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1.
Chinese Journal of Biotechnology ; (12): 4358-4375, 2023.
Article in Chinese | WPRIM | ID: wpr-1008030

ABSTRACT

Yeast surface display (YSD) is a technology that fuses the exogenous target protein gene sequence with a specific vector gene sequence, followed by introduction into yeast cells. Subsequently, the target protein is expressed and localized on the yeast cell surface by using the intracellular protein transport mechanism of yeast cells, whereas the most widely used YSD system is the α-agglutinin expression system. Yeast cells possess the eukaryotic post-translational modification mechanism, which helps the target protein fold correctly. This mechanism could be used to display various eukaryotic proteins, including antibodies, receptors, enzymes, and antigenic peptides. YSD has become a powerful protein engineering tool in biotechnology and biomedicine, and has been used to improve a broad range of protein properties including affinity, specificity, enzymatic function, and stability. This review summarized recent advances in the application of YSD technology from the aspects of library construction and screening, antibody engineering, protein engineering, enzyme engineering and vaccine development.


Subject(s)
Saccharomyces cerevisiae/metabolism , Protein Engineering , Biotechnology , Antibodies/metabolism , Amino Acid Sequence
2.
Chinese Journal of Biotechnology ; (12): 3710-3723, 2023.
Article in Chinese | WPRIM | ID: wpr-1007987

ABSTRACT

As central players in cellular structure and function, proteins have long been central themes in life science research. Analyzing the impact of protein sequence variation on its structure and function is one of the important means to study proteins. In recent years, a technology called deep mutational scanning (DMS) has been widely used in the field of protein research. It introduces thousands of mutations in parallel in specific regions of proteins through high-abundance DNA libraries. After screening, high-throughput sequencing is employed to score each mutation, revealing sequence-function correlations. Due to its high-throughput, fast and easy, and labor-saving features, DMS has become an important method for protein function research and protein engineering. This review briefly summarizes the principle of DMS technology, highlighting its applications in mammalian cells. Moreover, this review analyzes the current technical bottlenecks, aiming to facilitate relevant research.


Subject(s)
Animals , Mutation , Proteins/chemistry , Protein Engineering , High-Throughput Nucleotide Sequencing/methods , Mammals/genetics
3.
Chinese Journal of Biotechnology ; (12): 912-929, 2023.
Article in Chinese | WPRIM | ID: wpr-970413

ABSTRACT

Chitosanases represent a class of glycoside hydrolases with high catalytic activity on chitosan but nearly no activity on chitin. Chitosanases can convert high molecular weight chitosan into functional chitooligosaccharides with low molecular weight. In recent years, remarkable progress has been made in the research on chitosanases. This review summarizes and discusses its biochemical properties, crystal structures, catalytic mechanisms, and protein engineering, highlighting the preparation of pure chitooligosaccharides by enzymatic hydrolysis. This review may advance the understandings on the mechanism of chitosanases and promote its industrial applications.


Subject(s)
Chitosan/chemistry , Chitin , Glycoside Hydrolases/genetics , Protein Engineering , Oligosaccharides/chemistry , Hydrolysis
4.
Chinese Journal of Biotechnology ; (12): 632-649, 2022.
Article in Chinese | WPRIM | ID: wpr-927733

ABSTRACT

The redox biosynthesis system has important applications in green biomanufacturing of chiral compounds. Formate dehydrogenase (FDH) catalyzes the oxidation of formate into carbon dioxide, which is associated with the reduction of NAD(P)+ into NAD(P)H. Due to this property, FDH is used as a crucial enzyme in the redox biosynthesis system for cofactor regeneration. Nevertheless, the application of natural FDH in industrial production is hampered by low catalytic efficiency, poor stability, and inefficient coenzyme utilization. This review summarized the structural characteristics and catalytic mechanism of FDH, as well as the advances in protein engineering of FDHs toward improved enzyme activity, catalytic efficiency, stability and coenzyme preference. The applications of using FDH as a coenzyme regeneration system for green biomanufacturing of chiral compounds were summarized.


