164
6.6
Conclusions
The era of ‘protein engineering’ started in the early 1980s. The period of 35 years
of protein engineering has culminated in a remarkable collection of genetic engineering and computational tools as well as several concrete outcomes in altering
and improving the characteristics of enzymes. On the one hand, we have learned to
recognize the phenomenon of functional proteins in the sequence space and, on the
other hand, the large diverse potential of biotechnologically relevant protein functions. We only need to develop strategies to excavate them out. We know superficially in what way or manner a huge number of life-like proteins look like;
nonetheless, we are still far from understanding how life-like proteins are designed.
In spite of the advancements in the field of enzyme engineering, we still lack a
general theory on how a sequence generates a specific structure and how a structure
resolves its function. Enzyme engineering has an important function in the three
main fields of chemical biotechnology: metabolic engineering, single-step biocatalysis, and enzymatic cascades. It not only represents a mechanism for process optimization but is also essential for establishing successful strategies for developing
modified enzymes. It has helped in realizing new and efficient pathways and opened
up entry to a variety of diverse products. Enzyme engineering has a great influence
in biocatalysis and will remain to do so in the future as it promotes the catalysis of
novel non-physiological reactions, the design of innovative pathways, the synthesis
of novel products, and the optimization of the required processes. Furthermore, supplementary methods like the de novo design of enzymes and the utilization of catalytic promiscuity will help to fight against one of the greatest defect of directed
evolution approaches: the missing initial activity. Even though previously reported
de novo enzymes exhibited very limited catalytic features, Hilvert and co-workers
demonstrated that their activities could be greatly enhanced through enzyme engineering (Hilvert 2001). The impact of enzyme engineering and de novo design, particularly in combination, will be responsible for the progress of unique reactions and
will establish new chemoenzymatic and biosynthetic strategies.
Major advances in the field of DNA technologies and in bioinformatics over the
past decade have contributed critical assistance to the field of biocatalysis. The
exploration of novel enzymes in natural resources was promoted by these tools, and
they have also extensively accelerated the redesign of the existing biocatalysts.
Next-generation DNA sequencing technology (NGS) has allowed sequence analysis on an enormous scale and at greatly lowered cost. Isolation of genomic DNA has
become the first step for protein engineering due to the low-cost in DNA synthesis.
Whole-genome DNA synthesis favours the codons to be optimized for the host
organism and gene structures such as promoters, terminators, enhancers, and restriction sites to be inserted at convenient sites.
Bioinformatics tools have developed into an indispensable part of modern protein
engineering accompanied with the experimental advances (Bornscheuer et al. 2012).
Genes with similar catalytic activities have been identified using multiple sequence
alignments across immense enzyme families and homology searches, leading to
novel, potent biocatalysts (Hohne et al. 2010). Multiple sequence alignments help in
S.M. Basheer and S. Chellappan
6.6
Conclusions
The era of ‘protein engineering’ started in the early 1980s. The period of 35 years
of protein engineering has culminated in a remarkable collection of genetic engineering and computational tools as well as several concrete outcomes in altering
and improving the characteristics of enzymes. On the one hand, we have learned to
recognize the phenomenon of functional proteins in the sequence space and, on the
other hand, the large diverse potential of biotechnologically relevant protein functions. We only need to develop strategies to excavate them out. We know superficially in what way or manner a huge number of life-like proteins look like;
nonetheless, we are still far from understanding how life-like proteins are designed.
In spite of the advancements in the field of enzyme engineering, we still lack a
general theory on how a sequence generates a specific structure and how a structure
resolves its function. Enzyme engineering has an important function in the three
main fields of chemical biotechnology: metabolic engineering, single-step biocatalysis, and enzymatic cascades. It not only represents a mechanism for process optimization but is also essential for establishing successful strategies for developing
modified enzymes. It has helped in realizing new and efficient pathways and opened
up entry to a variety of diverse products. Enzyme engineering has a great influence
in biocatalysis and will remain to do so in the future as it promotes the catalysis of
novel non-physiological reactions, the design of innovative pathways, the synthesis
of novel products, and the optimization of the required processes. Furthermore, supplementary methods like the de novo design of enzymes and the utilization of catalytic promiscuity will help to fight against one of the greatest defect of directed
evolution approaches: the missing initial activity. Even though previously reported
de novo enzymes exhibited very limited catalytic features, Hilvert and co-workers
demonstrated that their activities could be greatly enhanced through enzyme engineering (Hilvert 2001). The impact of enzyme engineering and de novo design, particularly in combination, will be responsible for the progress of unique reactions and
will establish new chemoenzymatic and biosynthetic strategies.
Major advances in the field of DNA technologies and in bioinformatics over the
past decade have contributed critical assistance to the field of biocatalysis. The
exploration of novel enzymes in natural resources was promoted by these tools, and
they have also extensively accelerated the redesign of the existing biocatalysts.
Next-generation DNA sequencing technology (NGS) has allowed sequence analysis on an enormous scale and at greatly lowered cost. Isolation of genomic DNA has
become the first step for protein engineering due to the low-cost in DNA synthesis.
Whole-genome DNA synthesis favours the codons to be optimized for the host
organism and gene structures such as promoters, terminators, enhancers, and restriction sites to be inserted at convenient sites.
Bioinformatics tools have developed into an indispensable part of modern protein
engineering accompanied with the experimental advances (Bornscheuer et al. 2012).
Genes with similar catalytic activities have been identified using multiple sequence
alignments across immense enzyme families and homology searches, leading to
novel, potent biocatalysts (Hohne et al. 2010). Multiple sequence alignments help in
S.M. Basheer and S. Chellappan