Enlarging the substrate binding pocket of penicillin G acylase from Achromobacter sp. for highly efficient biosynthesis of ß-lactam antibiotics.

Penicillin G acylase (PGA) is a key biocatalyst for the enzymatic production of ß-lactam antibiotics, which can not only catalyze the synthesis of ß-lactam antibiotics but also catalyze the hydrolysis of the products to prepare semi-synthetic antibiotic intermediates. However, the high hydrolysis and low synthesis activities of natural PGAs severely hinder their industrial application. In this study, a combinatorial directed evolution strategy was employed to obtain new PGAs with outstanding performances. The best mutant ßF24G/ßW154G was obtained from the PGA of Achromobacter sp., which exhibited approximately a 129.62-fold and a 52.55-fold increase in specific activity and synthesis/hydrolysis ratio, respectively, compared to the wild-type AsPGA. Thereafter, this mutant was used to synthesize amoxicillin, cefadroxil, and ampicillin; all conversions > 99% were accomplished in 90-135 min with almost no secondary hydrolysis byproducts produced in the reaction. Molecular dynamics simulation and substrate pocket calculation revealed that substitution of the smallest glycine residue at ßF24 and ßW154 expanded the binding pocket, thereby facilitating the entry and release of substrates and products. Therefore, this novel mutant is a promising catalyst for the large-scale production of ß-lactam antibiotics.

Immobilized penicillin G acylase with enhanced activity and stability using glutaraldehyde-modified polydopamine-coated Fe3 O4 nanoparticles.

In this work, Fe3 O4 nanoparticles (NPs) were coated with polydopamine (PDA) to structure Fe3 O4 @PDA NPs by the spontaneous oxygen-mediated self-polymerization of dopamine (DA) in an aqueous solution of pH = 8.5. The fabricated Fe3 O4 @PDA NPs were grafted by glutaraldehyde to realize the immobilization of penicillin G acylase (PGA) under mild conditions. The carriers of each stage were characterized and investigated by transmission electron microscopy, X-ray diffraction, Fourier transform infrared, and vibrating sample magnetometry. To improve the catalytic activity and stability of immobilized PGA, the immobilization conditions were investigated and optimized. Under the optimal immobilization conditions, the enzyme loading capacity, enzyme activity, and enzyme activity recovery of immobilized PGA were 114 mg/g, 26,308 U/g, and 78.5%, respectively. In addition, the immobilized PGA presented better temperature and pH stability compared with free PGA. The reusability study ensured that the immobilized PGA showed an excellent repeating application performance. In particular, the recovery rate of immobilized PGA could reach 94.8% and immobilized PGA could retain 73.0% of its original activity after 12 cycles, indicating that the immobilized PGA exhibited a high operation stability and broad application potential in the biocatalysis field.

Penicillin G acylase production by Mucor griseocyanus and the partial genetic analysis of its pga gene.

Penicillin acylases (penicillin amidohydrolase, EC 3.5.1.11) are a group of enzymes with many applications within the pharmaceutical industry, and one of them is the production of semi-synthetic beta-lactam antibiotics. This enzyme is mainly produced by bacteria but also by some fungi. In the present study, the filamentous fungus Mucor griseocyanus was used to produce penicillin acylase enzyme (PGA). Its ability to express PGA enzyme in submerged fermentation process was assessed, finding that this fungal strain produces the biocatalyst of interest in an extracellular way at a level of 570 IU/L at 72 h of fermentation; in this case, a saline media using lactose as carbon source and penicillin G as inducer was employed. In addition, a DNA fragment (859 bp) of the pga from a pure Mucor griseocyanus strain was amplified, sequenced, and analyzed in silico. The partial sequence of pga identified in the fungi showed high identity percentage with penicillin G acylase sequences deposited in NCBI through BLAST, especially with the ß subunit of PGA from the Alcaligenes faecalis bacterium¸ which is a region involved in the catalytic function of this protein. Besides, the identification of domains in the penicillin G acylase sequence of Mucor griseocyanus showed three conserved regions of this protein. The bioinformatic results support the identity of the gen as penicillin G acylase. This is the first report that involves sequencing and in silico analysis of Mucor griseocyanus strain gene encoding PGA.

