共查询到20条相似文献,搜索用时 15 毫秒
1.
José Luis Martínez-Hernández Marco Arnulfo Mata-Gómez Cristóbal Noé Aguilar-González Anna Ilyina 《Applied biochemistry and biotechnology》2010,160(7):2045-2053
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. 相似文献
2.
Qi Wu Chun-Xiu Chen Li-Li Du Xian-Fu Lin 《Applied biochemistry and biotechnology》2010,160(7):2026-2035
A cascade reaction combining the enzymatic hydrolysis of Penicillin G potassium salt (PGK) with the kinetically controlled
enzymatic coupling of in situ formed 6-aminopenicillanic acid (6-APA) with p-hydroxyphenylglycine methyl ester (D-HPGM) to give amoxicillin as the final product by using a single enzyme has been demonstrated
successfully. Ethylene glycol (EG) was employed as a component of reaction buffer to improve the synthesis yield. Reaction
parameters, including different cosolvents, EG content, the loading of immobilized penicillin G acylase (IPA), and reaction
temperature and time were studied to evaluate their effects on the reaction. The best result of 55.2% yield was obtained from
the reaction which was carried out in the mixed media containing 40% sodium dihydrogen phosphate buffer (apparent pH 6.0)
and 60% EG (v/v), with the initial concentration 150 mM and 450 mM of PGK and D-HPGM, respectively, catalyzed by 50 IU/mL IPA at 25 °C for
10 h. The IPA could be recycled for nine batches without obviously losing of catalytic activity. The important strategy will
have potential application in the β-lactam antibiotics industry due to the advantages of saving the effort of isolating 6-APA,
reducing usual enzymatic steps and the industrial cost of amoxicillin synthesis. 相似文献
3.
不同介孔材料固定青霉素酰化酶的稳定性研究 总被引:9,自引:0,他引:9
介孔材料由于具有在2~30nm之间可调的纳米级规则孔道、大比表面积和强吸附性能而成为固定化酶的优良载体.将酶固定于介孔材料的孔道中制备成的固定化酶与溶液酶相比,有易于与产物分离,并可回收和反复使用,可降低生产成本,减少酶的自水解和保持酶的活性.青霉素酰化酶(Penicillin acylase,PGA,EC.3.5.1.11)又称为青霉素酰胺酶或青霉素氨基水解酶,该酶属于球蛋白,分子量较大,由2个亚基组成:分子量为19500的含有侧链结合位点的亚基和分子量为60000的含有催化位点的亚基. 相似文献
4.
介孔材料的修饰及固定青霉素酰化酶的稳定性研究 总被引:4,自引:0,他引:4
利用扩孔剂的作用合成出较大孔径(12 nm)的介孔材料SBA-15, 并进行表面氨基修饰, 以此为载体, 以戊二醛为交联剂, 对青霉素酰化酶进行组装固定, 并对固定化青霉素酰化酶(PGA)的稳定性进行了深入的研究. 实验结果表明, PGA与载体交联后仍保持活性. 热稳定性研究结果表明, 制备的固定化青霉素酰化酶在低于60 ℃时保持稳定; pH在6~11范围内保持稳定; 固定化酶重复使用10次之后, 仍具有高达90%的残留活力. 相似文献
5.
E. Boccù M. Carenza S. Lora G. Palma F. M. Veronese 《Applied biochemistry and biotechnology》1987,15(1):1-10
The immobilization ofEscherichia coli penicillin acylase (EC 3.5.1.11) was investigated by radiation-induced polymerization of 2-hydroxyethyl methacrylate at low
temperature. A leak-proof composite that does not swell in water was obtained by adding the crosslinking agent trimethylolpropane
trimethacrylate to the monomer-aqueous enzyme mixture. Penicillin acylase, which was immobilized with greater than 70% yield,
possessed a higherK
m
value toward the substrate 6-nitro-3-phenylacetamidobenzoic acid than the free enzyme form (K
m
= 1.7 × 10−5 and 1 × 10−5M, respectively). The structural stability of immobilized penicillin acylase, as assessed by heat, guanidinium chloride, and
pH denaturation profiles, was very similar to that of the free-enzyme form, thus suggesting that penicillin acylase was entrapped
in its native state into aqueous free spaces of the polymer matrix. 相似文献
6.
