Creatinine levels in biological fluids are important indicators for the clinical evaluation of renal function. Creatinase (CRE, EC3.5.3.3) is one of the key enzymes in the enzymatic measurement of creatinine concentration, and it is also the rate-limiting enzyme in the whole enzymatic cascade system. The poor catalytic activity of CRE severely limits its clinical and industrial applications. To address this issue, a semi-rational design is applied to increase the activity of a creatinase from Alcaligenes sp. KS-85 (Al-CRE). By high-throughput screen of saturation mutagenesis libraries on the selected hotspot mutations, multiple variant enzymes with increased activity are obtained. The five-point best variant enzyme (I304L/F395V/K351V/Y63S/Q88A) were further obtained by recombine the improved mutations sites that to showed a 2.18-fold increased specific activity. Additionally, structure analysis is conducted to understand the mechanism of the activity change. This study paves the way for a better practical application of creatinase and may help further understand its catalytic mechanism.
Creatinine ; Mutagenesis, Site-Directed ; Ureohydrolases/genetics* ; Catalysis

Arginine deiminase (ADI) has been studied as a potential anti-cancer agent for inhibiting arginine-auxotrophic tumors (such as melanomas and hepatocellular carcinomas) in phase III clinical trials. In this work, we studied the molecular mechanism of arginine deiminase activity by site-directed mutagenesis. Three mutation sites, A128, H404 and 1410, were introduced into wild-type ADI gene by QuikChange site-directed mutagenesis method, and four ADI mutants M1 (A128T), M2 (H404R), M3 (I410L), and M4 (A128T, H404R) were obtained. The ADI mutants were individually expressed in Escherichia coli BL21 (DE3), and the enzymatic properties of the purified mutant proteins were determined. The results show that both A128T and H404R had enhanced optimum pH, higher activity and stability of ADI under physiological condition (pH 7.4), as well as reduced K(m) value. This study provides an insight into the molecular mechanism of the ADI activity, and also the experimental evidence for the rational protein evolution in the future.
Escherichia coli ; metabolism ; Hydrolases ; genetics ; metabolism ; Mutagenesis, Site-Directed

Mutants of proteins are the basis for studying their structure and function, this work aimed to establish an efficient and rapid method for constructing multi-site mutants. When four or more adjacent amino acid residues need to be mutated, firstly, two long and two short primers (long primers Ⅰ/Ⅰ, short primersⅡ/Ⅱ) were designed: the long primers contain mutated sites, and the number of mutant bases is ≤20 bp, the short primers do not contain mutated sites; GC contents of the long and short primers are ≤80%, and the difference of annealing temperature is ≤40 °C. Then two sets of reverse PCR amplifications were performed using primer pairs (Ⅰ/Ⅱand Ⅰ/Ⅱ) and templates, respectively. After amplification, each system can obtain non-methylated linear plasmids which contain mutated sites, and the breakpoints of the two sets of linear plasmids amplified by primers Ⅰ/Ⅱ and Ⅲ/Ⅳ were distributed on both sides of the mutated sites. Followed by digested by DpnⅠ to remove the methylated templates, the recovered PCR products, which were mixed in an equimolar ratio, were performed another round of denaturation and annealing: the two sets of linear plasmids were denatured at 95 °C and then annealed with each other's single-stranded DNA as templates to form open-loop plasmids, and then the transformants containing the mutations will be obtained after transformed the open-loop plasmids into Escherichia coli competent cells. Results showed that, this method can mutate 4 to 11 consecutive amino acid residues (8-20 bp) simultaneously, which will greatly simplify the construction of multi-site mutants, Thereby improve the efficiency of protein structure and function research further.
DNA Primers ; genetics ; Escherichia coli ; Mutagenesis, Site-Directed ; methods ; Plasmids ; genetics ; Polymerase Chain Reaction

OBJECTIVETo investigate the molecular mechanism of haemophilia B caused by the novel mutation of Arg327Ile (R327I) in FIX gene.

METHODSThe R327I, R327Ala(A), R327Lys(K), R327Asn(N) and a replacement mutant (FIXβFVII), in which FIX β strand 324-329 was replaced by that of FVII 298-303, expression plasmids were constructed with site-directed mutagenesis method based on the wild-type (WT) FIX expression plasmid. The HEK293 cell was transiently transfected, then the activity of FIX (FIX:C) was assayed by one stage method in the conditioned medium, while the FIX:Ag in both the conditioned media and the cell lysates was measured by ELISA. The molecular weight and the semi-quantity of expressed FIX were analyzed by Western blot. Fluorescent protein expression plasmid was constructed to investigate the synthesis and secretion of the FIX R327I mutation in the viable cells.