Subject(s)
Catalysis , Coenzymes/metabolism , Formate Dehydrogenases/metabolism , NAD/metabolism , Protein Engineering
5.
Article in Spanish | LILACS, CUMED | ID: biblio-1341785

ABSTRACT

La levadura metilotrófica Pichia pastoris (clasificada actualmente como Komagataella phaffii) es una de las más importantes para la producción de proteínas heterólogas. En el trabajo se presenta un análisis de las principales características que se ponen de manifiesto en la expresión de proteínas recombinantes expresadas en este microorganismo. Se describen las cepas disponibles para la transformación y producción de proteínas recombinantes expresadas en Pichia pastoris, los principales vectores comerciales para la expresión, los promotores más eficientes, los marcadores seleccionables, la señal de secreción, los métodos usados en las transformaciones genéticas y los patrones de glicosilación que se presentan. Se brindan recomendaciones generales acerca de los parámetros de bioprocesos como la composición del medio, el pH, la temperatura, la velocidad de aireación, la inducción y las estrategias de alimentación para alcanzar altos valores de productividad. Se presentan los resultados de las aplicaciones de Pichia pastoris en la producción de dos vacunas en Cuba, la vacuna contra la hepatitis B y la vacuna para el control de la garrapata(AU)


Pichia pastoris metylotrofic yeast (currently classified as Komagataella phaffii) is one of the most important yeast for the production of heterologous proteins. The work presents an analysis of the main characteristics that are marked in the production of recombinant proteins expressed in Pichia pastoris. It describes the strains available for the transformation and production of recombinant proteins expressed in P. pastoris, the main commercial vectors for expression, the most efficient promoters, selectable markers, the secretion signal, the methods used in genetic transformations and glycosylation patterns that occur. General recommendations are provided on bioprocess parameters such as media composition, pH, temperature, aeration velocity, induction, and feeding strategies to achieve high productivity values. The results of Pichia pastoris applications for the production of two vaccines in Cuba, the hepatitis B vaccine and the tick control vaccine are shown(AU)


Subject(s)
Pichia , Yeasts , Recombinant Proteins , Protein Engineering , Tick Control/methods , Hepatitis B Vaccines/therapeutic use , Cuba
6.
Chinese Journal of Biotechnology ; (12): 4415-4429, 2021.
Article in Chinese | WPRIM | ID: wpr-921517

ABSTRACT

The zearalenone hydrolase (ZHD101) derived from Clonostachys rosea can effectively degrade the mycotoxin zearalenone (ZEN) present in grain by-products and feed. However, the low thermal stability of ZHD101 hampers its applications. High throughput screening of variants using spectrophotometer is challenging because the reaction of hydrolyzing ZEN does not change absorbance. In this study, we used ZHD101 as a model enzyme to perform computation-aided design followed by experimental verification. By comparing the molecular dynamics simulation trajectories of ZHD101 at different temperatures, 32 flexible sites were selected. 608 saturated mutations were introduced into the 32 flexible sites virtually, from which 12 virtual mutants were screened according to the position specific score and enzyme conformation free energy calculation. Three of the mutants N156F, S194T and T259F showed an increase in thermal melting temperature (ΔTm>4 °C), and their enzyme activities were similar to or even higher than that of the wild type (relative enzyme activity 95.8%, 131.6% and 169.0%, respectively). Molecular dynamics simulation analysis showed that the possible mechanisms leading to the improved thermal stability were NH-π force, salt bridge rearrangement, and hole filling on the molecular surface. The three mutants were combined iteratively, and the combination of N156F/S194T showed the highest thermal stability (ΔTm=6.7 °C). This work demonstrated the feasibility of engineering the flexible region to improve enzyme performance by combining virtual computational mutations with experimental verification.


Subject(s)
Computer-Aided Design , Edible Grain , Enzyme Stability , Hydrolases/metabolism , Hypocreales/enzymology , Protein Engineering , Zearalenone
7.
Chinese Journal of Biotechnology ; (12): 3268-3275, 2021.
Article in Chinese | WPRIM | ID: wpr-921423

ABSTRACT

Polyethylene terephthalate (PET) is a synthetic polymer consisting of ester bond-linked terephthalate and ethylene glycol. Tremendous amounts of PET have been produced and majority of them enters terrestrial and marine environment as wastes, posing serious threats to the global ecosystems. In 2016, a PET hydrolase from a PET-assimilating bacterium Ideonalla sakaiensis was reported and termed as IsPETase. This enzyme outperforms other PET-hydrolyzing enzymes in terms of its PET hydrolytic activity at ambient temperature, thus holds a great promise for PET biodegradation. In order to improve IsPETase activity, we conducted structure-based engineering to modify the putative substrate-binding tunnel. Among the several variants to the N233 residue of IsPETase, we discovered that the substitution of N233 with alanine increases its PET hydrolytic activity, which can be further enhanced when combined with a R280A mutation. We also determined the X-ray crystal structure of the IsPETase N233A variant, which shares nearly identical fold to the WT protein, except for an open end of subsite Ⅱ. We hypothesized that the smaller side chain of N233A variant might lead to an extended subsite Ⅱ for PET binding, which subsequently increases the enzymatic activity. Thus, this study provides new clues for further structure-based engineering of PETase.


