SbPL1CE8 from Segatella bryantii combines with SbGH28GH105 in a multi-enzyme cascade for pectic biomass utilization.

Pectinases are useful biocatalysts for pectic biomass processing and are extensively used in the food/feed, textile and papermaking industries. Two pectinase genes, a pectate lyase (SbPL1CE8) and a polygalacturonase (SbGH28GH105) were isolated from Segatella bryantii and functionally characterized. Recombinant rSbPL1CE8 was most active against polygalacturonic acid (PGA) and pectin with a 60 % degree of esterification, with kcat/Km values of 721.18 ± 64.77 and 327.02 ± 22.44 mL/s/mg, respectively. Truncated rSbPL1 acted as a mesophilic alkaline pectate lyase, which was highly resistant to inactivation by methanol and ethanol. The rSbPL1CE8 exclusively digested PGA and pectin into unsaturated digalacturonate (uG2), which was further converted into galacturonic acid by rSbGH28GH105. The rSbPL1CE8 was highly effective for saccharification of waste materials from Zea mays, Oryza sativa and Arachis hypogaea processing, and for ramie fiber degumming. This novel pectate lyase has great potential for application in industrial pectic biomass processing.

Expression and characterization of a novel microbial GH9 glucanase, IDSGLUC9-4, isolated from sheep rumen.

OBJECTIVE: This study aimed to identify and characterize a novel endo-ß-glucanase, IDSGLUC9-4, from the rumen metatranscriptome of Hu sheep. METHODS: A novel endo-ß-glucanase, IDSGLUC9-4, was heterologously expressed in Escherichia coli and biochemically characterized. The optimal temperature and pH of recombinant IDSGLUC9-4 were determined. Subsequently, substrate specificity of the enzyme was assessed using mixed-linked glucans including barley ß-glucan and Icelandic moss lichenan. Thin-layer chromatography (TLC), high-performance liquid chromatography (HPLC), matrix assisted laser desorption ionization time of flight mass spectrometry analyses were conducted to determine the products released from polysaccharides and cello-oligosaccharides substrates. RESULTS: The recombinant IDSGLUC9-4 exhibited temperature and pH optima of 40°C and pH 6.0, respectively. It exclusively hydrolyzed mixed-linked glucans, with significant activity observed for barley ß-glucan (109.59±3.61 µmol/mg min) and Icelandic moss lichenan (35.35±1.55 µmol/mg min). TLC and HPLC analyses revealed that IDSGLUC9-4 primarily released cellobiose, cellotriose, and cellotetraose from polysaccharide substrates. Furthermore, after 48 h of reaction, IDSGLUC9-4 removed most of the glucose, indicating transglycosylation activity alongside its endo-glucanase activity. CONCLUSION: The recombinant IDSGLUC9-4 was a relatively acid-resistant, mesophilic endo-glucanase (EC 3.2.1.4) that hydrolyzed glucan-like substrates, generating predominantly G3 and G4 oligosaccharides, and which appeared to have glycosylation activity. These findings provided insights into the substrate specificity and product profiles of rumen-derived GH9 glucanases and contributed to the expanding knowledge of cellulolytic enzymes and novel herbivore rumen enzymes in general.