Lysostaphin (Lysn) is an antibacterial metalloendopeptidase that cleaves the pentaglycin bridges in the cell wall of Staphylococci. Although many studies have demonstrated its high activity in vitro, the medical application of Lysn has been hampered by its short half-life in vivo. In order to enhance its stability in vivo without significantly suppressing the enzymatic activity, we designed and tested eight single cysteine substitutions in Lysn for covalent attachment of polyethylene glycol chains (PEGylation). The purified mutants, fully reduced by Dithiothreitol (DTT), were treated with mPEG-MAL(20 kDa). The PEG modification efficiency was above 70% as determined by reverse-phase high-pressure liquid chromatography (HPLC) analysis. The PEG-Lysn proteins were further purified by cation exchange chromatography (MacroCap SP), reaching at least 95% purity. The activities of the PEG-Lysn proteins were determined by the turbidity and minimum inhibitory concentration (MIC) assays. We found that the PEGylated V240C and T244C mutants retained about 50% of the original antibacterial activity of Lysn. Overall, this study will help develop highly stable and active PEG-Lysn to treat systemic S. aureus infections.
Amino Acid Substitution ; Lysostaphin ; chemistry ; Polyethylene Glycols ; chemistry ; Protein Engineering ; Recombinant Proteins ; chemistry ; Staphylococcus aureus

We engineered a disulphide bridge between two adjacent double-layered beta-sheet at the N-terminal region of Trichoderma reesei endo-1,4-beta-xylanase II(XYN II) by site-directed mutagenesis. The native xylanase XYN-OU and the mutated xylanase XYN-HA12 (T2C, T28C and S156F) were separately expressed in Pichia pastoris. Both xylanases were purified and characterized. The optimum temperature of XYN-HA12 was increased from 50 degrees C to 60 degrees C, relative to XYN-OU. At 70 degrees C, the halftime of inactivation for XYN-OU and XYN-HA12 were 1 min and 14 min, respectively. The optimum pH of XYN-HA12 was 5.0, similar to XYN-OU. However, XYN-HA12 could retain over 50% activity from pH 3.0 to 10.0 at 50 degrees C for 30 min. As for XYN-OU, it could retain over 50% activity from the pH value 4.0 to 9.0 at 50 degrees C in 30 min. The result of the mutated xylanase indicated that constructed disulphide bridge could improve its thermostability at relatively higher temperature.
Amino Acid Substitution ; Disulfides ; chemistry ; metabolism ; Endo-1,4-beta Xylanases ; biosynthesis ; chemistry ; genetics ; Enzyme Stability ; genetics ; Mutagenesis, Site-Directed ; Pichia ; genetics ; metabolism ; Protein Engineering ; methods ; Recombinant Proteins ; biosynthesis ; chemistry ; Trichoderma ; enzymology ; genetics

A mesophilic xylanase from Aspergillus oryzae, abbreviated to AoXyn11A, belongs to glycoside hydrolase family 11. Using AoXyn11A as the parent, the thermotolerant hybrid xylanase, we constructed AEx11A by substituting its N-terminus with the corresponding region of a hyperthermostable family 11 xylanase, EvXyn11(TS). AoXyn11A- and AEx11A-encoding genes were expressed in Pichia pastoris GS115 separately, and effects of temperatures on expressed products were determined and compared. The optimum temperature (T(opt)) of AEx11A was 75 degrees C and its half-life at 70 degrees C (t1/2(70)) was 197 min, improved as compared with those (T(opt) = 50 degrees C, t1/2(70) = 1.0 min) of AoXyn11A. Homology modeling of the AEx11A's structure and comparison between structures of AEx11A and AoXyn11A revealed that one disulfide bridge (Cys5-Cys32) was introduced into AEx11A resulted from N-terminus substitution. To explore the effect of the disulfide bridge on the thermostability of AEx11A, it was removed from AEx11A by site-directed mutagenesis (C5T). Analytical results show that the T(opt) of the mutant AEx11A (AEx11A(C5T)) dropped to 60 degrees C from 75 degrees C of AEx11A, and its t1/2(70) and t1/2(80) also decreased to 3.0 and 1.0 min from 197 and 25 min.
Amino Acid Sequence ; Amino Acid Substitution ; Aspergillus oryzae ; enzymology ; Base Sequence ; Disulfides ; chemistry ; metabolism ; Endo-1,4-beta Xylanases ; biosynthesis ; chemistry ; genetics ; Enzyme Stability ; genetics ; Molecular Sequence Data ; Mutagenesis, Site-Directed ; methods ; Pichia ; genetics ; metabolism ; Protein Engineering ; methods ; Recombinant Proteins ; biosynthesis ; chemistry ; genetics

