Cloning and high expression of glutaryl 7-aminocephalosporanic acid acylase gene from Pseudomonas diminuta

The gene coding for the glutaryl 7-aminocephalosporanic acid (GL 7-ACA) acylase from Pseudomonas diminuta KAC-1 was cloned and expressed in Escherichia coli. The acylase gene was composed of 2160 base pairs and encoded a polypeptide of 720 amino acid residues. The E. coli BL21 carrying pET2, the plasmid construct for high expression of GL 7-ACA acylase gene, produced this enzyme at approx. 30% of the total proteins with 3.2 units activity mg protein^–1. Growth at temperature below 31 °C and deletion of signal peptide increased the processing of precursor acylase to active enzyme in the recombinant E. coli cells.     

Cloning and characterization of Rv0621 gene related to surfactant stress tolerance in <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis>

To understand how Mycobacterium tuberculosis (M. tuberculosis) could survive in human lung, Genomic expression library of M. tuberculosis in Escherichia coli (E. coli) had been prepared. Taking advantage of the genetic simplicity of E. coli and the functional conservation of some prokaryote proteins, a surfactant stress resistant gene Rv0621 was identified, which encodes a 37 kDa putative membrane protein. The E. coli colony with the partial Rv0621 gene insert, named S1, was able to grow in medium containing 0.4% sodium dodecyl sulfate, while the strain carried empty vector was unable to grow. The full length of the Rv0621 gene was then cloned into plasmid pET32a (+) expressed in E. coli BL21 (DE3). Using gas chromatographic–mass spectrometric (GC–MS), the fatty acid composition of the E. coli BL21 (DE3) carrying Rv0621–pET32a (+) and the E. coli BL21 (DE3) carrying empty vector pET32a (+) were compared. E. coli BL21 (DE3) carrying Rv0621–pET32a (+) contained more oleic acid. This suggests the gene may be involved in regulation of fatty acid synthesis and M. tuberculosis resistance to the surfactant defense of its host.     

Enhanced production of penicillin G acylase from a recombinant Escherichia coli

Penicillin G acylase (pac) gene was cloned into a stable asd ⁺ vector (pYA292) and expressed in Escherichia coli. This recombinant strain produced 1000 units penicillin G acylase g^–1 cell dry wt, which is 23-fold more than that produced by parental Escherichia coli ATCC11105. This enzyme was purified to 16 units mg^–1 protein by a novel two-step process.     

雷氏普罗威登斯菌青霉素G酰化酶基因在大肠杆菌中的克隆与表达

利用PCR和分子克隆技术从雷氏普罗威登斯菌（Prouidencia rettgeri)(ATCC29944)的基因组DNA中获得一个青霉素G酰化酶（penicillinGacylase,PGA)基因并将其装入表达质粒pET24a。携带有重组质粒pETPGA的Escherichia coli基因工程菌BL21（DE3）/pETPGA实现了PGA的高效表达,对发酵条件的研究表明基因工程菌在24℃,添加5g/L甘油条件下以1.0mmol/LIPTG诱导1.5h酶活力即达到993.4U/L,比野生菌酶活力（15U/L）提高了66倍。     

Heterologous expression of leader-less <Emphasis Type="Italic">pga</Emphasis> gene in <Emphasis Type="Italic">Pichia pastoris</Emphasis>: intracellular production of prokaryotic enzyme

Background  

Penicillin G acylase of Escherichia coli (PGA_Ec) is a commercially valuable enzyme for which efficient bacterial expression systems have been developed. The enzyme is used as a catalyst for the hydrolytic production of β-lactam nuclei or for the synthesis of semi-synthetic penicillins such as ampicillin, amoxicillin and cephalexin. To become a mature, periplasmic enzyme, the inactive prepropeptide of PGA has to undergo complex processing that begins in the cytoplasm (autocatalytic cleavage), continues at crossing the cytoplasmic membrane (signal sequence removing), and it is completed in the periplasm. Since there are reports on impressive cytosolic expression of bacterial proteins in Pichia, we have cloned the leader-less gene encoding PGA_Ec in this host and studied yeast production capacity and enzyme authenticity.     

