Heterologous expression of Pycnoporus cinnabarinus cellobiose dehydrogenase in Pichia pastoris and involvement in saccharification processes

Abstract

Background: Cellobiose dehydrogenase (CDH) is an extracellular hemoflavoenzyme produced by lignocellulose-degrading fungi including Pycnoporus cinnabarinus. We investigated the cellulolytic system of P. cinnabarinus, focusing on the involvement of CDH in the deconstruction of lignocellulosic biomass.

Results: First, P. cinnabarinus growth conditions were optimized for CDH production. Following growth under cellulolytic conditions, the main components secreted were cellulases, xylanases and CDH. To investigate the contribution of P. cinnabarinus secretome in saccharification processes, the Trichoderma reesei enzymatic cocktail was supplemented with the P. cinnabarinus secretome. A significant enhancement of the degradation of wheat straw was observed with (i) the production of a large amount of gluconic acid, (ii) increased hemicellulose degradation, and (iii) increased overall degradation of the lignocellulosic material. P. cinnabarinus CDH was heterologously expressed in Pichia pastoris to obtain large amounts of pure enzyme. In a bioreactor, the recombinant CDH (rCDH) expression level reached 7800 U/L. rCDH exhibited values of biochemical parameters similar to those of the natural enzyme, and was able to bind cellulose despite the absence of a carbohydrate-binding module (CBM). Following supplementation of purified rCDH to T. reesei enzymatic cocktail, formation of gluconic acid and increased hemicellulose degradation were observed, thus confirming the previous results observed with P. cinnabarinus secretome.

Conclusions: We demonstrate that CDH offers an attractive tool for saccharification process enhancement due to gluconic acid production from raw lignocellulosic material.

Figure 1

SDS-PAGE and zymogram on the supernatant of P. cinnabarinus ss3 grown in cellulolytic conditions. 1, pre-stained molecular weight marker; 2, SDS-PAGE with 10 μg of protein supernatant after ultrafiltration step; 3, CDH zymogram; 4, zymogram with ABTS; 5, soluble xylan birchwood (xylanase) zymogram; 6, CMC (endoglucanase) zymogram and 7, locust bean gum (mannanase) zymogram.

Figure 2

SDS-PAGE, zymogram, immunoblot analysis, deglycosylation and cleavage by papain of purified recombinant rCDH. 1, pre-stained molecular weight marker; 2, 10 μg of purified CDH; 3, CDH zymogram on the sample 2; 4, Western blot analysis using anti-His antibodies; 5, CDH deglycosylated; 6, CDH digested by papain.

Figure 3

Kinetics of the binding of rCDH. Experiments were performed on Avicel at 30°C, pH 4.5. Error bars indicate standard deviations from triplicate measures performed independently.

Figure 4

Effect of temperature on the activity of purified rCDH using DCPIP as electron acceptor. Activity was determined at pH 4.5 except for determination of optimal pH. (A) Optimal pH of purified rCDH using DCPIP as electron acceptor. Activity was determined at 30°C in citrate-phosphate buffer pH 2.7-7. (B) Temperature optimum. (C) Temperature stability at 45°C(■), 50°C(♦), 55°C(▲) and 65°C(□). Measured were performed at least three times.

Figure 5

Analysis of total reducing sugar yield after 96 h of enzymatic treatment on wheat straw. Reducing sugars (mM) release during hydrolysis were quantified spectrophotometrically using the DNS method as described in Navarro et al., 2010 [69]. Assay conditions for hydrolysis were conducted in 20 mL of 50 mM sodium phosphate buffer (pH 4.8) containing 5% (w/v) wheat straw. (A) P. cinnabarinus secretome containing CDH or (B) purified rCDH were assayed in the presence of cellulose cocktail at 45°C with an orbital agitation (140 rpm) during 96 h. C: control reaction without CDH. Error bars are for triplicate, each measured once.

Figure 6

Determination of sugar yield in mg of sugars formed/g of wheat straw after 96 h of enzymatic treatment on wheat straw. Saccharification assays were performed in 20 mL of 50 mM sodium phosphate buffer (pH 4.8) containing 5% (w/v) wheat straw. (A) P. cinnabarinus secretome containing CDH or (B) purified rCDH were assayed in the presence of cellulose cocktail at 45°C with an orbital agitation (140 rpm) during 96 h. C: control reaction without CDH. Sugars were quantified by high-performance anion exchange chromatography (HPAEC) coupled with amperometric detection (PAD) equipped with a Carbo-Pac PA-1 analytical column. These experiments were repeated at least three times with similar results. For all-sugars analysis in (A) and (B) statistical significance was P < 0.1 and P < 0.05 respectively.