Efficient enzymatic synthesis of cephalexin in suspension aqueous solution system.

An efficient method for the enzymatic synthesis of cephalexin (CEX) from 7-amino-3-deacetoxycephalosporanic acid (7-ADCA) and d-phenylglycine methyl ester (PGME) using immobilized penicillin G acylase (IPGA) as catalyst in a suspension aqueous solution system was developed, where the reactant 7-ADCA and product CEX are mainly present as solid particles. The effects of key factors on the enzymatic synthesis were investigated. Results showed that continuous feeding of PGME was more efficient for the synthesis of CEX than the batch mode. Under the optimized conditions, the maximum 7-ADCA conversion ratio of 99.3% and productivity of 200 mmol/L/H were achieved, both of which are much superior to the homogeneous aqueous solution system. Besides, IPGA still retained 95.4% of its initial activity after 10 cycles of enzymatic synthesis, indicating the excellent stability of this approach. The developed approach shows great potential for the industrial production of CEX via an enzyme-based route.

Genetic Encoding and Enzymatic Deprotection of a Latent Thiol Side Chain to Enable New Protein Bioconjugation Applications.

The thiol group of the cysteine side chain is arguably the most versatile chemical handle in proteins. To expand the scope of established and commercially available thiol bioconjugation reagents, we genetically encoded a second such functional moiety in form of a latent thiol group that can be unmasked under mild physiological conditions. Phenylacetamidomethyl (Phacm) protected homocysteine (HcP) was incorporated and its latent thiol group unmasked on purified proteins using penicillin G acylase (PGA). The enzymatic deprotection depends on steric accessibility, but can occur efficiently within minutes on exposed positions in flexible sequences. The freshly liberated thiol group does not require treatment with reducing agents. We demonstrate the potential of this approach for protein modification with conceptually new schemes for regioselective dual labeling, thiol bioconjugation in presence of a preserved disulfide bond and formation of a novel intramolecular thioether crosslink.

Incorporating Lanthanum into Mesoporous Silica Foam Enhances Enzyme Immobilization and the Activity of Penicillin G Acylase Due to Lewis Acid-Base Interactions.

Penicillin G acylase (PGA) has been immobilized on a lanthanum-incorporated mesostructured cellular foam (La-MCF) support by using the interaction between the strong Lewis acid sites on the surface of La-MCF and the free amino groups of lysine residues of PGA. The La-MCF support was successfully synthesized in situ through the addition of a citric acid (CA) complexant. The results of pyridine-IR spectroscopy show the presence of strong Lewis acid sites on the surface of the prepared La-MCF (with CA), attributed to the incorporation of lanthanum species into the framework of MCF. Through interaction with the strong Lewis acid sites, the enzymes can be firmly immobilized on the surface of the support. The results indicate that PGA/La-MCF (with CA) exhibits a high specific activity and greatly enhanced operational stability. For the hydrolysis of penicillin G potassium salt, the initial specific activity of PGA/La-MCF (with CA) reaches 10023 U/g. Even after being recycled 10 times, PGA/La-MCF (with CA) retains 89 % of its initial specific activity, much higher than the 77 % of PGA/Si-MCF.

Efficient synthesis of ß-lactam antibiotics with in situ product removal by a newly isolated penicillin G acylase.

