Deletion combined with saturation mutagenesis of N-terminal residues in transglutaminase from Streptomyces hygroscopicus results in enhanced activity and thermostability

Transglutaminase (TGase) is an important industrial enzyme that catalyzes the cross-linking of proteins. In this study, the N-terminal residues were deleted and substituted to improve the activity and thermostability of Streptomyces hygroscopicus TGase. Seven N-terminal residues of TGase were chosen to be deleted individually. The mutated TGase missing the first four residues showed an increase in specific activity of 32.92%. The fifth residue (E5) in the N-terminus was then selected for substitution with the 19 other amino acids. The mutant replacing the fifth residue with an aspartic acid exhibited a 1.85-fold higher specific activity and a 2.7-fold longer half-life at 50 °C when compared with the wild-type enzyme. The melting temperature of the mutated TGase increased from 68.9 to 79.1 °C by circular dichroism spectroscopy analysis. This study showed that substitution combined with deletion of the N-terminal amino acids could enhance the activity and thermostability of TGase.     

Improvement of the thermostability and enzymatic activity of cholesterol oxidase by site-directed mutagenesis

Site-directed mutagenesis was applied to enhance the thermostability and enzymatic activity of cholesterol oxidase (ChOx) isolated from Brevibacterium sp. Three amino acid residues (Q153E, F128L, and S143H) located near the FAD-binding site of the enzyme were substituted based on structural analysis. The specific activity of the two-sites mutant Q153E/F128L increased by 11.6% and the relative activity increased by 47% when grown for 2 h at 50°C. This mutant is a potential industrial strain for producing ChOx.     

定点突变提高N-酰基高丝氨酸内酯酶酶活和温度稳定性

【目的】提高N-酰基高丝氨酸内酯酶(N-acylhomoserine lactonase,AiiA)酶活及温度稳定性。【方法】本研究基于AiiA同源蛋白的三维结构对AiiA进行定点突变,分析野生型AiiA及其突变蛋白酶活和温度稳定性。【结果】野生型AiiA较不稳定,在45℃下温浴30 min,或4℃储存5 d后均失去降解N-酰基高丝氨酸内酯(N-acylhomoserine lactone,AHL)的活性。但是突变AiiA蛋白(N65K,T195R和A206E)的酶活力较野生型AiiA均提高了20%以上,且4℃储存时间延长到7 d。此外,突变株N65K比野生型AiiA对高温具有更强的耐受性,在45℃温浴后剩余酶活力达到45%以上,55℃温浴30 min后仍保留5.0%的酶活力。【结论】通过定点突变改造AiiA蛋白结构,提升了AiiA蛋白的酶活和温度稳定性。     

Improvement of thermostability of fungal xylanase by using site-directed mutagenesis

Replacing several serine and threonine residues on the Ser/Thr surface of the xylanase from Aspergillus niger BCC14405 with four and five arginines effectively increases the thermostability of the enzyme. The modified enzymes showed 80% of maximal activity after incubating in xylan substrate for 2h at 50 degrees C compared to only 15% activity for wild-type enzyme. The half-life of the mutated enzymes increased to 257+/-16 and 285+/-10 min for the four- and five-arginine mutants, respectively, compared to 14+/-1 min for the wild-type enzyme. Thus, the arginine substitutions effectively increase stability by 18-20-fold. Kinetic parameters of the four-arginine-substitution enzyme were maintained at the level of the wild-type enzyme with the K(m) and V(max) values of 8.3+/-0.1 mgml(-1) and 9556+/-66 (n=3) U mg(-1) protein, respectively. The five-arginine-substitution enzyme showed only slight alteration in K(m) and V(max) with K(m) of 11.7+/-1.7 mgml(-1) and V(max) of 8502+/-65 Umg(-1) protein, indicating lower substrate affinity and catalytic rate. Our study demonstrated that properly introduced arginine residues on the Ser/Thr surface of xylanase family 11 might be very effective in improvement of enzyme thermostability.     

