Residue Tyr224 is critical for the thermostability of Geobacillus sp. RD-2 lipase

A thermophilic lipase (lipGRD) from Geobacillus sp. RD-2, isolated from a hot spring in Yunnan, China, was cloned and over-expressed in Escherichia coli. The function of the conserved residue, Tyr224, near the presumed temperature switch site was analyzed by site-directed saturation mutagenesis. The activity of the wild type lipGRD was optimal at 55??C and pH?7.5, but that from mutant Y224C was optimally active at 35??C, whereas Y224P lipase was optimally active at 65??C. Furthermore, the latter lipase retained 60% of its activity after incubation at 65??C for 5?h. The conserved residue Tyr224, which is close to the lid helix, is the key amino acid residue determining the thermostability of the thermostable lipase.     

K55R与ep8叠加突变对扩展青霉脂肪酶热稳定性的改善

利用重叠延伸PCR对扩展青霉脂肪酶(PEL)基因进行体外定点突变,构建了K55R与随机突变体ep8叠加突变的重组质粒pAO815-ep8-K55R。将该质粒电转化引入毕赤酵母(Pichia pastoris)GS115,进行异源表达。实验结果表明:该叠加突变体在毕赤酵母中获得了活性表达,得到表达产物脂肪酶PEL-ep8-K55R-GS。其表达量为508u/mL,分别约为野生型脂肪酶PEL-GS(627u/mL)的81%,随机突变脂肪酶PEL-ep8-GS(924u/mL)的55%;其比活力为2309.1u/mg,与随机突变脂肪酶PEL-ep8-GS和野生型脂肪酶PEL-GS的相仿。叠加突变脂肪酶PEL-ep8-K55R-GS的最适作用温度为37℃,与野生型脂肪酶PEL-GS和随机突变脂肪酶PEL-ep8-GS一致;其Tm值为41.0℃,比野生型脂肪酶PEL-GS提高了2.3℃,比随机突变脂肪酶PEL-ep8-GS提高了0.8℃。表明叠加突变脂肪酶PEL-ep8-K55R-GS的热稳定性有了进一步的提高。     

基于易错PCR定向进化扩展青霉 Penicillium expansum FS1884碱性脂肪酶

为改善扩展青霉FS1884碱性脂肪酶的活性及酶学性质,利用连续两轮易错PCR对扩展青霉FS1884脂肪酶基因PEL进行随机突变,在大肠杆菌JM109中构建突变文库。含突变脂肪酶基因的重组质粒电击转化巴斯德毕赤酵母GS115,经过YPOM板初筛和橄榄油检验板复筛,获得一株酶活性提高的脂肪酶突变体:PEL-ep25-GS。与野生型脂肪酶PEL-GS相比,在最适温度40℃、pH9.4时突变体的酶活力是野生型酶的1.3倍。测序结果表明:该突变体第253位氨基酸发生了突变,由赖氨酸变成蛋氨酸。     

Mutational analysis of Thermus caldophilus GK24 beta-glycosidase: role of His119 in substrate binding and enzyme activity

Three amino acid residues (His119, Glu164, and Glu338) in the active site of Thermus caldophilus GK24 beta- glycosidase (Tca beta-glycosidase), a family 1 glycosyl hydrolase, were mutated by site-directed mutagenesis. To verify the key catalytic residues, Glu164 and Glu338 were changed to Gly and Gln, respectively. The E164G mutation resulted in drastic reductions of both beta-galactosidase and beta-glucosidase activities, and the E338Q mutation caused complete loss of activity, confirming that the two residues are essential for the reaction process of glycosidic linkage hydrolysis. To investigate the role of His119 in substrate binding and enzyme activity, the residue was substituted with Gly. The H119G mutant showed 53-fold reduced activity on 5 mM p-nitrophenyl beta-Dgalactopyranoside, when compared with the wild type; however, both the wild-type and mutant enzymes showed similar activity on 5 mM p-nitrophenyl beta-D-glucopyranoside at 75degreeC. Kinetic analysis with p-nitrophenyl beta-D-galactopyranoside revealed that the kcat value of the H119G mutant was 76.3-fold lower than that of the wild type, but the Km of the mutant was 15.3-fold higher than that of the wild type owing to the much lower affinity of the mutant. Thus, the catalytic efficiency (kcat/Km) of the mutant decreased to 0.08% to that of the wild type. The kcat value of the H119G mutant for p-nitrophenyl beta- D-glucopyranoside was 5.1-fold higher than that of the wild type, but the catalytic efficiency of the mutant was 2.5% of that of the wild type. The H119G mutation gave rise to changes in optima pH (from 5.5-6.5 to 5.5) and temperature (from 90 degrees C to 80-85 degrees C). This difference of temperature optima originated in the decrease of H119G's thermostability. These results indicate that His119 is a crucial residue in beta- galactosidase and beta-glucosidase activities and also influences the enzyme's substrate binding affinity and thermostability.     

