Directed evolution of alpha-aspartyl dipeptidase from Salmonella typhimurium.

Model-free approaches (error-prone PCR to introduce random mutations, DNA shuffling to combine positive mutations, and screening of the resultant mutant libraries) have been used to enhance the catalytic activity and thermostability of alpha-aspartyl dipeptidase from Salmonella typhimurium, which is uniquely able to hydrolyze Asp-X dipeptides (where X is any amino acid) and one tripeptide (Asp-Gly-Gly). Under double selective pressures of activity and thermostability, through two rounds of error-prone PCR and three sequential generations of DNA shuffling, coupled with screening, a mutant pepEM3074 with approximately 47-fold increased enzyme activity compared with its wild-type parent was obtained. Moreover, the stability of pepEM3074 is increased significantly. Three amino acid substitutions (Asn89His, Gln153Glu, and Leu205Arg), two of them are near the active site and substrate binding pocket, were identified by sequencing the genes encoding this evolved enzyme. The mechanism of the enhancement of activity and stability was analyzed in this paper.     

The hybrid enzymes from alpha-aspartyl dipeptidase and L-aspartase

With combinative functionalities as well as the improved activity and stability, the novel hybrid enzymes (HEs) from the heterogeneous enzymes of alpha-aspartyl dipeptidase (PepE, monomer) and l-aspartase (l-AspA, tetramer) were constructed successfully by gene random deletion strategy. The wild-type hybrid enzyme (WHE) and the evolved hybrid enzyme (EHE) were selected, respectively, upon the phenotype and the enzyme activity. The relative activity of the WHE tested was about 110% of the wild-type PepE and 26% of the wild-type l-AspA, whilst the activity of EHE was about 340% of the PepE and 87% of the l-AspA. In comparison to its individual wild-type enzymes, the EHE exhibited an improved thermostability, when examined at the enzyme concentration of 10(-7)mol/L, but the WHE showed a reduced thermostability. The activity of the EHE was about 3-fold compared to that of the WHE. The current results give a good example that the hybridization of enzymes could be attained between the monomer and multimer enzymes. In addition, they also indicate that construction hybrid enzyme from evolved enzymes is feasible.     

Cloning, expression, and nucleotide sequence of glgC gene from an allosteric mutant of Escherichia coli B.

The Escherichia coli B mutant strain CL1136 accumulates glycogen at a 3.4- to 4-fold greater rate than the parent E. coli B strain and contains an ADPglucose synthetase with altered kinetic and allosteric properties. The enzyme from CL1136 is less dependent on the allosteric activator, fructose 1,6-bisphosphate, for activity and less sensitive to inhibition by AMP than the parent strain enzyme. The structural gene, glgC, for the allosteric mutant enzyme was selected by colony hybridization and cloned into the bacterial plasmid pBR322 by insertion of the chromosomal DNA at the PstI site. One recombinant plasmid, designated pKG3, was isolated from the genomic library of CL1136 containing glgC. The cloned ADPglucose synthetase from the mutant CL1136 was expressed and characterized with respect to kinetic and allosteric properties and found to be identical to the enzyme purified from the CL1136 strain. The mutant glgC was then subcloned into pUC118/119 for dideoxy sequencing of both strands. The mutant glgC sequence was found to differ from the wild-type at the deduced amino acid residue 67 where a single point mutation resulted in a change from arginine to cysteine.     

