Study on a Specific Site Tyr~(444) on a Hyperthermophilic Enzyme APE1547

Hyperthermophilic enzyme APE1547 is an extremely thermostable recombinant protein from thermophilic archaeon Aeropyrum pernix K1. The Tyr444 located in the catalytic domain adjacent to the catalytic amino acid Ser445 and formed hydrogen bond with Ile567. To study the effect of Tyr444 on the activity of APE1547,site-directed mutagenesis was applied. Two mutant enzymes T444S and T444G were created. Comparison of the mutant enzymes with wide enzyme,the thermostability of mutants T444S and T444G decreased by 10...     

Enzyme catalytic promiscuity: asymmetric aldol addition reaction catalyzed by a novel thermophilic esterase in organic solvent

The asymmetric aldol addition of 2-butanone and 4-nitrobenzaldehyde catalyzed by a novel thermophilic esterase (APE1547) from the archaeon Aeropyrum pernix K1 was successfully conducted in organic solvents. APE1547 exhibited a good enzyme activity and enantioselectivity in the reaction. The effects of organic solvent, temperature, water content, and substrate concentration were investigated. The reaction provided optically active secondary alcohol with satisfying enantioselectivity (71.2 %ee) and enzyme activity (38.1 µmol/g/h) under the optimum conditions. A high yield (68.7%) could be obtained when the reaction time was approximately 120 h.     

Denaturing Effects of Urea and Guanidine Hydrochloride on Hyperthermophilic Esterase from Aeropyrum pernix K1

Introduction Esterases(EC3.1.1.x)representadiversegroup ofhydrolasescatalyzingthecleavageandformationof esterbonds.Theyarewidelydistributedinanimals,plantsandmicroorganisms.Becauseoftheiractivities inbothaqueousandnonaqueoussolventsystems,ester aseshavebe…     

超嗜热酯酶APE1547中特殊位置氢键对酶活力和热稳定性的影响

探讨了APE1547蛋白的β-推进器结构中第3和第4“叶片”间的侧链氢键(Thr127-Gly154, Leu182-Arg145-Glu122)对蛋白质的作用.     

古细菌Aeropyrum pernix K1超嗜热酯酶APE1547的稳定性

研究了纯化的超嗜热酯酶APE1547的稳定性. 结果表明, 该酶的稳定性非常好, 蛋白的质量浓度为0.4 mg/mL时, 90 ℃的半衰期为20 h, 0.2 mg/mL时的半衰期为12 h; 而蛋白的质量浓度为0.04 mg/mL时, 保温2.5 h时残余活力仍在50%以上. 同时还研究了热变性时该酶表面疏水氨基酸的变化. 该酶的pH稳定性也很好, pH在6.5-9.0范围内作用24 h, 酶依然很稳定, 残余酶活力大于93％; 同时该酶还具有很强的耐有机溶剂的特性.     

Directed evolution of epoxide hydrolase from A. radiobacter toward higher enantioselectivity by error-prone PCR and DNA shuffling

The enantioselectivity of epoxide hydrolase from Agrobacterium radiobacter (EchA) was improved using error-prone PCR and DNA shuffling. An agar plate assay was used to screen the mutant libraries for activity. Screening for improved enantioselectivity was subsequently done by spectrophotometric progress curve analysis of the conversion of para-nitrophenyl glycidyl ether (pNPGE). Kinetic resolutions showed that eight mutants were obtained with up to 13-fold improved enantioselectivity toward pNPGE and at least three other epoxides. The large enhancements in enantioselectivity toward epichlorohydrin and 1,2-epoxyhexane indicated that pNPGE acts as an epoxyalkane mimic. Active site mutations were found in all shuffled mutants, which can be explained by an interaction of the affected amino acid with the epoxide oxygen or the hydrophobic moiety of the substrate. Several mutations in the shuffled mutants had additive effects.     

Molecular Basis for Stereospecific Hydrolysis of Ethyl Mandelate by Thermophilic Esterase

The stereospecific hydrolysis of mandelate can be effectively catalyzed by hyperthermophilic acylpeptide esterase APE 1547(Aeropyrum pernix esterase 1547). APE 1547 used in this reaction showed a remarkable stereodiscrimination in favour of R-mandelic acid(99% e.e.) with an enantiomeric ratio E>200. The results of computer simulation are consistent with the experimental results. It can be inferred that the R-substrate adopted a binding mode productive of the reaction due to the formation of the hydrogen bond at the active site of APE 1547.     