Engineering of <Emphasis Type="Italic">Bacillus</Emphasis> lipase by directed evolution for enhanced thermal stability: effect of isoleucine to threonine mutation at protein surface

A lip gene from a Bacillus isolate was cloned and expressed in E. coli. By thermal denaturation analysis, T_1/2 of lipase was observed to be 7 min at 50°C with less than 10% activity after 1 h incubation at 50°C. To expand the functionality of cloned lipase, attempts have been made to create thermostable variants of lip gene. A lipase variant with an isoleucine to threonine amino acid substitution at the protein surface was isolated that demonstrated higher thermostability than its wild type predecessor. To explore the structure–function relationship, the lip gene product of wild type (WT) and mutant was characterized in detail. The mutation enhanced the specific activity of enzyme by 2-folds when compared with WT. The mutant enzyme showed enhanced T_1/2 of 21 min at 50°C. The kinetic parameters of the mutant enzyme were significantly altered. The mutant enzyme displayed higher affinity for substrate (decreased K _m ) in comparison to the wild type. The k _cat and catalytic efficiency (k _cat/K _m ) of mutant were also enhanced by two and five times, respectively, as compared with the WT. The mutation resides on the part of helix which is exposed to the solvent and away from the catalytic triad. The replacement of a solvent exposed hydrophobic residue (Ile) in WT with a hydrophilic residue (Thr) in mutant might impart thermostability to the protein structure.     

嗜热酯酶APE1547催化活性的定向进化研究

对来源于嗜热古菌Aeropyrum pernix的酯酶(APE1547)催化活性进行定向进化研究。利用APE1547特殊的稳定性,建立了准确的高通量高温酯酶筛选方法。对第一代随机突变库筛选获得了催化活性较野生型提高1.5倍的突变体M010,序列分析表明其氨基酸突变为R526S。从第二代突变库中筛选出的总活力提高5.8倍突变体M020,突变位点为R526S/E88G/A200T/I519L,其比活力与M010一致,但表达量比野生型提高约4倍。对M020酶学性质表征发现,其最适pH为8.5,比野生型向碱性偏移0.5;活性中心残基酸性基团的解离常数(pK1)由野生型的7.0提高至7.5。晶体结构分析表明,突变位点R526距离活性中心较近,将其突变为Ser降低了活性中心的极性,抑制了催化残基His的解离,使酸性基团的解离常数升高。     

Improvement of the Optimum Temperature of Penicillium expansum Lipase by Site-Directed Mutagenesis

In order to improve the optimum temperature of lipases,the Penicillum expansum lipase (PEL) gene was mutated by site-directed mutagenesis using overlap extension PCR technique.The recombinant plasmid containing mutant E83 V pPIC3.5K-lip-E83V was expressed in Pichia pastoris GS 115.Comparison experiments of the mutant PEL-E83 V-GS and the wild-type PEL-GS showed that the optimum temperature (45℃) of the mutant was 5℃ higher than that of the wild type.The thermostability of the mutant was similar to that of the wild type.The enzymatic activity of the mutant was 188 U/ml at 37℃,which was 80% that of the wild type in the same conditions.Hydrophobic interaction may be enhanced in the surface region by the hydrophilic amino acid Glu substituted with the hydrophobic amino acid Val,and may be responsible for the improvement of the optimum temperature.     

