Thermostable Sites and Catalytic Characterization of Xylanase XYNB of Aspergillus niger SCTCC 400264

Abstract

In order to improve the expression of heat-resistant xylanase XYNB from Aspergillus niger SCTCC 400264, XynB has been cloned into Pichia pastoris secretary vector pPIC9K. The XynB production of recombinant P. pastoris was four times that of E. coli, and the $V_{max}$ and specific activity of XynB reached $2,547.7{\mu}mol/mg$ and 4,757 U/mg, respectively. XynB still had 74% residual enzyme activity after 30 min of heat treatment at $80^{\circ}C$. From the van der Waals force analysis of XYNB (ACN89393 and AAS67299), there is one more oxygen radical in AAS67299 in their catalytic site, indicating that the local cavity is much more free, and it is more optimal for substrate binding, affinity reaction, and proton transfer, etc, and eventually increasing enzyme activity. The H-bonds analysis of XYNB indicated that there are two more H-bonds in the 33rd Ser of XYNB (AAS67299) than in the 33rd Ala(ACN89393 ), and two H-bonds between Ser70 and Asp67.

Introduction

Xylans are hemicellulose compounds in plants, which are the second largest renewable resource in nature, accounting for more than 30% of dry cell weight [5, 9, 19]. Completed degradation of xylan requires participation of several different enzymes. However, endo-β-1,4-D-xylanase (E.C. 3.2.1.8), referred to as xylanase, plays a critical role in this process, degrading the linear polysaccharide β-1,4-xylan into xylose [15]. In the present study, the specificity of XYNB towards different national substrates was evaluated. XYNB was highly specific towards xylans tested but exhibited low activities towards lichenan (19%), gellan gum (7.3%), laminarin (3.4%), and carboxymethycellulose (CMC, 1.4%). These are of real commercial importance for industrial applications [11]. As to the hydrolyzed products of xylan, several studies showed that the XYNB hydrolyzed xylooligosaccharides to yield predominantly xylobiose (X2) or xylotriose (X3), and the conversion of X2 to xylose (X1), and conversion of X3 to X2 were observed. The hydrolysates of xylan by XYNB suggested that the enzyme had great value in the preparation of xylooligosaccharide [4, 13, 23]. Xylanase has not only broad application prospects in biotechnological and industrial applications such as animal feed, textile, paper industries, and functional xylooligosaccharides production [14, 21], but also great potential in the bioconversion of lignocellulosic feedstocks to fuel-grade ethanol, which has attracted tremendous interests in the past few years. Two approaches have been successfully tried to obtain thermophilic adaption xylanase [24]. One approach is to screen thermophilic adaption strains from natural microorganisms, and the reported thermophilic strains are mostly from bacteria and actinomycetes [2]. The other approach is to use genetic engineering on enzyme molecules through in vitro transformation [18]. In the present studies, hundreds of xylanase genes from various microorganisms, including bacteria and fungi, have been cloned and expressed in eukaryotic and prokaryotic hosts [7, 17, 28, 29]. We have been working on xylanase for 10 years; however, most of the studies were limited to the natural strain screening [1, 27], which makes the use of genetic engineering with industrial production of xylanase preparation a new field of study in China. The cDNA fragment of β-1,4-D-xylanase was cloned and the sequence was analyzed in Aspergillus niger [22]. Unfortunately, no research-related heterologous expression of this gene has been reported, and the eukaryotic expression of the xylanase gene xynB from Aspergillus niger is hardly reported.

A heat-resistant xylanase gene xynB from Aspergillus niger SCTCC 400264 strain was cloned and heterologously expressed in P. pastoris. High yield of xylanase was achieved in this expression system. We also made a study of the enzymatic properties of the recombinant xylanase.

 

Materials and Methods

Strains, Plasmids, Reagents, and Culturing Conditions

The xylanase gene xynB (FJ772090)was cloned from A. niger SCTCC 400264 in the Sichuan Type Culture Collection Center (SCTCC, China) [26]. E. coli JM109 was used as a cloning host and DNA propagation, and was grown in LB medium. P. pastoris GS115 strain (Invitrogen, USA) was used as a host for expression of xylanase and was grown in YPD. P. pastoris transformants were selected in MD (minimal dextrose) and MM (minimal methanol) medium. The fermentation of recombinant P. pastoris was used in BMGY and BMMY medium. The pMD19-T vector (TaKaRa, Japan) was used for cloning PCR fragments, and pPIC9K vector (TaKaRa, Japan) was used for protein expression in P. pastoris.