A penicillin G acylase (PGA) from Achromobacter xylosoxidans PX02 was newly isolated, and site-directed mutagenesis at three important positions αR141, αF142, ßF24 was carried out for improving the enzymatic synthesis of ß-lactam antibiotics. The efficient mutant ßF24A was selected, and the (Ps/Ph)ini (ratio between the initial rate of synthesis and hydrolysis of the activated acyl donor) dramatically increased from 1.42-1.50 to 23.8-24.1 by means of the optimization of reaction conditions. Interestingly, the efficient enzymatic synthesis of ampicillin (99.1% conversion) and amoxicillin (98.7% conversion) from a high concentration (600 mM) of substrate 6-APA in the low acyl donor/nucleus ratio (1.1:1) resulted in a large amount of products precipitation from aqueous reaction solution. Meanwhile, the by-product D-phenylglycine was hardly precipitated, and 93.5% yield of precipitated ampicillin (561 mM) and 94.6% yield of precipitated amoxicillin (568 mM) were achieved with high purity (99%), which significantly simplified the downstream purification. This was the first study to achieve efficient ß-lactam antibiotics synthesis process with in situ product removal, with barely any by-product formation. The effect enzymatic synthesis of antibiotics in aqueous reaction solution with in situ product removal provides a promising model for the industrial semi-synthesis of ß-lactam antibiotics.

Production and secretion dynamics of prokaryotic Penicillin G acylase in Pichia pastoris.

To take full advantage of recombinant Pichia pastoris (Komagataella phaffii) as a production system for heterologous proteins, the complex protein secretory process should be understood and optimised by circumventing bottlenecks. Typically, little or no attention has been paid to the fate of newly synthesised protein inside the cell, or its passage through the secretory pathway, and only the secreted product is measured. However, the system's productivity (i.e. specific production rate qp), includes productivity of secreted (qp,extra) plus intracellularly accumulated (qp,intra) protein. In bioreactor cultivations with P. pastoris producing penicillin G acylase, we studied the dynamics of product formation, i.e. both the specific product secretion (qp,extra) and product retention (qp,intra) as functions of time, as well as the kinetics, i.e. productivity in relation to specific growth rate (µ). Within the time course, we distinguished (I) an initial phase with constant productivities, where the majority of product accumulated inside the cells, and qp,extra, which depended on µ in a bell-shaped manner; (II) a transition phase, in which intracellular product accumulation reached a maximum and productivities (intracellular, extracellular, overall) were changing; (III) a new phase with constant productivities, where secretion prevailed over intracellular accumulation, qp,extra was linearly related to µ and was up to three times higher than in initial phase (I), while qp,intra decreased 4-6-fold. We show that stress caused by heterologous protein production induces cellular imbalance leading to a secretory bottleneck that ultimately reaches equilibrium. This understanding may help to develop cultivation strategies for improving protein secretion from P. pastoris.Key Points• A novel concept for industrial bioprocess development.• A Relationship between biomass growth and product formation in P. pastoris.• A Three (3) phases of protein production/secretion controlled by the AOX1-promoter.• A Proof of concept in production of industrially relevant penicillin G acylase.

Crystal structures and protein engineering of three different penicillin G acylases from Gram-positive bacteria with different thermostability.

Penicillin G acylase (PGA) catalyzes the hydrolysis of penicillin G to 6-aminopenicillanic acid and phenylacetic acid, which provides the precursor for most semisynthetic penicillins. Most applications rely on PGAs from Gram-negative bacteria. Here we describe the first three crystal structures for PGAs from Gram-positive Bacilli and their utilization in protein engineering experiments for the manipulation of their thermostability. PGAs from Bacillus megaterium (BmPGA, Tm = 56.0 °C), Bacillus thermotolerans (BtPGA, Tm = 64.5 °C), and Bacillus sp. FJAT-27231 (FJAT-PGA, Tm = 74.3 °C) were recombinantly produced with B. megaterium, secreted, purified to apparent heterogeneity, and crystallized. Structures with resolutions of 2.20 Å (BmPGA), 2.27 Å (BtPGA), and 1.36 Å (FJAT-PGA) were obtained. They revealed high overall similarity, reflecting the high identity of up to approx. 75%. Notably, the active center displays a deletion of more than ten residues with respect to PGAs from Gram-negatives. This enlarges the substrate binding site and may indicate a different substrate spectrum. Based on the structures, ten single-chain FJAT-PGAs carrying artificial linkers were produced. However, in all cases, complete linker cleavage was observed. While thermostability remained in the wild-type range, the enzymatic activity dropped between 30 and 60%. Furthermore, four hybrid PGAs carrying subunits from two different enzymes were successfully produced. Their thermostabilities mostly lay between the values of the two mother enzymes. For one PGA increased, enzyme activity was observed. Overall, the three novel PGA structures combined with initial protein engineering experiments provide the basis for establishment of new PGA-based biotechnological processes.

