One-step purification of two novel thermotolerant β-1,4-glucosidases from a newly isolated strain of Fusarium chlamydosporum HML278 and their characterization

Abstract

A newly identified cellulase-producing Fusarium chlamydosporum HML278 was cultivated under solid-state fermentation of sugarcane bagasse, and two new β-glucosides enzymes (BG FH1, BG FH2) were recovered from fermentation solution by modified non-denaturing active gel electrophoresis and gel filtration chromatography. SDS-PAGE analysis showed that the molecular weight of BG FH1 and BG FH2 was 93 kDa and 52 kDa, respectively, and the enzyme activity was 5.6 U/mg and 11.5 U/mg, respectively. The optimal reaction temperature of the enzymes was 60 ℃, and the enzymes were stable with a temperature lower than 70 ℃. The optimal pH of the purified enzymes was 6.0, and the enzymes were stable between pH 4–10. Km and Vmax values ​​were 2.76 mg/mL and 20.6 U/mg for pNPG, respectively. Thin-layer chromatography and high-performance liquid chromatography analysis showed that BG FH1and BG FH2 had hydrolysis activity toward cellobiose and could hydrolyze cellobiose into glucose. In addition, both enzymes exhibited transglycoside activity, which could use glucose to synthesize cellobiose and cellotriose, and preferentially synthesize alcohol. In conclusion, our study demonstrated that F. chlamydosporum HML278 produces heat-resistant β-glucosidases with both hydrolytic activity and transglycosidic activity, and these β-glucosidases have potential application in bioethanol and papermaking industries.

Purification and Characterization of a Glucose tolerant β-Glucosidase from a Newly Isolated Neofusicoccum Parvum strain F7: Production Optimization using Plackett Burman and Box Behnken

“…After anion exchange chromatography the total protein (4.20 mg) and fold purity (12%) decreased, the speci c activity and fold purity increased to 29.79 U/mg and 2.57, respectively. Qin et al [39] also observed similar results in their attempts to purify β-glucosidases from F. Chlyamydosporum. Size exclusion chromatography with a 250 kDa molecular weight cut-off was carried out with 50 mM Tris (pH 8.0) buffer, however, there was no protein or β-glucosidase activity detected in any of the eluted fractions.…”

“…The three active bands displaying black precipitation indicative of β-glucosidase activity were cut out of the gel, ground in a precooled mortar and the enzymes were leached with 50 mM sodium phosphate buffer (pH 6.0) at 4°C for 12 hours. Thereafter the leachate was centrifuged at 4000 g for 10 minutes and the supernatant containing the enzyme was collected, concentrated, and dialysed in the same buffer [39] . Protein concentration was determined by the Bradford method using Bovine albumin serum as the standard [8] .…”

“…Isoforms are produced at different periods of incubation, various types and concentrations of carbon and different temperatures for maximal β-glucosidase activity [56] . Qin et al [39] reported that two β-glucosidase isoforms were produced by Fusarium chlamydosporum HML278 after four days at 4°C in solid substrate fermentation. Different forms of β-glucosidases differ in stability, catalytic e ciency, absorption, and activity on various substrates [26] .…”

“…7) indicating that the enzyme is both thermophilic and able to withstand acidic environments [3] . Qin et al [39] reported optimal β-glucosidase activities at 60°C for Fusarium chlamydosporum and pH 4.0 for F. moniliforme. The Bgl3 was stable at pH 4.0, retaining 80% activity for 120 minutes.…”

“…Numerous microbes including bacteria, fungi and actinomycetes are ubiquitous in nature and the endo and exogenous microbial enzymes from many of these organisms have been widely explored [4,10,15,39] . Speci cally, β-glucosidases from several fungal species including Aspergillus, Fusarium, and Trichoderma have been explored for glucose-tolerant β-glucosidases [15,39,54] . β-Glucosidases from Aspergillus and Trichoderma sp.…”