RESUMO
A new protease gene (pro1437)was separated from an oil-polluted Mud flat metagenomic library. Pro1437 belongs to a peptidase M48 superfamily according to the results of sequence analysis, and it showed very low identities compared to other known proteases or peptidases. The error-prone PCR was used to introduce random mutations and improve the expression of pro1437. After two rounds of mutagenesis and screening, a mutant (Pro2T21) with a 6.6-fold higher activity and a 4.8-fold higher expression level than Pro1437 was obtained. Sequence analysis found three amino acid substitutions (A54V, L192H, F224L) in Pro2T21. 3D structure modelling analysis indicated A54V and L192H probably played a crucial role in the improvement of enzymatic activity and soluble expression level of Pro2T21. Furthermore, Pro2T21opti displayed a 5.8-fold higher expression level than the wild type under optimal pH 8.0 at 50°C after codon-optimization. Also, Pro2T21opti represented robust compatibility with several popular laundry detergents, and blood stains on white cloth pieces were completely washed away when endogenous protease-inactivated Tide and Pro2T21opti were used together. Therefore, Pro2T21opti has great potential for use as an additive in detergents after further study.
Assuntos
Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Endopeptidases/genética , Endopeptidases/metabolismo , Metagenoma , Poluição por Petróleo , Microbiologia do Solo , Substituição de Aminoácidos , Proteínas de Bactérias/isolamento & purificação , Manchas de Sangue , Detergentes , Evolução Molecular Direcionada , Endopeptidases/isolamento & purificação , Escherichia coli/enzimologia , Escherichia coli/genética , Humanos , Concentração de Íons de Hidrogênio , Cinética , Modelos Moleculares , Mutagênese , Filogenia , Conformação Proteica , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Solubilidade , TemperaturaRESUMO
Functional screening of a metagenomic library of termite hindgut identified an overlapping gene cluster encoding the phosphotransferase system (PTS) components, which consisted of a glucoside specific PTS enzyme II gene (glu1923) and a glycoside hydrolase gene (glu1392). Hydrolytic experiments revealed that the combined effect of Glu1923 and Glu1392 was responsible for the utilization of glucosidic substrates in recombinant Escherichia coli (E. coli) strains. Yeast two hybrid analysis suggested that there was an interaction between Glu1923 and Glu1392, and the domain EIIA of Glu1923 played an important role for the interaction. In addition, the hydrolase Glu1392 displayed hydrolysis ability toward cellobiose and maltose, and had a very high tolerance to glucose with a Ki value of 2.25M. These properties make Glu1392 an interesting candidate in biotechnology applications after further study.