Chitinase Chit62J4 Essential for Chitin Processing by Human Microbiome Bacterium Clostridium paraputrificum J4

Commensal bacterium Clostridium paraputrificum J4 produces several extracellular chitinolytic enzymes including a 62 kDa chitinase Chit62J4 active toward 4-nitrophenyl N,N′-diacetyl-β-d-chitobioside (pNGG). We characterized the crude enzyme from bacterial culture fluid, recombinant enzyme rChit62J4, and its catalytic domain rChit62J4cat. This major chitinase, securing nutrition of the bacterium in the human intestinal tract when supplied with chitin, has a pH optimum of 5.5 and processes pNGG with Km = 0.24 mM and kcat = 30.0 s−1. Sequence comparison of the amino acid sequence of Chit62J4, determined during bacterial genome sequencing, characterizes the enzyme as a family 18 glycosyl hydrolase with a four-domain structure. The catalytic domain has the typical TIM barrel structure and the accessory domains—2x Fn3/Big3 and a carbohydrate binding module—that likely supports enzyme activity on chitin fibers. The catalytic domain is highly homologous to a single-domain chitinase of Bacillus cereus NCTU2. However, the catalytic profiles significantly differ between the two enzymes despite almost identical catalytic sites. The shift of pI and pH optimum of the commensal enzyme toward acidic values compared to the soil bacterium is the likely environmental adaptation that provides C. paraputrificum J4 a competitive advantage over other commensal bacteria.

Chitosan Oligosaccharide Ameliorates Metabolic Syndrome Induced by Overnutrition via Altering Intestinal Microbiota

Chitosan oligosaccharides (COS) play a prebiotic role in many ways, whereas its function on microbiota is not fully understood. In this study, the effects of COS on metabolic syndrome were initially investigated by testing changes in the physiological indicators after adding COS to the diet of mice with high fat (group H) and low fat (group L). The results showed that COS markedly inhibited the accumulation of body weight and liver fat induced by high-fat diet, as well as restored the elevated concentration of blood glucose and fasting insulin to normal levels. Next, changes of the murine intestinal microbiota were examined. The results exhibited that COS reduced with-in-sample diversity, while the between-sample microbial diversity enhanced. Specifically, COS enriched Clostridium paraputrificum and Clostridium ramosum in the mice on a high-fat diet, while the abundance of Clostridium cocleatum was reduced. As a comparison, Parabacteroides goldsteinii and Bacteroides uniformis increased their abundance in response to COS in the low-fat diet group. Noticeably, a large amount of Akkermansia muciniphila was enriched in both high-fat or low-fat diet groups. Among the differential fecal bacteria, Clostridium ramosume was found to be positively interacted with Faecalibacterim prausnitzii and Clostridium paraputrificum; Clostridium paraputrificum had a positive interactions with Lactococcus chungangensis and Bifidobacterium mongoliense, suggesting that COS probably ameliorate metabolic syndrome through the microbiota in view of the lipid-lowering effects of these interacted bacteria. Furthermore, the gene expression data revealed that COS improved the functions related to intestinal barrier and glucose transport, which could be the trigger and consequence of the variations in gut microbiota induced by COS. Additionally, correlation analysis found that intestinal bacteria are related to physiological parameters, which further supports the mediating role of gut microbiota in the beneficial effect of COS. In summary, our research results provide new evidence for the prebiotic effects of COS.

Identification and characterization of a gene encoding NADP(H)-dependent bile acid 12β-hydroxysteroid dehydrogenase from Clostridium paraputrificum ATCC 25780

ABSTRACTBile acids are detergent molecules that solubilize dietary lipids and lipid-soluble vitamins. Humans synthesize bile acids with α-orientation hydroxyl groups which can be biotransformed by gut microbiota to toxic, hydrophobic bile acids, such as deoxycholic acid (DCA). Gut microbiota are also capable of converting hydroxyl groups from the α-orientation through an oxo-intermediate to the β-orientation, resulting in more hydrophilic and less toxic bile acids. This interconversion is catalyzed by regio- (C-3 vs. C-7) and stereospecific (α vs. β) hydroxysteroid dehydrogenases (HSDHs). Recently, multiple human gut clostridia have been reported to encode 12α-HSDH, which interconverts DCA and 12-oxolithocholic acid (12-oxoLCA). Bile acid 12β-HSDH activity completes the epimerization of DCA by converting 12-oxoLCA to the 12β-bile acid known as epiDCA. While 12β-HSDH activity has been shown in cell extracts of Clostridium paraputrificum, the gene has not yet been reported. In order to identify the first gene encoding this activity, 6 candidate oxidoreductase genes from C. paraputrificum ATCC 25780 were cloned, overexpressed, purified, and screened for activity with 12-oxoLCA and epiDCA. LC-MS analysis was performed on reaction products from the enzyme encoded by DR024_RS09610, confirming the first 12β-HSDH gene discovered. The enzyme was more specific for bile acids lacking a 7-hydroxyl group than cholic acid derivatives containing a 7-hydroxyl. Phylogenetic analysis revealed previously unknown diversity for bile acid 12β-HSDH by experimentally validating two additional 12β-HSDHs within the tree from Eisenbergiella sp. OF01-20 and Olsenella sp. GAM18.