Evaluating the ability of polyhydroxyalkanoate synthase mutants to produce P(3HB-co-3HA) from soybean oil

Polyhydroxyalkanoate (PHA) synthase from Pseudomonas sp 61-3 (PhaC1(Ps)) is able to synthesize P(3HB-co-3HA), consisting of a 3HB unit and medium-chain-length 3HA units of 6-12 carbon atoms. Expression vectors encoding 76 PhaC1(Ps) mutants with an amino acid replacement at position 130, 325, 477 or 481 were individually introduced into Ralstonia eutropha. The mutant enzyme genes were evaluated in terms of their abilities to synthesize P(3HB-co-3HA) using soybean oil as a carbon source. 20 mutants showed significantly high accumulation levels of PHA exceeding 30 wt.-% and as high as 57 wt.-%. It was found that hydrophobic amino acids at the positions are more likely to enhance accumulation of PHA in R. eutropha.     

Mutation effects of a conserved alanine (Ala510) in type I polyhydroxyalkanoate synthase from Ralstonia eutropha on polyester biosynthesis

Type I polyhydroxyalkanoate (PHA) synthases, as represented by Ralstonia eutropha enzyme (PhaC(Re)), have narrow substrate specificity toward (R)-3-hydroxyacyl-coenzyme A with acyl chain length of C3-C5 to yield PHA polyesters. In this study, saturation point mutagenesis of a highly conserved alanine at position 510 (A510) in PhaC(Re) was carried out to investigate the effects on the polymerization activity and the substrate specificity for in vivo PHA biosynthesis in bacterial cells. A series of saturation mutants were first applied for poly[(R)-3-hydroxybutyrate] homopolymer synthesis in Escherichia coli and R. eutropha PHB(-)4 (PHA negative mutant) cells to assess the polymerization activity. All mutants showed quantitatively similar polymerization activities when R. eutropha PHB(-)4 was used for assay, whereas several mutants such as A510P showed low activities in E. coli. Further analysis has revealed that majority of mutants synthesize polyesters with higher molecular weights than the wild-type. In particular, substitution by acidic amino acids, A510D(E), led to remarkable increases in molecular weights. Subsequently, PHA copolymer synthesis from dodecanoate (C12 fatty acid) was examined. The copolymer compositions were varied depending on the mutants used. Significant increased fractions of long monomer units (C6 and C8) in PHA copolymers were observed for three mutants [A510M(Q,C)]. From these results, the mutations at this potion are beneficial to change the molecular weight of polyesters and the substrate specificity of PhaC(Re). Molecular weight distributions of PHA polymers synthesized by the wild-type enzyme (PhaC(Re)) and its mutants.     

Biosynthesis and compositional regulation of poly[(3-hydroxybutyrate)-co-(3-hydroxyhexanoate)] in recombinant ralstonia eutropha expressing mutated polyhydroxyalkanoate synthase genes

A new strategy for bacterial polyhydroxyalkanoate (PHA) production by recombinant Ralstonia eutropha PHB(-)4 harboring mutated PHA synthase genes (phaC(Ac)) from Aeromona caviae was investigated. The strain harboring wild-type phaC(Ac) gene produced a PHA copolymer consisting of (R)-3-hydroxybutyrate and (R)-3-hydroxyhexanoate [P(3HB-co-3HHx)] with 3.5 mol-% of 3HHx fraction from soybean oil. When the mutants of phaC(Ac) gene were applied to this production system, 3HHx fraction in copolymers was varied in the range of 0-5.1 mol-%. Thus, the regulation of PHA copolymer compositions has been achieved by the use of mutated PHA synthase genes.     

Characterization and properties of G4X mutants of Ralstonia eutropha PHA synthase for poly(3-hydroxybutyrate) biosynthesis in Escherichia coli

Modification of the type I polyhydroxyalkanoate synthase of Ralstonia eutropha (PhaC(Re)) was performed through systematic in vitro evolution in order to obtain improved PhaC(Re) having an enhanced activity of poly(3-hydroxybutyrate) (PHB) synthesis in recombinant Escherichia coli. For the first time, a beneficial G4D N-terminal mutation important for the enhancement of both PHB content in dry cells and PhaC(Re) level in vivo was identified. Site-directed saturation mutagenesis at the G4 position enabled us to identify other mutations conferring similar enhanced characteristics. In addition, the PHB homopolymer synthesized by most G4X single mutants also had higher molecular weights than that of the wild-type. In vitro enzymatic assays of purified G4D mutant PhaC(Re) revealed that the mutant enzyme exhibited slightly lower activity and reaction efficiency compared to the wild-type enzyme. [diagram in text].     