Cloning of penicillin G acylase-encoding gene from Escherichia coli high-producing strain RE3

Summary Pga gene from the industrial strain of E. coli RE3 hyperproducing penicillin G acylase (PGA) was cloned using a simple and rapid NIPAB-based chromogenic method for the detection of PGA-positive recombinant clones. Heterogeneous genetical material was prepared by subcloning pga in vectors pACYC184, pBR322-2 and pK19. The highest constitutive expression of pga was observed in a strain bearing the recombinant plasmid pKA18 synthesizing 2.5 times more of enzyme than the parent strain.     

Improvement of the catalytic properties of penicillin G acylase from Escherichia coli ATCC 11105 by selection of a new substrate specificity

Cloned penicillin G acylase (PGA) from Escherichia coli ATCC 11105 was mutagenized in vivo using N-methyl-N-nitrosoguanidine. Mutants of PGA were selected by their ability to allow growth of the host strain E. coli M8820 with the new substrates phenylacetyl--alanyl-l-proline (PhAc-Ala-Pro) phthalyl-l-leucine (Pht-Leu) or phthalylglycyl-l-proline (Pht-Gly-Pro) as sole source of proline and leucine respectively. PGA mutants were purified and immobilized onto spherical methacrylate (G-gel). The immobilized form of mutant PGA selected with (PhAc-gbAla-Pro) hydrolyzed 95% of 9 mmol penicillin G 30% faster than wild-type PGA using the same specific activities. The specific activity of the soluble enzyme was 2.7-fold, and inhibition by phenylacetic acid was halved. Immobilized PGA mutant selected with Pht-Gly-Pro hydrolyzed penicillin G 20% faster than wild-type PGA. The K _m of the soluble enzyme was increased 1.7-fold. Furthermore, the latter two mutants were also 3.6-fold more stable at 45° C than wild-type PGA. The specific activity of the mutant selected with Pht-Leu was 6.3-fold lower, and inhibition by phenylacetic acid was increased 13-fold.     

Monitoring bioreactors using principal component analysis: production of penicillin G acylase as a case study

The complexity of biological processes often makes impractical the development of detailed, structured phenomenological models of the cultivation of microorganisms in bioreactors. In this context, data pre-treatment techniques are useful for bioprocess control and fault detection. Among them, principal component analysis (PCA) plays an important role. This work presents a case study of the application of this technique during real experiments, where the enzyme penicillin G acylase (PGA) was produced by Bacillus megaterium ATCC 14945. PGA hydrolyzes penicillin G to yield 6-aminopenicilanic acid (6-APA) and phenyl acetic acid. 6-APA is used to produce semi-synthetic β-lactam antibiotics. A static PCA algorithm was implemented for on-line detection of deviations from the desired process behavior. The experiments were carried out in a 2-L bioreactor. Hotteling’s T ² was the discrimination criterion employed in this multivariable problem and the method showed a high sensibility for fault detection in all real cases that were studied.     

Functional Overexpression and Purification of a Codon Optimized Synthetic Glucarpidase (Carboxypeptidase G2) in Escherichia coli

Glucarpidase (former name: carboxypeptidase G2, or CPG2) is a bacterial enzyme that is widely used in detoxification of the cytotoxic drug, methotrexate, and in Antibody Directed Enzyme Prodrug Therapy for cancer treatment. The glucarpidase gene of Pseudomonas sp. strain RS-16 was previously cloned in E coli, but expresses at a level that is approximately 100-fold lower than in the native strain. In this study, a synthetic gene coding for glucarpidase was codon-optimised and synthesized for maximum expression in E. coli using the vector pET28a. Our work indicated that the enzyme was expressed to ~60% of the total host protein and that purification of the recombinant His-tagged protein could be achieved in a single step by Ni²⁺ charged column chromatography. The synthetic recombinant glucarpidase expressed within this system was biologically active and zinc dependant. Our study showed that Mg²⁺ as well as Mn²⁺ ions inhibit the activity of the recombinant enzyme.     