Aspergillus fumigatus wild-type phytase has many favorable properties, such as a good thermorstability and a broad pH optimum. However, the specific activity of the enzyme is relative low. A. fumigatus Q23L phytase resulted in a remarkable increase in specific activity around pH4.5 - 7.0, but the pH stability of Q23L was lower than A. fumigatus wild-type phytase. To increase the pH stability of Q23L, the mutant Q23LG272E was constructed by site-directed mutagenesis with PCR. The gene of A. fumigatus wild-type phytase and the mutant genes encoding the Q23LG272E and the Q23L were correctly expressed in Pichia pastoris GS115. Enzymes were purified and their enzymatic properties were determined. The results revealed that the specific activity of the Q23L improved remarkably, which increased from 51 u/mg of the wild type to 109 u/mg at pH5.5. Meanwhile, the pH stability of Q23L, decreased evidently, especially from pH3.0 to pH4.0.The pH stability of Q23LG272E in pH3.0 - 4.5 and pH6.5 - 7.0 has been improved compared with Q23L. The specific activity of Q23LG272E basically maintained at the level of Q23L. Analysis of 3-D structure and sequence similarity were used to reveal the presumable factors influencing the enzymatic properties of Q23LG272E, and discussion for the relationship between structure and function of phytase was given.
6-Phytase ; chemistry ; genetics ; metabolism ; Amino Acid Substitution ; Aspergillus fumigatus ; enzymology ; genetics ; Biocatalysis ; Electrophoresis, Polyacrylamide Gel ; Fungal Proteins ; chemistry ; genetics ; metabolism ; Hydrogen-Ion Concentration ; Models, Molecular ; Mutagenesis, Site-Directed ; Mutant Proteins ; genetics ; metabolism ; Mutation ; Pichia ; genetics ; Polymerase Chain Reaction ; Protein Conformation ; Protein Engineering ; methods ; Recombinant Proteins ; metabolism ; Structure-Activity Relationship ; Substrate Specificity

The experiment was conducted by directed evolution strategy (error-prone PCR) to improve the activity of aflatoxin detoxifzyme with the high-throughput horse radish peroxidas and recessive brilliant green (HRP-RBG) screening system. We built up a mutant library to the order of 10(4). Two rounds of EP-PCR and HRP-RBG screening were used to obtain three optimum mutant strains A1773, A1476 and A2863. We found that mutant A1773 had upper temperature tolerance of 70 degrees C and that its enzyme activity was 6.5 times higher than that of the parent strain. Mutant strains A1476 worked well at pH 4.0 and its enzyme activity was 21 times higher than that of the parent strain. Mutant A2863 worked well at pH 4.0 and pH 7.5, and its enzyme activity was 12.6 times higher than that of the parent strain. With DNA sequencing we found that mutant A1773 revealed two amino acid substitutions, Glu127Lys and Gln613Arg. Mutant A1476 revealed four amino acid substitutions: Ser46Pro, Lys221Gln, Ile307Leu and Asn471lle. Mutant A2863 revealed four amino acid substitutions: Gly73Ser, Ile307Leu, Va1596Ala and Gln613Arg. The results provided a useful illustration for the deep understanding of the relationship between the function and structure of aflatoxin detoxifzyme.
Aflatoxin B1 ; antagonists & inhibitors ; chemistry ; Amino Acid Substitution ; Directed Molecular Evolution ; Enzyme Activation ; Enzyme Stability ; Multienzyme Complexes ; genetics ; metabolism ; Mutant Proteins ; genetics ; metabolism ; Point Mutation ; Polymerase Chain Reaction ; methods ; Protein Engineering

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

Glycosidases are widely used in food and pharmaceutical industries due to its ability to hydrolyze the glycosidic bonds of various sugar-containing compounds including glycosides, oligosaccharides and polysaccharides to generate derivatives with important physiological and pharmacological activity. While glycosidases often need to be used under high temperature to improve reaction efficiency and reduce contamination, most glycosidases are mesophilic enzymes with low activity under industrial production conditions. It is therefore critical to improve the thermo-stability of glycosidases. This review summarizes the recent advances achieved in engineering the thermo-stability of glycosidases using strategies such as directed evolution, rational design and semi-rational design. We also compared the pros and cons of various techniques and discussed the future prospects in this area.
Glycoside Hydrolases/genetics* ; Oligosaccharides ; Polysaccharides ; Protein Engineering