Polyhydroxyalkanoate (PHA) Biosynthesis from Whey Lactose

Summary: The potential of three different microbial wild type strains as polyhydroxyalkanoate (PHA) producers from whey lactose is compared. Homopolyester and co-polyester biosynthesis was investigated by the archaeon Haloferax mediterranei and the eubacterial strains Pseudomonas hydrogenovora and Hydrogenophaga pseudoflava. H. mediterranei accumulated 50 wt.-% of poly-3-(hydroxybutyrate-co-6%-hydroxyvalerate) in cell dry mass from hydrolyzed whey without addition of 3-hydroxyvalerate (3HV) precursors (specific productivity q_p: 2.9 mg/g h). Using P. hydrogenovora, the final percentage of poly-3-hydroxybutyrate (PHB) amounted to 12 wt.-% (q_p: 0.03 g/g h); co-feeding of valeric acid resulted in the production of 12 wt.-%. P-3(HB-co-21%-HV) (q_p: 0.02 g/g h). With H. pseudoflava, it was possible to reach 40 wt.-% P-3 (HB-co-5%-HV) on not-hydrolyzed whey lactose plus valeric acid as 3HV precursor (q_p: 9.1 mg/g h); on hydrolyzed whey lactose without addition of valeric acid, the strain produced 30 wt.-% of PHB (q_p: 0.16 g/g h). The characterization of the isolated biopolyesters completes the study.     

Potential of various archae- and eubacterial strains as industrial polyhydroxyalkanoate producers from whey

Three different microbial wild-type strains are compared with respect to their potential as industrial scale polyhydroxyalkanoate (PHA) producers from the feed stock whey lactose. The halophilic archaeon Haloferax mediterranei as well as two eubacterial strains (Pseudomonas hydrogenovora and Hydrogenophaga pseudoflava) are investigated. H. mediterranei accumulated 50 wt.-% of poly-3-(hydroxybutyrate-co-8%-hydroxyvalerate) from hydrolyzed whey without addition of 3-hydroxyvalerate (3HV) precursors (specific productivity q(p): 9.1 mg x g(-1) x h(-1)). Using P. hydrogenovora, the final percentage of poly-3-hydroxybutyrate (PHB) amounted to 12 wt.-% (q(p): 2.9 mg x g(-1) x h(-1)). With H. pseudoflava, it was possible to reach 40 wt.-% P-3(HB-co-5%-HV) on non-hydrolyzed whey lactose plus addition of valeric acid as 3HV precursor (q(p): 12.5 mg x g(-1) x h(-1)). A detailed characterization of the isolated biopolyesters and an evaluation with regard to the economic feasibility completes the study.     

Construction of pha-operon-defined knockout mutants of Pseudomonas putida KT2442 and their applications in poly(hydroxyalkanoate) production

Pseudomonas putida KT2442 could accumulate medium-chain-length poly(hydroxyalkanoate)s (PHA) consisting of 3-hydroxyhexanoate, 3-hydroxyoctanoate, 3-hydroxydecanoate, and 3-hydroxydodecanoate from a wide range of carbon sources. In this study, the PHA synthase pha operon (phaC1-phaZ-phaC2) was knocked out and the vgb gene encoding vitreoscilla hemoglobin protein (VHb), which could enhance oxygen uptake rate especially at low oxygen concentration, was integrated into the P. putida KT2442 genome to replace the deleted fragment. The resulting mutant P. putida KTOY01 or gene-replaced mutant KTOY02 was used as the host to study PHA synthase properties and PHA production. Different PHA polymerase (PhaC) genes, phaC(Re) from Rastonia eutropha H16, phaC(Ac) from Aeromonas cavie, and phaC2(Ps) from Pseudomonas stutzeri 1317, were expressed in the mutant strains to test the PhaC enzyme substrate specificity. The result showed P. putida KTOY01 or KTOY02 could provide not only mcl PHA monomers but also 3-hydroxybutyrate from fatty acids, which may allow the production of copolyesters poly(3HB-co-mcl 3HA). Plasmid pCJY10 containing phaC2(Ps), phbA, and phbB genes encoding PHA polymerase, beta-ketothiolase, and acetoacetyl-CoA reductase, respectively, were transformed into P. putida KTOY01 and KTOY02. Shake-flask culture showed P. putida KTOY01 or KTOY02 (pCJY10) could accumulate poly(3HB-co-mcl 3HA) from glucose. The above result showed pha operon knockout mutant of P. putida KT2442 was a very useful host of great potential not only for studying PhaC synthase, but also for microbial production of copolyesters poly(3HB-co-mcl 3HA), which is very difficult to obtain.     