Optimization of penicillin G acylase immobilized on glutaraldehyde-modified titanium dioxide.

In this work, TiO2 , which was modified by glutaraldehyde, was adopted as the carrier; the penicillin G acylase (PGA) was immobilized and the influence of immobilized conditions, such as pH of solution, the concentration of PGA, the immobilization temperature, and the reaction time, on the catalytic performance of the immobilized PGA was investigated and optimized. During this process, potassium penicillin G (PG) was chosen as substrate, and the quantity of 6-aminopenicillanic acid (6-APA) produced by PG at the temperature of 25 °C for 3 Min in neutral solution was conscripted as the evaluation foundation, indexes, containing the loading capacity (ELC), the activity (EA), and activity retention rate (EAR), were calculated based on quantities of produced 6-APA and compared with finding out the suitable conditions. Results showed that when the solution pH, PGA concentration, immobilization temperature, and reaction time were 8.0, 2.5% (v/v), 35 °C, and 24 H, respectively, ELC, EA, and EAR presented optimal values of 9,190 U, 14,969 U/g, and 88.5% relatedly. After that, the stability and reusability of immobilized PGA were studied, and the results documented that the pH resistance, thermal stability, and storage stability of immobilized PGA were significantly improved. This work provided technique support for the practical application of immobilized PGA carrier.

Efficient synthesis of ß-lactam antibiotics with very low product hydrolysis by a mutant Providencia rettgeri penicillin G acylase.

Penicillin G acylase (PGA) was isolated from Providencia rettgeri PX04 (PrPGApx04) and utilized for the kinetically controlled synthesis of ß-lactam antibiotics. Site-directed mutagenesis was performed to increase the process efficiency. Molecular docking was carried out to speculate the key mutant positions corresponding with synthetic activity, which resulted in the achievement of an efficient mutant, ßF24G. It yielded higher conversions than the wild-type enzyme in the synthesis of amoxicillin (95 versus 17.2%) and cefadroxil (95.4 versus 43.2%). The reaction time for achieving the maximum conversion decreased from 14 to 16 h to 2-2.5 h. Furthermore, the secondary hydrolysis of produced antibiotics was hardly observed. Kinetic analysis showed that the (kcat/Km)AD value for the activated acyl donor D-hydroxyphenylglycine methyl ester (D-HPGME) increased up to 41 times. In contrast, the (kcat/Km)Ps values for the products amoxicillin and cefadroxil decreased 6.5 and 21 times, respectively. Consequently, the α value (kcat/Km)Ps/(kcat/Km)AD, which reflected the relative hydrolytic specificity of PGA for produced antibiotics with respect to the activated acyl donor, were only 0.028 and 0.043, respectively. The extremely low hydrolytic activity for the products of the ßF24G mutant enabled greater product accumulation to occur during synthesis, which made it a promising enzyme for industrial applications.

Fitting replacement of signal peptide for highly efficient expression of three penicillin G acylases in E. coli.

