γ-谷氨酰转肽酶(γ-glutamyltranspeptidase,GGT)可催化苦味氨基酸形成氨基酸衍生物,从而降低食物的苦味,在食品行业具有很好的应用前景。为了提高GGT表达量,基于NH+4,邻苯二甲醛(O-phthalaldehyde, OPA),三氯乙酸(trichloroacetic acid,TCA)与二甲基亚砜(dimethyl sulfoxide,DMSO)之间的显色反应,建立一种用于获得GGT高表达量菌株的高通量筛选方法。易错PCR对HpaII-10区上游的7个碱基进行随机突变,筛选得到EF3HpaII突变体,使GGT表达量较原始启动子提高了63%。建立核糖体结合位点(ribosome binding site, RBS)文库,筛选得到RBS362,与EF3HpaII共同调控ggt表达,可使重组菌GGT表达量提高至60.25 U/mL,是原始菌的2.02倍。GGT催化L-苯丙氨酸(L-phenylalanine,L-Phe)合成γ-谷氨酰苯丙氨酸(γ-glutamylphenylalanine,γ-Glu-Phe),在最优条件下:底物浓度为50 mmol/L L-谷氨酰胺(L-glutamine,L-Gln)和200 mmol/L L-Phe,酶浓度为1 U/mL,初始反应温度35 ℃,pH 10.0,γ-Glu-Phe最终产率高达94%。
γ-glutamyltranspeptidase (GGT) can catalyze bitter amino acid into its derivative and reduce the bitterness of foods, thus showing great potential for the application in food industry. To enhance GGT production, a robust high-throughput screening process for isolating GGT high-producers was developed based on a colorimetric reaction among NH+4, O-phthalaldehyde (OPA), trichloroacetic acid (TCA) and dimethyl sulfoxide (DMSO). Random mutations on the seven bases located in the upstream of the -10 region of promoter HpaII were introduced by error-prone PCR. A mutant promoter EF3HpaII was isolated and identified as the most efficient for GGT production, which increased GGT production by 63% compared to that of the original promoter. Subsequently, from an RBS library, a suitable RBS362 was selected to co-regulate the expression of ggt gene with promoter EF3HpaII and the activity of GGT reached 60.25 U/mL, which was 2.02 times higher than that of the original strain. The obtained GGT was further utilized for transformation of L-phenylalanine(L-Phe) to γ-glutamylphenylalanine(γ-Glu-Phe). Under optimal conditions, the highest yield of γ-Glu-Phe reached 94 % with a reaction mixture of 50 mmol/L L-glutamine (L-Gln), 200 mmol/L L-Phe and 1 U/mL GGT at 35 °C and pH 10.0.
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