Biosynthesis of High Quality Polyhydroxyalkanoate Co- and Terpolyesters for Potential Medical Application by the Archaeon Haloferax mediterranei

Summary : Haloferax mediterranei was investigated for the production of two different high-performance polyhydroxyalkanoates (PHAs). A copolyester containing 6 mol-% 3-hydroxyvalerate (3HV) was produced from whey sugars as sole carbon source. The maximum specific growth rate (µ_max.) and the maximum specific PHA production rate (q_{p max.}) were determined with 0.10 1/h and 0.15 1/h, respectively. The cells contained 72.8 wt.-% of P-(3HB-co-6%-3HV) which featured low melting points between 150 and 160 °C and narrow molecular mass distribution (polydispersity PDI = 1.5). Further, a PHA terpolyester with an increased 3HV fraction as well as 4-hydroxybutyrate (4HB) building blocks was accumulated by feeding of whey sugars plus 3HV - and 4HB precursors. Kinetic analysis of the process reveals a µ_max. of 0.14 1/h and a q_{p max.} of 0.23 1/h, respectively. The final percentage of P-(3HB-co-21.8%-3HV-co-5.1%-4HB) in biomass amounted to 87.5 wt.-%. Also this material showed a narrow molecular mass distribution (PDI = 1.5) and a high difference between the two melting endotherms of the material (between 140 and 150 °C) and the onset of decomposition at 236 °C. The accomplished work provides viable strategies to obtain different high-quality PHAs which might be potential candidates for application in the medical and pharmaceutical field.     

Variation in copolymer composition and molecular weight of polyhydroxyalkanoate generated by saturation mutagenesis of Aeromonas caviae PHA synthase

Amino acid substitutions at two residues downstream from the active-site histidine of polyhydroxyalkanoate (PHA) synthases are effective for changing the composition and the molecular weight of PHA. In this study, saturation mutagenesis at the position Ala505 was applied to PHA synthase (PhaCAc) from Aeromonas caviae to investigate the effects on the composition and the molecular weight of PHA synthesized in Ralstonia eutropha. The copolymer composition and molecular weight of PHA were varied by association with amino acid substitutions. There was a strong relationship between copolymer composition and PHA synthase activity of the cells. This finding will serve as a rationale for producing tailor-made PHAs.     

Metabolic engineering for the production of copolyesters consisting of 3-hydroxybutyrate and 3-hydroxyhexanoate by Aeromonas hydrophila

Aeromonas hydrophila 4AK4 was able to synthesize copolyesters consisting of 3-hydroxybutyrate (3HB) and about 15 mol-% 3-hydroxyhexanoate (3HHx) (PHBHHx) when grown in long chain fatty acids such as dodecanoate regardless of growth conditions. To regulate the unit fraction in PHBHHx, phbA and phbB genes encoding beta-ketothiolase and acetoacetyl-CoA reductase in Ralstonia eutropha, were introduced into A. hydrophila 4AK4. When gluconate was used as cosubstrate of dodecanoate, the recombinant produced PHBHHx containing 3-12 mol-% 3HHx, depending on the gluconate concentration in media. Vitreoscilla hemoglobin gene, vgb, was also introduced into the above recombinant, resulting in improved PHBHHx content from 38 to 48 wt.-% in shake flask study. Fermentor studies also showed that increased gluconate concentration in medium containing dodecanoate promoted the recombinant strain harboring phbA and phbB genes to incorporate more 3HB unit into PHBHHx, resulting in reduced 3HHx fraction. Recombinant A. hydrophila harboring phbA, phbB and vgb genes demonstrated better PHBHHx productivity and higher conversion efficiency from dodecanoate to PHBHHx than those of the recombinant without vgb in fermentation study. Combined with the robust growth property and simple growth requirement, A. hydrophila 4AK4 appeared to be a useful organism for metabolic engineering.     