A dodecane/thermosensitive polymer/water three-liquid-phase system was introduced for enzymatic hydrolysis of penicillin G
(Pen G) for 6-aminopenicillanic acid (6-APA). The enzyme was covalently attached to the terminal of PEO–PPO–PEO polymer (L63),
which would be transferred into a polymer coacervate phase at high temperature above its “cloud point”. 6-APA was primarily
resided in the aqueous phase due to its zwitterionic nature. More than 70% phenylacetic acid (PAA) was transferred into the
organic phase using trioctylmethylammonium hydroxide and trihexyl-(tetradecyl)phosphonium bis 2,4,4-trimethylpentylphosphinate
ionic liquids (Cyphos IL-104) mixture at pH 5.5, while most of Pen G resided in water. As a result, high operational pH was
permitted in three-liquid-phase system, which leads to higher enzymatic activity (120 IU at 40°C) and stability (enzymatic
half-time up to 55 h at 60°C) in comparison with the value in butyl acetate/water two-phase system. On the other hand, two
products in three-liquid-phase system might be automatically separated from the enzyme sphere into different phases at the
same time, which facilitated the reaction equilibrium towards the product’s side with 6-APA productivity of 80% at 42°C, pH 5.5.
Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. 相似文献
7.
A mathematical model that describes the heterogeneous reaction–diffusion process involved in penicillin G hydrolysis in a
batch reactor with immobilized penicillin G acylase is presented. The reaction system includes the bulk liquid phase containing
the dissolved substrate (and products) and the solid biocatalyst phase represented by glyoxyl-agarose spherical porous particles
carrying the enzyme. The equations consider reaction and diffusion components that are presented in dimensionless form. This
is a complex reaction system in which both products of reaction and the substrate itself are inhibitors. The simulation of
a batch reactor performance with immobilized penicillin G acylase is presented and discussed for the internal diffusional
restrictions impact on effectiveness and productivity. Increasing internal diffusional restrictions, through increasing catalyst
particle size and enzyme loading, causes impaired catalyst efficiency expressed in a reduction of effectiveness factor and
specific productivity. High penicillin G initial concentrations decrease the impact of internal diffusional restrictions by
increasing the mass transfer towards porous catalyst until product inhibition becomes significant over approximately 50 mM
of initial penicillin G, where a drop in conversion rate and a maximum in specific productivity are then obtained. Results
highlight the relevance of considering internal diffusional restrictions, reactor performance, and productivity analysis for
proper catalyst and reactor design. 相似文献
8.
Massolini G Calleri E De Lorenzi E Pregnolato M Terreni M Félix G Gandini C 《Journal of chromatography. A》2001,921(2):147-160
In this paper, the use of penicillin G acylase (PGA) as a biocatalyst and as a chiral selector is described. Penicillin G-acylase is an interesting enzyme used in the manufacture of semisynthetic antibiotics and, in particular, in the production of 6-APA by hydrolysis of penicillin G. Five PGA-based HPLC columns have been prepared by using two different silica supports by employing two immobilization methods, namely "in situ" and "in batch". The effects of the immobilization techniques and of different silica pore size on the catalytic properties of the enzyme as well as the applicability of the PGA-bonded stationary phases as chiral selectors for a number of chiral drugs have been investigated. The HPLC columns based on immobilized PGA combine the hydrolytic activity and the chiral recognition properties of PGA, therefore they have been used for the development of a combined reaction-separation system for chiral and achiral substrates. 相似文献
9.
