Constitutive expression of barley α-amylase in Pichia pastoris by high-density cell culture

Abstract

α-amy gene amplified from barley genome was cloned into MCS of pGAP9K to generate pGAP9K-α-amy which was then transformed into Pichia pastoris GS115 by electroporation. Transformants with multi-copies and high expression for the foreign gene were selected on G418 containing plate and expression analysis. The fermentation was carried out in a 50 l bioreactor with 20 l working volume, using a high-density cell culture method by continuously feeding with 50% glycerol-0.8% PTM4 to the growing culture for 54 h at 30°C. Under the control of GAP promoter (pGAP), α-amy gene was constitutively expressed. At the end of the fermentation, the α-AMY expression reached 125 mg/l, while the biomass growth was 186 as measured by absorption of 600 nm. The secreted α-AMY was purified to 97.5% by SP-Sepharose FF ion-exchange chromatography and affinity purification. The recombinant α-AMY showed activity on hydrolysis of starch.

Expression ofAspergillus nigerglucose oxidase in yeastPichia pastorisSMD1168

“…[25] Therefore, construction of the P. pastoris expression system by using the pGAP promoter attracts more attention. Until now, angiostatin, [26] thermophilic actinomycetes a-amylase, [27] b-mannanase, [28] laccase, [29] barley a-amylase, [30] lipase LIP2 [24] and b-galactosidase, [31] etc. have been expressed under the promoter of pGAP.…”

“…The enzyme kinetics of recombinant GOD were studied in the solution with oxygen saturation, pH 6.0 at 30 C. The reaction rate of recombinant GOD and commercial GOD standard to substrate glucose with different concentration (10,12,14,16,18,20,25,30,35,40, 50, 60, 80 and 100 mmol/L) was measured. The kinetic constants of two GOD proteins were measured by double reciprocal plot method.…”

Industrial Production of Proteins with Pichia pastoris—Komagataella phaffii

“…As generally recognised, the separation of biomass from high-density cultures is also a challenging task during downstream processing [ 18 , 120 ]. Several target proteins have been successfully obtained with K. phaffii by exploiting P GAP and P AOX1 in continuous cultures at laboratory bench-scale [ 18 , 135 , 142 , 143 , 144 , 145 , 146 , 147 , 148 , 149 , 150 , 151 ]. The variation of operational mode from fed-batch to continuous is considered as a successful strategy to boost the efficiency of the bioprocess; the FDA has even encouraged the development of continuous processing to manufacture biopharmaceuticals [ 18 , 152 , 153 , 154 , 155 ].…”

Rational development of bioprocess engineering strategies for recombinant protein production in Pichia pastoris (Komagataella phaffii) using the methanol-free GAP promoter. Where do we stand?

“…For P. pastoris, several recombinant proteins have been successfully produced in continuous cultures at laboratory bench-scale for both constitutive (P GAP ) and methanol-inducible (P AOX1 ) expression [38,88,[130][131][132][133]. A summary of several continuous processes under P GAP is presented in Table 3 [76,[134][135][136]. The change of operational mode from fed-batch to continuous should be considered an effective strategy for improving the bioprocess efficiency.…”

“…Indeed, the FDA has encouraged the development of continuous processing for biopharmaceuticals manufacturing [126,137,138]. α-amylase GS115 20 125 --------------- [135] *Some of the presented values were estimated from the published available data.…”