Active-site residues of the transpeptidase domain of penicillin-binding protein 2 from Escherichia coli: similarity in catalytic mechanism to class A beta-lactamases.

By means of amino acid sequence alignment with class A beta-lactamases, the residues essential for the catalytic activity of the peptidoglycan transpeptidase of penicillin-binding protein 2 (PBP2) have been predicted to be Lys333, Asp447, and Lys544, in addition to the acylation site residue for the acyl-enzyme mechanism, Ser330. Accordingly, these residues were replaced by site-directed mutagenesis, and the resultant mutants were examined as to penicillin-binding activity and genetic complementation, which represent only the acylation step and the total reaction during transpeptidation, respectively. All the mutants at position 333 showed the complete loss of both the binding and complementation activities. Most of the mutants at position 447 retained the binding activity but lost the complementation activity, the exception being the D447E mutant, which retained both. The binding rates for various penicillins of the D447N mutant, which had lost the complementation activity, were almost identical to those of the wild type. The binding of the mutants at position 544 tended to require a higher penicillin concentration, and that of the K544H mutant required a lower pH. When the roles of the counterpart residues, Lys73, Glu166, and Lys234, in class A beta-lactamases were considered, the results suggested that Lys333 and Asp447 are essential for the acylation and acyl-transfer steps, respectively, and that Lys544 stabilizes the Michaelis complex through its side-chain positive charge.     

Effect of a charged residue at the 213th site of thermolysin on the enzymatic activity

Considering the electrostatic potential of active site, four mutants of thermolysin (EC 3.4.24.4) are designed in an attempt to change the optimum pH of the hydrolytic activity toward acidic regions. On the basis of the numerical calculation of the electrostatic potential in the thermolysin molecule, Asp213 is targeted to be replaced by a basic residue, His, Lys, Arg or a neutral one, Asn. The mutant enzymes are produced inBacillus subtilis as a host using the method of site-directed mutagenesis and their optimum pH values for hydrolyzing a synthetic substrate furylacryloyl-Gly-l-Leu-NH₂ are found to be lowered by 0.2–0.4 pH units with reference to the wild type enzyme. The pl shifts of the mutants are evaluated. Neither optimum pH nor pl shift can be explained by the contribution of the pK change only at the mutation site. We find a clear negative correlation between the activities at pH 7.0 and the pI values among the four mutants and wild-type enzyme. It suggests that the contribution of pK shift of other residues must be taken into account in order to explain the activity change. Little change of thermal stability is observed among the mutants and wild type enzymes.     

Mutational effects on thermostable superoxide dismutase from Aquifex pyrophilus: understanding the molecular basis of protein thermostability

We designed two mutants of superoxide dismutase (SOD), one is thermostable and the other is thermolabile, which provide valuable insight to identify amino acid residues essential for the thermostability of the SOD from Aquifex pyrophilus (ApSOD). The mutant K12A, in which Lys12 was replaced by Ala, had increased thermostability compared to that of the wild type. The T(1/2) value of K12A was 210 min and that of the wild type was 175 min at 95 degrees C. However, the thermostability of the mutant E41A, which has a T(1/2) value of 25 min at 95 degrees C, was significantly decreased compared to the wild type of ApSOD. To explain the enhanced thermostability of K12A and thermolabile E41A on the structural basis, the crystal structures of the two SOD mutants have been determined. The results have clearly shown the general significance of hydrogen bonds and ion-pair network in the thermostability of proteins.     