Directed evolution to increase camptothecin sensitivity of human DNA topoisomerase I

BACKGROUND: Human DNA topoisomerase I (top1) relaxes DNA supercoiling during basic nuclear processes. The enzyme is the main target of antitumor agents, such as camptothecins (CPT), that transform top1 into a DNA-damaging agent. RESULTS: By directed evolution of a C-terminal portion, we selected human top1 mutants that were 22-28-fold more CPT-sensitive than wild-type top1 in Saccharomyces cerevisiae cells. The evolved enzymes showed unique mutation patterns and were more processive in plasmid relaxation assays. A top1 mutant had only two amino acid changes in the linker domain, one of which may change a linker/core domain contact surface. The mutant stimulated DNA cleavage to higher levels than the wild-type enzyme and was more sensitive to CPT in a cleavage assay. Moreover, the mutant was more CPT-sensitive than wild-type top1 in a repair-deficient yeast strain. CONCLUSIONS: Mutations in the linker domain can affect DNA binding and CPT sensitivity of human top1. Such drug-hypersensitive topoisomerases may be useful in developing DNA cutters with high cell lethality and in new drug discovery programs.     

Evaluation of the metal binding properties of a histidine-rich fusogenic peptide by electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry

Electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (ESI-FTICRMS) was used to investigate metal ion interactions of the 18 amino acid peptide fragment B18 (LGLLLRHLRHHSNLLANI), derived from the membrane-associated protein bindin. The peptide sequence B18 represents the minimal membrane-binding motif of bindin and resembles a putative fusion peptide. The histidine-rich peptide has been shown to self-associate into distinct supramolecular structures, depending on the presence of Zn(2+) and Cu(2+). We examined the binding of B18 to the metal ions Cu(2+), Zn(2+), Mg(2+), Ca(2+), Mn(2+) and La(3+). For Cu(2+), we compared the metal binding affinities of the wild-type B18 peptide with those of its mutants in which one, two or three histidine residues have been replaced by serines. Upon titration of B18 with Cu(2+) ions, we found sequential binding of two Cu(2+) ions with dissociation constants of approximately 34 and approximately 725 micro M. Mutants of B18, in which one histidine residue is replaced by serine, still exhibit sequential binding of two copper ions with affinities for the first Cu(2+) ion comparable to that of wild-type B18 peptide, but with a greatly reduced affinity for the second Cu(2+) ion in mutants H112S and H113S. For mutants in which two histidines are replaced by serines, the affinity for the first Cu(2+) ion is reduced approximately 3-10 times in comparison with B18. The mutant in which all three histidine residues are replaced by serines exhibits an approximately 14-fold lower binding for the first Cu(2+) ion compared with B18. For the other metal ions under investigation (Zn(2+), Mg(2+), Ca(2+), Mn(2+) and La(3+)), a modest affinity to B18 was detected binding to the peptide in a 1 : 1 stoichiometry. Our results show a high affinity of the wild-type fusogenic peptide B18 for Cu(2+) ions whereas the Zn(2+) affinity was found to be comparable to that of other di- and trivalent metal ions.     

Altered expression of polyhydroxyalkanoate synthase gene and its effect on poly[(R)-3-hydroxybutyrate] synthesis in recombinant Escherichia coli

Polyhydroxyalkanoate (PHA) synthase (PhaC) from Wautersia eutropha was expressed in a wide range of production level in Escherichia coli XL1-Blue cells and its effects on PhaC activity, poly[(R)-3-hydroxybutyrate] [P(3HB)] production and its molecular weights were investigated. The production level of PhaC was controlled both by the amount of chemical inducer (isopropyl-β-d-thiogalactopyranoside, IPTG) added into the medium and the use of different copy number of plasmids. In a flask experiment, as PhaC production level in the cells increased, the PhaC activity also increased in the range of low PhaC concentration. However, PhaC activity did not further increase in the range of high PhaC concentration, probably due to the formation of inclusion body in the cells. The molecular weight of P(3HB) was found to decrease with increasing PhaC activity. This trend was also verified in high cell density cultivation using 10-l jar fermentor. Furthermore, we demonstrated that the use of low copy number plasmid and appropriate induction of PhaC expression were effective in achieving both high productivity and high molecular weight of P(3HB).     