The hydroxyalkylation of the penicillin nucleus at C-6 with benzaldehyde and formaldehyde is reported. 6-aminopenicillanic acid (6-APA) ( 2 ) was used as starting material. Protection of the functional groups and activation of the C-6 position were effected by converting 6-APA successively into the Schiff base 13 , the methyl ester 14 and the copper complex 15 . The latter gave with benzaldehyde the C-6 monosubstituted complex 17 , and with formaldehyde the disubstituted complex 18 . The direct α-hydroxybenzylation of 6-APA at C-6 was also carried out with an excess of benzaldehyde at pH 7,5 leading to the SCHIFF base 19 . Mild hydrolysis of 19 gave 6-amino-6-(α-hydroxybenzyl)-penicillanic acid ( 21 ). Phenylacetylation of the latter yielded the penicillin G analogue 22 . Direct reaction of 6-APA with formaldehyde took place only in the presence of salicylaldehyde, giving the oxazolidine 24 , from which the amino acid 25 could not be obtained. The new compounds showed only weak antibacterial activity as compared with penicillin G. 相似文献
10.
Jesús Torres-Bacete Miguel Arroyo Raquel Torres-Guzmán Isabel de La Mata Carmen Acebal M. Pilar Castillón 《Applied biochemistry and biotechnology》2005,126(2):119-131
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. 相似文献
11.
In this paper, an efficient method was established for continuous kinetic resolution of racemic 2-aminobutanol by selective hydrolysis of N-phenylacetyl (±)-2-aminobutanol over immobilized penicillin G acylase (PGA) in a fixed-bed reactor. Several N-acylated derivatives of 2-aminobutanol were screened in batch experiments, and it was found that the hydrolysis of N-phenylacetyl (±)-2-aminobutanol proceeded smoothly in the presence of immobilized penicillin G acylase with satisfied enantioselectivity. Thus, the reaction parameters were optimized in a fixed-bed reactor. Under the optimized conditions, 39.3% conversion of N-phenylacetyl (±)-2-aminobutanol and 98.2% ee value of S-2-aminobutanol were obtained. This fixed-bed system was operated continuously for 40 h without significant decrease of enzyme activity. It has been demonstrated to be more efficient compared to the batch experiments. 相似文献
12.
V. K. Svedas E. V. Kozlova D. A. Mironenko V. P. Kukhar T. N. Kasheva V. A. Solodenko 《Phosphorus, sulfur, and silicon and the related elements》2013,188(1-4)
Abstract Penicillin acylase from E. coli (FC 3.5.1.11) was found to hydrolyse N-phenylacetylated 1-aminoalkylphosphonic acids and their esters. Enzyme preferentially converts the R-form of the substrates: the ratios of the bimolecular rate constants of penicillin acylase-catalysed hydrolysis of R-and S- forms of 1-(N-phenylacetaminol-ethylphosphonic acid and its dimethyl- and diisopropyl- esters are 58000, 2600, 1800; these derivatives were shown to have the greatest values of the catalytic constants for enzymatic hydrolysis of all known substrates of penicillin acylase: 237, 148, and 134 s; corresponding values of Michaelis constants are 3.7×10?5, 6.8×10?4, and 6.2×10?4 M. The kinetics of the enzymatic hydrolysis of 1-(N-phenylacetaminol-ethylphosphonic acid was investigated up to high degrees of conversion. The inhibition of penicillin acylase by high concentrations of the R-form of the substrate (with substrate inhibition constant 0.07 Ml and competitive inhibition by the reaction product phenylacetic acid (Ki=3.5×10?5 M) was observed. Penicillin acylase was shown to possess quite broad substrate specificity among N-acylated 1-aminoalkylphosphonic acids and was found to be capable of hydrolysing 1-(N-phenylacetaminol-substituted 2-phenylethyl-, 1-phenylmethyl- and 3-methylbutylphosphonic acids with high efficiency and enantioselectivity. 相似文献
13.
