Expression of Thermobifida fusca thermostable raw starch digesting alpha-amylase in Pichia pastoris and its application in raw sago starch hydrolysis

Abstract

A gene encoding the thermostable raw starch digesting alpha-amylase in Thermobifida fusca NTU22 was amplified by PCR, sequenced and cloned into Pichia pastoris X-33 host strain using the vector pGAPZalphaA, allowing constitutive expression and secretion of the protein. Recombinant expression resulted in high levels of extracellular amylase production, as high as 510 U/l in the Hinton flask culture broth. The purified amylase showed a single band at about 65 kDa by SDS-polyacrylamide gel electrophoresis after being treated with endo-beta-N-acetylglycosaminidase H, and this agrees with the predicted size based on the nucleotide sequence. About 75% of the original activity remained after heat treatment at 60 degrees C for 3 h. The optimal pH and temperature of the purified amylase were 7.0 and 60 degrees C, respectively. The purified amylase exhibited a high level of activity with raw sago starch. After 48-h treatment, the DPw of raw sago starch obviously decreased from 830,945 to 378,732. The surface of starch granules was rough, and some granules displayed deep cavities.

Cloning, expression, and purification of insect (Sitophilus oryzae) alpha-amylase, able to digest granular starch, in Yarrowia lipolytica host

“…Noteworthy, we were still able to recover the α-amylase from the end-point culture medium after the purification procedure, through ammonium sulfate precipitation and affinity chromatography, which demonstrates that the protein was still present in the medium. Also, previous reports on heterologous enzyme production within the Y. lipolytica -hp4d promoter-based expression systems showed continued accumulation of the proteins during the stationary growth phase rather than a peak value followed by a decrease in activity (Madzak et al 2005; Yang et al 2010a). According to the characteristics provided by Madzak et al (2004), hp4d recombinant promoter is almost independent from environmental conditions and is able to drive a strong expression in virtually any medium, promoting growth-phase-dependent gene expression.…”

“…(Jeang et al 2002), Rhizopus oryzae (Li et al 2011), and Thermobifida fusca (Yang and Liu 2007). The latter gene was also expressed in other hosts, namely Pichia pastoris (Yang et al 2010a) and Yarrowia lipolytica (Yang et al 2010b), showing superior performance of the latter host.…”

Heterologous expression of Thermobifida fusca thermostable alpha-amylase in Yarrowia lipolytica and its application in boiling stable resistant sago starch preparation

“…Each protein is rarely produced in several hosts. In the case of the a-amylases from T. fusca NTU22, we had the opportunity to compare the production of a same protein in three different host systems: E. coli [6], P. pastoris [9], and Y. lipolytica. The E. coli is the first choice for heterologous protein expression due to the ease of genetic manipulation, availability of efficient genetic tools, high transformation efficiency, and rapid growth rates.…”

“…Recombinant expression resulted in the extracellular amylase production, as high as 510 U/l in the Hinton flask culture broth. The amylase activity was higher than that expressed in E. coli DH5a (pAMY13H8) [9], but the activity was still lower than that produced by the original strain, T. fusca NTU22.…”

Bioprocess for efficient production of recombinant Pichia anomala phytase and its applicability in dephytinizing chick feed and whole wheat flat Indian breads

“…The inclusion of phytase in the diets of monogastrics mitigates anti-nutrient effects of phytastes, ameliorates the assimilation of phytate phosphorus and other nutrients and growth, besides reducing the addition of phosphate to their diets. The recombinant Pichia pastoris strains have been extensively used for the production of industrially important enzymes and other products [5,40,41]. During the last few decades, inducible alcohol oxidase (AOX1) and constitutive GAP promoters have been used for recombinant protein expression in P. pastoris [10,13,19,38].…”