The synthesis of poly(3,4-dihydroxystyrene) and poly[(sodium 4-styrenesulfonate)-co-(3,4-dihydroxystyrene)]

Poly(2-methoxy-4-vinylphenol), polyMVP, and poly[(sodium 4-styrenesulfonate)-co-(2-methoxy-4-vinylphenol)], poly(SSS/MVP), were synthesized by radical polymerization using Vazo-64 or tributylborane as initiator. Poly(3,4-dihydroxystyrene) and poly[(sodium 4-styrenesulfonate)-co-(3,4-dihydroxystyrene)] were obtained by demethylation of polyMVP and poly(SSS/MVP) using HBr and trimethylsilyl iodide, respectively. (Co)polymer structures were confirmed by ¹H and ¹³C NMR spectra. About 30 wt.-% gel formed in the polyMVP polymerizations, whereas only a small amount (0.5 mol-%) of gel formed in the copolymerizations.     

Greatly reduced amino acid alphabets in directed evolution: making the right choice for saturation mutagenesis at homologous enzyme positions

Enantioselective mutants of the thermally robust phenyl acetone monooxygenase (PAMO) as catalysts in Baeyer-Villiger reactions have been evolved by utilizing saturation mutagenesis in which drastically reduced amino acid alphabets are employed at homologous enzyme positions.     

Biobased contents of biodegradable poly(epsilon-caprolactone) composites polymerized and directly molded using aluminium triflate from caprolactone with cellulose and inorganic filler

Poly(epsilon-caprolactone) (PCL) composite samples were prepared by polymerization and direct molding. The starting compound was epsilon-caprolactone monomer liquid combined with cellulose and inorganic fillers, using aluminium triflate as a catalyst at 80 degrees C, for 6 or 24 h. Cylinder-shaped PCL composite samples with a homogeneously dispersed cellulose filler were prepared with (-)M(n) = 4 600 ((-)M(w)/(-)M(n) = 2.9). The mechanical properties of the PCL composite samples were studied using compression test methods. The strength of a PCL composite with 50 wt.-% cellulose filler (10.8 MPa) was found to be lower than the PCL sample without fillers (19.2 MPa). The biobased content of the PCL composite with 50 wt.-% cellulose filler (51.67%) measured using accelerated mass spectrometry (AMS) was slightly higher than the carbon ratio of cellulose in the starting powder samples (41.3 mol-%). The biobased content of the polymer composite powders by AMS was found not to be affected by the presence of inorganic fillers, such as talc. The rate and extent of biodegradation, caused by Amano Lipase PS, of the PCL composite sample with cellulose filler (40% degradation in 4 d) was the same as that of a PCL sample without the cellulose filler.     

Roles of the proximal hydrogen bonding network in cytochrome P450cam-catalyzed oxygenation

Structural and functional roles of the hydrogen bonding network that surrounds the heme-thiolate coordination of P450(cam) from Pseudomonas putida were investigated. A hydrogen bond between the side chain amide of Gln360 and the carbonyl oxygen of the axial Cys357 was removed in Q360L. The side chain hydrogen bond and the electrostatic interaction between the polypeptide amide proton of Gln360 and the sulfur atom of Cys357 were simultaneously removed in Q360P. The increased electron donation of the axial thiolate in Q360L and Q360P was evidenced by negative shifts of their reduction potentials by 45 and 70 mV, respectively. Together with the results on L358P in which the amide proton at position 358 was removed (Yoshioka, S., Takahashi, S., Ishimori, K., Morishima, I. J. Inorg. Biochem. 2000, 81, 141-151), we propose that the side chain hydrogen bond and the electrostatic interaction of the amide proton with the thiolate ligand cause approximately 45 and approximately 35 mV of positive shifts, respectively, of the redox potential of the heme in P450(cam). The resonance Raman spectra of the ferrous-CO form of the Q360 mutants showed a downshifted Fe-CO stretching mode at 482 approximately 483 cm(-)(1) compared with that of wild-type P450(cam) at 484 cm(-)(1). The Q360 mutants also showed the upshift by 4 approximately 5 cm(-)(1) of the Fe-NO stretching mode in the ferrous-NO form. These Raman results indicate the increase in the sigma-electron donation of the thiolate ligand in the reduced state of the Q360 mutants and were in contrast to the increased pi-back-donation of the thiolate in L358P having an upshifted Fe-CO stretching mode at 489 cm(-)(1). The catalytic activities of the Q360 mutants for the unnatural substrates were similar to those of the wild-type enzyme, indicating that the increased sigma-electron donation does not promote the O-O bond heterolysis in the Q360 mutants, although the increased pi-electron donation in L358P promoted the heterolysis of the O-O bond. We conclude that the functions of the proximal hydrogen bonding network in P450(cam) are to stabilize the heme-thiolate coordination, and to regulate the redox potential of the heme iron. Furthermore, we propose that the pi-electron donation, not the sigma-electron donation, of the thiolate ligand promotes the heterolysis of the O-O bond of dioxygen.     