Structure-based redesign of GST A2-2 for enhanced catalytic efficiency with azathioprine

Glutathione transferase (GST) A2-2 is the most efficient human enzyme in the biotransformation of the prodrug azathioprine (Aza). The activation of Aza has therapeutic potential for possible use of GSTs in targeted enzyme-prodrug treatment of diseases. Based on the assumed catalytic mechanism and computational docking of Aza to the active site of the enzyme, active-site residues were selected for construction of focused mutant libraries, which were thereafter screened for Aza activity. Mutants with elevated Aza activity were identified, DNA sequenced, and the proteins purified. The two most active mutants showed up to 70-fold higher catalytic efficiency than the parental GST A2-2. The structure of the most active triple mutant (L107G/L108D/F222H) enzyme was determined by X-ray crystallography demonstrating significant changes in the topography of the active site facilitating productive binding of Aza as a substrate.     

High-throughput Screening of the Enantioselectivity of Hyperthermophilic Mutant Esterases from Archaeon Aeropyrum pernix K1 for Resolution of （ R, S ） -2-Octanol Acetate

To identify the desired hyperthermophilic variants within a mutant esterase library for the resolution of (R,S)-2-octanol acetate, a simple, reliable, and versatile method was developed in this study. We built a screening strategy including two steps, first we selected agar plate with substrate to screen the enzymatic activity; secondly we used a pH indicator to screen the enantioselectivity. This method could rapidly detect favorable mutants with high activity and enantioselectivity. A total of 96.2% of tedious screening work can be precluded using this screening strategy. It is an effective screening for alkyl ester and can be applied to relative screening researches. The four improved mutants were screened from the mutant esterase library. Their enantioselectivities, activities, and structures were investigated at different temperatures.     

Acylation of Quercetin with a Novel Thermophilic Esterase as Biocatalyst

The regioselective acylation of quercetin catalyzed by a novel thermophilic esterase(APE1547)from the archaeon Aeropyrum pernix K1 was successfully conducted in organic solvents.The effects of acyl don...     

Focusing mutations into the P. fluorescens esterase binding site increases enantioselectivity more effectively than distant mutations

Rational design of enzymes with improved properties, such as enantioselectivity, usually focuses mutations within the substrate binding site. On the other hand, directed evolution of enzymes usually targets the entire protein and discovers beneficial mutations far from the substrate binding site. In this paper, we propose an explanation for this discrepancy and show that a combined approach--random mutagenesis within the substrate binding site--is better. To increase the enantioselectivity (E) of a Pseudomonas fluorescens esterase (PFE) toward methyl 3-bromo-2-methylpropionate, we focused mutagenesis into the substrate binding site at Trp28, Val121, Phe198, and Val225. Five of the catalytically active mutants (13%) showed better enantioselectivity than wild-type PFE. The increases in enantioselectivity were higher (up to 5-fold, reaching E = 61) than with mutants identified by random mutagenesis of the entire enzyme.     

Engineering P450 Peroxygenase to Catalyze Highly Enantioselective Epoxidation of cis‐β‐Methylstyrenes

P450 119 peroxygenase and its site‐directed mutants are discovered to catalyze the enantioselective epoxidation of methyl‐substituted styrenes. Two new site‐directed P450 119 mutants, namely T213Y and T213M, which were designed to improve the enantioselectivity and activity for the epoxidation of styrene and its methyl substituted derivatives, were studied. The T213M mutant is found to be the first engineered P450 peroxygenase that shows highly enantioselective epoxidation of cis‐β‐methylstyrenes, with up to 91 % ee. Molecular modeling studies provide insights into the different catalytic activity of the T213M mutant and the T213Y mutant in the epoxidation of cis‐β‐methylstyrene. The results of the calculations also contribute to a better understanding of the substrate specificity and configuration control for the regio‐ and stereoselective peroxygenation catalyzed by the T213M mutant.     

An Enhanced Process for the Production of a Highly Purified Extracellular Lipase in the Non-conventional Yeast <Emphasis Type="Italic">Yarrowia lipolytica</Emphasis>

Yarrowia lipolytica LgX64.81 is a non-genetically modified mutant that was previously identified as a promising microorganism for extracellular lipase production. In this work, the development of a fed-batch process for the production of this enzyme in this strain was described. A lipolytic activity of 2,145 U/mL was obtained after 32 h of batch culture in a defined medium supplemented with 10 g/L of tryptone, an enhancer of lipase expression. To maximize the volumetric productivity, two different fed-batch strategies had been investigated. In comparison to batch process, the intermittent fed-batch strategy had not improved the volumetric lipase productivity. In contrast, the stepwise feeding strategy combined with uncoupled cell growth and lipase production phases resulted in a 2-fold increase in the volumetric lipase productivity, namely, the lipase activity reached 10,000 U/mL after 80 h of culture. Furthermore, this lipase was purified to homogeneity by anion exchange chromatography on MonoQ resin followed by gel filtration on Sephacryl S-100. This process resulted in an overall yield of 72% and a 3.5-fold increase of the specific lipase activity. The developed process offers a great potential for an economic production of Lip2 at large scale in Y. lipolytica LgX64.81.     

