ABSTRACT
Cellulose is the most abundant renewable polysaccharide with a high potential for degradation to useful end products. In nature, most cellulose is produced as crystalline cellulose. Therefore, cellulases with high hydrolytic activity against crystalline cellulose are of great interest. In this study, a crystalline cellulose degradation enzyme was investigated. The cDNA encoding a ß-glucanase, CbhYW23-2, was cloned from the ruminal fungus Piromyces rhizinflatus. To examine the enzyme activities, CbhYW23-2 was expressed in Escherichia coli as a recombinant His(6) fusion protein and purified by immobilized metal ion-affinity chromatography. Response surface modeling (RSM) combined with central composite design (CCD) and regression analysis was then employed for the planned statistical optimization of the ß-glucanase activities of CbhYW23-2. The optimal conditions for the highest ß-glucanase activity of CbhYW23-2 were observed at 46.4°C and pH 6.0. The results suggested that RSM combined with CCD and regression analysis were effective in determining optimized temperature and pH conditions for the enzyme activity of CbhYW23-2. CbhYW23-2 also showed hydrolytic activities toward Avicel, carboxymethyl cellulose (CMC), lichenan, and pachyman. The results also proved that the high activity of CbhYW23-2 on crystalline cellulose makes it a promising candidate enzyme for biotechnological and industrial applications.
Subject(s)
Cellulases/metabolism , Fungal Proteins/metabolism , Glucans/metabolism , Piromyces/enzymology , Amino Acid Sequence , Cellulases/genetics , Cellulose/metabolism , Cloning, Molecular , Escherichia coli/genetics , Escherichia coli/metabolism , Fungal Proteins/genetics , Hydrogen-Ion Concentration , Hydrolysis , Molecular Sequence Data , Piromyces/genetics , Recombinant Proteins/biosynthesis , Recombinant Proteins/genetics , Substrate Specificity , TemperatureABSTRACT
The aim of this study was to display a rumen bacterial ß-glucanase on the cell surface of a probiotic Lactobacillus reuteri strain. The ß-glucan degrading ability and the adhesion capability of the genetically modified strain were evaluated. The ß-glucanase (Glu) from Fibrobacter succinogenes was fused to the C-terminus of collagen-binding protein (Cnb) from L. reuteri and then expressed by L. reuteri Pg4 as a recombinant Cnb-Glu-His(6) fusion protein. Confocal immunofluorescence microscopy and flow cytometric analysis of the transformed strain L. reuteri pNZ-cnb/glu demonstrated that Cnb-Glu-His(6) fusion protein was displayed on its cell surface. In addition, L. reuteri pNZ-cnb/glu acquired the capacity to break down barley ß-glucan and showed higher adhesion capability, in comparison with the parental strain L. reuteri Pg4. To the best of the authors' knowledge, this is the first report of successful display of fibrolytic enzymes on the cell surface of intestinal lactobacilli.
Subject(s)
Fibrobacter/enzymology , Gene Expression , Glycoside Hydrolases/genetics , Limosilactobacillus reuteri/enzymology , Animals , Bacterial Adhesion , Caco-2 Cells , Fluorescent Antibody Technique , Glycoside Hydrolases/metabolism , Humans , Microscopy, Confocal , Plasmids/genetics , Probiotics , Recombinant Fusion Proteins , Rumen/microbiology , beta-Glucans/metabolismABSTRACT
The collagen-binding protein gene cnb was cloned from the probiotic Lactobacillus reuteri strain Pg4. The DNA sequence of the cnb gene (792 bp) has an open reading frame encoding 263 amino acids with a calculated molecular weight of 28.5 kDa. The cnb gene was constructed so as to constitutively express under the control of the Lactococcus lactis lacA promoter and was transformed into Lactobacillus casei ATCC 393, a strain isolated from dairy products with poor ability to adhere to intestinal epithelial cells. Confocal immunofluorescence microscopic and flow cytometric analysis of the transformed strain Lb. casei pNZ-cnb indicated that Cnb was displayed on its cell surface. Lb. casei pNZ-cnb not only showed a higher ability to adhere to Caco-2 cells but also exhibited a higher competition ability against Escherichia coli O157:H7 and Listeria monocytogenes adhesion to Caco-2 cells than Lb. casei ATCC 393.
Subject(s)
Bacterial Adhesion , Bacterial Proteins/genetics , Gene Expression , Lacticaseibacillus casei/physiology , Lactobacillus/genetics , Bacterial Proteins/metabolism , Caco-2 Cells , Epithelial Cells/microbiology , Humans , Intestines/microbiology , Lactobacillus/metabolism , Lacticaseibacillus casei/genetics , Open Reading Frames , Up-RegulationABSTRACT
This study aimed to evaluate the hypocholesterolaemic property of milk-kefir and soyamilk-kefir. Male hamsters were fed on a cholesterol-free or cholesterol-enriched diet containing 10 % skimmed milk, milk-kefir, soyamilk or soyamilk-kefir for a period of 8 weeks. The soyamilk, milk-kefir and soyamilk-kefir diets all tended towards a lowering of serum triacylglycerol and total cholesterol concentrations, and a reduction of cholesterol accumulation in the liver, the decrease in serum cholesterol concentration being mainly in the non-HDL fraction. The soyamilk-kefir diet led to a significant increase in the faecal excretion of neutral sterols and bile acids compared with the other two diets. The soyamilk-kefir diet also elicited a significant decrease in the serum ratio of non-HDL-cholesterol to HDL-cholesterol, compared with the control, than was the case for the other diets. These findings demonstrate that soyamilk-kefir may be considered to be among the more promising food components in terms of preventing CVD through its hypocholesterolaemic action.