Xylanase production from Penicillium citrinum isolate HZN13 using response surface methodology and characterization of immobilized xylanase on glutaraldehyde-activated calcium-alginate beads

Abstract

The present study reports the production of high-level cellulase-free xylanase from Penicillium citrinum isolate HZN13. The variability in xylanase titers was assessed under both solid-state (SSF) and submerged (SmF) fermentation. SSF was initially optimized with different agro-waste residues, among them sweet sorghum bagasse was found to be the best substrate that favored maximum xylanase production (9643 U/g). Plackett-Burman and response surface methodology employing central composite design were used to optimize the process parameters for the production of xylanase under SSF. A second-order quadratic model and response surface method revealed the optimum conditions for xylanase production (sweet sorghum bagasse 25 g/50 ml; ammonium sulphate 0.36 %; yeast extract 0.6 %; pH 4; temperature 40 °C) yielding 30,144 U/g. Analysis of variance (ANOVA) showed a high correlation coefficient (R ² = 97.63 %). Glutaraldehyde-activated calcium-alginate-immobilized purified xylanase showed recycling stability (87 %) up to seven cycles. Immobilized purified xylanase showed enhanced thermo-stability in comparison to immobilized crude xylanase. Immobilization kinetics of crude and purified xylanase revealed an increase in K _m (12.5 and 11.11 mg/ml) and V _max (12,500 and 10,000 U/mg), respectively. Immobilized (crude) enzymatic hydrolysis of sweet sorghum bagasse released 8.1 g/g (48 h) of reducing sugars. Xylose and other oligosaccharides produced during hydrolysis were detected by High-Performance Liquid Chromatography. The biomass was characterized by Scanning Electron Microscopy, Energy Dispersive X-ray and Fourier Transformation Infrared Spectroscopy. However, this is one of the few reports on high-level cellulase-free xylanase from P. citrinum isolate using sweet sorghum bagasse.

Keywords: Enzymatic hydrolysis; Immobilization; Penicillium citrinum; Response surface methodology; Sweet sorghum bagasse; Xylanase.

Fig. 1

A consensus tree representing phylogenetic analysis of the 18S rDNA gene sequence analysis of isolated fungal culture [diamond Penicillium citrinum isolate HZN13 (Gene bank accession no. KP119605)]. Isolate sequence was used for BLASTn analysis in NCBI and the nearest neighbor sequences of other fungal cultures were chosen for Phylogenetic tree construction using MEGA 6 software with neighbor-joining method. Numbers at branches are bootstrap values of 100 replications

Fig. 2

Production of cellulase-free xylanase in SmF and SSF using various agro-waste residues by Penicillium citrinum isolate HZN13 (a) and SDS-PAGE and zymogram analysis showing differential expression of xylanase from sweet sorghum bagasse in SmF and SSF [lane 1 zymogram analysis for xylanase production in SSF; lane 2 SDS-PAGE with silver nitrate staining of proteins produced in SSF; lane 3 SDS-PAGE with silver nitrate staining of proteins produced in SmF] (b)

Fig. 3

3D response surface and contour plots showing interactions between independent variables. Sweet sorghum bagasse vs ammonium sulphate (a), sweet sorghum bagasse vs pH (b) and temperature vs yeast extract (c) affecting the xylanase production (U/g)

Fig. 4

pH optima and its stability (a), temperature optima and its stability (b), enzyme kinetics (c) and recycling stability (d) of glutaraldehyde-activated calcium-alginate immobilized crude and purified xylanase

Fig. 5

Enzymatic hydrolysis of sweet sorghum bagasse with glutaraldehyde-activated calcium-alginate immobilized xylanase (a), reusability of glutaraldehyde-activated calcium-alginate immobilized xylanase for enzymatic hydrolysis (b) HPLC chromatogram showing the profile of hydrolyzed products from sweet sorghum bagasse (c) and HPLC chromatogram for standard xylose (d)