Construction and Transformation of Expression Plasmid

Based on the nucleotide sequence of XynB (FJ772090), the entire coding region without signal peptides was amplified by PCR. P1: 5’-GGAATTCTCGACCCCGACCGGCGAGAA-3’; P2: 5’-ATA GCGGCCGCTTACTGAACAGTGATGGAGGAAGA-3’. The PCR products were gel-purified and digested with EcoRI and NotI before being cloned into pPIC9K. For P. pastoris integration, 10 μg of recombinant plasmid was linearized with SalI, and transfered into P. pastoris as described by the manufacturer. The transformants were selected at 28℃ on the MD and MM agar plates, and then the positive transformants were selected at 28℃ on the YPD agar plates containing different concentrations of G418 (0.25, 0.50, 1.00, 1.50, 2.00, 2.50, 3.00, 4.00, 5.00, and 8.00 mg/ml). The integration of the xylanase gene into the genome of P. pastoris was confirmed by PCR using the primers described above.

Xylanase Activity Assays and Protein Concentration Determination

The xylanase activity was evaluated by measuring the reducing groups liberated from 0.8% (w/v) oat spelt xylan by the DNS method using xylose as the standard [8]. One unit of enzyme activity (1 U) was defined as the amount of enzyme that produced reducing sugars equivalent to 1 μmol of xylose per minute. Protein was estimated by the method of Bradford [3]. The determination of activity of the enzyme was carried out at each optimal temperature of enzymes for 10 min by the DNS method. Each data point represents a mean of triplicate determinations.

Genetic Stability Test of Recombinant P. pastoris

The recombinant P. pastoris was passaged 50 times, taking the kept strain and culture every 10th generation with methanol induction. In this process, the cell density and xylanase expression were measured and the genetic stability was confirmed by PCR as well.

Computer Analysis Software

DeepView, the Swiss-PdbViewer for the analysis of mutation sites of hydrogen bonds and van der Waals force.

 

Results

Recombinant Enzyme Specific Activity and Enzyme Kinetics Parameters

XYNB was expressed in P. pastoris, and analysis of SDSPAGE (Fig. 1) showed that the XYNB produced a specific clear protein band with a molecular mass of about 21 kDa. The enzyme production of recombinant P. pastoris was four times more than E. coli; the Vmax and specific activity of enzyme reached 2,547.7 μmol/mg.min and 4,757 U/mg (Table 1), respectively, but the substrate affinity had reduced to 29.4 mg/ml (18.7 mg/ml in E. coli) [29].

Fig. 1.SDS-PAGE analysis of the His-tag XYNB. XYNB was expressed in P. pastoris successfully, and the results were showed in SDS-PAGE.

Table 1.Recombinant enzyme specific activity and enzyme kinetics parameters.

Characterization of Enzymatic Properties

Characterization of the recombinant enzyme revealed that the optimum reaction temperature and pH of the recombinant XYNB were 55°C and 5.0 (Figs. 2 and 3), respectively. It is consistent with the wild xylanase obtained from the culture of Aspergillus niger [12]. The optimal temperature was determined by incubating the enzyme in 0.1 M citrate buffer, pH 5.0, for 10 min at different temperatures, as indicated in the graph. The maximal activity was defined as 100% relative activity. The optimal pH was determined by incubating the enzyme at 50°C for 10 min at different pH buffers. To determine the optimal pH, pH values ranging from 2 to 10 were used with the following (100mM) buffers: KCl-HCl (pH 2), sodium citrate (pH 3-7), Na2HPO4-NaH2PO4 (pH 8.0), and glycine-NaOH (pH 9-10). The maximal enzyme activity was defined as 100% relative activity.

Fig. 2.Optimum temperature of the recombinant xylanase. The optimal temperature was determined by Ghose’s [8] method by incubating the enzyme in 0.1 M citrate buffer, pH 5.0, for 10 min at different temperatures, as indicated in the graph.

Fig. 3.The optimum pH and acid-alkali stability of the recombinant xylanase. The optimal pH was determined by incubating the enzyme at 50℃ for 10 min at different pHs by Ghose’s method [8]; pH range from 2 to 10.

Fig. 4.Thermal stability of the recombinant xylanase. The recombinant protein was determined by incubation at 80℃ for 10 min.

Moreover, it was worth stressing the effects of temperature on xylanase activity and the thermostability of the recombinant enzyme. The recombinant XYNB showed 86% of maximal activity after incubation at 80℃ for 10 min (Fig.4). The results of the pH effects on the recombinant xylanase revealed that the XYNB was relatively stable after being incubated at pH 2.0-10.0 for 30 min at 37℃. All results showed more than 70% of maximal activity, and the highest remained at 92% at pH 5.0 (Fig. 3).

Xylanase Activity Influenced by Different Metal Ions

Various metal ions had different effects on xylanase activity. K+, Mg2+, Fe2+, and Co2+ could improve the enzyme activity, whereas Mn2+, and Cu2+ obviously inhibited the activity of the enzyme. Other metal ions, such as Ca2+, Fe3+, and Zn2+, did not influence the xylanase activity (Fig. 5).

