RESUMO
Epithelioid glioblastoma is a rare subtype of glioblastoma, but the coexistence of a sarcomatous component is even rarer. An 80-year-old woman was admitted to our hospital with somnolence. Magnetic resonance imaging revealed a cystic lesion with a solid component in the left temporal-parietal lobe. Histopathological examination of the resected tumor revealed three components; namely, typical glioblastoma, sarcomatous and epithelioid components at a ratio of about 5:3:2. All components were immunohistochemically positive for vimentin and mutated BRAF (V600E) and showed focal expression of glial fibrillary acidic protein and cytokeratin AE1/AE3, but they were negative for isocitrate dehydrogenase 1. Genetic analysis revealed that both the sarcomatous and epithelioid components harbored BRAF T1799A (V600E) mutation and homozygous deletion of cyclin-dependent kinase inhibitor 2A/B. We diagnosed this tumor as epithelial glioblastoma with a sarcomatous component. Our results indicate that even when the epithelial component is not dominant, immunohistochemical and genetic investigation of BRAF mutations is useful for the diagnosis of glioblastoma subtypes. In particular, although the prognosis of epithelial glioblastoma is poor, potentially effective targeted therapies for BRAF V600E-mutated tumors are available.
Assuntos
Neoplasias Encefálicas/diagnóstico por imagem , Gliossarcoma/diagnóstico por imagem , Proteínas Proto-Oncogênicas B-raf/genética , Idoso de 80 Anos ou mais , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patologia , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Epitélio/diagnóstico por imagem , Epitélio/patologia , Feminino , Gliossarcoma/genética , Gliossarcoma/patologia , Homozigoto , Humanos , Imageamento por Ressonância Magnética , Mutação , Prognóstico , Deleção de Sequência , Lobo Temporal/diagnóstico por imagem , Lobo Temporal/patologia , Vimentina/metabolismoRESUMO
Recent studies have identified that high glucose-induced renal tubular cell damage. We previously demonstrated that high glucose treatment induced oxidative stress in human renal proximal tubular epithelial cells (RPTECs), and angiotensin II type 1 (AT1) receptor blockers reduce high glucose-induced oxidative stress in RPTEC possibly via blockade of intracellular as well as extracellular AT1 receptor. However, exact roles of tumor necrosis factor (TNF)-α and AT1 receptor on high glucose-induced renal tubular function remain unclear. N-acetyl-beta-glucosaminidase (NAG), concentrations of TNF-α/angiotensin II and p22(phox) protein levels after high glucose treatment with or without AT1 receptor blocker or thalidomide, an inhibitor of TNF-α protein synthesis, were measured in immortalized human renal proximal tubular epithelial cells (HK2 cells). AT1 receptor knockdown was performed with AT1 receptor small interfering RNA (siRNA). High glucose treatment (30 mM) significantly increased NAG release, TNF-α/angiotensin II concentrations in cell media and p22(phox) protein levels compared with those in regular glucose medium (5.6 mM). Candesartan, an AT1R blocker, showed a significant reduction on high glucose-induced NAG release, TNF-α concentrations and p22(phox) protein levels in HK2 cells. In addition, significant decreases of NAG release, TNF-α concentrations and p22(phox) protein levels in HK2 cells were observed in high glucose-treated group with thalidomide. AT1R knockdown with siRNA markedly reversed high glucose, angiotensin II or TNF-α-induced p22(phox) protein levels in HK2 cells. TNF-α may be involved in high glucose-induced renal tubular damage in HK2 cells possibly via AT1 receptor signaling.
