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Protein Engineering: Different Biotechnology Applications

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A special issue of Molecules (ISSN 1420-3049). This special issue belongs to the section "Bioorganic Chemistry".

Deadline for manuscript submissions: closed (1 June 2020) | Viewed by 19955

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Special Issue Editor

Dr. Benevides C. Pessela

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Departamento de Biotecnología y Microbiología de Alimentos, Instituto de Investigación en Ciencias de la Alimentación CIAL (CSIC-UAM), Universidad Autonoma de Madrid, Cantoblanco, Calle Nicolás Cabrera 9, CP. 28049 Madrid, Spain
Interests: protein engineering; chimeric proteins applications; protein production; modification of active centers for different industrial applications; proteins for hydrocarbon biosulfurization; synthetic proteins; design of matrices for protein purification; proteins and different industrial applications; multienzyme proteins
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Dear Colleagues,

Protein Engineering is an emerging branch of engineering that applies knowledges of mathematics and molecular biology to the design of proteins. It operates iteratively, following a cyclic process that alternates stages in which the changes to be made are planned and executed with others in which the result of the changes is evaluated. There are two methods for the design of proteins: rational design and directed evolution. In the "rational design" method, changes are introduced in certain amino acids through directed mutagenesis, on the basis of the hypothesis that some specific changes will cause the desired functional effect. In some occasions, these changes occur when domains or structural motifs of different proteins are combined, generating a hybrid protein or chimera. This is a simple and cheap method. In the “directed evolution” method, random mutations are introduced in the protein under study, and only those variants that exhibit the desired properties are selected. Generally, two molecular biology techniques are used to perform random mutagenesis of isolated genes. One is known as "error-prone PCR" and consists in the amplification by PCR (polymerase chain reaction) of the gene that codes for the protein of interest in conditions that induce the DNA polymerase to make mistakes. The other procedure is called "DNA-shuffling" and consists in the fragmentation of the sequence to mutagenize by digestion with DNase, followed by a reassembly of the same sequence through PCR. Several rounds of mutation and selection give rise to a collection of modified proteins having the desired characteristics; however, some functional assays can be considerably complex, and the attainment of the mutant protein containing the desired modification can involve a very high number of assays. To solve these difficulties, the use of robotic processes or "high-throughput screening" is gaining momentum. These two techniques are inspired by natural evolution and sexual reproduction, respectively.The final or biochemical phase of the design cycle has the immediate objective of purifying the protein, as a preliminary stage for the resolution of its three-dimensional structure, since the determination of the structure of a protein is the best available tool to explain how it performs its function. The close link between the structure of a protein and its function makes solid structural information essential for the success of any approach to protein engineering. It is common for researchers to apply both techniques in the design of a given protein: first the rational design is applied, and then the product is subjected to directed evolution, following the design cycle.

Dr. Benevides C. Pessela
Guest Editor

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Keywords

Proteins engineering
Synthetic proteins
Different industrial applications
Chimeric proteins applications
Protein preparation by modification of active centers
Hydrocarbon biosulfurization
Protein synthesis
Polymerase chain reaction (PCR)
Rational design and directed evolution
Directed mutagenesis

Published Papers (4 papers)

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Research

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17 pages, 4792 KiB

Open AccessArticle

Single Residue Substitution at N-Terminal Affects Temperature Stability and Activity of L2 Lipase

by Noramirah Bukhari, Adam Thean Chor Leow, Raja Noor Zaliha Raja Abd Rahman and Fairolniza Mohd Shariff

Molecules 2020, 25(15), 3433; https://doi.org/10.3390/molecules25153433 - 28 Jul 2020

Cited by 8 | Viewed by 2608

Abstract

Rational design is widely employed in protein engineering to tailor wild-type enzymes for industrial applications. The typical target region for mutation is a functional region like the catalytic site to improve stability and activity. However, few have explored the role of other regions which, in principle, have no evident functionality such as the N-terminal region. In this study, stability prediction software was used to identify the critical point in the non-functional N-terminal region of L2 lipase and the effects of the substitution towards temperature stability and activity were determined. The results showed 3 mutant lipases: A8V, A8P and A8E with 29% better thermostability, 4 h increase in half-life and 6.6 °C higher thermal denaturation point, respectively. A8V showed 1.6-fold enhancement in activity compared to wild-type. To conclude, the improvement in temperature stability upon substitution showed that the N-terminal region plays a role in temperature stability and activity of L2 lipase. Full article

(This article belongs to the Special Issue Protein Engineering: Different Biotechnology Applications)

