To address the deficient activity of TrCel5A in naturally secreted cellulase preparation,this study used the GAP promoter to induce constitutive expression of Trichoderma reesei TrCel5A in Pichia pastoris.A recombinan...To address the deficient activity of TrCel5A in naturally secreted cellulase preparation,this study used the GAP promoter to induce constitutive expression of Trichoderma reesei TrCel5A in Pichia pastoris.A recombinant TrCel5A was screened out after gene optimization,synthesis,and expression.The biochemical and enzymatic properties of the new recombinant were characterized.As a result,optimization of shake-flask fermentation of the recombinant was obtained at 28℃,2%inoculum volume,an initial pH of 6.0,as well as glycerol and Tween-80 additions of 30 g/L and 6 g/L,respectively.Under the above-optimized conditions,the recombinant produced 14.8 U/mL of the enzyme activity at 96 h of fermentation.To further enhance enzyme production,pilot-scale cultivation was evaluated using 5-L bioreactors.Using high-cell-density fermentation,the recombinant strain increased enzyme activity to 130.4 U/ml and protein content to 2.49 g/L.In addition,the kinetic factors,including K_(m) and V_(max) values for TrCel5A,were detected to be 5.1 mg/mL and 265.9μmol/(min.mg),respectively.Thus,TrCel5A was effectively expressed in P.pastoris under the GAP promoter,and it demonstrated its potential in commercially relevant enzyme hydrolysis of lignocellulosic biomass.展开更多
采用同源克隆、反转录聚合酶链式反应(reverse transcription polymerase chain reaction,RT-PCR)、融合引物嵌套PCR(fusion primer and nested integrated PCR,FPNI-PCR)与3’-c DNA末端快速扩增(rapid amplification of c DNA end,RA...采用同源克隆、反转录聚合酶链式反应(reverse transcription polymerase chain reaction,RT-PCR)、融合引物嵌套PCR(fusion primer and nested integrated PCR,FPNI-PCR)与3’-c DNA末端快速扩增(rapid amplification of c DNA end,RACE)技术相结合,从油橄榄(Olea europaea)中克隆得到苯丙氨酸解氨酶(phenylalanine ammonia lyase,PAL)基因全长,命名为Oe PAL。序列分析表明,Oe PAL的DNA全长2 970 bp(Gen Bank登录号KJ511867),含一个内含子(393~1 220 bp);全长c DNA有2 142 bp(Gen Bank登录号KJ511868),开放阅读框编码713个氨基酸,与其他植物有较高的同源性。利用该基因构建重组质粒p PICZαA-Oe PAL,且在毕赤酵母X33中进行诱导表达。十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(sodium dodecyl sulfate polyacrylamide gelelectrophoresis,SDS-PAGE)检测显示该重组酶的分子质量在77.5 k D左右,纯化后的酶比活力为196.3 U/mg。酶学性质研究表明:该重组酶的最适反应条件为40℃,p H 8.8;在供试范围内Cu2+与Na+可以提高该酶活性;以L-苯丙氨酸为底物,在最适条件下该酶的Km为4.89×10-4 mol/L。结果表明成功地克隆到Oe PAL,构建了表达载体,并进行了功能验证。展开更多
基金supported by the National Key Research and Development Program of China(2019YFE0114600)National Natural Science Foundation of China(21776114).
文摘To address the deficient activity of TrCel5A in naturally secreted cellulase preparation,this study used the GAP promoter to induce constitutive expression of Trichoderma reesei TrCel5A in Pichia pastoris.A recombinant TrCel5A was screened out after gene optimization,synthesis,and expression.The biochemical and enzymatic properties of the new recombinant were characterized.As a result,optimization of shake-flask fermentation of the recombinant was obtained at 28℃,2%inoculum volume,an initial pH of 6.0,as well as glycerol and Tween-80 additions of 30 g/L and 6 g/L,respectively.Under the above-optimized conditions,the recombinant produced 14.8 U/mL of the enzyme activity at 96 h of fermentation.To further enhance enzyme production,pilot-scale cultivation was evaluated using 5-L bioreactors.Using high-cell-density fermentation,the recombinant strain increased enzyme activity to 130.4 U/ml and protein content to 2.49 g/L.In addition,the kinetic factors,including K_(m) and V_(max) values for TrCel5A,were detected to be 5.1 mg/mL and 265.9μmol/(min.mg),respectively.Thus,TrCel5A was effectively expressed in P.pastoris under the GAP promoter,and it demonstrated its potential in commercially relevant enzyme hydrolysis of lignocellulosic biomass.
