. For example, a half‐life time of 23 h was obtained in 1‐ethyl‐3‐methylimidazolium bis{(trifluoromethyl)sulfonyl}imide, [emim⁺][Tf₂N^?], which was about 2000‐fold higher than that in 2‐propanol. An enhancement of the PGA stability was observed by the presence of substrate in ionic liquids based on tetrafluoroborate and hexafluorophosphate anions, achieving the highest increase of the half‐life time in 1‐butyl‐3‐methylimidazolium hexafluorophosphate ([bmim⁺][PF₆^?]), which was about 9‐fold higher than the half‐life time in the absence of substrate. Copyright © 2007 Society of Chemical Industry     

Efficient Biocatalytic Cleavage and Recovery of Organic Substrates Supported on Soluble Polymers

The applicability of novel solution‐phase supports in combination with enzymes for biocatalytic transformations is reported. Ex novo designed styrene‐based copolymers, bearing a phenylacetic residue in variable loadings and linked as a pendant group to the macromolecular backbone, through a spacer of variable length, have been synthesized and characterized. These derivatives are compatible and can be used as soluble supports in combination with immobilized penicillin G acylase (PGA – EC 3.5.1.11) for the biocatalytic cleavage of the covalently anchored organic substrate in quantitative yields, in water or water/dimethylformamide solvent mixtures, with recovery of the immobilized enzyme with negligible losses in activity.     

青霉素酰化酶纳米凝胶的制备与性质

提高青霉素酰化酶的耐热性和耐有机溶剂性对于其工业应用具有重要的意义.采用E.coli Top10F'/pGEMKT-TacPGA-Tag为表达菌株发酵生产青霉素酰化酶,经金属鳌合层析得到比酶活为23200 U · L-1的青霉素酸化酶样品,将青霉素酰化酶样品与丙烯酰琥珀酰亚胺反应在酶分子表面修饰上丙烯酰基,然后加入丙烯...     

Influence of Substrate Structure on PGA‐Catalyzed Acylations. Evaluation of Different Approaches for the Enzymatic Synthesis of Cefonicid

The influence of the substrate structure on the catalytic properties of penicillin G acylase (PGA) from Escherichia coli in kinetically controlled acylations has been studied. In particular, the affinity of different β‐lactam nuclei towards the active site has been evaluated considering the ratio between the rate of synthesis (v_s) and the rate of hydrolysis of the acylating ester (v_h1). 7‐Aminocephalosporanic acid (7‐ACA) and 7‐amino‐3‐(1‐sulfomethyl‐1,2,3,4‐tetrazol‐5‐yl)thiomethyl‐3‐cephem‐4‐carboxylic acid (7‐SACA) showed a good affinity for the active centre of PGA. The enzymatic acylation of these nuclei with R‐methyl mandelate has been studied in order to evaluate different approaches for the enzymatic synthesis of cefonicid. The best results have been obtained in the acylation of 7‐SACA. Cefonicid ( 8 ) was recovered from the reaction mixture as the disodium salt in 65% yield and about 95% of purity. Furthermore, through acylation of 7‐ACA, a “one‐pot” chemo‐enzymatic synthesis was carried out starting from cephalosporin C using three enzymes in sequence: D ‐amino acid oxidase (DAO), glutaryl acylase (GA) and PGA. Cefonicid disodium salt was obtained in three steps, avoiding any intermediate purification, in 35% overall yield and about 94% purity. This approach presents several advantages compared with the classical chemical processes.     