Cloning and expression of an amylase gene from Streptococcus bovis in Escherichia coli

An amylase gene was identified in a Streptococcus bovis 033 gtWESB genomic library. Using a starch overlay and a Congo red-iodine staining procedure, amylase positive clones could be identified by zones of clearing. Ten amylase positive clones were identified using this procedure. The clone chosen for further study, SBA105, contained an insert of approximately 7.5 kb. The insert was mapped, and subcloning localized the amylase gene to a region of approximately 3.1 kb. Cloning of the 3.1 kb amylase fragment into pUC18 in both orientations revealed that the amylase gene was transcribed from its own promoter. Amylase activity was expressed by the Escherichia coli subclones and was found to be largely associated with the cytoplasmic fraction. Southern hybridization of genomic DNA from the amylolytic strains, S. bovis 033, S. bovis 077, Butyrivibrio fibrisolvens 194 and 195 revealed a single hybridizing band in S. bovis 033 DNA only. This indicates that the amylase gene from S. bovis may differ from the amylases of these other amylolytic bacteria.     

磷脂酰丝氨酸合成酶基因pss的克隆与表达

磷脂酰丝氨酸合成酶能催化转酯反应,是定向合成特定磷脂类物质特别是磷脂酰丝氨酸的工具酶,但出发菌株产量低,很大程度上限制了酶法合成磷脂酰丝氨酸的工业化应用。利用表达载体pET-22b,实现了大肠杆菌磷脂酰丝氨酸合成酶基因在大肠杆菌BL21(DE3)中的同源高效表达。利用镍亲和柱对表达产物进行纯化,并用HPLC法对纯化后的重组酶的活力进行检测。结果表明,目的蛋白可在短时间内进行大量表达,蛋白含量是出发菌株的100倍,同时经6h的转酯反应转化率达到33%,重组磷脂酰丝氨酸合成酶活力达到69U/mg蛋白。     

Purification and properties of dipeptidase from Escherichia coli AJ005

Summary A Co²⁺-dependent dipeptidase from E. coli strain AJ005, a peptidase-deficient mutant, was purified with streptomycin sulfate, ammonium sulfate and DEAE-cellulose. The purified dipeptidase increased by about 106-fold in specific activity, with dilysine as a substrate. The dipeptidase cleaved dilysine to two lysines among the lysine homopolymers, the possibility remaining that it is active toward peptides other than dilysine, since it was investigated in the present study only for activity toward lysine homopolymers. Activity was inhibited 54% by 10^–3 M KCN and completely by 10^–3 M PCMB, EDTA and benzethonium chloride, but not at all by soybean trypsin inhibitors. 78% and 95% of its activity was lost with 30 minutes' treatment at 45°C and 50°C, respectively. The apparent Km value was 6.7 × 10^–4 M for dilysine. It is probable that the dipeptidase differs from dipeptidase DP.Abbreviations EDTA Ethylenediaminetetraacetate - PCMB pchloromercuribenzoate     

High-resolution X-ray structure of isoaspartyl dipeptidase from Escherichia coli

Isoaspartyl dipeptidase from Escherichia coli functions in protein degradation by catalyzing the hydrolysis of beta-L-isoaspartyl linkages in dipeptides. The best substrate for the enzyme reported thus far is iso-Asp-Leu. Here we report the X-ray analysis of the enzyme in its resting state and complexed with aspartate to 1.65 and 2.1 A resolution, respectively. The quaternary structure of the enzyme is octameric and can be aptly described as a tetramer of dimers. Each subunit folds into two distinct domains: the N-terminal region containing eight strands of mixed beta-sheet and the C-terminal motif that is dominated by a (beta,alpha)(8)-barrel. A binuclear zinc center is located in each subunit at the C-terminal end of the (beta,alpha)(8)-barrel. Ligands to the binuclear metal center include His 68, His 70, His 201, His 230, and Asp 285. The two zincs are bridged by a carboxylated lysine residue (Lys 162) and a solvent molecule, most likely a hydroxide ion. The product of the reaction, aspartate, binds to the enzyme by displacing the bridging solvent with its side chain functional group. From this investigation it is proposed that the reaction mechanism of the enzyme proceeds through a tetrahedral intermediate and that the bridging solvent attacks the re face of the carbonyl carbon of the scissile peptide bond. This structural analysis confirms the placement of isoaspartyl dipeptidase into the urease-related amidohydrolase superfamily.