Assuntos
Células Epiteliais , Glucose , Túbulos Renais Proximais , Receptor Tipo 1 de Angiotensina/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Acetilglucosaminidase/metabolismo , Angiotensina II/metabolismo , Bloqueadores do Receptor Tipo 1 de Angiotensina II/farmacologia , Células Cultivadas , Relação Dose-Resposta a Droga , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Glucose/efeitos adversos , Glucose/metabolismo , Humanos , Túbulos Renais Proximais/efeitos dos fármacos , Túbulos Renais Proximais/metabolismo , Túbulos Renais Proximais/patologia , Estresse Oxidativo/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacosRESUMO
We describe an efficient method for producing both enantiomers of chiral alcohols by asymmetric hydrogen-transfer bioreduction of ketones in a 2-propanol (IPA)-water medium with E. coli biocatalysts expressing phenylacetaldehyde reductase (PAR: wild-type and mutant enzymes) from Rhodococcus sp. ST-10 and alcohol dehydrogenase from Leifsonia sp. S749 (LSADH). We also describe the detailed properties of mutant PARs, Sar268, and HAR1, which were engineered to have high activity and productivity in media composed of polar organic solvent and water, and the construction of three-dimensional structure of PAR by homology modeling. The K(m) and V(max) values for some substrates and the substrate specificity of mutant PARs were quite different from those of wild-type PAR. The results well explained the increased productivity of engineered PARs in IPA-water medium.
Assuntos
2-Propanol , Álcool Desidrogenase/metabolismo , Oxirredutases do Álcool/metabolismo , Álcoois/metabolismo , Meios de Cultura/química , Engenharia Genética/métodos , 2-Propanol/análise , Álcool Desidrogenase/química , Álcool Desidrogenase/genética , Oxirredutases do Álcool/química , Oxirredutases do Álcool/genética , Álcoois/química , Biocatálise , Biotecnologia/métodos , Escherichia coli/enzimologia , Escherichia coli/genética , Hidrogênio/metabolismo , Modelos Moleculares , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Estereoisomerismo , Especificidade por Substrato , Água/análiseRESUMO
BACKGROUND: Antineutrophil cytoplasmic antibodies (ANCA) and Anti-glomerular basement membrane (GBM) antibodies often induce rapidly progressive glomerulonephritis (RPGN). Some reports have demonstrated RPGN with the sequential appearance of ANCA then anti-GBM antibodies, suggesting that ANCA may induce the development of anti-GBM antibodies. Whereas, many reports have shown that the development of ANCA is associated with various infectious diseases, such as non-tuberculous mycobacterial infection. CASE PRESENTATION: A 65-year-old woman with pulmonary non-tuberculous mycobacterial (NTM) infection was monitored without treatment. One year later, serum myeloperoxidase (MPO)- ANCA were elevated (14.1 U/mL (normal value < 3.0 U/ml)). A high fever and RPGN appeared 1 year later, and serum MPO-ANCAs were 94.1 U/mL. Anti-GBM antibodies were also detected. A renal biopsy revealed crescentic glomerulonephritis with linear deposits of IgG and C3c along the GBM and interstitial inflammation with endarteritis of arterioles. The diagnosis was RPGN associated with anti-GBM nephritis and ANCA-associated vasculitis. CONCLUSION: This report shows that preceding NTM infection may have induced ANCA and anti-GBM antibodies and caused the development of RPGN.
RESUMO
BACKGROUND/AIMS: Proteinuria is not merely a marker of chronic nephropathies, but may also be involved in the progression to end-stage renal failure. We investigated the effect of angiotensin II type 1 receptor blockers (ARBs) on albumin-induced cell damage in human renal proximal tubular epithelial cells (RPTEC). METHODS: The N-acetyl-beta-D-glucosaminidase (NAG) and 8-hydroxy-2'-deoxyguanosine (8-OHdG) levels in the medium after albumin treatment with ARBs were determined by commercially available kits. The levels of p22(phox) protein in RPTEC were measured using Western blotting after albumin treatment with ARBs. Angiotensin II concentrations in cell media and cell lysates were assayed with a commercially available kit. RESULTS: Human albumin (0.1-10 mg/ml) dose-dependently increased NAG release and olmesartan or valsartan (10(-9)-10(-7) mol/l) showed a significant reduction on albumin (1 mg/ml)-induced NAG release in RPTEC. Albumin treatment (1 mg/ml) showed significant increases in p22(phox) protein levels in RPTEC and ARBs significantly decreased albumin-induced p22(phox) protein levels. Significant increases in 8-OHdG levels were observed in the albumin (1 mg/ml)-treated group and ARBs markedly reduced albumin-induced 8-OHdG levels in RPTEC. Human albumin dose-dependently increased angiotensin II concentrations in both cell media and lysates. CONCLUSION: These observations suggest renal tubular cell-protective properties of ARBs related to decreased oxidative stress during proteinuria.