High level expression of penicillin G acylase (PGA) in Escherichia coli is generally constricted by a complex maturation process and multiple limiting steps. In this study, three PGAs isolated from Providencia rettgeri (PrPGA), Alcaligenes faecalis (AfPGA), and Achromobacter xylosoxidans (AxPGA) were efficiently expressed in E. coli by replacing with applicable signal peptide. Different bottlenecks of the expression process were analyzed for PrPGA, AfPGA, and AxPGA. Subsequently, five efficient signal peptides, including OmpA, pelB, Lpp, PhoA, and MalE, were used to replace the original signal peptides of the PGAs. With respect to AfPGA and AxPGA, translocation was the primary limitation, and the use of pelB signal peptide effectively overcame this barrier. For PrPGA, which was almost not expressed in wild type, the translation initiation efficiency was optimized by replacing with MalE signal peptide. In addition, low temperature (20 °C) slowed down the transcription and translation, thereby facilitating the posttranslational process and preventing the formation of inclusion bodies. Furthermore, combined induction with IPTG and arabinose not only enhanced the cell density but also remarkably improved the expression of PGAs. Final specific activities of the three PGAs reached 2100 (PrPGA), 9200 (AfPGA), and 1400 (AxPGA) U/L/OD600, respectively. This simple and robust strategy by fitting replacement of signal peptide might dramatically improve the expression of PGAs from various bacteria, which was significant in the production of many valuable ß-lactam antibiotics.

Immobilization and property of penicillin G acylase on amino functionalized magnetic Ni0.3Mg0.4Zn0.3Fe2O4 nanoparticles prepared via the rapid combustion process.

Penicillin G acylase plays an important role in the biocatalytic process of semi-synthetic penicillin. In order to overcome the disadvantages of free enzymes and improve the catalytic performance of enzymes, it is a new method to immobilize enzymes on carrier materials. And magnetic materials have the characteristics of easy separation. In the present study, the Magnetic Ni0.3Mg0.4Zn0.3Fe2O4 nanoparticles were successfully prepared by a rapid-combustion method and calcined at 400°C for 2 h. The surface of the nanoparticles was modified with sodium silicate hydrate, and the PGA was covalently bound to the carrier particles through the cross-linking of glutaraldehyde. The results showed that the activity of immobilized PGA reached 7121.00 U/g. The optimum pH for immobilized PGA was 8 and the optimum temperature was 45°C, the immobilized PGA exhibited higher stability against changes in pH and temperature. The Michaelis-Menten constant (Km) values of the free and immobilized PGA were 0.00387 and 0.0101 mol/L and the maximum rate (Vmax) values were 0.387 and 0.129 µmol/min. Besides, the immobilized PGA revealed excellent cycling performance. The immobilization strategy presented PGA had the advantages of reuse, good stability, cost saving and had considerable practical significance for the commercial application of PGA.

Analytical expression for the model that describes the heterogeneous reaction-diffusion process with immobilized enzyme (penicillin G acylase).

This research article examines the reaction-diffusion process in an immobilized enzyme batch reactor. The model incorporates strongly non-linear factors that are associated with standard Michaelis-Menten kinetics. The non-linear reaction-diffusion equations for substrate and product concentrations have been approximated analytically. Employing two different semi-analytical methods, Akbari-Ganji's method (AGM) and the modified Adomian decomposition method (MADM), to compute the dimensionless steady-state solutions to the system of non-linear differential equations for all values of reaction parameters. In addition, the dynamics of the mean integrated effectiveness factor of penicillin acylase in porous spherical particles have been presented for the determination of the local effectiveness factor. In order to gauge the potency of our proposed solution, we compare two semi-analytical results with a numerical result that are in good agreement across the whole concentration range. The proposed formulation aims to simulate the dynamic performance of the system utilizing the parameters and would enhance the determination of the optimum particle size for enzyme catalysts.

The study of Fe3O4@SiO2-NH2 nano-magnetic composite modified by glutaraldehyde to immobilized penicillin G acylase.

