Periplasmic Export of Bile Salt Hydrolase in Escherichia coli by the Twin-Arginine Signal Peptides.

Bile salt hydrolase (BSH, EC 3.5.1.24) is considered as an ideal way with lower cost and less side effects to release the risk of coronary heart disease caused by hypercholesterolemia. As bile salt hydrolase from Lactobacillus plantarum BBE7 could not be efficiently exported by PelB signal peptide of the general secretory (Sec) pathway, three twin-arginine signal peptides from twin-arginine translocation (Tat) pathway were synthesized, fused with bsh gene, inserted into expression vectors pET-20b(+) and pET-22b(+), and transformed into four different Escherichia coli hosts, respectively. Among the 24 recombinant bacteria obtained, E. coli BL21 (DE3) pLysS (pET-20b(+)-dmsA-bsh) showed the highest BSH activity in periplasmic fraction, which was further increased to 1.21 ± 0.03 U/mL by orthogonal experimental design. And, signal peptide dimethyl sulfoxide reductase subunit DmsA (DMSA) had the best activity of exported BSH. More importantly, the presence of BSH in the periplasm had proven to be caused by the export rather than cell leakage. For the first time, we report the periplasmic expression of BSH by signal peptides from the Tat pathway. This will lay a solid foundation for the purification and biochemical characterization of BSH from the supernatant, and strategies adopted here could be used for the periplasmic expression of other proteins in E. coli.

Codon and Propeptide Optimizations to Improve the Food-grade Expression of Bile Salt Hydrolase in Lactococcus lactis.

To achieve the food-grade expression of bile salt hydrolase (BSH, EC 3.5.1.24) from Lactobacillus plantarum BBE7, the nisin controlled gene expression system (NICE), food-grade selection maker and signal peptide of Lactococcus lactis were used in this study. The open reading frame of BSH was optimized based on the codon bias of L. lactis, resulting in 12-fold and 9.5% increases in the intracellular and extracellular BSH activities, respectively. Three synthetic propeptides, LEISSTCDA (acidic), LGISSTCNA (neutral) and LKISSTCHA (basic) were also fused with signal peptide SPusp45 of vector pNZ8112 and introduced into the food-grade expression vector pNZ8149, respectively. Among these propeptides, acidic propeptide was effective in increasing the secretion efficiency and yield of BSH in recombinant bacteria, while neutral propeptide had no significant effect on the secretion of BSH. In contrast, basic propeptide strongly reduced the extracellular expression of BSH. By using codon optimization and the acidic propeptide together, the extracellular BSH activity was increased by 11.3%, reaching its maximum of 3.56 U/mg. To the best of our knowledge, this is the first report on the intracellular and extracellular expression of BSH using food-grade expression system, which would lay a solid foundation for large-scale production of BSH and other heterologous proteins in L. lactis.

Mutation studies in the active site of ß-glycosidase from Pyrococcus furiosus DSM 3638.

Sequence alignments and homology modeling of Pyrococcus furiosus thermostable glycosidase (PFTG) showed that the residue 150 is conserved as tryptophan in ß-glycosidase and in other related enzymes such as ß- mannosidase and ß-galactosidase. To elucidate the relationship between the substrate size and geometric shape of the catalytic site of thermophilic ß-glycosidase and category of PFTG, the Q77, Q150 and D206 located at the interface of the dimer were replaced with Trp and Asn. Also, to confirm the role of active sites of PFTG, the Q77R/Q150W double mutant was created through subcloning. Temperature and pH optima of both mutants and native enzyme were same at 100 °C and pH 5.0 in sodium citrate buffer, respectively. The catalytic efficiencies (k(cat)/K(m)) of the mutants on synthetic and natural substrates by Isothermal Titration Calorimetry were slightly changed, but indicated the characteristics of ß-glycosidase activity. Kinetic parameters of the mutant enzymes indicated that they possess characteristics of both ß- galactosidase and ß-mannosidase activities. Although the mutant enzymes showed similar substrate specificities compared to the recombinant enzyme, they had more affinity (K(m)) to substrates with low turnover number (k(cat)).

Overexpression and characterization of a novel transgalactosylic and hydrolytic ß-galactosidase from a human isolate Bifidobacterium breve B24.

After the complete gene of a ß-galactosidase from human isolate Bifidobacterium breve B24 was isolated by PCR and overexpressed in E. coli, the recombinant ß-galactosidase was purified to homogeneity and characterized for the glycoside transferase (GT) and glycoside hydrolase (GH) activities on lactose. One complete ORF encoding 691 amino acids (2,076 bp) was the structural gene, LacA (galA) of the ß-gal gene. The recombinant enzyme shown by activity staining and gel-filtration chromatography was composed of a homodimer of 75 kDa with a total molecular mass of 150 kDa. The K(m) value for lactose (95.58 mM) was 52.5-fold higher than the corresponding K(m) values for the synthetic substrate ONPG (1.82 mM). This enzyme with the optimum of pH 7.0 and 45°C could synthesize approximately 42.00% of GOS from 1M of lactose. About 97.00% of lactose in milk was also quickly hydrolyzed by this enzyme (50 units) at 45°C for 5h to produce 46.30% of glucose, 46.60% of galactose and 7.10% of GOS. The results suggest that this recombinant ß-galactosidase derived from a human isolate B. breve B24 may be suitable for both the hydrolysis and synthesis of galacto-oligosaccharides (GOS) in milk and lactose processing.