Improvement in thermostability and psychrophilicity of psychrophilic alanine racemase by site-directed mutagenesis

A psychrophilic alanine racemase from Bacillus psychrosaccharolyticus has a higher catalytic activity than a thermophilic alanine racemase from Bacillus stearothermophilus even at 60 °C in the presence of pyridoxal 5′-phosphate (PLP), although the thermostability of the former enzyme is lower than that of the latter one [FEMS Microbial. Lett. 192 (2000) 169]. In order to improve the thermostability of the psychrophilic enzyme, two hydrophilic amino acid residues (Glu150 and Arg151) at a surface loop surrounding the active site of the enzyme were substituted with the corresponding residues (Val and Ala) in the B. stearothermophilus alanine racemase. The mutant enzyme (ER150,151VA) showed a higher thermostability, and a markedly lower K_m value for PLP, than the wild type one. In addition, the catalytic activities at low temperatures and kinetic parameters of the two enzymes indicated that the mutant enzyme was more psychrophilic than the wild type one. Thus, the psychrophilic alanine racemase was improved in both psychrophilicity and thermostability by the site-directed mutagenesis. The mutant enzyme may be useful for the production of stereospecifically deuterated NADH and various -amino acids.     

Mutant subtilisin E with enhanced protease activity obtained by site-directed mutagenesis

The specific activity of subtilisin E, an alkaline serine protease of Bacillus subtilis, was substantially increased by optimizing the amino acid residue at position 31 (Ile in the wild-type enzyme) in the vicinity of the catalytic triad of the enzyme. Eight uncharged amino acids (Cys, Ser, Thr, Gly, Ala, Val, Leu, and Phe) were introduced at this site, which is next to catalytic Asp32, using site-directed mutagenesis. Mutant enzymes were expressed in Escherichia coli and were prepared from the periplasmic space. Only the Val and Leu substitutions gave active enzyme, and the Leu31 mutant was found to have a greatly increased activity compared to the wild-type enzyme. The other six mutant enzymes showed a marked decrease in activity. This result indicates that a branched-chain amino acid at position 31 is essential for the expression of subtilisin activity and that the level of the activity depends on side chain structure. The purified Leu31 mutant enzyme was analyzed with respect to substrate specificity, heat stability, and optimal temperature. It was found that the Leu31 replacement caused a prominent 2-6-fold increase in catalytic efficiency (kcat/Km) due to a larger kcat for peptide substrates.     

Crystal structure and site-directed mutagenesis of enzymatic components from Clostridium perfringens iota-toxin

Iota-toxin from Clostridium perfringens type E is an ADP-ribosylating toxin (ADPRT) that ADP-ribosylates actin, which is lethal and dermonecrotic in mammals. It is a binary toxin composed of an enzymatic component (Ia) and a binding component (Ib). Ia ADP-ribosylates G-actin at arginine 177, resulting in the depolymerization of the actin cytoskeleton. Here, we report on studies of the structure-function relationship by the crystal structures of Ia complexed with NADH and NADPH (at 1.8 A and 2.1 A resolution, respectively) and mutagenesis that map the active residues. The catalytic C-domain structure was similar to that of Bacillus cereus vegetative insecticidal protein (VIP2), which is an insect-targeted toxin, except for the EXE loop region. However, a significant structural difference could be seen in the N-domain, which interacts with Ib, suggesting an evolutionary difference between mammalian-targeted and insect-targeted ADPRT. The high resolution structure analysis revealed specific NAD conformation (a ring-like conformation of nicotinamide mononucleotide (NMN)) supported by Arg295, Arg296, Asn335, Arg352 and Glu380. Additionally, the mutagenesis study showed that the residues Tyr251, Arg295, Glu301, Ser338, Phe349, Arg352 and Glu380, including a newly identified one, are essential for NAD(+)-glycohydrolase (NADase) activity. At least one residue, Glu378, is an essential residue for ADP-ribosyltransferase (ARTase), but not for NADase. Consequently, the structural feature and these mutagenesis findings suggest that the catalytic mechanism of Ia proceeds via an Sn1-type reaction.     

