Table 1.

List of the enzymes engineered by protein engineering.

Enzyme	Organism	Improved property	Method	Application	Reference
Hydantoinase	Arthrobacter sp.	Enantioselective hydantoinase and 5-fold more productivity	Saturation mutagenesis, screening	Production of l-Met (l-amino acids)	[7]
Cyclodextrin glucanotransferase	Bacillus stearothermophilus ET1	Modulation of cyclizing activity and thermostability	Site-directed mutagenesis	Bread industry	[8]
Lipase B	Candida antarctica	20-fold increase in half-life at 70 °C	epPCR	Resolution and desymmetrization of compound	[9]
Tagatose-1,6-Bisphosphate aldolase	E. coli	80-fold improvement in kcat/Km and 100-fold change in stereospecificity	DNA shuffling and screening	Efficient syntheses of complex stereoisomeric products	[10]
Xylose isomerase	Thermotoga neapolitana	High activity on glucose at low temperature and low pH	Random Mutagenesis and screening	Used in preparation of high fructose syrup	[11]
Amylosucrase	Neisseria polysaccharea	5-fold increased activity	Random mutagenesis, gene shuffling, and directed evolution	Synthesis or the modification of polysaccharides	[12]
Galactose oxidase	F. graminearum	3.4–4.4 fold greater Vmax/Km and increased specificity	epPCR and screening	Derivatization of guar gum	[13]
Fructose bisphosphate aldolase	E. coli	Increased thermostablity and stability to treatment with organic solvent	DNA shuffling	Use in organic synthesis	[14]
1,3-1,4-α-d-glucanase	Fibrobacter succinogenes	3–4-fold increase in the turnover rate (k)	PCR-based gene truncation	Beer industry	[15]
Lipase	P. aeruginosa	2-fold increase in amidase activity	Random mutagenesis and screening	Understanding lipase inability to hydrolyze amides	[16]
Protease BYA	Bacillus sp. Y	Specific activity1.5-fold higher	Site-directed mutagenesis	Detergents products	[17]
p-Hydroxybenzoate hydroxylase	Pseudomonas fluorescens NBRC 14160	Activity, reaction specificity, and thermal stability	Combinatorial mutagenesis	Degrading various aromatic compounds in the environment	[18]
Endo-1,4-β-xylanase II	Trichoderma reesei	Increased alkali stability	Site-directed mutagenesis	Sulfate pulp bleaching	[19]
Xylose isomerase	Thermotoga neapolitana	2.3-fold increases in catalytic efficiency	Random mutagenesis	Production of high fructose corn syrup	[11]
α-Amylase	Bacillus sp. TS-25	10 °C enhancement in thermal stability	Directed evolution	Baking industry	[20]
Xylanase		Tm improved by 25 °C	Gene site-saturation mutagenesis	Degradation of hemicellulose	[21]
Fructosyl peptide oxidase	Coniochaeta sp	79.8-fold enhanced thermostability	Directed evolution and site-directed mutagenesis	Clinical diagnosis	[22]
Endo-β-1,4-xylanase	Bacillus subtilis	Acid stability	Rational protein engineering	Degradation of hemicellulose	[23]
Subtilase	Bacillus sp.	6-fold increase in caseinolytic activity at 15–25 °C	Directed evolution and site-directed mutagenesis	Detergent additives and food processing	[24]
CotA laccase	B. subtilis	120-fold more specific for ABTS	Directed evolution	Catalyze oxidation of polyphenols	[25]
Pyranose 2-oxidase	Trametes multicolor	Altered substrate selectivity for d-galactose, d-glucose	Semi-rational enzyme engineering approach	Food industry	[26]
Xylanase XT6	Geobacillus stearothermophilus	52-fold enhancement in thermostability; increased catalytic efficiency	Directed evolution and site-directed mutagenesis	Degradation of hemicellulose	[27]
Lipase	Bacillus pumilus	Thermostability and 4-fold increase in kcat	Site-directed mutagenesis	Chemical, food, leather and detergent industries	[28]
Bgl-licMB	Bacillus amyloliquefaciens (Bgl) and Clostridium thermocellum (licMB)	2.7 and 20-fold higher kcat/Km than that of the parental Bgl and licMB, respectively	Splicing-by-overlap extension	Brewing and animal-feed industries	[29]
β-agarase AgaA	Zobellia galactanivorans	Catalytic activity and thermostability	Site-directed mutagenesis	Production of functional neo-agarooligosaccharides	[30]
Prolidase	Pyrococcus horikoshii	Thermostability	Random mutagenesis	Detoxification of organophosphorus nerve agents	[31]
Lipases	Geobacillus sp. NTU 03	79.4-fold increment in activity; 6.3–79-fold enhanced thermostability	Error-prone PCR and site-saturation mutagenesis	Transesterification	[32]