Overexpression and characterization of an extremely thermostable maltogenic amylase, with an optimal temperature of 100 degrees C, from the hyperthermophilic archaeon Staphylothermus marinus.

A gene encoding a hyperthermostable maltogenic amylase of Staphylothermus marinus (SMMA) was cloned and overexpressed in Escherichia coli. SMMA consisted of 696 amino acids with a predicted molecular mass of 82.5 kDa. The enzyme was active in acidic conditions (pH 3.5-5.0), with an optimal pH of 5.0, and was extremely thermostable, with a temperature optimum of 100 degrees C and a melting temperature of 109 degrees C, both of which extremely favored the starch conversion process. SMMA hydrolyzed linear malto-oligosaccharides, starch, cyclodextrins, and cycloamylose, primarily to maltose and glucose, and showed highest activity toward acarbose and pullulan, hydrolyzed to acarviosine-glucose and panose, respectively. Investigation of the cleavage mode using (14)C-maltoheptaose revealed that SMMA preferentially hydrolyzed the first and second glycosidic bonds from the reducing end. To our knowledge, this enzyme is the most thermostable maltogenic amylase yet reported, and might be of potential value in the food and starch industries.

Characterization of a novel debranching enzyme from Nostoc punctiforme possessing a high specificity for long branched chains.

A novel debranching enzyme from Nostoc punctiforme PCC73102 (NPDE) exhibits hydrolysis activity toward both alpha-(1,6)- and alpha-(1,4)-glucosidic linkages. The action patterns of NPDE revealed that branched chains are released first, and the resulting maltooligosaccharides are then hydrolyzed. Analysis of the reaction with maltooligosaccharide substrates labeled with (14)C-glucose at the reducing end shows that NPDE specifically liberates glucose from the reducing end. Kinetic analyses showed that the hydrolytic activity of NPDE is greatly affected by the length of the substrate. The catalytic efficiency of NPDE increased considerably upon using substrates that can occupy at least eight glycone subsites such as maltononaose and maltooctaosyl-alpha-(1,6)-beta-cyclodextrin. These results imply that NPDE has a unique subsite structure consisting of -8 to +1 subsites. Given its unique subsite structure, side chains shorter than maltooctaose in amylopectin were resistant to hydrolysis by NPDE, and the population of longer side chains was reduced.

Changes in the catalytic properties of Pyrococcus furiosus thermostable amylase by mutagenesis of the substrate binding sites.

Pyrococcus furiosus thermostable amylase (TA) is a cyclodextrin (CD)-degrading enzyme with a high preference for CDs over maltooligosaccharides. In this study, we investigated the roles of four residues (His414, Gly415, Met439, and Asp440) in the function of P. furiosus TA by using site-directed mutagenesis and kinetic analysis. A variant form of P. furiosus TA containing two mutations (H414N and G415E) exhibited strongly enhanced alpha-(1,4)-transglycosylation activity, resulting in the production of a series of maltooligosaccharides that were longer than the initial substrates. In contrast, the variant enzymes with single mutations (H414N or G415E) showed a substrate preference similar to that of the wild-type enzyme. Other mutations (M439W and D440H) reversed the substrate preference of P. furiosus TA from CDs to maltooligosaccharides. Relative substrate preferences for maltoheptaose over beta-CD, calculated by comparing k(cat)/K(m) ratios, of 1, 8, and 26 for wild-type P. furiosus TA, P. furiosus TA with D440H, and P. furiosus TA with M439W and D440H, respectively, were found. Our results suggest that His414, Gly415, Met439, and Asp440 play important roles in substrate recognition and transglycosylation. Therefore, this study provides information useful in engineering glycoside hydrolase family 13 enzymes.

Enzymatic preparation of maltohexaose, maltoheptaose, and maltooctaose by the preferential cyclomaltooligosaccharide (cyclodextrin) ring-opening reaction of Pyrococcus furiosus thermostable amylase.

Specific-length maltooligosaccharides, particularly maltohexaose, maltoheptaose, and maltooctaose, were prepared from cyclomaltooligosaccharides (cyclodextrins, CDs) by the preferential cyclodextrin ring-opening reaction of an amylolytic enzyme from Pyrococcus furiosus. The enzyme primarily produces maltohexaose, maltoheptaose, and maltooctaose by hydrolyzing alpha-, beta-, and gamma-CD, respectively. This study aims to develop a high-efficiency synthesis of specific maltooligosaccharides at high-purity. [formula: see text]