Preparation of biocatalyst dependent on immobilized penicillin G acylase (PGA) was of substantial importance for proteomic research, organic synthesis, and industrial applications. Herein, we developed an easy method for nano-magnetic composite to immobilize PGA. Fe3O4 nano-magnetic particles were co-precipitated with Fe3+ and Fe2+ in an ammonia solution (NH3) and treated with silicon dioxide (SiO2), which was developed using the sol-gel process. Thereafter, 3-aminopropyltriethoxysilane (APTES) was used to modify the silica-coated Fe3O4, which would result in the attachment of the primary amine groups to the particle surface. After that, the attachment of primary amine group was reacted with glutaraldehyde (Glu) to immobilize PGA; the products related to each step were confirmed by X-ray diffraction (XRD), Fourier-transform infrared spectroscopy (FTIR), vibration sample magnetometer (VSM), and scanning electron microscope-energy spectroscopy of dispersive x-rays (SEM-EDS). Condition investigation results revealed that the suitable pH value, reaction time, and immobilization temperature were 8.0, 6 h, and 40 °C, respectively, under optimal conditions. Enzyme loading capacity (ELC), enzyme activity (EA), and enzyme activity retention ratio (EAR) of PGA were 9198 U, 14602 U/g, and 87.7% respectively. Reusability findings showed that the immobilization PGA preserved 79% of its activity after 11 cycles of repeating.

Strategies to Improve the Biosynthesis of ß-Lactam Antibiotics by Penicillin G Acylase: Progress and Prospects.

ß-Lactam antibiotics are widely used anti-infection drugs that are traditionally synthesized via a chemical process. In recent years, with the growing demand for green alternatives, scientists have turned to enzymatic synthesis. Penicillin G acylase (PGA) is the second most commercially used enzyme worldwide with both hydrolytic and synthetic activities toward antibiotics, which has been used to manufacture the key antibiotic nucleus on an industrial level. However, the large-scale application of PGA-catalyzed antibiotics biosynthesis is still in the experimental stage because of some key limitations, such as low substrate concentration, unsatisfactory yield, and lack of superior biocatalysts. This paper systematically reviews the strategies adopted to improve the biosynthesis of ß-lactam antibiotics by adjusting the enzymatic property and manipulating the reaction system in recent 20 years, including mining of enzymes, protein engineering, solvent engineering, in situ product removal, and one-pot reaction cascade. These advances will provide important guidelines for the future use of enzymatic synthesis in the industrial production of ß-lactam antibiotics.

Proteomic analysis of Penicillin G acylases and resulting residues in semi-synthetic ß-lactam antibiotics using liquid chromatography - tandem mass spectrometry.

Penicillin G acylase (PGA), as a key enzyme, is increasingly used in the commercial production of semi-synthetic ß-lactam antibiotics (SSBAs). With the substitution of conventional chemical synthesis by emerging bioconversion processes, more and more PGAs fermented from different types of strains such as Escherichia coli (E. coli, ATCC 11105), Achromobacter sp. CCM 4824 and Providencia rettgeri (ATCC 31052) have been used in this kind of enzymatic processes. As an intermediate reaction catalyst, PGA protein and its presence in the final products may cause a potential risk of human allergic reaction and bring challenges for both quality and process controls. To achieve qualitative and quantitative analysis of PGAs and their residues in SSBAs, a tryptic digestion coupled with liquid chromatography - tandem mass spectrometry (LC-MS/MS) method was developed and proposed because of advantages like high selectivity and sensitivity. A suitable filter aided sample preparation (FASP) method was also used to remove matrix interference and to enrich the target PGA retained in the ultrafiltration membrane for an efficient enzymatic hydrolysis and subsequent accurate MS detection. Finally, twelve batches of PGAs from eight companies were identified and categorized into two types of strains (E. coli and Achromobacter sp. CCM 4824) using proteomic analysis. In total nine batches of five types of SSBAs (amoxicillin, cephalexin, cefprozil, cefdinir and cefaclor) from eight manufacturers were selected for investigation. Trace levels of PGA residual proteins ranging from 0.01 to 0.44 ppm were detected in six batches of different SSBAs which were far lower than the safety limit of 35 ppm reported by DSM, a manufacturer with expertise in the production of SSBAs by enzymatic processes. The developed FASP with LC-MS/MS method is superior to traditional protein assays in terms of selectivity, sensitivity and accuracy. Moreover, it could provide in-depth analysis of amino acid sequences and signature peptides contributing to assignment of the strain sources of PGAs. This method could become a promising and powerful tool to monitor enzymatic process robustness and reliability of this kind of SSBAs manufacturing.

Rsn-2-mediated directed foam enrichment of ß-lactamase.