Pinotti Laura M. Silva Rosineide G. Giordano Roberto C. Giordano Raquel L. C. 《Applied biochemistry and biotechnology》2002,98(1-9):679-686
This article reports studies concerning the production of penicillin G acylase (PGA) by Bacillus megaterium. This enzyme has industrial use in the hydrolysis of penicillin G to obtain 6-aminopenicillanic acid, an essential intermediate
for the production of semisynthetic β-lactam antibiotics. Although most microorganisms produce the enzyme intracellularly,
B. megaterium provides extracellular PGA. The enzyme production by microorganisms involves several steps, resulting in a many operational
variables to be studied. The study of the inoculum is an important step to be accomplished, before addressing other issues
such as culture optimization and downstream processing. In this study, using a standard inoculum as reference, several runs
were performed aiming at the definition of operational conditions in the PGA production. Cell concentration and PGA activity
in the production medium were measured after 24, 48, and 72 h of the beginning of the production phase. This study encompasses
the duration of the inoculum germination phase and the concentration of cells used to startup the germination. Based on these
results, PGA productivity during the production phase was maximized. The selected values for these variables were 1.5 × 107 spores/mL of germination medium, germination during 24 h, and 72 h for the production phase. 相似文献
14.
<正>Penicillin G acylase(PGA) was immobilized on the magnetic hydrophilic polymer microspheres with average pore size of 17.1 nm,specific surface area of 128.2 m~2/g and saturate magnetization of 6.4 emu/g.The 96.7%ampicillin yield with 1.60 of the synthesis/hydrolysis(S/H) ratio from 6-aminopenicillanic acid(6-APA) and D-(-)-alpha-phenylglycine methyl ester(D-PGME) can be achieved using the resultant magnetic biocatalyst in ethylene glycol,where only 82.1%yield with 1.40 of the S/H ratio was obtained using the free PGA under the identical reaction conditions.The immobilized PGA can be separated magnetically and recycled for five times without obvious loss of its catalytic activity. 相似文献
15.
介孔材料MCFs的合成及组装青霉素酰化酶的性质研究 总被引:4,自引:0,他引:4
介孔材料由于具有纳米级规则孔道和巨大的比表面积而在催化、吸附及分离等方面存在较大的应用价值.近年来,由介孔分子筛如MCM-41和SBA-15州等组装功能性材料已成为研究的热点.酶作为高效催化剂有许多优点,但在溶液中易失活,使用后无法回收,有的酶在溶液中还存在自水解问题:将酶组装在介孔材料中制成固定化酶则可解决上述问题.目前已成功地将辣根过氧化物酶 相似文献
16.
Cross-linked enzyme aggregates: a simple and effective method for the immobilization of penicillin acylase 总被引:2,自引:0,他引:2
[reaction--see text] Penicillin G acylase (penicillin amidohydrolase, E.C. 3.5.1.11) was immobilized in a simple and effective way by physical aggregation of the enzyme, using a precipitant, followed by chemical cross-linking to form insoluble cross-linked enzyme aggregates (CLEAs). These had the same activity in the synthesis of ampicillin as cross-linked crystals of the same enzyme, but the accompanying hydrolysis of the side-chain donor was much less. Penicillin G acylase CLEAs also catalyzed the synthesis of ampicillin in a broad range of organic solvents. 相似文献
17.
Karthikeyan Rajendran Sudharshan Sekar Surianarayanan Mahadevan Bhuvanesh Kumar Shanmugam Rajendhran Jeyaprakash Gunasekaran Paramasamy Asit Baran Mandal 《Applied biochemistry and biotechnology》2014,172(8):3736-3747
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. 相似文献
18.
19.
N. A. Lozinskaya S. E. Sosonyuk Yu. N. Firsova M. V. Proskurnina N. S. Zefirov 《Russian Chemical Bulletin》2009,58(1):152-155
N,N’-Bis(arylidene)methanediamines regiospecifically react with 2,3-diphenylcyclo- propenone to form stable adducts of both the
mono- and the diaddition. Ammonium acetate serves as the catalyst of the process. During hydrolysis of these adducts, an oxidative
coupling of 1,2-dihydro-3H-pyrrol-3-ones occurs with the formation of 2,2’,4,4’,5,5’-hexaaryl-l,1’,2,2’- tetrahydro-H,3’-2,2’-bipyrrole-3,3’-diones. 相似文献
20.
《Tetrahedron: Asymmetry》1999,10(23):4495-4500
Racemic 2-amino-1-butanol has been resolved to obtain (S)-2-amino-1-butanol with >99% e.e. via enantioselective hydrolysis of its N-phenylacetyl derivative with penicillin G acylase immobilised on Eupergit C. 相似文献