Improved thermostability and the optimum temperature of Rhizopus arrhizus lipase by directed evolution

To expand the functionality of lipase from Rhizopus arrhizus (RAL) we have used error-prone PCR and DNA shuffling methods to create RAL mutants with improved thermostability and the optimum temperature. One desirable mutant with three amino acids substitution was obtained. The mutated lipase was purified and characterized. The optimum temperature of the mutant lipase was higher by 10 °C than that of the wild-type RAL (WT-RAL). In addition, the thermostability characteristic of the mutant was also improved as the result of directed evolution. The half-life (T_1/2) at 50 °C of the mutant exceeded those of WT-RAL by 12-fold. To confirm which substitution contributed to enhance thermostability and the optimum temperature for lipase activity, three chimeric lipases: chimeric lipase 1(CL-1; A9T), chimeric lipase 2 (CL-2; E190V) and chimeric lipase 3 (CL-3; M225I) from the WT-RAL gene were constructed. Each of the chimeric enzymes was purified and characterized. Amino acid substitution at position 190 was determined to be critical for lipase thermostability and the optimum temperature, while the residue at position 9 and 225 had only marginal effect. The mutational effect is interpreted according to a simulated three-dimensional structure for the mutant lipase.     

Combinatorial reshaping of a lipase structure for thermostability: Additive role of surface stabilizing single point mutations

Thermostable lipases are of high priority for industrial applications. In the present study, targeted improvement of the thermostability of a lipase from metagenomic origin was examined by using a combinatorial protein engineering approach exploring additive effects of single amino acid substitutions. A variant (LipR5) was generated after combination of two thermostabilizing mutations (R214C & N355K). Thermostability of the variant enzyme was analyzed by half-life measurement and circular dichroism (CD). To assess whether catalytic properties were affected by mutation, the optimal reaction conditions were determined. The protein LipR5, displayed optimum activity at 50 °C and pH 8.0. It showed two fold enhancement in thermostability (at 60 °C) as compared to LipR3 (R214C) and nearly 168 fold enhancement as compared to parent enzyme (LipR1). Circular dichroism and fluorescence study suggest that the protein structure had become more rigid and stable to denaturation. Study of 3D model suggested that Lys355 was involved in formation of a Hydrogen bond with OE1 of Glu284. Lys355 was also making salt bridge with OE2 of Glu284.     

易错PCR提高华根霉脂肪酶的热稳定性

为了提高华根霉Rhizopus chinensis CCTCC M201021脂肪酶的热稳定性,运用定向进化-易错PCR的方法,经两轮易错PCR引入突变,利用fast-blue RR顶层琼脂法对突变文库进行筛选,第一轮易错PCR后筛选到2株突变菌株,第二轮筛选到4株突变株。第二轮最佳突变株Ep2-4,其中3个氨基酸发生了突变：A129S、P168L和V329A。该突变酶ep2-4在60 ℃下半衰期相对原始酶r27RCL提高5.4倍,T50值提高7.8 ℃。酶学性质研究表明,突变酶ep2-4在热稳定性提高的基础上,仍保有良好的催化活性。蛋白质三维结构模拟显示,突变A129S可以和Gln133形成氢键,增加了酶表面的亲水性和极性;P168L可以与邻近的Leu164形成疏水键,导致突变酶的热稳定性提高。     

Thermostability of manganese- and iron-superoxide dismutases from Escherichia coli is determined by the characteristic position of a glutamine residue.

The structurally homologous mononuclear iron and manganese superoxide dismutases (FeSOD and MnSOD, respectively) contain a highly conserved glutamine residue in the active site which projects toward the active-site metal centre and participates in an extensive hydrogen bonding network. The position of this residue is different for each SOD isoenzyme (Q69 in FeSOD and Q146 in MnSOD of Escherichia coli). Although site-directed mutant enzymes lacking this glutamine residue (FeSOD[Q69G] and MnSOD[Q146A]) demonstrated a higher degree of selectivity for their respective metal, they showed little or no activity compared with wild types. FeSOD double mutants (FeSOD[Q69G/A141Q]), which mimic the glutamine position in MnSOD, elicited 25% the activity of wild-type FeSOD while the activity of the corresponding MnSOD double mutant (MnSOD[G77Q/Q146A]) increased to 150% (relative to wild-type MnSOD). Both double mutants showed reduced selectivity toward their metal. Differences exhibited in the thermostability of SOD activity was most obvious in the mutants that contained two glutamine residues (FeSOD[A141Q] and MnSOD[G77Q]), where the MnSOD mutant was thermostable and the FeSOD mutant was thermolabile. Significantly, the MnSOD double mutant exhibited a thermal-inactivation profile similar to that of wild-type FeSOD while that of the FeSOD double mutant was similar to wild-type MnSOD. We conclude therefore that the position of this glutamine residue contributes to metal selectivity and is responsible for some of the different physicochemical properties of these SODs, and in particular their characteristic thermostability.     

