A novel GH10 xylanase from Penicillium sp. accelerates saccharification of alkaline-pretreated bagasse by an enzyme from recombinant Trichoderma reesei expressing Aspergillus β-glucosidase

Abstract

Background Trichoderma reesei is considered a candidate fungal enzyme producer for the economic saccharification of cellulosic biomass. However, performance of the saccharifying enzymes produced by T. reesei is insufficient. Therefore, many attempts have been made to improve its performance by heterologous protein expression. In this study, to increase the conversion efficiency of alkaline-pretreated bagasse to sugars, we conducted screening of biomass-degrading enzymes that showed synergistic effects with enzyme preparations produced by recombinant T. reesei.Results Penicillium sp. strain KSM-F532 produced the most effective enzyme to promote the saccharification of alkaline-pretreated bagasse. Biomass-degrading enzymes from strain KSM-F532 were fractionated and analyzed, and a xylanase, named PspXyn10, was identified. The amino acid sequence of PspXyn10 was determined by cDNA analysis: the enzyme shows a modular structure consisting of glycoside hydrolase family 10 (GH10) and carbohydrate-binding module family 1 (CBM1) domains. Purified PspXyn10 was prepared from the supernatant of a recombinant T. reesei strain. The molecular weight of PspXyn10 was estimated to be 55 kDa, and its optimal temperature and pH for xylanase activity were 75 °C and pH 4.5, respectively. More than 80% of the xylanase activity was maintained at 65 °C for 10 min. With beechwood xylan as the substrate, the enzyme had a K m of 2.2 mg/mL and a V max of 332 μmol/min/mg. PspXyn10ΔCBM, which lacked the CBM1 domain, was prepared by limited proteolysis. PspXyn10ΔCBM showed increased activity against soluble xylan, but decreased saccharification efficiency of alkaline-pretreated bagasse. This result indicated that the CBM1 domain of PspXyn10 contributes to the enhancement of the saccharification efficiency of alkaline-pretreated bagasse. A recombinant T. reesei strain, named X2PX10, was constructed from strain X3AB1. X3AB1 is an Aspergillus aculeatus β-glucosidase-expressing T. reesei PC-3-7. X2PX10 also expressed PspXyn10 under the control of the xyn2 promoter. An enzyme preparation from X2PX10 showed almost the same saccharification efficiency of alkaline-pretreated bagasse at half the enzyme dosage as that used for an enzyme preparation from X3AB1.ConclusionsOur results suggest that PspXyn10 promotes the saccharification of alkaline-pretreated bagasse more efficiently than TrXyn3, a GH10 family xylanase from T. reesei, and that the PspXyn10-expressing strain is suitable for enzyme production for biomass saccharification.Electronic supplementary materialThe online version of this article (10.1186/s13068-017-0970-2) contains supplementary material, which is available to authorized users.

Selective fluorescence labeling: time-lapse enzyme visualization during sugarcane hydrolysis

“…As previously reported [22], Xyn10 demonstrated a higher activity than Xyn III against alkali-pretreated sugarcane bagasse powder (Fig. 1), reconfirming the excellent performance of Xyn10.…”

“…Xyn III of T. reesei was reported to be initially devoid of a CBM and Xyn III, expressed with a xylan-binding domain from Streptomyces olivaceoviridis E-86, showed higher adsorption toward insoluble xylan by a factor of two [28]. Furthermore, when Xyn10 that initially possessed a CBM [22] was modified to be devoid of a CBM, Xyn10 was no longer observed to adsorb onto the substrates (data not shown). Therefore, the CBM of xylanase was found to be critical for the interaction with xylan molecules that are closely associated with cellulose molecules at the substrate surface, which may improve the total activity of the cellulase system toward biomass.…”

“…As xylanase is known to be a key enzyme for the saccharification of biomass, the investigation herein studied two endo-β-xylanases, Xyn III and Xyn10, both of which belong to the glycoside hydrolase family GH-10 in the CAZy database. Xyn III was observed by Xu et al [27] to be a highly active xylanase enzyme, while Xyn10, developed by Kao Corporation, Tokyo, Japan [22], exhibits an even higher activity. Xyn III has no carbohydrate-binding module (CBM) and shows a high affinity to soluble xylan [28], while Xyn10 is an endo-type xylanase with a CBM 1 that demonstrates a high affinity to the surface of crystalline cellulose [22].…”

“…To prepare Xyn10 from Penicillium sp., the pspxyn10 gene was amplified from pUC-Pcbh1-pspxyn10-amdS plasmid [22] by a polymerase chain reaction and expressed in A. oryzae cells under the control of an improved enoA promoter (PenoA142f ) [23] that harbored 12 tandem repeats of the cis-acting element (region III) of the agdA promoter [24]. A. oryzae cells expressing the pspxyn10 gene were cultured in a DP medium (2% dextrin hydrate, 1% peptone) containing 0.5% potassium dihydrogen phosphate, 0.05% magnesium sulfate, 0.187% l-glutamic acid monosodium salt, and 0.003% l-methionine at 30 °C, 105 rpm for 3 days.…”

Insights into the cellulose degradation mechanism of the thermophilic fungus Chaetomium thermophilum based on integrated functional omics

“…G0SBF1 is a special xylanase with CBM1. A study showed that the CBM1 of PspXyn10 could bind to cellulose [50]. Therefore, it was hypothesized that the CBM1 of G0SBF1 could bind to the surface of cellulose.…”