Subject(s)
Burkholderiales/enzymology , Hydrolases/genetics , Polyethylene Terephthalates/metabolism , Protein Engineering
8.
Chinese Journal of Biotechnology ; (12): 3242-3252, 2021.
Article in Chinese | WPRIM | ID: wpr-921421

ABSTRACT

L-asparaginase hydrolyzes L-asparagine to produce L-aspartic acid and ammonia. It is widely distributed in microorganisms, plants and serum of some rodents, and has important applications in the pharmaceutical and food industries. However, the poor thermal stability, low catalytic efficiency and low yield hampered the further application of L-asparaginase. In this paper, rational design and 5' untranslated region (5'UTR) design strategies were used to increase the specific enzyme activity and protein expression of L-asparaginase derived from Rhizomucor miehei (RmAsnase). The results showed that among the six mutants constructed through homology modeling combined with sequence alignment, the specific enzyme activity of the mutant A344E was 1.5 times higher than the wild type. Subsequently, a food-safe strain Bacillus subtilis 168/pMA5-A344E was constructed, and the UTR strategy was used for the construction of recombinant strain B. subtilis 168/pMA5 UTR-A344E. The enzyme activity of B. subtilis 168/pMA5 UTR-A344E was 7.2 times higher than that of B. subtilis 168/pMA5-A344E. The recombinant strain B. subtilis 168/pMA5 UTR-A344E was scaled up in 5 L fermenter, and the final yield of L-asparaginase was 489.1 U/mL, showing great potential for industrial application.


Subject(s)
Asparaginase/genetics , Bacillus subtilis/genetics , Industrial Microbiology , Protein Engineering , Rhizomucor/enzymology , Sequence Alignment
9.
Chinese Journal of Biotechnology ; (12): 2105-2115, 2021.
Article in Chinese | WPRIM | ID: wpr-887784

ABSTRACT

Triterpenoids are a class of natural products of great commercial value that are widely used in pharmaceutical, health care and cosmetic industries. The biosynthesis of triterpenoids relies on the efficient synthesis of squalene epoxide, which is synthesized from the NADPH dependent oxidation of squalene catalyzed by squalene epoxidase. We screened squalene epoxidases derived from different species, and found the truncated squalene epoxidase from Rattus norvegicus (RnSETC) showed the highest activity in engineered Escherichia coli. Further examination of the effect of endogenous cytochrome P450 reductase like (CPRL) proteins showed that overexpression of NADH: quinone oxidoreductase (WrbA) under Lac promoter in a medium-copy number plasmid increased the production of squalene epoxide by nearly 2.5 folds. These results demonstrated that the constructed pathway led to the production of squalene epoxide, an important precursor for the biosynthesis of triterpenoids.


Subject(s)
Animals , Rats , Escherichia coli/genetics , NADPH-Ferrihemoprotein Reductase , Protein Engineering , Repressor Proteins , Squalene , Squalene Monooxygenase/genetics
10.
Chinese Journal of Biotechnology ; (12): 1919-1930, 2021.
Article in Chinese | WPRIM | ID: wpr-887772

ABSTRACT

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.


Subject(s)
Glycoside Hydrolases/genetics , Oligosaccharides , Polysaccharides , Protein Engineering
11.
Chinese Journal of Biotechnology ; (12): 1845-1857, 2021.
Article in Chinese | WPRIM | ID: wpr-887767

ABSTRACT

Non-ribosomal peptide synthetases catalyze the biosynthesis of structurally and functionally diverse non-ribosomal peptide natural products, which have broad applications in pharmaceutical, agricultural, and industrial sectors. Engineered non-ribosomal peptide synthetases can be used to produce novel non-ribosomal peptides through combinatorial biosynthesis. This conforms to the concept of green chemistry, thus attracts increasing attention across the world. Herein, three different engineering strategies were summarized, and recent advances in this field were reviewed.


Subject(s)
Biological Products , Peptide Synthases/genetics , Peptides , Protein Engineering
12.
Chinese Journal of Biotechnology ; (12): 891-898, 2020.
Article in Chinese | WPRIM | ID: wpr-826887

ABSTRACT

Unnatural amino acid orthogonal translation machinery can insert unnatural amino acids at desired sites of protein through stop codon by means of foreign orthogonal translation system composed of aminoacyl-tRNA synthetase and orthogonal tRNA genes. This new genetic engineering technology is not only a new tool for biochemical researches of proteins, but also an epoch-making technology for the development of new-type live viral vaccines. The mutated virus containing premature termination codon in genes necessary for replication can be propagated in transgenic cells harboring unnatural amino acid orthogonal translation machinery in media with corresponding unnatural amino acid, but it cannot replicate in conventional host cells. This replication-deficient virus is a new-type of live viral vaccine that possesses advantages of high efficacy of traditional attenuated vaccine and high safety of killed vaccine. This article reviews the application and prospect of unnatural amino acid orthogonal translation machinery in the development of novel replication-deficient virus vaccines.