Improving the thermostability of raw-starch-digesting amylase from a Cytophaga sp. by site-directed mutagenesis

A heat-stable raw-starch-digesting amylase (RSDA) was generated through PCR-based site-directed mutagenesis. At 65 degrees C, the half-life of this mutant RSDA, which, compared with the wild-type RSDA, lacks amino acids R178 and G179, was increased 20-fold. While the wild type was inactivated completely at pH 3.0, the mutant RSDA still retained 41% of its enzymatic activity. The enhancement of RSDA thermostability was demonstrated to be via a Ca(2+)-independent mechanism.     

Improvement in the thermostability of D-psicose 3-epimerase from Agrobacterium tumefaciens by random and site-directed mutagenesis

The S213C, I33L, and I33L S213C variants of D-psicose 3-epimerase from Agrobacterium tumefaciens, which were obtained by random and site-directed mutagenesis, displayed increases of 2.5, 5, and 7.5°C in the temperature for maximal enzyme activity, increases of 3.3-, 7.2-, and 29.9-fold in the half-life at 50°C, and increases of 3.1, 4.3, and 7.6°C in apparent melting temperature, respectively, compared with the wild-type enzyme. Molecular modeling suggests that the improvement in thermostability in these variants may have resulted from increased putative hydrogen bonds and formation of new aromatic stacking interactions. The immobilized wild-type enzyme with and without borate maintained activity for 8 days at a conversion yield of 70% (350 g/liter psicose) and for 16 days at a conversion yield of 30% (150 g/liter psicose), respectively. After 8 or 16 days, the enzyme activity gradually decreased, and the conversion yields with and without borate were reduced to 22 and 9.6%, respectively, at 30 days. In contrast, the activities of the immobilized I33L S213C variant with and without borate did not decrease during the operation time of 30 days. These results suggest that the I33L S213C variant may be useful as an industrial producer of D-psicose.     

Identification of essential amino acid residues for catalytic activity and thermostability of novel chitosanase by site-directed mutagenesis

The functional importance of a conserved region in a novel chitosanase from Bacillus sp. CK4 was investigated. Each of the three carboxylic amino acid residues (Glu-50, Glu-62, and Asp-66) was changed to Asp and Gln or Asn and Glu by site-directed mutagenesis, respectively. The Asp-66-->Asn and Asp-66-->Glu mutation remarkably decreased kinetic parameters such as Vmax and kcat to approximately 1/1,000 those of the wild-type enzyme, indicating that the Asp-66 residue was essential for catalysis. The thermostable chitosanase contains three Cys residues at positions 49, 72, and 211. The Cys-49-->Ser/Tyr and Cys-72-->Ser/Tyr mutant enzymes were as stable to thermal inactivation and denaturating agents as the wild-type enzyme. However, the half-life of the Cys-211-->Ser/Tyr mutant enzyme was less than 10 min at 80 degrees C, while that of the wild-type enzyme was about 90 min. Moreover, the residual activity of Cys-211-->Ser/Tyr enzyme was substantially decreased by 8 M urea; and it lost all catalytic activity in 40% ethanol. These results show that the substitution of Cys with any amino acid residues at position 211 seems to affect the conformational stability of the chitosanase.     

