The design of enzyme catalytic stability is of great significance in medicine and industry. However, traditional methods are time-consuming and costly. Hence, a growing number of complementary computational tools have been developed, e.g. ESMFold, AlphaFold2, Rosetta, RosettaFold, FireProt, ProteinMPNN. They are proposed for algorithm-driven and data-driven enzyme design through artificial intelligence (AI) algorithms including natural language processing, machine learning, deep learning, variational autoencoder/generative adversarial network, message passing neural network (MPNN). In addition, the challenges of design of enzyme catalytic stability include insufficient structured data, large sequence search space, inaccurate quantitative prediction, low efficiency in experimental validation and a cumbersome design process. The first principle of the enzyme catalytic stability design is to treat amino acids as the basic element. By designing the sequence of an enzyme, the flexibility and stability of the structure are adjusted, thus controlling the catalytic stability of the enzyme in a specific industrial environment or in an organism. Common indicators of design goals include the change in denaturation energy (ΔΔG), melting temperature (ΔTm), optimal temperature (Topt), optimal pH (pHopt), etc. In this review, we summarized and evaluated the enzyme design in catalytic stability by AI in terms of mechanism, strategy, data, labeling, coding, prediction, testing, unit, integration and prospect.

BACKGROUND Mesophilic α‐amylases function effectively at low temperatures with high rates of catalysis and require less energy for starch hydrolysis. Bacillus amyloliquefaciens is an essential producer of mesophilic α‐amylases. However, because of the existence of the restriction‐modification system, introducing exogenous DNAs into wild‐type B. amyloliquefaciens is especially tricky. RESULTS α‐Amylase producer B. amyloliquefaciens strain Z3 was screened and used as host for endogenous α‐amylase gene expression. In vitro methylation was performed in recombinant plasmid pWB980‐amyZ3. With the in vitro methylation, the transformation efficiency was increased to 0.96 × 102 colony‐forming units μg–1 plasmid DNA. A positive transformant BAZ3‐16 with the highest α‐amylase secreting capacity was chosen for further experiments. The α‐amylase activity of strain BAZ3‐16 reached 288.70 ± 16.15 U mL−1 in the flask and 386.03 ± 16.25 U mL−1 in the 5‐L stirred‐tank fermenter, respectively. The Bacillus amyloliquefaciens Z3 expression system shows excellent genetic stability and high‐level extracellular production of the target protein. Moreover, the synergistic interaction of AmyZ3 with amyloglucosidase was determined during the hydrolysis of raw starch. The hydrolysis degree reached 92.34 ± 3.41% for 100 g L−1 raw corn starch and 81.30 ± 2.92% for 100 g L−1 raw cassava starch after 24 h, respectively. CONCLUSION Methylation of the plasmid DNA removes a substantial barrier for transformation of B. amyloliquefaciens strain Z3. Furthermore, the exceptional ability to hydrolyze starch makes α‐amylase AmyZ3 and strain BAZ3‐16 valuable in the starch industry. © 2020 Society of Chemical Industry

α-Amylase is the most extensively applied enzyme in industry. There is an urgent need for improvement on the yield of α-amylases currently. Herein, a strategy which combined Atmospheric and Room Temperature Plasma (ARTP) mutagenesis tool for construction of mutant library of Bacillus amyloliquefaciens with a 24-well plates screening technique was adopted to improve the yield of recombinant Bacillus amyloliquefaciens α-amylases (BAA). A mutant strain named B. amyloliquefaciens ZN mut-7# was obtained, and the activity of BAA produced by this mutant strain was 86.92% higher than that of the original strain. B. amyloliquefaciens ZN mut-7# has an unchanged BAA gene and genetic stability. This successful application proved that ARTP can be applied to the genetically engineering strains that contain recombinant plasmid. Furthermore, response surface methodology offers an achievable and efficient strategy to optimize the composition of medium used to generate BAA in B. amyloliquefaciens ZN mut-7#. A 1.28-fold increase had been obtained compared to the production of non-optimized fermentation medium. This study demonstrates that ARTP mutagenesis and medium optimization are efficient and feasible methods for increasing recombinant enzyme production in the genetically engineering strains.

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BACKGROUND: 2 0 -Fucosyllactose (2 0 -FL) is the most abundant human milk oligosaccharide (HMO) in human milk and has important physiological functions. The market demand of 2 0 -FL is continuing to grow, but high production cost has limited its availability. To solve the dilemma, biosynthesis of 2 0 -FL has been proposed and is considered the most promising pathway for massive production. ⊍-1,2-Fucosyltransferase is one of the key elements involved in its biosynthesis, but the limited intracellular accumulation and unstable properties of ⊍-1,2-fucosyltransferases when expressed in host strains have become a major hurdle for the effective biosynthesis of 2 0 -FL. RESULTS: A combinatorial engineering strategy of synergic modification of ribosome binding site, fusion peptide and enzyme gene was leveraged to enhance the soluble expression of ⊍-1,2-fucosyltransferases and promote enzyme activity. The preferable combination was to employ an optimized ribosome binding site region to drive 3 × FLAG as a fusion partner along with the ⊍-1,2-fucosyltransferase for expression in Escherichia coli (DE3) PlySs, and protein yield and enzyme activity were remarkably improved by 11.51-fold and 13.72-fold, respectively. CONCLUSION: After finely tuning the synergy among different elements, the abundant protein yield and high enzyme activity confirmed that the drawbacks of heterologous expression in ⊍-1,2-fucosyltransferase had been properly addressed. A suitable external environment further drives the efficient synthesis of ⊍-1,2-fucosyltransferases. To our knowledge, this is the first report of a systematic and effective modification of ⊍-1,2-fucosyltransferase expression, which could potentially serve as a guideline for industrial application.