Derepressed 2-deoxyglucose-resistant mutants of Aspergillus niger with altered hexokinase and acid phosphatase activity in hyperproduction of β-fructofuranosidase

Aspergillus niger NRRL330 produces extracellular β-fructofuranosidase (Ffase), and its production is subject to repression by hexoses in the medium. After ultraviolet mutagenization and selection, seven derepressed mutants resistant to 2-deoxyglucose (2-DG) were isolated on Czapek’s minimal medium containing glycerol. One of the mutants, designated DGRA-1, produced higher levels of Ffase. A considerable difference occurred in the mutants with reference to hexokinase and intracellular acid phosphatase activities. The hexokinase activity of the mutant DGRA-1 (0.69 U/mg) was 1.8-fold higher than the wild type (0.38U/mg). Intracellular acid phosphatase activity of the mutant DGRA-1 (0.83 U/g of mycelia) was twofold higher than that of the wild type (0.42U/g of mycelia), suggesting that phosphorylation and dephosphorylation steps could attribute to the 2-DG resistance of A. niger. However, additional mutations could account for the increased production of Ffase in the mutant DGRA-1.     

去甲万古霉素键合毛细管电色谱硅胶整体柱的制备及应用

采用溶胶-凝胶技术制备了具有高机械强度和良好通透性的毛细管硅胶整体柱。以国产大环抗生素去甲万古霉素为 手性选择试剂对所制备的整体柱进行化学衍生,成功地制备了去甲万古霉素键合手性硅胶整体柱。在反相和极性有机相模 式下考察了所制备柱的手性识别能力,并详细考察了流动相条件对分离的影响。研究结果表明,β-受体阻滞剂类药物在极 性有机流动相组成为甲醇-乙腈-乙酸-三乙胺(体积比为80∶20∶0.1∶0.1)时,可获得最佳分离。在反相色谱条件下,电 渗流仍主要由整体硅胶基质材料产生,而手性选择试剂的贡献甚小。在反相色谱条件下,多种不同结构类型的手性药物在 所制备的色谱柱上获得了分离。     

Effects of Val34Leu and Val35Leu polymorphism on the enzyme activity of the coagulation factor XIII-A

Change in fibrin stabilizing activity of factor XIII A subunit (FXIII-A) caused by a specific mutation, Val34Leu, is recently implicated to incidences of pathophysiology of thrombosis. In an effort to understand the effect of Val34Leu on enhanced catalytic role of FXIII-A, wild type human factor XIII A (HFXIII-A) and mutant HFXIII-A: HFXIII-A (V34L), HFXIII-A (V35L) and HFXIII-A (V34L/V35L) cDNA were expressed in E.coli system where the purified recombinant FXIII-A (gammaFXIII-A) showed a similar specific transglutaminase activity comparable to the human native FXIII-A from platelet. Using these gammaFXIII-A mutants, the activation kinetics by thrombin and the enzymatic properties of the activated gammaFXIII-A were characterized. gammaFXIII-A (V34L) and gammaFXIII-A (V34L/V35L) mutants were activated by thrombin much faster than those of wild type gammagFXIII-A and V35L variant. However, the activated gammaFXIII-A and mutants showed the identical catalytic efficiency as measured by in vitro assay. These results suggest that ready activation caused by a specific mutation of neighboring thrombin cleavage site(s) in the activation peptide of FXIII-A like V34L resulted in the real-time amount of the activated factor XIII-A that could influence the outcome of fibrin stabilization in vivo such as alpha2-plasmin inhibitor crosslinking to fibrin, a reaction known to be dependent on the initial concentration of active factor-XIII-A.     

Sodium Dodecyl Sulphate, a Strong Inducer of Thermostable Glucanhydrolase Secretion from a Derepressed Mutant Strain of Bacillus alcalophilus GCBNA-4

In the present study, we report the optimisation of batch conditions for improved α-1,4-glucan-glucanohydrolase (GGH) secretion by a nitrous acid (NA)-treated Bacillus alcalophilus. The wild (isolate GCB-18) and NA-derivative (mutant GCBNA-4) were grown in a medium containing 10 g/L nutrient broth, 10 g/L starch, 5 g/L lactose, 2 g/L ammonium sulphate, 2 g/L CaCl₂ and phosphate buffer (pH 7.6). Sodium dodecyl sulphate (SDS) was used as an enzyme inducer while batch fermentations were carried out at 40 °C. The mutant produced GGH in 40 h which was 15-fold higher than the wild in presence of SDS. Thermodynamic studies revealed that the mutant culture exhibited the capability for improved enzyme activity over a broad range of temperature (35–70 °C). The enzyme was purified by cation-exchange column chromatography with ～80 % recovery. The performance of fuzzy-logic system control was found to be highly promising for the improved substrate conversion rate. The correlation (1.045E?+?0025) among variables demonstrated the model terms as highly significant indicating commercial utility of the culture used (P?<?0.05).     