Indigenous plasmids in a production line of strains for penicillin G acylase derived fromEscherichia coli W

Three indigenous plasmids designated pRK1, pRK2 and pRK3 were identified among producers of penicillin G acylase, (PGA) derived from the strainEscherichia coli W ATCC 9637. Their size and copy number (CN) inE. coli W were determined (kb; CN); pRK1 (80; 3.4), pRK2 (5.1; 71), and pRK3 (4.8; 13.7). StrainE. coli RE2 harboring these plasmids was used for selection of strains with reduced number of plasmids: the strain RE3 without plasmid pRK1 and the plasmid-less strain cERE3 were isolated. Indigenous plasmids did not code for the resistance determinants against 23 antibiotics and 10 heavy metals.     

Enzymatic synthesis of cephalosporins. The immobilized acylase from <Emphasis Type="Italic">Arthrobacter viscosus</Emphasis>: A new useful biocatalyst

The acylase from Arthrobacter viscosus was immobilized, studied in the enzymatic synthesis of some cephalosporins by kinetically controlled N-acylation (kcNa) of different cephem nuclei, and compared with the penicillin G acylase (PGA) from Escherichia coli. The reaction outcomes were dependent on the acylase microbial source and on the type of immobilization support. Generally, both enzymes, when immobilized onto hydrophilic resins such as glyoxyl-agarose (activated with aldehyde groups), displayed higher synthetic performances in comparison with hydrophobic acrylic epoxy-supports like Eupergit C. The kcNa of 7-amino cephalosporanic acid catalyzed by A. viscosus immobilized on glyoxyl-agarose afforded a quantitative conversion in 7-[(1-hydroxy-1-phenyl)-acetamido]-3-acetoxymethyl-Δ³-cephem-4-carboxylic acid, a useful intermediate for the synthesis of Cefamandole and Cefonicid. Similar results were obtained in the synthesis of these cephalosporins by direct acylation of the corresponding 3′-functionalized nucleus. In these reactions, A. viscosus displayed higher synthetic performances than the PGA from E. coli.     

Stabilization and functional properties of Escherichia coli penicillin G acylase by covalent conjugation of anionic polysaccharide carboxymethylcellulose

The stabilization of Escherichia coli penicillin G acylase (PGA) conjugated with carboxymethylcellulose (CMC) against temperature and pH was studied. The 2,3-dialdehyde derivative of CMC obtained by periodate oxidation was covalently conjugated to PGA via Schiff's base formation. The inactivation mechanism of both native and CMC-conjugated PGA appeared to obey first order inactivation kinetics during prolonged incubations at 40–60 °C and in the pH range 4–9. Inactivation rate constants of conjugated enzyme were always lower, and half-life times were always higher than that of native PGA. The activation free energy of inactivation (G _i values) of CMC-conjugated enzyme were found to be always higher than that of native PGA at all temperatures and pH values studied as another indicator of enzyme stabilization. Highest stability of CMC-conjugated enzyme was observed as nearly four-fold at 40 °C and pH 8.0. No changes were observed on the temperature and pH profiles of PGA after CMC conjugation. Lower K _m and higher k _cat values of PGA obtained after CMC conjugation indicates the improved effect of conjugation on the substrate affinity and catalytic performance of the enzyme.     

Comparing the performance of multilayer perceptrons networks and neuro-fuzzy systems for on-line inference of <Emphasis Type="Italic">Bacillus megaterium</Emphasis> cellular concentrations

Penicillin G acylase (PGA) is one of the most important enzymes for the pharmaceutical industry. Bacillus megaterium has the advantage of producing extra-cellular PGA. This work compares two neural networks (NNs) architectures for on-line inference of B. megaterium cell mass in an aerated stirred tank bioreactor, during the production of PGA. Nowadays, intelligent computing tools such as artificial NNs and fuzzy logic are commonly applied for state inference and modeling of bioreactors. Combining these two approaches in hybrid, neuro-fuzzy systems, may be advantageous. Our results indicate that a neuro-fuzzy inference system showed a better performance to infer cell concentrations, when compared to multilayer perceptrons networks.     

Expression and characterization of a thermostable penicillin G acylase from an environmental metagenomic library

One clone (ACPGA001) exhibiting penicillin G acylase (PGA) activity was screened from a metagenomic library by using a medium containing penicillin G. A novel PGA gene from the inserted fragment of ACPGA001 was obtained by sequencing. The amino acid sequence of ACPGA001 PGA exhibited <33 % similarity to PGAs retrieved from GenBank. This gene was expressed in Escherichia coli M15 and the recombinant protein was purified and characterized. The ACPGA001 PGA exhibited a maximum activity at 60 °C and showed high activity at pH 4–10 with an optimum pH of 8.0. This enzyme was stable at 40 °C for 70 min with a half-life of 60 min at 55 °C. These beneficial characteristics of ACPGA001 PGA provide some advantages for the potential application of ACPGA001 PGA in industry.     