Microbial transglutaminase, which could catalyze the cross-linking of many proteins or non-protein materials, has been widely used in food, pharmaceutical and textile industry. To enhance the yield of the enzyme and establish corresponding platform for molecular modification, the researchers of Japanese Ajinomoto began to construct the recombinant strain producing transglutaminase in the 1990s. So far, the enzyme has been successfully expressed in different expression systems. Some of the recombinant strains are more productive than wild strains. Recently, progress has been made in the molecular modification of microbial transglutaminase, and the activity, thermo-stability and specificity of the enzyme are improved. This review briefly summarized and analyzed the strategies involved in these studies, and noted its trends.
Bacterial Proteins ; biosynthesis ; genetics ; Catalysis ; Enzyme Stability ; Mutagenesis, Site-Directed ; Mutant Proteins ; genetics ; metabolism ; Protein Engineering ; methods ; Streptomyces ; enzymology ; Substrate Specificity ; Transglutaminases ; biosynthesis ; genetics

In order to improve the thermostability of the Penicillium expansum Lipase (PEL), the lipase encoding genes was mutated by site-directed mutagenesis. A recombinant vector pAO815-ep8-K55R which contain double mutant genes was constructed by overlap extension PCR using the cDNA of a random-mutant lipase ep8 (a single site mutant) as the template and two special primers were used to generate another mutation site K55R. The recombinant vector was transformed into Pichia pastoris GS115 by electroporation and the recombinant mutant GS-pAO815-ep8- K55R can secret double-mutant lipase PEL-ep8-K55R-GS into the medium when it was induced by Methanol. The yield of the double-mutant lipase is 508 u/mL, which is 81% that of the wild type lipase PEL-GS (627 u/mL) and 55% that of random-mutant PEL-ep8-GS (924 u/mL). The specific activity of double-mutant lipase is 2309.1 u/mg, which is similar to random-mutant lipase PEL-ep8-GS and the wild type lipase PEL-GS. The optimum temperature of the double-mutant lipase is same with the wild type lipase PEL-GS and random-mutant lipase PEL-ep8-GS. While the Tm of the double-mutant lipase is 41.0 degrees C, 2.3 degrees C higher than the wild type lipase PEL-GS and 0.8% higher than the random-mutant lipase PEL-ep8-GS, indicating that the double-mutant lipase PEL-ep8-K55R-GS has higher thermostability.
Electroporation ; Enzyme Stability ; Hot Temperature ; Lipase ; genetics ; metabolism ; Mutagenesis, Site-Directed ; Mutant Proteins ; metabolism ; Penicillium ; enzymology ; Pichia ; genetics ; metabolism ; Protein Engineering ; methods ; Recombinant Proteins ; biosynthesis ; genetics

In proteins of thermophilic bacteria, Gly is tend to be replaced by Ala and Lys is tend to be replaced by Arg to adapt the high temperature. In order to improve the thermal stability of phenylalanine hydroxylase (PAH) from Chromobacterium violaceum, all the Gly on PAH were mutated to Ala and Lys to Arg. Positive mutant enzymes with improved thermal stability were selected, followed by combined mutation and characterization. The results revealed that half-lives of K94R and G221A mutants at 50 °C were 26.2 min and 16.8 min, which were increased by 1.9-times and 0.9-times than the parent enzyme (9.0 min). The residual activity of K94R/G221A mutant was improved to 65.6% after keeping at 50 °C for 1 h, which was 6.6 time higher than the parent enzyme (8.6%). Circular dichroism (CD) spectroscopy revealed that Tm values of the parent enzyme, K94R, G221A and K94R/G221A were 51.5 ℃, 53.8 ℃, 53.1 ℃ and 54.8 ℃, respectively. According to the protein structure simulation, the two mutations were located on flexible loop. In the K94R mutant, the mutated Arg94 on the surface of the enzyme formed an extra hydrogen bond with Ile95, which stabilized the located loop. In the G221A mutant, the mutated Ala221 formed hydrophobic interaction with Leu281, which could stabilize both the loop and flexible area of the C-terminus of G221A. The results not only provided a reference for protein modification on thermal stability, but also laid the foundation for application of phenylalanine hydroxylase in the field of functional foods.
Bacterial Proteins ; biosynthesis ; genetics ; Chromobacterium ; enzymology ; Enzyme Stability ; Hot Temperature ; Kinetics ; Mutagenesis, Site-Directed ; Mutation ; Phenylalanine Hydroxylase ; biosynthesis ; genetics ; Protein Engineering