Thermostability enhancement of Escherichia coli phytase by error-prone polymerase chain reaction (epPCR) and site-directed mutagenesis

Abstract

Phytase efficiently hydrolyzes phytate to phosphate; thus, it is widely used to increase phosphorus availability in animal feeds and reduce phosphorus pollution through excretion. Phytase is easily inactivated during feed pelleting at high temperature, and sufficient thermostability of phytase is essential for industrial applications. In this study, directed evolution was performed to enhance phytase thermostability. Variants were initially expressed in Escherichia coli BL21 for screening, then in Pichia pastoris for characterization. Over 19,000 clones were generated from an error-prone Polymerase Chain Reaction (epPCR) library; 5 mutants (G10, D7, E3, F8, and F9) were obtained with approximately 9.6%, 10.6%, 11.5%, 11.6%, and 12.2% higher residual activities than the parent after treatment at 99°C for 60 min. Three of these mutants, D7, E3, and F8, exhibited 79.8%, 73.2%, and 92.6% increases in catalytic efficiency (kcat/Km), respectively. In addition, the specific activities of D7, E3, and F8 were 2.33-, 1.98-, and 2.02-fold higher than parental phytase; they were also higher than the activities of all known thermostable phytases. Sequence analysis revealed that all mutants were substituted at residue 75 and was confirmed that the substitution of cysteine at position 75 was the main contribution to the improvement of thermostability of mutants by saturation mutagenesis, indicating that this amino acid is crucial for the stability and catalytic efficiency of phytase. Docking structure analysis revealed that substitution of the C75 residue allowed the mutants to form additional hydrogen bonds in the active pocket, thereby facilitating binding to the substrate. In addition, we confirmed that the intrinsic C77-C108 disulfide bond in E. coli phytase is detrimental to its stability.

Komagataella phaffii as a Platform for Heterologous Expression of Enzymes Used for Industry

“…So the data outlined above are evidence of the successful use of K. phaffii for the heterologous production of almost all the major enzymes important for increasing the nutri- The cited studies also point to the high potential of K. phaffii for industrial applications in terms of its secretion capacity and some advantages from the standpoint of protein folding and glycosylation, which positively affect enzymes' properties and thermal stability. Thus, several of the enzymes analyzed above, produced using K. phaffii, (i) exhibit higher thermostability compared to when other expression systems are used, as demonstrated for phytase AppA E. coli [198], glucoamylase from Aspergillus awamori [235], β-endoglucanase from B. subtilis [317]; and (ii) exhibit higher production levels, as demonstrated for α-amylase from Alkalimonas amylolytica [283], endoglucanase E1 from Acidothermus cellulolyticus [218], and endoglucanase II and cellobiohydrolase II from Trichoderma reesei [319].…”

“…A series of enzyme preparations with high activity and unique stability at high temperatures and low pH were obtained using K. phaffii , which is important for their application in pelleted animal feed. These are phytase E. coli [ 198 ], and xylanases from the fungus Bispora sp. [ 247 ] and T. maritima [ 253 ].…”

“…So the data outlined above are evidence of the successful use of K. phaffii for the heterologous production of almost all the major enzymes important for increasing the nutritional value of feed, including the most popular ones: thermostable phytases [ 198 ], glucoamylases [ 229 , 230 , 231 , 232 , 233 , 234 ], and α-amylases [ 223 , 224 , 225 ], including the co-expression of glucoamylase and α-amylase genes [ 230 ] as well as for the heterologous synthesis of xylanase [ 246 , 247 , 253 ], mannanase [ 276 , 277 , 279 , 281 , 282 , 283 , 285 , 332 ], laccase [ 314 ], cellulases [ 316 ], and proteases [ 326 , 327 , 328 , 329 , 330 , 331 ].…”

“…This enzyme shows maximal activity at pH 2.0, retains more than 90% of its activity at 60 °C for 120 h, and has manifested a strong resistance to pepsin and trypsin [ 191 ]. Today, approved feed phytases are manufactured using fungal or bacterial genes from Aspergillus niger , E. coli , Citrobacter braakii, Buttiauxella spp., and others [ 194 , 197 , 198 ].…”

“…Recently, genes of phytase from other organisms have been identified, the expression of which in K. phaffii has allowed for the production of highly active acidic phytase preparations. Moreover, the stability of some phytases, including AppA from E. coli , at high temperatures was significantly higher when K. phaffii was used as an expression platform compared to E. coli [ 198 , 203 ], partly due to the ability to optimize glycosylation sites on the phytase. For example, optimization of the number and localization of glycosylation sites of phytase from Yersinia intermedia produced by K. phafii GS115 increased the protein half-life from 3.32 min at 65 °C to 25 min at 100 °C [ 204 ].…”