Assuntos
Albuminúria/tratamento farmacológico , Bloqueadores do Receptor Tipo 1 de Angiotensina II/farmacologia , Imidazóis/farmacologia , Túbulos Renais Proximais/efeitos dos fármacos , Tetrazóis/farmacologia , Valina/análogos & derivados , 8-Hidroxi-2'-Desoxiguanosina , Acetilglucosaminidase/metabolismo , Albuminas/farmacologia , Albuminúria/metabolismo , Albuminúria/patologia , Angiotensina II/metabolismo , Células Cultivadas , Desoxiguanosina/análogos & derivados , Desoxiguanosina/metabolismo , Relação Dose-Resposta a Droga , Humanos , Túbulos Renais Proximais/metabolismo , Túbulos Renais Proximais/patologia , NADPH Oxidases/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Valina/farmacologia , ValsartanaRESUMO
AIM: We investigated the effect of angiotensin II (AII) type 1 (AT1) and angiotensin II type 2 (AT2) receptor blockers on tumour necrosis factor alpha (TNF-alpha)-induced cell damage in human renal proximal tubular epithelial cells (RPTEC). METHODS: The lactate dehydrogenase (LDH) and N-acetyl-beta-glucosaminidase (NAG) release into the medium after TNF-alpha treatment in RPTEC were determined using modified commercial procedures. In addition, the levels of caspase 3/7 activity in RPTEC were measured after TNF-alpha treatment with AlphaTau1 or AT2 receptor blockers. Finally we investigated the change of p22phox protein levels after TNF-alpha with AlphaTau1 or AT2 receptor blockers in RPTEC. RESULTS: Tumour necrosis factor alpha (10(-8) mol/L) significantly increased LDH and NAG release into the medium from RPTEC. AlphaTau1 receptor blockers, olmesartan and valsartan (10(-9)-10(-6) mol/L) showed a significant reduction on TNF-alpha-induced LDH and NAG release in RPTEC. AT2 receptor blocker, PD123319 (10(-7)-10(-5) mol/L) also decreased TNF-alpha-induced LDH and NAG release in RPTEC. Blockade of both AlphaTau1 and AT2 receptor indicated additional reduction on TNF-alpha-induced LDH and NAG release. TNF-alpha (10(-8) mol/L) treatment showed small but significant increases of caspase 3/7 activity in RPTEC, and AT1 and AT2 receptor blockers (10(-8) mol/L) comparably decreased TNF-alpha-induced caspase 3/7 activity. Significant increases of p22phox protein levels were observed in TNF-alpha-treated group in RPTEC. However, only AlphaTau1 (10(-8) mol/L) but not AT2 (10(-5) mol/L) receptor blocker significantly decreased TNF-alpha-induced p22phox protein levels. CONCLUSION: The present study demonstrates that TNF-alpha induces renal tubular cell damage in RPTEC and AT1/AT2 receptor blockers showed cytoprotective effects probably via at least partly different mechanism.
Assuntos
Bloqueadores do Receptor Tipo 1 de Angiotensina II/farmacologia , Bloqueadores do Receptor Tipo 2 de Angiotensina II , Células Epiteliais/efeitos dos fármacos , Imidazóis/farmacologia , Túbulos Renais Proximais/efeitos dos fármacos , Piridinas/farmacologia , Tetrazóis/farmacologia , Fator de Necrose Tumoral alfa/metabolismo , Valina/análogos & derivados , Apoptose/efeitos dos fármacos , Caspase 3/metabolismo , Caspase 7/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Citoproteção , Relação Dose-Resposta a Droga , Ativação Enzimática , Células Epiteliais/enzimologia , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Glucosamina/análogos & derivados , Glucosamina/metabolismo , Humanos , Túbulos Renais Proximais/enzimologia , Túbulos Renais Proximais/metabolismo , Túbulos Renais Proximais/patologia , L-Lactato Desidrogenase/metabolismo , NADPH Oxidases/metabolismo , Receptor Tipo 2 de Angiotensina/metabolismo , Receptores Tipo I de Fatores de Necrose Tumoral/metabolismo , Valina/farmacologia , ValsartanaRESUMO
In order to examine the formation of a neural network and the functional development of a visual pathway, we performed in vitro reconstruction of the retinotectal pathway using organotypic explants and co-culture methods. Retinas and superior colliculus (SC) slices obtained from embryonic rats were co-cultured on microelectrode array (MEA) substrates for four weeks. We observed retinal ganglion cell neurites innervating SC slices that evoked responses in retinas or SC slices after applying electrical stimulation. Functional connections between retinas and SC slices were formed in the cultures. At the same time, spontaneous electrical activities were recorded from both the retinas and SC slices over the four weeks. In the co-cultured SC slices, sporadic firings were initially observed at 3-4 days in vitro (DIV), and thereafter the frequency of spontaneous firing increased and synchronized activities occurred after two weeks in vitro (WIV). In most of the single-cultured SC slices, however, only sporadic firings were observed over four weeks. In addition, the retinas and SC slices were co-cultured to enable the exchange of soluble factors with each other via culture medium but not via direct neural connections. The activity patterns resembled ones of single-cultured SC slices. These results suggest that signal inputs from retinas through direct neural connections affect the development of SCs in the retinotectal pathway.