Subject(s)
Amino Acids , Genetics , Amino Acyl-tRNA Synthetases , Genetic Engineering , Protein Engineering , RNA, Transfer , Viral Vaccines
13.
Chinese Journal of Biotechnology ; (12): 920-931, 2020.
Article in Chinese | WPRIM | ID: wpr-826884

ABSTRACT

The capacity for thermal tolerance is critical for industrial enzyme. In the past decade, great efforts have been made to endow wild-type enzymes with higher catalytic activity or thermostability using gene engineering and protein engineering strategies. In this study, a recently developed SpyTag/SpyCatcher system, mediated by isopeptide bond-ligation, was used to modify a rumen microbiota-derived xylanase XYN11-6 as cyclized and stable enzyme C-XYN11-6. After incubation at 60, 70 or 80 ℃ for 10 min, the residual activities of C-XYN11-6 were 81.53%, 73.98% or 64.41%, which were 1.48, 2.92 or 3.98-fold of linear enzyme L-XYN11-6, respectively. After exposure to 60-90°C for 10 min, the C-XYN11-6 remained as soluble in suspension, while L-XYN11-6 showed severely aggregation. Intrinsic and 8-anilino-1-naphthalenesulfonic acid (ANS)-binding fluorescence analysis revealed that C-XYN11-6 was more capable of maintaining its conformation during heat challenge, compared with L-XYN11-6. Interestingly, molecular cyclization also conferred C-XYN11-6 with improved resilience to 0.1-50 mmol/L Ca²⁺ or 0.1 mmol/L Cu²⁺ treatment. In summary, we generated a thermal- and ion-stable cyclized enzyme using SpyTag/SpyCatcher system, which will be of particular interest in engineering of enzymes for industrial application.


Subject(s)
Animals , Cyclization , Endo-1,4-beta Xylanases , Chemistry , Metabolism , Enzyme Stability , Industrial Microbiology , Methods , Microbiota , Protein Engineering , Rumen , Microbiology , Temperature
14.
Chinese Journal of Biotechnology ; (12): 1234-1246, 2019.
Article in Chinese | WPRIM | ID: wpr-771805

ABSTRACT

1,3-1,4-β-glucanase (E.C.3.2.1.73) is an important industrial enzyme which cleave β-glucans into oligosaccharides through strictly cutting the β-1,4 glycosidic bonds in 3-O-substituted glucopyranose units. Microbial 1,3-1,4-β-glucanase belongs to retaining glycosyl hydrolases of family 16 with a jellyroll β-sandwich fold structure. The present paper reviews the industrial application and protein engineering of microbial β-glucanases in the last decades and forecasts the research prospects of microbial β-glucanases.


Subject(s)
Amino Acid Sequence , Glycoside Hydrolases , Models, Molecular , Protein Engineering , Substrate Specificity
15.
Chinese Journal of Biotechnology ; (12): 1806-1818, 2019.
Article in Chinese | WPRIM | ID: wpr-771751

ABSTRACT

Industrial enzymes are the "chip" of modern bio-industries, supporting tens- and hundreds-fold of downstream industries development. Elucidating the relationships between enzyme structures and functions is fundamental for industrial applications. Recently, with the advanced developments of protein crystallization and computational simulation technologies, the structure-function relationships have been extensively studied, making the rational design and de novo design become possible. This paper reviews the progress of structure-function relationships of industrial enzymes and applications, and address future developments.


Subject(s)
Biocatalysis , Biotechnology , Enzymes , Chemistry , Genetics , Metabolism , Metabolic Engineering , Protein Engineering , Structure-Activity Relationship
16.
Chinese Journal of Biotechnology ; (12): 1819-1828, 2019.
Article in Chinese | WPRIM | ID: wpr-771750

ABSTRACT

We review major computational chemistry techniques applied in industrial enzyme studies, especially approaches intended for guiding enzyme engineering. These include molecular mechanics force field and molecular dynamics simulation, quantum mechanical and combined quantum mechanical/molecular mechanical approaches, electrostatic continuum models, molecular docking, etc. These approaches are essentially introduced from the following two angles for viewing: one is about the methods themselves, including the basic concepts, the primary computational results, and potential advantages and limitations; the other is about obtaining valuable information from the respective calculations to guide the design of mutants and mutant libraries.