Purification of Aspergillus carbonarius polygalacturonase using polymeric membranes

BACKGROUND: Microfiltration (MF: 70–450 nm) and ultrafiltration (UF: 10–500 kDa) membranes were used to eliminate carbohydrates and other non‐protein impurities from Aspergillus carbonarius culture broth containing polygalacturonase enzyme (EC 3.2.1.15) that would otherwise interfere with the purification processes and lead to enzyme loss. Further, diafiltration was attempted to improve the elimination of impurities as well as recovery of enzymes. RESULTS: MF resulted in removal of 2–25% carbohydrates with an enzyme recovery of 69–82% from the crude culture broth owing to the secondary layer formation. UF with 10 kDa membrane eliminated most of the carbohydrates (96%), phosphate salts and total acids with a recovery of 96% polygalacturonase and resulted in greater productivity. Using the above procedure, the enzyme was concentrated nearly 10‐fold while the purity improved from 4.6 to 49.4 U mg^?1 of dry matter. CONCLUSIONS: The results of this study focused on the elimination of carbohydrates and other non‐protein impurities showed that UF could be used efficiently as a primary purification step during downstream processing of microbial culture broths containing enzymes. The present approach will ensure complete elimination of non‐protein impurities thereby reducing the losses and difficulties in the subsequent purification steps. Copyright © 2008 Society of Chemical Industry     

Application of Plackett–Burman screening design to the modeling of grafted alginate–carrageenan beads for the immobilization of penicillin G acylase

Grafted alginate–carrageenan beads were used to immobilize the industrial enzyme penicillin G acylase (PGA). Sixteen factors were screened with the Plackett–Burman design (PBD) to test their significance on the gel beads formation and enzyme immobilization process. The results of PBD showed a wide variation of 30‐fold in the amount of immobilized penicillin G acylase (iPGA) from 11.9 to 354.16 U/g of beads; this reflected the importance of the optimizing process. Among the 16 tested factors, only 3 were proven to be significant. These factors were the enzyme buffer pH (N), enzyme soaking time (Q) with the gel beads, and enzyme concentration (P). The Pareto chart revealed that both Q and P exerted significant positive effects on the amount of iPGA, whereas N had a negative effect. We recommend further study to optimize only these three significant, distinctive enzyme factors. The PGA covalent attachment to the gel beads were proven by Fourier transform infrared spectroscopy, elemental analysis, and NaCl and reusability tests. The best gel bead formula succeeded in the immobilization of 354.16 U/g of beads and proved to be reusable 14 times, retaining 84% of the initial enzyme activity. © 2013 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2014 , 131, 40295.     

High‐Throughput Feasible Screening Tool for Determining Enzyme Stabilities against Organic Solvents Directly from Crude Extracts

Biotransformations in organic chemistry frequently suffer from limitations caused by low water‐solubility of substrates and product inhibition. Both, usually are addressed by the addition of organic cosolvents, which often accompanies at the expense of enzyme stability. A common method for measuring enzyme stability is to determine the melting temperature (T_m) of the enzyme. However, current methods are limited to the application of purified enzymes. Herein, for the first time, an easy and fast (<1 h) high‐throughput feasible method to determine enzyme stabilities directly from crude extracts is reported. In pure buffer, the T_m value measured in the crude extract was identical to that obtained for the purified enzyme. Through the addition of different organic compounds, the T_m values in the crude extract differed by up to 2.4 °C from that of the purified enzymes due to the presence of the host‐cell proteins. Thus, the measurement of enzyme stabilities in crude extracts appears to represent conditions in whole‐cell catalysts even better. The applied nano differential scanning fluorimetry technology is further proven to be suitable for whole‐cell catalysts with two overexpressed enzymes; thus representing a tool for the rapid screening of natural and mutant enzyme libraries in terms of process stability for challenging biotransformations.     

Enzyme flow microcalorimetry—a useful tool for screening of immobilized penicillin G acylase

Screening of a representative series of immobilized penicillin G acylase biocatalysts (enzyme, cells) using enzyme flow microcalorimetry is described. Immobilized penicillin G acylase biocatalysts were either prepared in the laboratory by various techniques or obtained from four commercial manufacturers. An industrial strain of Escherichia coli was entrapped in (poly)acrylamide gel or hardened calcium pectate gel. Semi-purified enzyme was immobilized in various ways—either by covalent binding to oxirane-acrylic beads or chlorotriazine bead cellulose or by entrapment in (poly)acrylamide gel. The validity of the enzyme flow microcalorimetry results was corroborated by a pH-stat method, showing enzyme flow microcalorimetry to be a suitable method for rapid screening of immobilized biocatalysts regardless of the immobilization technique, carrier type or the biocatalyst source. © 1998 Society of Chemical Industry     