Xylanase	Hypocrea jecorina	Thermostability	Look-through mutagenesis (LTMTM) and combinatorial beneficial mutagenesis (CBMTM)	Degradation of hemicellulose	[33]
Amylase	Bacillus sp. US149	Thermostability	Site-directed mutagenesis	Bread industry	[34]
Cholesterol oxidase	Brevibacterium sp.	Thermostability and enzymatic activity	Site-directed mutagenesis	Detection and conversion of cholesterol	[35]
Lipase B	Candida antarctica	Enhancement of thermostability	Molecular dynamics (MD) simulation and site-directed mutagenesis	Detergent industries	[36]
Laccase	Bacillus HR03	3-fold improved kcat and thermostability	Directed mutagenesis	Catalyze oxidation of polyphenols, and polyamines	[37]
d-psicose 3-epimerase	Agrobacterium tumefaciens	Thermostability	Random and site-directed mutagenesis	Industrial producer of d-psicose	[38]
1,3-1,4-β-d-glucanase	Fibrobacter succinogenes	Thermostability and specific activity	Rational mutagenesis	Widely used as a feed additive	[39]
α-Amylase	Bacillus licheniformis	Acid stability	Direct evolution	Starch hydrolysis	[40]
Alkaline amylase	Alkalimonas amylolytica	Oxidative stability	Site-directed mutagenesis	Detergent and textile industries	[41]
Endoglucanase	Thermoascus aurantiacus	4-fold increase in kcat and 2.5-fold improvement in hydrolytic activity on cellulosic substrates	Site-directed mutagenesis	Bioethanol production	[42]
d-glucose 1-dehydrogenase isozymes	Bacillus megaterium	Substrate specificity	Site-directed mutagenesis	Measurements of blood glucose level	[43]
Glycerol dehydratase	Klebsiella pneumoniae	2-fold pH stability; enhanced specific activity	Rational design	Synthesis of 1,3-Propanediol	[44]
Cyclodextrin Glucanotransferase	Bacillus sp. G1	Enhancement of thermostability	Rational mutagenesis	Starch is converted into cyclodextrins	[45]
Cellobiose phosphorylase	Clostridium thermocellum	Enhancement of thermostability	Combined rational and random approaches	Phosphorolysis of cellobiose	[46]
Superoxide dismutase	Potentilla atrosanguinea	Thermostability	Site-directed mutagenesis	Scavenging of O2−	[47]
Endoglucanase Cel8A	Clostridium thermocellum	Thermostability	Consensus-guided mutagenesis	Conversion of cellulosic biomass to biofuels	[48]
Endo β-glucanase EgI499	Bacillus subtilis JA18	Increase in half life from 10 to 29 mins at 65 °C	Deletion of C-terminal region	Animal feed production	[49]
Pyranose 2-oxidase	Trametes multicolor	Increase half life from 7.7 min to 10 h (at 60 °C)	Designed triple mutant	Food industry	[50]
Xylanase XT6	Geobacillus stearothermophilus	52× increase in thermal stability, kopt increase by 10 °C, catalytic efficiency increase by 90%	Directed evolution and site-directed mutagenesis	Biobleaching	[27]
Tyrosine phenol-lyase	Symbiobacterium toebi	Improved thermal stability and activity (Increase in Tm up to 11.2 °C)	Directed evolution (random mutagenesis, reassembly and activity screening)	Industrial production of l-tyrosine and its derivatives	[51]
Phytase	Penicilium sp.	Increased thermal stability	Random mutation and selection	Feed additives	[52]
l-Asparaginase	Erwinia carotovora	Increase in half-life from 2.7 to 159.7 h	In vitro directed evolution	Therapeutic agent	[53]
Endoglucanase CelA	Clostridium thermocellum	10-fold increase in half-life of inactivation at 86 °C	Saturation mutagenesis	Bioconversion of cellulosic biomass	[54]
β-glucosidase BglC	Thermobifida fusca	Increase in half-life from 12 to 1244 min	Family shuffling, site saturation, and site-directed mutagenesis	Bioconversion of cellulosic biomass	[55]
Phospholipase D	Streptomyces	Improved thermal stability and activity	Semi-rational, site-specific saturation mutagenesis	Phosphatidylinositol synthesis	[56]
β-glucosidase	Trichoderma reesei	Enhanced kcat/Km and kcat values by 5.3- and 6.9-fold	Site-directed mutagenesis	Hydrolysis of cellobiose and cellodextrins	[57]
Lipases		144-fold enhanced thermostability	Error prone PCR	Synthesis and hydrolysis of long chain fatty acids	[58]
Laccase	Pycnoporus cinnabarinus	8000-fold increase in kcat/Km	Directed evolution and semi-rational engineering	Lignocellulose biorefineries, organic synthesis, and bioelectrocatalysis	[59]
Feruloyl esterase A	Aspergillus niger	Increase in half-life from 15 to >4000 min	Random and site-directed mutagenesis	Degradation of lignocellulose	[60]