Assuntos
Técnicas de Cocultura/métodos , Rede Nervosa/fisiologia , Plasticidade Neuronal/fisiologia , Técnicas de Cultura de Órgãos/métodos , Células Ganglionares da Retina/fisiologia , Colículos Superiores/fisiologia , Vias Visuais/fisiologia , Animais , Células Cultivadas , Ratos , Ratos WistarRESUMO
2-Acetamido-2-deoxy-D-galactose (GalNAc) is a common monosaccharide found in biologically functional sugar chains, but its availability is often limited due to the lack of abundant natural sources. In order to produce GalNAc from abundantly available sugars, 2-acetamido-2-deoxy-D-glucose (GlcNAc) was converted to GalNAc by a one-pot reaction using three enzymes involved in the galacto-N-biose/lacto-N-biose I pathway of bifidobacteria. Starting the reaction with 600 mM GlcNAc, 170 mM GalNAc was produced at equilibrium in the presence of catalytic amounts of ATP and UDP-Glc under optimized conditions. GalNAc was separated from GlcNAc using water-eluting cation-exchange chromatography with a commonly available cation-exchange resin.
Assuntos
Acetilgalactosamina/síntese química , Acetilglucosamina/química , Biocatálise , Acetilgalactosamina/isolamento & purificação , Acetilglucosamina/isolamento & purificação , Trifosfato de Adenosina/química , Proteínas de Bactérias/química , Bifidobacterium/enzimologia , Cromatografia por Troca Iônica , Ensaios Enzimáticos , Cinética , Fosfotransferases (Aceptor do Grupo Álcool)/química , UDPglucose 4-Epimerase/química , UDPglucose-Hexose-1-Fosfato Uridiltransferase/química , Uridina Difosfato Glucose/química , Uridina Difosfato N-Acetilglicosamina/químicaRESUMO
BACKGROUND: Recent studies have identified high glucose as a potent stimulus for the intracellular synthesis of angiotensin II. However, the exact roles of angiotensin II and angiotensin II type 1 receptor blockers (ARB) in high-glucose-induced renal tubular function remain unclear. METHODS: N-Acetyl-beta-glucosaminidase (NAG), angiotensin II and 8-hydroxy-2'-deoxyguanosine (8-OHdG) concentrations in renal proximal tubular epithelial cells (RPTECs) with or without high glucose/ARB were determined using a modified commercial procedure. The changes of p22phox and cytoplasmic inhibitory kappa B (IkB) protein levels in RPTECs were measured using Western blotting. RESULTS: High-glucose treatment (4x10-2 mol/L) significantly increased NAG release, angiotensin II concentrations in cell lysates and 8-OHdG and p22phox protein levels compared with those in regular glucose medium (1.75x10(-2) mol/L). ARBs (candesartan, olmesartan or valsartan; 1x10(-9)-10(-7) mol/L) showed a significant reduction in high-glucose-induced NAG, 8-OHdG and p22phox protein levels in RPTECs. Significant decreases of cytoplasmic IkB protein levels were observed in the high-glucose-treated group in RPTECs. ARBs markedly reversed high-glucose-induced reduction of IkB protein levels in RPTECs. CONCLUSIONS: ARBs reduce high-glucose-induced oxidative stress in RPTECs possibly via blockade of intracellular as well as extracellular AT1 receptor signaling, which possibly protects renal tubular cell function during diabetic nephropathy.