Improved thermostability and PCR efficiency of Thermococcus celericrescens DNA polymerase via site-directed mutagenesis

The Thermococcus celericrescens (Tcel) DNA polymerase gene, which contains a 2328-bp open reading frame that encodes 775 amino acid residues, was expressed in the Escherichia coli strain Rosetta(DE3)pLysS. The expressed enzyme was purified through heat treatment, HisTrap™ HP column chromatography and then HiTrap™ SP HP column chromatography. Tcel DNA polymerase has poor thermostability and PCR efficiency compared to those of other family B DNA polymerases. To improve thermostability and PCR efficiency, mutant Tcel DNA polymerases were created via site-directed mutagenesis. Specifically, we targeted the A752 residue for enhanced thermostability and the N213 residue for improved PCR efficiency. The mutant Tcel DNA polymerases all showed enhanced PCR efficiency and thermostability compared to those of the wild-type Tcel DNA polymerase. Specifically, the double mutant TcelA752K/N213D DNA polymerase had an approximately three-fold increase in thermostability over that of the wild-type enzyme and amplified a long 10-kb PCR product in an extension time of 2 min. However, there was a small change in the 3′ → 5′ exonuclease activity compared with that of the wild-type Tcel DNA polymerase, even though the mutation is in the ExoII motif. The double mutant TcelA752K/N213D DNA polymerase had a 2.6-fold lower error rate compared to that of Taq DNA polymerase. It seems that the double mutant TcelA752K/N213D DNA polymerase can be used in LA (long and accurate) PCR.     

Enhancement of a bacterial laccase thermostability through directed mutagenesis of a surface loop

Laccases (benzenediol oxygen oxidoreductases, EC 1.10.3.2) are used in many biotechnological processes, including removal of polyphenols in beverages, decolorizing and detoxifying effluents, drug analysis and bioremediation. In the present work, we have tried to increase thermal stability of laccase from Bacillus HR03 using site directed point mutations. Glu188 was substituted with 2 positive (Lys and Arg) and one hydrophobic (Ala) residues. All mutations showed improved thermal stability. Thermal activation of laccase was also increased after introducing the mutations. Remarkably, the Glu188Lys variant showed 3-fold higher thermal activation and higher T₅₀ (5 °C) with respect to the native enzyme. Furthermore steady-state k_cat and K_m values were influenced despite the distance between the mutated position and the catalytic site. In Glu188Arg mutation, the k_cat was improved 3-fold and K_m reduced by 25%. Interestingly, all three variants showed higher stability against urea as a chemical denaturant. Structural analyses of the native and mutated variants were carried out using fluorescence and far-UV circular dichroism.     

Improving the functional expression of a Bacillus licheniformis laccase by random and site-directed mutagenesis

Background  

Laccases have huge potential for biotechnological applications due to their broad substrate spectrum and wide range of reactions they are able to catalyze. These include, for example, the formation and degradation of dimers, oligomers, polymers, and ring cleavage as well as oxidation of aromatic compounds. Potential applications of laccases include detoxification of industrial effluents, decolorization of textile dyes and the synthesis of natural products by, for instance, dimerization of phenolic acids. We have recently published a report on the cloning and characterization of a CotA Bacillus licheniformis laccase, an enzyme that catalyzes dimerization of phenolic acids. However, the broad application of this laccase is limited by its low expression level of 26 mg l^-1 that was achieved in Escherichia coli. To counteract this shortcoming, random and site-directed mutagenesis have been combined in order to improve functional expression and activity of CotA.     

Improvement of stability and enzymatic activity by site-directed mutagenesis of E. coli asparaginase II

Bacterial asparaginases (EC 3.5.1.1) have attracted considerable attention because enzymes of this group are used in the therapy of certain forms of leukemia. Class II asparaginase from Escherichia coli (EcA), a homotetramer with a mass of 138 kDa, is especially effective in cancer therapy. However, the therapeutic potential of EcA is impaired by the limited stability of the enzyme in vivo and by the induction of antibodies in the patients. In an attempt to modify the properties of EcA, several variants with amino acid replacements at subunit interfaces were constructed and characterized. Chemical and thermal denaturation analysis monitored by activity, fluorescence, circular dichroism, and differential scanning calorimetry showed that certain variants with exchanges that weaken dimer–dimer interactions exhibited complex denaturation profiles with active dimeric and/or inactive monomeric intermediates appearing at low denaturant concentrations. By contrast, other EcA variants showed considerably enhanced activity and stability as compared to the wild-type enzyme. Thus, even small changes at a subunit interface may markedly affect EcA stability without impairing its catalytic properties. Variants of this type may have a potential for use in the asparaginase therapy of leukemia.     