Today, the availability of methods for the activity-preserving and cost-efficient downstream processing of enzymes forms a major bottleneck to the use of these valuable tools in technical processes. A promising technology appears to be foam fractionation, which utilizes the adsorption of proteins at a gas-liquid interface. However, the employment of surfactants and the dependency of the applicability on individual properties of the target molecules are considerable drawbacks. Here, we demonstrate that a reversible fusion of the large, surface-active protein Ranaspumin-2 (Rsn-2) to a ß-lactamase (Bla) enabled both surfactant-free formation of a stable foam and directed enrichment of the enzyme by the foaming. At the same time, Bla maintained 70% of its catalytic activity, which was in stark contrast to the enzyme without fusion to Rsn-2. Rsn-2 predominantly mediated adsorption. Comparable results were obtained after fusion to the structurally more complex penicillin G acylase (PGA) as the target enzyme. The results indicate that using a surface-active protein as a fusion tag might be the clue to the establishment of foam fractionation as a general method for enzyme downstream processing.

Construction and Application of a Plasmid-Based Signal Peptide Library for Improved Secretion of Recombinant Proteins with Priestia megaterium.

The secretion of recombinant proteins plays an important role in their economic production and purification. The secretion efficiency depends on the responsible signal peptide (SP) in combination with the target protein and the given host and cannot be predicted so far. Due to its high plasmid stability, the lack of alkaline extracellular proteases and only few contaminating extracellular host proteins, Priestia megaterium provides a promising alternative to common Bacillus species. For the development of an easy and fast cloning and screening system to identify the SP best suited to a distinct protein, a plasmid-based SP library containing all predicted 182 Sec-dependent SPs from P. megaterium was established. The splitting of the SPs into 10 groups of individual multi-SP plasmids (pMSPs) allows their grouped amplification and application in screening approaches. The functionality of the whole library was demonstrated by enhancing the amount of the already well-secreted α-amylase AmyE by 1.6-fold. The secretion of a novel penicillin G acylase, which remained as insoluble protein inside the cells, as its native SP is unsuitable for secretion in P. megaterium, could be enhanced even up to 29-fold. Overall, only around 170 recombinant P. megaterium clones based on 50 inserted SPs had to be screened to achieve sufficient amounts for further enzyme characterizations. Thus, this newly developed plasmid-based genetic tool applicable for P. megaterium and also other Bacillus species facilitates the identification of suitable SPs for secretion of recombinant proteins.

Combing multiple site-directed mutagenesis of penicillin G acylase from Achromobacter xylosoxidans PX02 with improved catalytic properties for cefamandole synthesis.

Penicillin G acylase (PGA) was an important biocatalyst for enzymatic production of second-generation cephalosporin. PGA from Achromobacter xylosoxidans PX02 (AxPGA) showed relatively lower identity to EcPGA (54.9% in α subunit and 51.7% in ß subunit), which could synthesize cefamandole in the kinetically controlled N-acylation (kcNa). Semi-rational design of AxPGA and "small and smart" mutant libraries were developed with minimal screening to improve cefamandole production. A triple mutant αR141A/αF142I/ßF24G by combining the mutational sites (ßF24, αR141, and αF142) from different subunits of AxPGA showed better performance in cefamandole production, with 4.2-fold of improvement in the (kcat/Km)AD value for activated acyl donor (R)-Methyl mandelate. Meanwhile, the (kcat/Km)Ps value for cefamandole by mutant αR141A/αF142I/ßF24G was sharply dropped by 25.5 times, indicating its highly synthetic activity and extremely low hydrolysis of cefamandole. Strikingly, the triple mutant αR141A/αF142I/ßF24G could form cefamandole with a yield of 85% at an economical substrate ratio (acyl donor/nucleophile) of 1.3:1 (82% at 1.1:1), which advanced the greener and more sustainable process of cefamandole production than the wild type. Furtherly, the improved synthetic ability and lower hydrolysis of cefamandole by mutant were rationalized using molecular docking.