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14 pages, 2936 KiB

Open AccessArticle

Thermostable Tannase from Aspergillus Niger and Its Application in the Enzymatic Extraction of Green Tea

by Yuan Shao, Yong-Hui Zhang, Feng Zhang, Qiu-Ming Yang, Hui-Fen Weng, Qiong Xiao and An-Feng Xiao

Molecules 2020, 25(4), 952; https://doi.org/10.3390/molecules25040952 - 20 Feb 2020

Cited by 36 | Viewed by 3442

Abstract

Tannase is widely used in tea beverage processing because of its ability to catalyze the hydrolysis of hydrolysable tannins or gallic acid esters and effectively improve the quality of tea extracts through enzymatic extraction. A new thermophilic tannase was cloned from Aspergillus niger FJ0118 and characterized. The tannase exhibited an optimal reaction temperature of 80 °C and retained 89.6% of the initial activity after incubation at 60 °C for 2 h. The enzymatic extraction of green tea at high temperature (70 °C) for a short time (40 min) was devised on the basis of the superior thermal stability of tannase. The enzymatic reaction significantly increased the total polyphenol content of green tea extract from 137 g·kg⁻¹ to 291 g·kg⁻¹. The enzymatic reaction effectively degraded the ester catechins into non-ester catechins compared with the water extraction method. Results suggested that the thermally stable tannase exhibited potential applications in the enzymatic extraction of green tea beverage. Full article

(This article belongs to the Special Issue Protein Engineering: Different Biotechnology Applications)

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13 pages, 2675 KiB

Open AccessArticle

A Generic Method for Fast and Sensitive Detection of Adeno-Associated Viruses Using Modified AAV Receptor Recombinant Proteins

by Mengtian Cui, Yabin Lu, Can Tang, Ran Zhang, Jing Wang, Yang Si, Shan Cheng and Wei Ding

Molecules 2019, 24(21), 3973; https://doi.org/10.3390/molecules24213973 - 3 Nov 2019

Cited by 3 | Viewed by 4301

Abstract

Adeno-Associated Viruses (AAV) are widely used gene-therapy vectors for both clinical applications and laboratory investigations. The titering of different AAV preparations is important for quality control purposes, as well as in comparative studies. However, currently available methods are limited in their ability to detect various serotypes with sensitivity and convenience. Here, we took advantage of a newly discovered AAV receptor protein with high affinity to multiple AAV serotypes, and developed an ELISA-like method named “VIRELISA” (virus receptor-linked immunosorbent assay) by adopting fusion with a streptavidin-binding peptide (SBP). It was demonstrated that optimized VIRELISA assays exhibited satisfactory performance for the titering of AAV2. The linear range of AAV2 was 1 × 10⁵ v.g. to 5 × 10⁹ v.g., with an LOD (limit of detection) of 5 × 10⁴ v.g. Testing of VIRELISA for the quantification of AAV1 was also successful. Our study indicated that a generic protocol for the quantification of different serotypes of AAVs was feasible, reliable and cost-efficient. The applications of VIRELISA will not only be of benefit to laboratory research due to its simplicity, but could also potentially be used for monitoring the circulation AAV loads both in clinical trials and in wild type infection of a given AAV serotype. Full article

(This article belongs to the Special Issue Protein Engineering: Different Biotechnology Applications)

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Review

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25 pages, 1661 KiB

Open AccessReview

Genetically Engineered Proteins to Improve Biomass Conversion: New Advances and Challenges for Tailoring Biocatalysts

by Lucas Ferreira Ribeiro, Vanesa Amarelle, Luana de Fátima Alves, Guilherme Marcelino Viana de Siqueira, Gabriel Lencioni Lovate, Tiago Cabral Borelli and María-Eugenia Guazzaroni

Molecules 2019, 24(16), 2879; https://doi.org/10.3390/molecules24162879 - 8 Aug 2019

Cited by 25 | Viewed by 7937

Abstract

Protein engineering emerged as a powerful approach to generate more robust and efficient biocatalysts for bio-based economy applications, an alternative to ecologically toxic chemistries that rely on petroleum. On the quest for environmentally friendly technologies, sustainable and low-cost resources such as lignocellulosic plant-derived biomass are being used for the production of biofuels and fine chemicals. Since most of the enzymes used in the biorefinery industry act in suboptimal conditions, modification of their catalytic properties through protein rational design and in vitro evolution techniques allows the improvement of enzymatic parameters such as specificity, activity, efficiency, secretability, and stability, leading to better yields in the production lines. This review focuses on the current application of protein engineering techniques for improving the catalytic performance of enzymes used to break down lignocellulosic polymers. We discuss the use of both classical and modern methods reported in the literature in the last five years that allowed the boosting of biocatalysts for biomass degradation. Full article

(This article belongs to the Special Issue Protein Engineering: Different Biotechnology Applications)

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