Cumulative improvements of thermostability and pH-activity profile of Aspergillus niger PhyA phytase by site-directed mutagenesis

Aspergillus niger phytase (PhyA) has been used as a feed supplement to reduce manure phosphorus excretion of swine and poultry but lacks sufficient thermostability for feed pelleting and appropriate pH-activity profile for phytate hydrolysis in the stomach of animals. Previously, a thermostable mutant PhyA18 and two pH-activity profile-improved mutants E228K and K300E were developed. In this study, the mutations were combined to determine if both improvements were cumulative. Four substitutions (S149P, F131L, K112R, and K195R) identified from random mutagenesis were added sequentially to the combined mutants to further improve their thermostability. Mutant E228K shifted the optimum pH of the parent one from 5.5 to 4.0 and increased (P < 0.05) its specific activity at pH 3.5, whereas mutant K300E eliminated the activity dip at pH 3.5 shown in the wild type. Mutant S149P further improved thermostability over PhyA18. Our results illustrate the feasibility and structural basis to improve thermostability and pH-activity profile of PhyA phytase by assembling mutations derived from rational design and random mutagenesis.     

利用定点突变法研究精氨酸脱亚胺酶活性的影响机制

精氨酸脱亚胺酶(ADI)是一种针对精氨酸缺陷型癌症(如:肝癌、黑素瘤)的新药,目前处于临床三期试验。文中通过定点突变技术分析了精氨酸脱亚胺酶的特定氨基酸位点对酶活力的影响机制。针对已报道的关键氨基酸残基A128、H404、I410,采用QuikChange法进行定点突变,获得ADI突变株M1(A128T)、M2(H404R)、M3(I410L)和M4(A128T/H404R)。将突变株在大肠杆菌BL21(DE3)中进行重组表达,并对纯化获得的突变蛋白进行酶学性质研究。结果表明,突变位点A128T和H404R对ADI最适pH的提高,生理中性(pH 7.4)条件下的酶活力和稳定性的提高,以及Km值的降低均具有显著的作用。研究结果为阐明ADI的酶活力影响机制和蛋白质的理性改造提供了一定的依据。     

Use of coagglutination for serotyping Shigella dysenteriae and Shigella boydii

The coagglutination test was used to identify Shigella boydii and Shigella dysenteriae. A trial was carried out with 13 native rabbit antisera to S. boydii and 10 antisera to S. dysenteriae, as well as with coagglutinating reagents prepared from these antisera. The use of coagglutinating reagents was shown to ensure the complete specificity of the results, to prevent the adsorption of diagnostic antisera and to decrease their consumption 50 times. The importance of the coagglutination test for the identification of shigellae is discussed.     

Characterization of purified Shiga toxin from Shigella dysenteriae 1

Shiga toxin was purified from the culture supernatant of Shigella dysenteriae 1 by ammonium sulfate fractionation, DEAE-cellulose column chromatography and repeated chromatofocusing column chromatography. About 1.6 mg of purified Shiga toxin was obtained from 15 liters of culture with a yield of about 27%. The molecular weight of purified Shiga toxin was estimated to be 62,000. The toxin consisted of A and B subunits with molecular weights of about 30,000 and 5,000-6,000, respectively. The isoelectric point of purified Shiga toxin was 7.0. Purified Shiga toxin showed the following biological activities: lethal toxicity to mice when injected intraperitoneally with an LD50 of 28 ng per mouse; cytotoxicity to Vero cells, killing about 50% of the cells at 1 pg and all of the cells at 10 pg; and fluid accumulation in rabbit ileal loops at a concentration of more than 1 microgram.