L-asparaginase (ASNase) from Escherichia coli (EcAII) is used in the treatment of acute lymphoblastic leukaemia (ALL). EcAII activity in vivo has been described to be influenced by the human lysosomal proteases asparaginyl endopeptidase (AEP) and cathepsin B (CTSB); these hydrolases cleave and could expose epitopes associated with the immune response against EcAII. In this work, we show that ASNase resistance to CTSB and/or AEP influences the formation of anti-ASNase antibodies, one of the main causes of hypersensitivity reactions in patients. Error-prone polymerase chain reaction was used to produce variants of EcAII more resistant to proteolytic cleavage by AEP and CTSB. The variants with enzymatic activity and cytotoxicity levels equivalent to or better than EcAII WT were submitted to in vivo assays. Only one of the mutants presented increased serum half-life, so resistance to these proteases is not the only feature involved in EcAII stability in vivo. Our results showed alteration of the phenotypic profile of B cells isolated after animal treatment with different protease-resistant proteoforms. Furthermore, mice that were exposed to the protease-resistant proteoforms presented lower anti-asparaginase antibodies production in vivo. Our data suggest that modulating resistance to lysosomal proteases can result in less immunogenic protein drugs.

In this study, the prokaryotic expression system of Escherichia coli was used to modify prolyl aminopeptidase derived from Aspergillus oryzae JN-412 (AoPAP) via random mutagenesis and site-directed saturation mutagenesis. A random mutant library with a capacity of approximately 3000 mutants was compiled using error-prone polymerase chain reaction, and nonconservative amino acids within 3 Å of the substrate L-proline-p-nitroaniline were selected as site-directed saturation mutagenesis sites via homologous simulation and molecular docking of AoPAP. Variants featuring high catalytic efficiency were screened by a high-throughput screening method. The specific activities of the variants of 3D9, C185V, and Y393W were 127 U mg-1, 156 U mg-1, and 120 U mg-1, respectively, which were 27%, 56%, and 20% higher than those of the wild type, with a value of 100 U mg-1. The half-life of thermostability of the mutant 3D9 was 4.5 h longer than that of the wild type at 50 °C. The mutant C185V improved thermostability and had a half-life 2 h longer than that of the wild type at a pH of 6.5. Prolyl aminopeptidase had improved stability within the acidic range and thermostability after modification, making it more suitable for a synergistic combination with various acidic and neutral endoproteases.

The variant Lip-T (S88T/A99N/V116D) of lipase Lip from Thermomyces lanuginosus has been proved to be a potential biocatalyst for kinetic resolution of 2-carboxyethyl-3-cyano-5-methylhexanoic acid ethyl ester (CNDE) to produce valuable chiral intermediate of Pregabalin. In this study, random, site-directed and site-saturation mutagenesis were performed to further enhance the activity of Lip-T, and the key residues responsible for catalytic efficiency were revealed. A mutant S63L/D232A with improved activity toward CNDE was obtained after screening of approximately 2500 clones from random-mutant libraries. Site-directed mutagenesis at site 63 and 232 demonstrated that the single-point mutants S63L and D232A showed opposite effect on activity. S63L exhibited a significant improvement on activity, whereas D232A exerted a slight inhibitory effect. Then a mutant S63M with a 4.5-fold higher catalytic efficiency than Lip-T was obtained by site-saturation mutagenesis. Structural changes resulting from the mutations were analyzed and the mechanisms responsible for the enhanced activity were discussed. Moreover, the engineered lipase catalyzed enantioselective hydrolysis of CNDE at a very high substrate loading (765 g/l). As only 5% (w/v) resting cells were used, the bioprocess is much more cost-effective than Pfizer\'s process using 8% (w/v) commercially available lipase Lipolase(®). These results provide not only new insights into lipase structure-function relationships but also a novel robust biocatalyst for the production of Pregabalin.

Genetic diversity creation is a core technology in directed evolution where a high quality mutant library is crucial to its success. Owing to its importance, the technology in genetic diversity creation has seen rapid development over the years and its application has diversified into other fields of scientific research. The advances in molecular cloning and mutagenesis since 2008 were reviewed. Specifically, new cloning techniques were classified based on their principles of complementary overhangs, homologous sequences, overlapping PCR and megaprimers and the advantages, drawbacks and performances of these methods were highlighted. New mutagenesis methods developed for random mutagenesis, focused mutagenesis and DNA recombination were surveyed. The technical requirements of these methods and the mutational spectra were compared and discussed with references to commonly used techniques. The trends of mutant library preparation were summarised. Challenges in genetic diversity creation were discussed with emphases on creating \"smart\" libraries, controlling the mutagenesis spectrum and specific challenges in each group of mutagenesis methods. An outline of the wider applications of genetic diversity creation includes genome engineering, viral evolution, metagenomics and a study of protein functions. The review ends with an outlook for genetic diversity creation and the prospective developments that can have future impact in this field.