RESULTSFIX:C of the R327I mutant protein was 4.49% of the level of the WT in the conditioned medium, and the FIX:Ag of the R327I mutant protein in the conditioned medium and the cell lysates was 31.02% and 129.29% compared to that of WT, respectively. The mutation was characterized as cross-reaction material reduced (CRMR). The viable cell fluorescent assays showed that the R327I protein was more in both the viable cells and in lysosome than that of WT. The FIX:C of the R327A, R327K, R327N and FIXβFVII mutants was reduced compared to that of WT, the reduction of FIX:C of FIXβFVII was the most significantly amount among all the mutants in medium. FIX:Ag of all the mutants in the medium, except that the R327K increased, was reduced. The result of Western blot showed that the molecular weight of R327I protein was the same as that of WT, but the amount of the protein was much less compared with WT in the conditioned medium.

CONCLUSIONThe abnormal synthesis and secretion as well as the abnormal function of the R327I mutant protein causes haemophilia B. The residue of R327 as well as the β strand domain of R327 located play important roles of the specific function of FIX.

Factor IX ; genetics ; HEK293 Cells ; Hemophilia B ; genetics ; pathology ; Humans ; Mutagenesis, Site-Directed ; Mutation ; Transfection

Functional improvement to one component of the cellulase, endo-beta-1, 4-glucanase, has been a focus of the recent research in this area. We report here the saturation mutagenesis of the active site of an endoglucanase (CfEG) from termite Coptotermes formosanus. First, three dimensional structure of CfEG was built via homology modeling by using a close-related (79% homology in sequence) endo-beta-1,4-glucanase (NtEG PDB id = 1ks8) from higher termite Nasutitermes takasagoensis as a template. Second, we identified three corresponding amino acid positions at the active site of CfEG by structural superposition onto NtEG. These three putative amino acids for the active site of CfEG, i.e., Asp53, Asp56 and Glu411, were subjected to saturation mutagenesis using degenerate primers. Among the mutants, Asp53Glu and Asp56Cys showed somewhow higher activities than the wildtype, with the latter having more than 3-fold decrease in Km. Double mutation Asp53Leu/Asp56IIe showed nearly 2-fold increase in specific activity and in the same time 2-fold decrease in Km. Saturation mutagenesis to the position Glu411 produced no active mutant, even changing Glu411 explicitly into its similar amino acids such as Glu411Asp and Glu411Gln could not result in any active mutant. These imply that position Glu411 could be extremely important and therefore indispensable for CfEG functionality.
Amino Acids ; genetics ; Animals ; Cellulase ; chemistry ; genetics ; metabolism ; Isoptera ; enzymology ; Mutagenesis, Site-Directed ; Mutation

In order to develop a simple and efficient site-directed mutagenesis solution, the Gibson assembly technique was used to clone the cyclin dependent kinase 4 gene with single or double site mutations, with the aim to simplify the overlap extension PCR. The gene fragments containing site mutations were amplified using a strategy similar to overlap extension PCR. Meanwhile, an empty plasmid was digested by double restriction endonucleases to generate a linearized vector with a short adaptor overlapping with the targeted gene fragments. The gene fragments were directly spliced with the linearized vector by Gibson assembly in an isothermal, single-reaction, creating a recombinant plasmid. After the recombinant plasmids were transformed into competent Escherichia coli DH5α, several clones were screened from each group. Through restriction analysis and DNA sequencing, it was found that the randomly selected clones were 100% target mutants. Since there was neither tedious multiple-round PCR amplification nor frequent DNA extraction operation, and there was no need to digest the original plasmid, this protocol circumvents many factors that may interfere with the conventional site-directed mutagenesis. Hence, genes with single or multiple mutations could be cloned easily and efficiently. In summary, the major defects associated with overlap extension PCR and rolling circle amplification were circumvented in this protocol, making it a good solution for site-directed mutagenesis.
Clone Cells ; Mutagenesis, Site-Directed ; Mutation ; Plasmids/genetics* ; Polymerase Chain Reaction/methods*

