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

Date Published: August 11, 2016

Publisher: Springer Berlin Heidelberg

Author(s): Zabin K. Bagewadi, Sikandar I. Mulla, Yogesh Shouche, Harichandra Z. Ninnekar.


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 (R2 = 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 Km (12.5 and 11.11 mg/ml) and Vmax (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.

Partial Text

Xylan is the major structural polysaccharide constituent of hard and soft wood and is the second most abundant renewable resource. This complex heteropolysaccharide is composed of β-(1,4)-linked d-xylopyranosyl residues with substitutions of l-arabinofuranose, d-glucuronic acid, and 4-O-methyl-d-glucuronic acid. Xylan degradation requires different xylanolytic enzymes, like xylanase (EC, β-xylosidase (EC, α-l-arabinofuranosidase (EC, α-d-glucuronidase (EC, and acetyl xylan esterase (EC (Beg et al. 2001). Crude enzyme preparations are cost effective as they bypass the high cost involved in downstream processing. Such preparations have been employed as cocktails for enzymatic hydrolysis of biomass which requires a pool of hemicellulolytic enzymes. A mixture of crude extracts from Trichoderma viride and commercial cellulolytic enzymes have been used to hydrolyze cellulose Avicel (Vintila et al. 2010). Crude xylanase have been used for the hydrolysis of xylan and hemicellulosic materials to several xylooligosaccharides. However, xylanase with specific characteristics like pH and thermo-stability, high specific activity, resistance to metal ions and chemicals are required to meet the desired needs of industries (Ramírez-Cavazos et al. 2014), which could be achieved by enzyme purification. Purified xylanases have been employed in feedstuff (Knob and Carmona 2010) and cellulase-free xylanase has gained importance in paper and pulp industry (Collins et al. 2005). Highly purified xylanase finds specific applications in synthetic chemistry, food and cosmetic industries, medical diagnostics (Rodriguez Couto and Toca Herrera 2006) and acts as inhibitory agent towards human HIV-1 reverse transcriptase (Xiao et al. 2003). Enzyme purification is essential for the determination of biochemical, molecular, accurate kinetic, structural and functional properties. Because, it helps to understand the molecular interactions, secondary structures of proteins and also reveals the occurrence of multiple isoforms of enzymes. Based on the amino acid sequence of purified enzymes, they have been classified into glycosyl hydrolase families (Henrissat and Davies 1997). For commercial production of enzymes, the focus is on utilization of agro-residual wastes along with development of efficient bioprocess strategies to obtain high enzyme titers. Hence, lot of emphasis has been given for screening of such agricultural residues like rice straw, wheat straw, and sugarcane bagasse. Moreover, sweet sorghum bagasse could be a potential substrate for production of higher enzyme titers. Xylanase has been reported from microbial sources like Aspergillus sp. and Trichoderma sp., as well as bacterial isolates (Sapag et al. 2002). However, less work on xylanase from Penicillium citrinum isolate has been reported and, moreover, cellulase-free xylanases are of considerable research interests due to their industrial significance (Walia et al. 2014). An attractive fermentation process is SSF for xylanase production as it involves the growth of fungi on moist substrates in the absence of free flowing water thereby mimicking the natural environment. Due to low water content in SSF, the microbe is in contact with gaseous oxygen and substrate, unlike in the case of SmF. Therefore, SSF offers several advantages over SmF, such as compactness, higher product yields, less investment and low energy demand. Hence, SSF has been widely employed in enzyme production, solid waste management, biomass energy conversion and in production of microbial secondary metabolites (Holker et al. 2004; Narang et al. 2001). To develop a successful fermentation process, one of the approaches is to optimize the process parameters to improve enzyme yields (El-Hadi et al. 2014). There are two ways by which optimization of fermentation process can be addressed: classical and statistical. The classical approach is based on the “one-factor-at-a-time” in which one independent variable is studied while fixing all the other factors at a constant level (Khucharoenphaisan et al. 2008), but this method seems to be time consuming, gives unreliable results and inaccurate conclusion. Hence, an alternate strategy is statistical experimental designs including Plackett–Burman design (PBD) and response surface methodologies (RSM) which can collectively eliminate these limitations of a single-factor optimization process and has been extensively used for optimization of fermentation factors for enzyme production using SSF (Trivedi et al. 2012). RSM involves a minimum number of experiments for a large number of variables and simultaneously solves multivariate equations, by which improvement in enzyme production has been demonstrated successfully (Khucharoenphaisan et al. 2008). RSM has been employed for modeling and optimization of process parameters for enzyme production, wastewater treatment as well as production of extracellular polysaccharides (Zambare and Christopher 2011) and many in biochemical as well as biotechnological processes (Bas and Boyaci 2007). Although RSM has been used to optimize the production of microbial xylanases, less work has been reported on xylanase production by SSF using P. citrinum isolate. Enzyme immobilization offers advantages like reusability and continuous processing. Enzyme immobilization methods vary for different enzymes and applications. Covalent immobilization of xylanase on glutaraldehyde-activated calcium-alginate beads has proved to be easy and economical (Pal and Khanum 2011). Enzymatic hydrolysis using immobilized enzymes has been demonstrated to produce xylooligosaccharides (Aragon et al. 2013b).

In the present study, the P. citrinum isolate HZN13 isolated from forest soil produced exceptionally high-level cellulase-free xylanase from a variety of agro-waste residues. Xylanase production using sweet sorghum bagasse was statistically optimized by PBD and RSM-CCD. Glutaraldehyde-activated calcium-alginate immobilized xylanase showed pH and temperature stability with increased kinetics as compared to free enzyme and was used efficiently for enzymatic hydrolysis of bagasse. Very few reports on high yields of xylanase by P. citrinum isolates are available. SEM, EDX and FTIR characterization of bagasse presents the insights into hydrolysis process. Xylose detection by HPLC from bagasse reveals the industrial significance of xylanase.




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