保加利亚乳杆菌ATCC11842 L-乳酸脱氢酶同工酶基因的克隆与表达分析

通过对保加利亚乳杆菌(Lactobacillus delbrueckii subsp.bulgaricus)L-乳酸脱氢酶(L-lactate dehydrogenase, L-LDH)同工酶基因的异源表达、酶活测定和摇瓶发酵研究L-LDH在乳酸合成中的作用。将保加利亚乳杆菌ATCC11842中L-乳酸脱氢酶基因ldb0120和ldb0094分别克隆至载体pET28a(+)中,构建重组表达载体pET28aldb0120和pET28aldb0094,并转化到大肠埃希菌(Escherichia coli) BL21(DE3)中进行表达。进一步对重组蛋白进行Ni-NTA柱亲和层析和酶学活性测定,结果显示,LDB0120和LDB0094的比活力分别为0和25 U/mg,表明LDB0094是具有低活性的L-乳酸脱氢酶,而LDB0120不具有活性。对两株重组菌分别进行好氧和微好氧发酵,重组菌E.coli BL21/pET28aldb0094在好氧和微好氧条件可以合成L-乳酸,浓度分别为41.9和227.9 mg/L,而菌株E.coli BL21/pET28aldb0120在两种培养条件下均基本不合...     

Stabilization of Immobilized Enzymes Against Water-Soluble Organic Cosolvents and Generation of Hyper-Hydrophilic Micro-Environments Surrounding Enzyme Molecules

Enzymes usually undergo rapid inactivation in the presence of organic media. In some cases, the mechanism is quite simple. For example, when an enzyme, fully dispersed and immobilized inside porous supports, is inactivated, at neutral p^H and moderate temperature, in the presence of medium-high concentrations of water-miscible organic cosolvents, the unique cause of inactivation is the interaction of the enzyme with cosolvent molecules and the only inactivating effect is the promotion of conformational changes on enzyme structure.

On this basis, two distinct strategies for stabilization of enzymes against organic solvents are proposed:

a. reduction of the causes of inactivation: generation of hyper-hydrophilic micro-environments having a very open structure and fully surrounding every enzyme molecule;

b. reduction of the effects of inactivation: “rigidification of enzymes” via multipoint covalent immobilization.

By using penicillin G acylase (PGA) as a model enzyme, both strategies have been evaluated and compared. Both stabilizing strategies had significant effects. In this case, hydrophilization of the enzyme nano-environment was found to be more effective than rigidification of the enzyme via multipoint covalent attachment. The combined effect of both stabilizing strategies was also tested: multipoint covalently immobilized enzyme molecules were completely surrounded by hyper-hydrophilic microenvironments. In this way, native PGA that was unstable against organic cosolvents (completely inactivated in less than 3 min in 95% dioxane) was transformed into a very stable immobilized derivative (preserving more than 80% of activity after 40 days under the same conditions).     

产GL-7-ACA酰化酶重组大肠杆菌的构建和表达

为了实现GL-7-ACA酰化酶在大肠杆菌中的成功表达,将GL-7-ACA酰化酶基因用PCR的方法去除其信号肽序列,并将其连接到质粒pET-28a,通过筛选得到了表达GL-7-ACA酰化酶的重组菌B121(DE3),pET-ACY。分别考察了诱导温度、菌浓(OD600)、诱导剂IFrG的用量等因素对重组菌表达GL-7-ACA酰化酶的影响。在优化条件下,GL-7-ACA酰化酶酶活可达266U／L。GL-7-ACA酰化酶经一步DEAE-Sepharose纯化即可达到80％的纯度,酶活收率为50％。     