Subject(s)
Enzymes , Chemistry , Genetics , Metabolism , Molecular Docking Simulation , Molecular Dynamics Simulation , Mutant Proteins , Chemistry , Genetics , Metabolism , Protein Engineering , Quantum Theory , Static Electricity
17.
Chinese Journal of Biotechnology ; (12): 1829-1842, 2019.
Article in Chinese | WPRIM | ID: wpr-771749

ABSTRACT

Industrial enzymes have become the core "chip" for bio-manufacturing technology. Design and development of novel and efficient enzymes is the key to the development of industrial biotechnology. The scientific basis for the innovative design of industrial catalysts is an in-depth analysis of the structure-activity relationship between enzymes and substrates, as well as their regulatory mechanisms. With the development of bioinformatics and computational technology, the catalytic mechanism of the enzyme can be solved by various calculation methods. Subsequently, the specific regions of the structure can be rationally reconstructed to improve the catalytic performance, which will further promote the industrial application of the target enzyme. Computational simulation and rational design based on the analysis of the structure-activity relationship have become the crucial technology for the preparation of high-efficiency industrial enzymes. This review provides a brief introduction and discussion on various calculation methods and design strategies as well as future trends.


Subject(s)
Biocatalysis , Biotechnology , Enzymes , Chemistry , Metabolism , Metabolic Engineering , Protein Engineering , Structure-Activity Relationship
18.
Chinese Journal of Biotechnology ; (12): 1843-1856, 2019.
Article in Chinese | WPRIM | ID: wpr-771748

ABSTRACT

By constructing mutant libraries and utilizing high-throughput screening methods, directed evolution has emerged as the most popular strategy for protein design nowadays. In the past decade, taking advantages of computer performance and algorithms, computer-assisted protein design has rapidly developed and become a powerful method of protein engineering. Based on the simulation of protein structure and calculation of energy function, computational design can alter the substrate specificity and improve the thermostability of enzymes, as well as de novo design of artificial enzymes with expected functions. Recently, machine learning and other artificial intelligence technologies have also been applied to computational protein engineering, resulting in a series of remarkable applications. Along the lines of protein engineering, this paper reviews the progress and applications of computer-assisted protein design, and current trends and outlooks of the development.


Subject(s)
Directed Molecular Evolution , High-Throughput Screening Assays , Protein Engineering , Proteins , Chemistry , Genetics , Metabolism , Substrate Specificity
19.
Chinese Journal of Biotechnology ; (12): 1857-1869, 2019.
Article in Chinese | WPRIM | ID: wpr-771747

ABSTRACT

Enzymes have a wide range of applications and great industrial potential. However, large-scale applications of enzymes are restricted by the harsh industrial environment, such as high temperature, strong acid/alkali, high salt, organic solvents, and high substrate concentration. Adaptive modification (such as rational or semi-rational design, directed evolution and immobilization) is the most common strategy to improve the catalysis of enzymes under industrial conditions. Here, we review the catalysis of enzymes in the industrial environment and various methods adopted for the adaptive modifications in recent years, to provide reference for the adaptive modifications of enzymes.


Subject(s)
Biocatalysis , Biotechnology , Enzymes , Chemistry , Metabolism , Hot Temperature , Hydrogen-Ion Concentration , Protein Engineering , Solvents , Chemistry , Pharmacology
20.
Chinese Journal of Biotechnology ; (12): 1-12, 2019.
Article in Chinese | WPRIM | ID: wpr-771405

ABSTRACT

The increasing atmospheric carbon dioxide levels have been correlated with global warming. Carbonic anhydrases (CA) are the fastest among the known enzymes to improve carbon capture. The capture of carbon dioxide needs high temperature and alkaline condition, which is necessary for CaCO₃ precipitation in the mineralization process. In order to use CAs for biomimetic carbon sequestration, thermo-alkali-stable CAs are, therefore, essential, and polyextremophilic microbes are one of the important sources of these enzymes. The current review focuses on both those isolated by thermophilic organisms from the extreme environments and those obtained by protein engineering techniques, and the industrial application of the immobilized CAs is also briefly addressed. To reduce the greenhouse effect and delay global warming, we think further research efforts should be devoted to broadening the scope of searching for carbonic anhydrase, modifying the technology of protein engineering and developing highly efficient immobilization strategies.


Subject(s)
Biomimetics , Carbon Dioxide , Carbon Sequestration , Carbonic Anhydrases , Protein Engineering
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