Enzymatic esterification of lactic acid,utilizing the basicity of particular polar organic solvents to suppress the acidity of lactic acid

BACKGROUND: Lipase‐catalyzed esterification of lactic acid with ethanol was investigated; however, some difficulties exist with such a system. Because of its high polarity, lactic acid is immiscible with non‐polar organic solvents, which have generally been used for non‐aqueous enzyme reactions. In addition, the strong acidity of lactic acid causes acid inactivation of enzymes and acid‐catalyzed, non‐enzymatic esterification. RESULT: In the present study, particular polar organic solvents, such as 1,4‐dioxane, were found to suppress the enzyme inactivation and non‐enzymatic esterification caused by the acidity. The magnitude of this effect varied with solvents and strongly correlated with the Kamlet‐Taft parameter β, which indicates the basicity of the solvents in non‐aqueous systems. An immobilized lipase from Candida antarctica was found to be the most active and stable enzyme for the reaction. Based on the findings, lipase‐catalyzed esterification of lactic acid (1.0 mol L^?1) could be continued for up to 4 weeks without any loss of enzyme activity. CONCLUSION: In addition to miscibility with lactic acid, the effect of these polar solvents, which is to suppress the acidity of lactic acid and is presumably due to the basicity, appears to play a very important role in the efficient enzymatic reaction of lactic acid. Copyright © 2008 Society of Chemical Industry     

Thermal inactivation kinetics of penicillin g acylase obtained from a mutant derivative of escherichia coli atcc 11105

Thermal inactivation kinetics of native and glutaraldehyde cross-linked forms of penicillin G acylase obtained from a mutant derivative of Escherichia coli ATCC 11105 were studied. Apparent activation energies for thermal inactivation of both native and cross-linked forms of enzyme were calculated to be [57-71 ± 8.46] and [67.11 ± 13.83] kcal mol^?1 respectively. This slight increase in activation energy-suggested that glutaraldehyde cross-linking did not markedly protect against thermal inactivation. Cross-linked enzyme did, however, have a significantly improved half-life at temperatures between 40°C and 50°C.     

Expression, purification and crystallization of penicillin G acylase from Escherichia coli ATCC 11105

The penicillin acylase gene (pac) from Escherichia coli ATCC11105 was cloned into pUC 9 and the resulting vector (pUPA-9),when transformed into E.coli strain 5K, allowed the constitutiveoverproduction of mature penicillin acylase when grown at 28°C.The enzyme ws purified from the periplasmic fraction of E.colipUPA-9 by hydrophobic interaction chromatography and anion exchange.Crystals of penicillin acylase were grown in batch using polyethyleneglycol 8000 as a precipitant. The crystals (space group P1)diffracted to beyond 2.3 Å.     

Improving Cell‐Free Protein Synthesis through Genome Engineering of Escherichia coli Lacking Release Factor 1

Site‐specific incorporation of non‐standard amino acids (NSAAs) into proteins opens the way to novel biological insights and applications in biotechnology. Here, we describe the development of a high yielding cell‐free protein synthesis (CFPS) platform for NSAA incorporation from crude extracts of genomically recoded Escherichia coli lacking release factor 1. We used genome engineering to construct synthetic organisms that, upon cell lysis, lead to improved extract performance. We targeted five potential negative effectors to be disabled: the nuclease genes rna, rnb, csdA, mazF, and endA. Using our most productive extract from strain MCJ.559 (csdA^? endA^?), we synthesized 550±40 μg mL^?1 of modified superfolder green fluorescent protein containing p‐acetyl‐L ‐phenylalanine. This yield was increased to ～1300 μg mL^?1 when using a semicontinuous method. Our work has implications for using whole genome editing for CFPS strain development, expanding the chemistry of biological systems, and cell‐free synthetic biology.