Genome sequencing projects revealed massive cryptic gene clusters encoding the undiscovered secondary metabolites in Streptomyces. To investigate the metabolic products of silent gene clusters in Streptomyces chattanoogensis L10 (CGMCC 2644), we used site-directed mutagenesis to generate ten mutants with point mutations in the highly conserved region of rpsL (encoding the ribosomal protein S12) or rpoB (encoding the RNA polymerase β-subunit). Among them, L10/RpoB (H437Y) accumulated a dark pigment on a yeast extract-malt extract-glucose (YMG) plate. This was absent in the wild type. After further investigation, a novel angucycline antibiotic named anthrachamycin was isolated and determined using nuclear magnetic resonance (NMR) spectroscopic techniques. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis and electrophoretic mobility shift assay (EMSA) were performed to investigate the mechanism underlying the activation effect on the anthrachamycin biosynthetic gene cluster. This work indicated that the rpoB-specific missense H437Y mutation had activated anthrachamycin biosynthesis in S. chattanoogensis L10. This may be helpful in the investigation of the pleiotropic regulation system in Streptomyces.
Anti-Bacterial Agents/pharmacology* ; Antioxidants/pharmacology* ; Bacterial Proteins/genetics* ; Multigene Family ; Mutagenesis, Site-Directed ; Streptomyces/metabolism*

It is well known that non-aqueous enzymatic catalysis has emerged as an important area of enzyme engineering with the advantages of higher substrate solubility, increased stereoselectivity, modified substrate specificity and suppression of unwanted water-dependent side reactions. As a result, non-aqueous enzymatic catalysis has been applied in the biocatalytic synthesis of important pharmaceuticals and nutriceuticals. With the advancement of non-aqueous enzymatic catalysis in recent years, the efforts have been centered on the discovery and modification of solvent-tolerant biocatalysts for non-aqueous environments. Additionally, with the inevitable trends of green chemistry and sustainable development, green solvents have been utilized for increased number of enzymatic reactions to replace conventional organic solvents. In this review, modification, immobilization and mutagenesis of various enzymes for non-aqueous catalysis are discussed. Recent progress of non-aqueous enzymatic catalysis in solvent-free environments, reverse micelles, supercritical liquid and ionic liquid are also presented. In particular, while direct evolution, high-throughput screening and site-directed mutagenesis are combined as powerful tools for protein engineering, vapor/solid/ice water mixture, sticky solid-state liquid crystal and high density salt suspension are the future directions for solvent engineering in order to broaden the utility and elevate the efficiency of non-aqueous enzymatic catalysis.
Animals ; Biocatalysis ; Enzymes ; genetics ; metabolism ; Enzymes, Immobilized ; Humans ; Mutagenesis, Site-Directed ; Solvents

In this study, a single base editing system was used to edit the FecB and GDF9 gene to achieve a targeted site mutation from A to G and from C to T in Ouler Tibetan sheep fibroblasts, and to test its editing efficiency. Firstly, we designed and synthesized sgRNA sequences targeting FecB and GDF9 genes of Ouler Tibetan sheep, followed by connection to epi-ABEmax and epi-BE4max plasmids to construct vectors and electrotransfer into Ouler Tibetan sheep fibroblasts. Finally, Sanger sequencing was performed to identify the target point mutation of FecB and GDF9 genes positive cells. T-A cloning was used to estimate the editing efficiency of the single base editing system. We obtained gRNA targeting FecB and GDF9 genes and constructed the vector aiming at mutating single base of FecB and GDF9 genes in Ouler Tibetan sheep. The editing efficiency for the target site of FecB gene was 39.13%, whereas the editing efficiency for the target sites (G260, G721 and G1184) of GDF9 gene were 10.52%, 26.67% and 8.00%, respectively. Achieving single base mutation in FecB and GDF9 genes may facilitate improving the reproduction traits of Ouler Tibetan sheep with multifetal lambs.
Animals ; Sheep/genetics* ; Gene Editing ; Tibet ; Mutation ; Phenotype ; Mutagenesis, Site-Directed

As an efficient and promising protein engineering strategy, directed evolution includes the construction of mutant libraries and screening of desirable mutants. A rapid and high-throughput screening method has played a critical role in the successful application of directed evolution strategy. We reviewed several high-throughput screening tools which have great potential to be applied in directed evolution. The development of powerful high-throughput screening tools will make great contributions to the advancement of protein engineering.
Directed Molecular Evolution ; methods ; High-Throughput Screening Assays ; methods ; Mutagenesis, Site-Directed ; methods ; Mutant Proteins ; genetics ; Protein Engineering ; methods