Improving a Natural Enzyme Activity through Incorporation of Unnatural Amino Acids

The bacterial phosphotriesterases catalyze hydrolysis of the pesticide paraoxon with very fast turnover rates and are thought to be near to their evolutionary limit for this activity. To test whether the naturally evolved turnover rate could be improved through the incorporation of unnatural amino acids and to probe the role of peripheral active site residues in nonchemical steps of the catalytic cycle (substrate binding and product release), we replaced the naturally occurring tyrosine amino acid at position 309 with unnatural L-(7-hydroxycoumarin-4-yl)ethylglycine (Hco) and L-(7-methylcoumarin-4-yl)ethylglycine amino acids, as well as leucine, phenylalanine, and tryptophan. Kinetic analysis suggests that the 7-hydroxyl group of Hco, particularly in its deprotonated state, contributes to an increase in the rate-limiting product release step of substrate turnover as a result of its electrostatic repulsion of the negatively charged 4-nitrophenolate product of paraoxon hydrolysis. The 8-11-fold improvement of this already highly efficient catalyst through a single rationally designed mutation using an unnatural amino acid stands in contrast to the difficulty in improving this native activity through screening hundreds of thousands of mutants with natural amino acids. These results demonstrate that designer amino acids provide easy access to new and valuable sequence and functional space for the engineering and evolution of existing enzyme functions.     

Posttranslational hydroxylation of human phenylalanine hydroxylase is a novel example of enzyme self-repair within the second coordination sphere of catalytic iron

Phenylalanine hydroxylase, a mononuclear non-heme iron enzyme, catalyzes the hydroxylation of phenylalanine to tyrosine in the presence of oxygen and reduced pterin cofactor. X-ray structural studies have established the coordination around the iron metal center and point to significant interactions within the second coordination sphere. One such interaction involves Tyr325 in human phenylalanine hydroxylase (hPAH), which forms a hydrogen-bonding network with an aqua ligand on iron and the pterin cofactor. The full-length tetramer (1-452) and truncated dimer (117-424) Tyr325Phe hPAH mutant enzymes showed similar kinetics, thermal stabilities, and oligomerization profiles as their corresponding wild-type proteins. The possibility of in vivo posttranslational hydroxylation that would restore the activity of hPAH was examined by mass spectrometry on the trypsin digested full-length (1-452) hPAH Tyr325Phe point mutant. The amino acid tags obtained by ESI-MS/MS confirmed the presence of a Phe325 in the peptide corresponding to the doubly charged precursor ion at m/z 916.4 (L A T I F W F T V E F G L C K), and its hydroxylated counterpart in the peptide corresponding to the m/z 924.4 (L A T I F-OH W F T V E F G L C K) byproduct ion series comprising the fragments y(5)-y(12). Furthermore, the point mutation Tyr325Ala resulted in an enzyme that was totally inactive and did not display any evidence of hydroxylation. These results demonstrate the importance of Tyr325 for proper conformation of the active site, substrate binding, and catalysis. The rescue of the Tyr325Phe mutant in hPAH via self-hydroxylation presents a novel example of oxidative repair on the molecular level.     

Expression and characterization of mutant forms of penicillin acylase from Alcaligenes faecalis

The sequencing of six plasmids carrying a gene of penicillin acylase from Alcaligenes faecalis VKM B1518 (AfPA) revealed the presence of random mutations in the gene; they occurred during a polymerase chain reaction. Six mutant AfPAs and a wild-type enzyme were expressed in E. coli cells. The activity assay of mutant AfPAs in E. coli cells indicated that several amino acid substitutions affect the expression level of the AfPA gene and the rate of cell growth. Four mutant AfPAs were purified; their catalytic properties and thermal stability were studied. It is shown that the amino acid substitutions under study do not affect the catalytic efficiency value. Within the experimental error, the βQ133R and βK184E (the AfPA M2 mutant) substitutions had no effect on the thermal stability of the enzyme; in the case of mutants AfPA M4 (βY90H), M5 (αD132G, βR97C), and M6 (αV5E, αN183S, and βE439G), the inactivation rate constant increased 2.4, 2.75, and 8.3 times, respectively, as compared to that of the wild-type enzyme.