Assuntos
Células Epiteliais/metabolismo , Células Epiteliais/patologia , Glucose/efeitos adversos , Túbulos Renais Proximais/metabolismo , Túbulos Renais Proximais/patologia , Receptores de Angiotensina/metabolismo , 8-Hidroxi-2'-Desoxiguanosina , Bloqueadores do Receptor Tipo 1 de Angiotensina II/farmacologia , Células Cultivadas , Desoxiguanosina/análogos & derivados , Desoxiguanosina/metabolismo , Relação Dose-Resposta a Droga , Células Epiteliais/efeitos dos fármacos , Glucose/farmacologia , Humanos , Proteínas I-kappa B/metabolismo , Túbulos Renais Proximais/efeitos dos fármacos , NADPH Oxidases/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , beta-N-Acetil-Galactosaminidase/metabolismoRESUMO
In recent years a diamond-like carbon (DLC) film and a 2-methacryloyloxyethyl phosphorylcholine (MPC) polymer have attracted attention as coating materials for implantable artificial organs or devices. When these materials are coated on vascular devices, compatibility to blood is an important problem. The present paper focuses on friction characteristics of erythrocytes to these coating materials in a medium. With an inclined centrifuge microscope developed by the authors, observation was made for erythrocytes moving on flat glass plates with and without coating in a medium of plasma or saline under the effect of inclined centrifugal force. Friction characteristics of erythrocytes with respect to these coating materials were then measured and compared to each other to characterize DLC and MPC as coating materials. The friction characteristics of erythrocytes in plasma using the DLC-coated and noncoated glass plates are similar, changing approximately proportional to the 0.5th power of the cell velocity. The cells stick to these plates in saline as well, implying the influence of plasma protein. The results using the MPC-coated plate in plasma are similar to those of the other plates for large cell velocities, but deviate from the other results with decreased cell velocity. The results change nearly proportional to the 0.75th power of the cell velocity in the range of small velocities. The results for the MPC-coated plate in saline are similar to that in plasma but somewhat smaller, implying that the friction characteristics for the MPC-coated plate are essentially independent of plasma protein.
Assuntos
Movimento Celular/fisiologia , Materiais Revestidos Biocompatíveis/química , Diamante/química , Eritrócitos/citologia , Eritrócitos/fisiologia , Vidro/química , Metacrilatos/química , Fosforilcolina/análogos & derivados , Adesão Celular/fisiologia , Fricção , Teste de Materiais , Fosforilcolina/química , Estresse MecânicoRESUMO
An asymmetric hydrogen-transfer biocatalyst consisting of mutated Rhodococcus phenylacetaldehyde reductase (PAR) or Leifsonia alcohol dehydrogenase (LSADH) was applied for some water-soluble ketone substrates. Among them, 4-hydroxy-2-butanone was reduced to (S)/(R)-1,3-butanediol, a useful intermediate for pharmaceuticals, with a high yield and stereoselectivity. Intact Escherichia coli cells overexpressing mutated PAR (Sar268) or LSADH were directly immobilized with polyethyleneimine or 1,6-diaminehexane and glutaraldehyde and evaluated in a batch reaction. This system produced (S)-1,3-butanediol [87% enantiomeric excess (e.e.)] with a space time yield (STY) of 12.5 mg h(-1) ml(-1) catalyst or (R)-1,3-butanediol (99% e.e.) with an STY of 60.3 mg h(-1) ml(-1) catalyst, respectively. The immobilized cells in a packed bed reactor continuously produced (R)-1,3-butanediol with a yield of 99% (about 49.5 g/l) from 5% (w/v) 4-hydroxy-2-butanoate over 500 h.