Enhancing thermostability of a Rhizomucor miehei lipase by engineering a disulfide bond and displaying on the yeast cell surface

To increase the thermostability of Rhizomucor miehei lipase, the software Disulfide by Design was used to engineer a novel disulfide bond between residues 96 and 106, and the corresponding double cysteine mutants were constructed. The R. miehei lipase mutant could be expressed by Pichia pastoris in a free secreted form or could be displayed on the cell surface. The new disulfide bond spontaneously formed in the mutant R. miehei lipase. Thermostability was examined by measuring of hydrolysis activity using 4-nitrophenyl caprylate as a substrate. The engineered disulfide bond contributed to thermostability in the free form of the R. miehei lipase variant. The variant displayed on the yeast cell surface had significantly increased residual hydrolytic activity in aqueous solution after incubation at 60°C for 5 h and increased synthetic activity in organic solvent at 60°C. These results indicated that yeast surface display might improve the stability of R. miehei lipase, as well as amplifying the thermostability through the engineered disulfide bond.     

Critical role of Lys212 and Tyr140 in porcine NADP-dependent isocitrate dehydrogenase

Lys212 and Tyr140 are close to the enzyme-bound isocitrate in the recently determined crystal structure of porcine NADP-specific isocitrate dehydrogenase (Ceccarelli, C., Grodsky, N. B., Ariyaratne, N., Colman, R. F., and Bahnson, B. J. (2002) J. Biol. Chem. 277, 43454-43462). We have constructed mutant enzymes in which Lys212 is replaced by Gln, Tyr, and Arg, and Tyr140 is replaced by Phe, Thr, Glu, and Lys. Wild type and mutant enzymes were each expressed in Escherichia coli and purified to homogeneity. At pH 7.4, the specific activity is decreased in K212Q, K212Y, and K212R, respectively, to 0.01-9% of wild type. The most striking change is in the pH-V(max) curves. Wild type depends on the deprotonated form of a group of pKaes 5.7, whereas this pKaes is increased to 7.4 in neutral K212Q and to 8.3 in K212Y. In contrast, the positive K212R has a pKaes of 5.9. These results indicate that (by electrostatic repulsion) a positively charged residue at position 212 lowers the pK of the nearby ionizable group in the enzyme-substrate complex. Lys212 may also stabilize the carbanion formed initially on substrate decarboxylation. The Tyr140 mutants have specific activities at pH 7.4 that are reduced to 0.2-0.5% of those of wild type, whereas their Km values for isocitrate and NADP are not increased. Most notable are the altered pH-V(max) profiles. V(max) is constant from pH 5.3 to 8 for Y140F and Y140T and increases as pH is decreased for Y140E and Y140K. These results suggest that in wild type enzyme, Tyr140 is the general acid that protonates the substrate after decarboxylation and that the carboxyl and ammonium forms of Y140E and Y140K provide partial substitutes. Relative to wild type, the Y140T enzyme is specifically activated 106-fold by exogenous addition of acetic acid and 88-fold by added phenol; and the K212Q enzyme is activated 4-fold by added ethylamine. These chemical rescue experiments support the conclusion that Tyr140 and Lys212 are required for the catalytic activity of porcine NADP-dependent isocitrate dehydrogenase.     