粪产碱杆菌青霉素G酰化酶在枯草芽孢杆菌中的表达、纯化及其性质

利用PCR技术克隆了粪产碱杆菌 (Alcaligenesfaecalis,CICCAS1.76 7)青霉素G酰化酶 (pencillinGacylase ,PGA)基因 (GenBank登录号AF4 5 5 35 6 )。通过构建工程菌E .coli(pETAPGA) ,该酶在大肠杆菌中获得了表达 ,表达产物分泌到周质空间。进一步构建的工程菌B .subtilis (pMAPGA)和B .subtilis(pBAPGA)实现了该酶的胞外分泌表达。分泌表达的最高表达量为 6 5 3u/L ,比野生型A .faecalis表达量高 10 9倍。表达产物经硫酸铵分级沉淀和DEAE SepharoseCL 6B两步纯化 ,纯度提高 86倍 ,活力回收率达到 81% ,纯化后的PGA活力为 1.4 6 9u/mg。研究表明 ,PGA家族成员中只有粪产碱杆菌PGA和巨大芽孢杆菌PGA可以在枯草芽孢杆菌中分泌表达。与巨大芽孢杆菌PGA相比 ,粪产碱杆菌PGA的最适pH值为 8.0 ,最适温度为 6 0°C ,而且在有机溶剂中具有更强的稳定性。该酶在水相中具有较低的头孢氨苄合成活力。本研究为粪产碱杆菌PGA的获得提供了新的途径。     

An approach for the improved immobilization of penicillin G acylase onto macroporous poly(glycidyl methacrylate‐co‐ethylene glycol dimethacrylate) as a potential industrial biocatalyst

The use of penicillin G acylase (PGA) covalently linked to insoluble carrier is expected to produce major advances in pharmaceutical processing industry and the enzyme stability enhancement is still a significant challenge. The objective of this study was to improve catalytic performance of the covalently immobilized PGA on a potential industrial carrier, macroporous poly(glycidyl methacrylate‐co‐ethylene glycol dimethacrylate) [poly(GMA‐co‐EGDMA)], by optimizing the copolymerization process and the enzyme attachment procedure. This synthetic copolymer could be a very promising alternative for the development of low‐cost, easy‐to‐prepare, and stable biocatalyst compared to expensive commercially available epoxy carriers such as Eupergit or Sepabeads. The PGA immobilized on poly(GMA‐co‐EGDMA) in the shape of microbeads obtained by suspension copolymerization appeared to have higher activity yield compared to copolymerization in a cast. Optimal conditions for the immobilization of PGA on poly(GMA‐co‐EGDMA) microbeads were 1 mg/mL of PGA in 0.75 mol/L phosphate buffer pH 6.0 at 25°C for 24 h, leading to the active biocatalyst with the specific activity of 252.7 U/g dry beads. Chemical amination of the immobilized PGA could contribute to the enhanced stability of the biocatalyst by inducing secondary interactions between the enzyme and the carrier, ensuring multipoint attachment. The best balance between the activity yield (51.5%), enzyme loading (25.6 mg/g), and stability (stabilization factor 22.2) was achieved for the partially modified PGA. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 32:43–53, 2016     

A novel mechanism involved in the metabolism of the tartaric acid stereoisomers in Rhodopseudomonas sphaeroides: enzymatic conversion of meso-tartaric acid to D(-)-glyceric acid and CO2

In cell extracts of Rhodopseudomonas sphaeroides grown on meso-tartrate the activities of the bifunctional L(+)-tartrate dehydrogenase-D(+)-malate dehydrogenase (decarboxylating) (EC 1.1.1.93 and 1.1.1.83, respectively) could be measured spectrophotometrically but not the activity of a meso-tartrate dehydrogenase or dehydratase. However, an enzyme activity was detected manometrically that catalyzed the stoichiometric release of CO₂ from mesotartrate in a molar ratio of 1:1. This reaction required catalytic amounts of NAD and the presence of both divalent (Mn²⁺ or Mg²⁺) and monovalent (NH ₄ ⁺ or K⁺) cations. Purification of the meso-tartrate decarboxylase showed that it was part of the bifunctional L(+)-tartrate dehydrogenase-D(+)-malate dehydrogenase (decarboxylating), which thus possessed a third catalytic function. The homogeneous enzyme catalyzed the stoichiometric conversion of incso-tartaric acid to D(-)-glyceric acid and CO₂. All interfering catalytic activities had been eliminated during the course of enzyme purification.