羊肝源穿山龙薯蓣皂苷-α-L-鼠李糖苷酶的分离及其动力学特性

对羊肝中含有的水解穿山龙薯蓣皂苷鼠李糖糖基的穿山龙薯蓣皂苷-α-L-鼠李糖苷酶进行了分离、纯化, 并对其动力学特性进行了研究. 结果表明, 粗酶液经DEAE-Cellulose离子交换层析柱纯化后, 其比活提高了19.9倍. 在pH＝6.8、反应温度为42 ℃、反应时间为8 h和底物浓度为23 mmol/L的条件下, 该酶达到其最高活力. 在10—200 mmol/L范围内, Fe3+和Cu2+对酶活力有明显的抑制作用, Mg2+和Zn2+对酶活力有微弱的激活作用, 而Ca2+对酶有较强的激活作用. 采用SDS-PAGE方法测得酶蛋白分子量为71000. 选择穿山龙薯蓣皂苷、人参皂苷Re和芦丁为酶反应底物, 进行酶的底物专一性研究发现, 穿山龙薯蓣皂苷-α-L-鼠李糖苷酶对其底物具有高度专一性.     

A Practical System for High-Throughput Screening of Mutants of <Emphasis Type="Italic">Bacillus fastidiosus</Emphasis> Uricase

For high-throughput screening (HTS) of Bacillus fastidiosus uricase mutants, a practical system was proposed. By error-prone PCR with final 1.5 mM MnCl₂, two focused libraries of mutants for A1-V158 and V150-D212 were generated separately. After induced expression of individual clones in 48-well microplates, Escherichia coli cells (BL21) were lyzed by 1.0 M Tris-HCl at pH 9.0 in 96-well microplates at 25 °C for 7.5 ~ 10.5 h; uricase reaction was continuously monitored with 0.15 mM uric acid in 96-well plates by absorbance at 298 nm to estimate V _m/K _m by kinetic analysis of reaction curve for comparison. V _m/K _m was resistant to initial uric acid levels with an upper limit 3-fold over that of initial rates. By receiver-operator-characteristic analysis of the recognition of the one of higher activity in uricase pair whose specific activity ratio was 1.8 or 3.3, the area-under-the-curve was comparable to that with cell lysates prepared by sonication treatment. A cutoff for the maximum Youden index was thus developed to recognize positive mutants of 1-fold higher activity. Indeed, mutant L171I/Y182F/Y187F/A193S of higher activity but lower thermostability at pH 7.4 and mutant V144A of higher activity and consistent thermostability were discovered. Therefore, the proposed system was practical for HTS of uricase mutants.     

Mutations in distant residues moderately increase the enantioselectivity of Pseudomonas fluorescens esterase towards methyl 3bromo-2-methylpropanoate and ethyl 3phenylbutyrate

Directed evolution combined with saturation mutagenesis identified six different point mutations that each moderately increases the enantioselectivity of an esterase from Pseudomonas fluorescens (PFE) towards either of two chiral synthons. Directed evolution identified a Thr230Ile mutation that increased the enantioselectivity from 12 to 19 towards methyl (S)-3-bromo-2-methylpropanoate. Saturation mutagenesis at Thr230 identified another mutant, Thr230Pro, with higher-than-wild-type enantioselectivity (E=17). Previous directed evolution identified mutants Asp158Asn and Leu181Gln that increased the enantioselectivity from 3.5 to 5.8 and 6.6, respectively, towards ethyl (R)-3-phenylbutyrate. In this work, saturation mutagenesis identified other mutations that further increase the enantioselectivity to 12 (Asp158Leu) and 10 (Leu181Ser). A homology model of PFE indicates that all mutations lie outside the active site, 12-14 A from the substrate and suggests how the distant mutations might indirectly change the substrate-binding site. Since proteins contain many more residues far from the active site than close to the active site, random mutagenesis is strongly biased in favor of distant mutations. Directed evolution rarely screens all mutations, so it usually finds the distant mutations because they are more common, but probably not the most effective.