Changes in Thermostability of Photosystem Ⅱ and Leaf Lipid Composition of Rice Mutant with Deficiency of Light-harvesting Chlorophyll Protein Complexes

We studied the difference in thermostability of photosystem Ⅱ (PSⅡ) and leaf lipid composition between a T-DNA insertion mutant rice (Oryza sativa L.) VG28 and its wild type Zhonghua11. Native green gel and SDS-PAGE electrophoreses revealed that the mutant VG28 lacked all light-harvesting chlorophyll a/b protein complexes. Both the mutant and wild type were sensitive to high temperatures, and the maximal efficiency of PSⅡ photochemistry (Fv/Fm) and oxygen-evolving activity of PSⅡ in leaves significantly decreased with increasing temperature. However, the PSⅡ activity of the mutant was markedly more sensitive to high temperatures than that of the wild type. Lipid composition analysis showed that the mutant had less phosphatidylglycerol and sulfoquinovosyl diacylglycerol compared with the wild type. Fatty acid analysis revealed that the mutant had an obvious decrease in the content of unsaturation of membrane lipids on the thermostability of PSll are discussed.     

Biochemical and Structural Characterization of Two Site-Directed Mutants of <Emphasis Type="Italic">Staphylococcus xylosus</Emphasis> Lipase

Staphylococcus xylosus AF208229 lipase was expressed in E. coli containing an histidine-tag (WT-Val). In the present work, in order to check the importance of the residue 309 in the specific activity, the amino acid side chain residue valine 309 was substituted by aspartate or lysine through site-directed mutagenesis. Both mutant lipases (MUT-Lys and MUT-Asp) were expressed in E. coli and the recombinant histidine-tagged lipases were purified by immobilized metal ion affinity chromatography. The enzyme activity was determined using p-nitrophenyl butyrate as substrate and secondary structure content was evaluated by circular dichroism. MUT-Lys and MUT-Asp presented significant increase of lipase activity (P < 0.05) in comparison to WT-Val, although highest activities for the three enzymes were observed at the same pH and temperature (pH 9.0 and 42°C). The wild type and mutant lipases presented high thermal stability, after 30 min of incubation at 80°C all enzymes retained their initial activities.     

Improvement of the Optimum Temperature of Penicillium expansum Lipase by Site-Directed Mutagenesis

In order to improve the optimum temperature of lipases, the Penicillum expansum lipase (PEL) gene was mutated by site-directed mutagenesis using overlap extension PCR technique. The recombinant plasmid containing mutant E83V pPIC3.5K-lip-E83V was expressed in Pichia pastoris GS115. Comparison experiments of the mutant PEL-E83V-GS and the wild-type PEL-GS showed that the optimum temperature (45°C) of the mutant was 5°C higher than that of the wild type. The thermostability of the mutant was similar to that of the wild type. The enzymatic activity of the mutant was 188 U/ml at 37°C, which was 80% that of the wild type in the same conditions. Hydrophobic interaction may be enhanced in the surface region by the hydrophilic amino acid Glu substituted with the hydrophobic amino acid Val, and may be responsible for the improvement of the optimum temperature. Translated from Microbiology, 2005, 32(1) (in Chinese)     

A thermostable variant of P. aeruginosa cold-adapted LipC obtained by rational design and saturation mutagenesis

Cold-adapted Pseudomonas aeruginosa LipC is a secreted lipase showing differential properties compared to its well-known counterpart LipA. LipC is fundamentally a cold-acting lipase, capable of tolerating high concentrations of ions and heavy metals, and showing a shift in substrate specificity when incubated at higher temperatures. These properties make LipC an interesting enzyme, well suited for biotechnological or environmental applications, where activity at low temperatures would be required. However, a relatively low thermal resistance constitutes the main drawback for using this enzyme in long-term operational processes. To overcome the lability of LipC, we developed a rational design system to modify specific sites on the enzyme structure to obtain an improved variant of the lipase bearing higher thermal stability, but without loss of its cold-adapted properties. Eight mutant libraries plus two point mutations were constructed affecting those amino acids showing the highest flexibility on the 3D model structure. After screening more than 3000 mutant clones, a LipC variant bearing two amino acid changes and the required thermostability and cold-adapted properties was obtained. The new variant D2_H8, with a 7-fold increased thermal stability in comparison to wild type LipC, will guarantee the use and maintenance of such a lipase in a number of processes being performed at low (4–20 °C) temperatures.