Assuntos
Reatores Biológicos , Butileno Glicóis/metabolismo , Escherichia coli/metabolismo , Hidrogênio/metabolismo , 2-Propanol/química , 2-Propanol/metabolismo , Butileno Glicóis/química , Células Imobilizadas , Conformação Molecular , Oxirredução , Especificidade por SubstratoRESUMO
The gene encoding Leifsonia alcohol dehydrogenase (LSADH), a useful biocatalyst for producing (R)-chiral alcohols, was cloned from the genomic DNA of Leifsonia sp. S749. The gene contained an opening reading frame consisting of 756 nucleotides corresponding to 251 amino acid residues. The subunit molecular weight was calculated to be 24,999, which was consistent with that determined by polyacrylamide gel electrophoresis. The enzyme was expressed in recombinant Escherichia coli cells and purified to homogeneity by three column chromatographies. The predicted amino acid sequence displayed 30-50% homology to known short chain alcohol dehydrogenase/reductases (SDRs); moreover, the NADH-binding site and the three catalytic residues in SDRs were conserved. The recombinant E. coli cells which overexpressed lsadh produced (R)-form chiral alcohols from ketones using 2-propanol as a hydrogen donor with the highest level of productivity ever reported and enantiomeric excess (e.e.).
Assuntos
Álcool Desidrogenase/metabolismo , Álcoois/química , Álcoois/metabolismo , Expressão Gênica/genética , Hidrogênio/química , Hidrogênio/metabolismo , Micrococcaceae/enzimologia , Álcool Desidrogenase/química , Álcool Desidrogenase/genética , Álcool Desidrogenase/isolamento & purificação , Sequência de Aminoácidos , Sistema Livre de Células , Clonagem Molecular , Sequência Conservada , Escherichia coli/química , Escherichia coli/metabolismo , Micrococcaceae/genética , Dados de Sequência Molecular , Peso Molecular , Oxirredução , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Estereoisomerismo , Transformação Genética/genéticaRESUMO
To find microorganisms that could reduce phenyl trifluoromethyl ketone (PTK) to (S)-1-phenyltrifluoroethanol [(S)-PTE], styrene-assimilating bacteria (ca. 900 strains) isolated from soil samples were screened. We found that Leifsonia sp. strain S749 was the most suitable strain for the conversion of PTK to (S)-PTE in the presence of 2-propanol as a hydrogen donor. The enzyme corresponding to the reaction was purified homogeneity, characterized and designated Leifsonia alcohol dehydrogenase (LSADH). The purified enzyme had a molecular weight of 110,000 and was composed of four identical subunits (molecular weight, 26,000). LSADH required NADH as a cofactor, showed little activity with NADPH, and reduced a wide variety of aldehydes and ketones. LSADH catalyzed the enantioselective reduction of some ketones with high enantiomeric excesses (e.e.): PTK to (S)-PTE (>99% e.e.), acetophenone to (R)-1-phenylethanol (99% e.e.), and 2-heptanone to (R)-2-heptanol (>99% e.e.) in the presence of 2-propanol without an additional NADH regeneration system. Therefore, it would be a useful biocatalyst.
Assuntos
Actinomycetales/enzimologia , Álcool Desidrogenase/isolamento & purificação , Hidrogênio/metabolismo , Cetonas/metabolismo , Microbiologia do Solo , 2-Propanol/metabolismo , Actinomycetales/crescimento & desenvolvimento , Actinomycetales/isolamento & purificação , Álcool Desidrogenase/química , Álcool Desidrogenase/metabolismo , Sequência de Aminoácidos , Meios de Cultura , Cinética , Dados de Sequência Molecular , NAD/metabolismo , Oxirredução , Estireno/metabolismo , Especificidade por SubstratoRESUMO
Phenylacetaldehyde reductase (PAR) is suitable for the conversion of various aryl ketones and 2-alkanones to corresponding chiral alcohols. 2-Propanol acts as a substrate solvent and hydrogen donor of coupled cofactor regeneration during the conversion of substrates catalyzed by PAR. To improve the conversion efficiency in high concentrations of substrate and 2-propanol, selection of a PAR mutant library and the subsequent rearrangement of mutations were attempted. With only a single selection round and following the manual combination of advantageous mutations, PAR was successfully adapted for the conversion of high concentrations of substrate with concentrated 2-propanol. This method will be widely applicable for the engineering of enzymes potentially valuable for industry.