Acidophilic β-xylanases have attracted considerable attention due to their excellent activity under extreme acidic environments and potential industrial utilizations. In this study, a novel β-xylanase gene (Xyl11) of glycoside hydrolase family 11, was cloned from Trichoderma asperellum ND-1 and efficiently expressed in Pichia pastoris (a 2.0-fold increase). Xyl11 displayed a maximum activity of 121.99 U/ml at pH 3.0 and 50°C, and exhibited strict substrate specificity toward beechwood xylan (Km = 9.06 mg/ml, Vmax = 608.65 μmol/min/mg). The Xyl11 retained over 80% activity at pH 2.0–5.0 after pretreatment at 4°C for 1 h. Analysis of the hydrolytic pattern revealed that Xyl11 could rapidly convert xylan to xylobiose via hydrolysis activity as well as transglycosylation. Moreover, the results of site-directed mutagenesis suggested that the Xyl11 residues, Glu127, Glu164, and Glu216, are essential catalytic sites, with Asp138 having an auxiliary function. Additionally, a high degree of synergy (15.02) was observed when Xyl11 was used in association with commercial β-xylosidase. This study provided a novel acidophilic β-xylanase that exhibits excellent characteristics and can, therefore, be considered a suitable candidate for extensive applications, especially in food and animal feed industries.
In this study, an acidophilic GH5 β-mannanase (TaMan5) from Trichoderma asperellum ND-1 was efficiently expressed in Pichia pastoris (a 2.0-fold increase, 67.5 ± 1.95 U/mL). TaMan5 displayed the highest specificity toward locust bean gum (Km = 1.34 mg/mL, Vmax = 749.14 μmol/min/mg) at pH 4.0 and 65°C. Furthermore, TaMan5 displayed remarkable tolerance to acidic environments, retaining over 80% of its original activity at pH 3.0–5.0. The activity of TaMan5 was remarkably decreased by Cu2+, Mn2+, and SDS, while Fe2+/Fe3+ improved the enzyme activity. A thin-layer chromatography (TLC) analysis of the action model showed that TaMan5 could rapidly degrade mannan/MOS into mannobiose without mannose via hydrolysis action as well as transglycosylation. Site-directed mutagenesis results suggested that Glu205, Glu313, and Asp357 of TaMan5 are crucial catalytic residues, with Asp152 playing an auxiliary function. Additionally, TaMan5 and commercial α-galactosidase displayed a remarkable synergistic effect on the degradation of galactomannans. This study provided a novel β-mannanase with ideal characteristics and can be considered a potential candidate for the production of bioactive polysaccharide mannobiose.
Ying Hu,Lu Shen,Yinan Zhang,Lingmin Lu,Haiyan Fu,Yuanbin She
Nitrite (NO 2 − ) is a widely used food additive and long-term aging of cooked leftovers may also contribute to the formation of NO 2 − , excessive consumption of NO 2 − is harmful to human health. Developing an effective sensing strategy for on-site monitoring of NO 2 − has attracted considerable attention. Herein, a novel colorimetric and fluorometric probe ND-1 based on photoinduced electron transfer effect (PET) was designed for highly selective and sensitive detection of nitrite (NO 2 − ) in foods. The probe ND-1 was strategically constructed by employing naphthalimide as the fluorophore and o -phenylendiamine as the specific recognition site for NO 2 − . The triazole derivative ND-1-NO 2 - could be produced exclusively by reacting with NO 2 − , leading to a visible colorimetric change from yellow to colorless accompanied by a significantly enhanced fluorescence intensity at 440 nm. The probe ND-1 exhibited promising sensing performances towards NO 2 − including high selectivity , rapid response time (within 7 min), low detection limit (47.15 nM) and wide quantitative detection range (0–35 μM). In addition, probe ND-1 was capable of quantitative detecting of NO 2 − in real food samples (including pickled vegetables and cured meat products) with satisfactory recovery rates (97.61%–103.08%). Moreover, the paper device loaded by probe ND-1 could be utilized for visual monitoring of NO 2 − levels variation of stir-fried greens. This study provided a feasible method for the accurate, traceable and rapid on-site monitoring NO 2 − in foods.
危险品化学品经营许可证（带存储）