Fig. 5.The influence of the recombinant enzyme activity by different metal ions. The substrate included K+, Mg2+, Fe2+, Mn2+, Cu2+, Ca2+, Fe3+, Co2+, Zn2+.

Genetic Stability of the Recombinant P. pastoris

The genetic experiment implied that the indicators in each generation remained stable. The PCR identification of the 10th, 20th, 30th, 40th, and 50th generation cells showed that there was a fragment of 567 bp of xynB gene obtained in every generation. After 50 passages, xynB remained stable in integration of genes in the P. pastoris genome. The results of the above experiment showed that the fourth recombinant strain had a good genetic stability, and achieved xylanase gene in P. pastoris of heterologous high expression.

 

Discussion

Thermostable Sites of Xylanase Gene from Aspergillus niger

Sriprang et al. [20] reconstructed XYLB (AY551187) of Aspergillus BCC14405, replacing Arg for Ser/Thr, and obtained two mutants, ST4 and ST5, which had higher thermostability. In this study, the protein encoded by the cloned gene of A. niger SCTCC 4000264 xynB (ACN89393) showed only one different amino acid in contrast to XYLB (AAS67299). The similarity was the site of 33rd (considering the signal peptide, the site is 70th); the difference was the amino acid in this site; in A. niger 400264 it was Ala, whereas in XYLB it was Ser (Fig. 6). These two recombinant enzymes showed different thermostabilities [24]. Besides this, the van der Waals force analysis in XYNB (ACN89393 and AAS67299), showed there is one more oxygen radical in AAS67299 in their catalytic site (Figs. 7A and 7B), indicating that the local cavity is much more free, and it is better for substrate binding, affinity reaction, and proton transfer, etc, and eventually increasing enzyme activity. The H-bonds analysis of XYNB (Figs. 7C and 7D) indicated that there are two more H-bonds in the 33rd Ser of XYNB (AAS67299) than the 33rd Ala (ACN89393), and two Hbonds between Ser70 and Asp67. Ser is more hydrophilic than Ala, and near the 70th site, the hydrophilic amino acids are more than hydrophobic amino acid, where only two Ala68 and Ile222 were hydrophobic among the nine amino acids. The hydrophobic interaction increases to maintain the two-layer structure and to improve the thermosability. Thus, it infers the amino acid of this site has effects on the thermostability of xylanase.

Fig. 6.Multiple sequence alignment of xynB in Aspergillus niger. I: Signal peptide, 1-37; II-IV: different site.

Fig. 7.Van der Waals force and internal H-bonds analyses of xynB (ACN89393 and AAS67299) in Aspergillus niger. (A, B) Van der Waals force analysis for XYNB (A: ACN89393; B: AAS67299); (C, D), Internal H-bonds analysis for XYNB (C: ACN89393; D: AAS67299).

Expression of Thermostable Xylanase in Pichia pastoris

To date, E. coli and P. pastoris are the most common expression platforms for industrial enzyme productions. Generally, bacteria are not perfect expression systems owing to complicated downstream processing and high purification cost. In contrast, recombinant proteins expressed in P. pastoris are secreted into the medium and can be purified easily at a lower cost. In addition, P. pastoris enables some posttranslation modifications, including the assembly of disulfide bonds, the exclusion of signal peptides, and glycosylation, etc. A variety of foreign proteins had been successful expressed in the host expression system [25].

The integration of xylanase gene xynB into AOX1 in the genome made the target gene highly secreted in P. pastoris. After optimizing the induction conditions, the production of recombinant strain pPIC9K-XynB xylanase reached 4,757 U/mg, increased by about 4 times as the initial strain. The expression product possessed biological activity and provided an optimal strain for further industrial fermentation. Since this study has not been carried out in the fermentation tank, the enzyme activity should also be further improved in the next fermentation tanks experiment.

Enzymatic Properties of Thermostable Xylanase Gene from Aspergillus niger

The optimum temperature of the recombinant xylanase was 55℃ and the optimal pH was 5.0, which was the same as XylB from A. niger described by Ruanglek et al. [16]. However, the recombinant xylanase from this work had a better thermal stability than the similar xylanase; our xylanase showed 74% of maximal activity after 30 min incubation in water at 80℃, but the xylanase XYNB from Streptomyces olivaceoviridis A1 only maintained 20% residual activity after incubating in water for 30 min at 70℃ [10].

The xylanase in animal feed applications requires high activity at body temperature (37℃) in the gastrointestinal tract [6]. This recombinant xylanase showed 82% residual activity at 37℃, and more than 50% relative activity remained in the range of pH 4-6, which met these requirements.

A short high-temperature treatment is necessary in the feed pelleting process, so the xylanase must have good thermal stability in the real application of animal feed. In this work, the recombinant xylanase showed 74% of maximal activity after incubating in water for 30 min at 80℃ and had good thermal stability as well as pH stability The excellent heat resistance of the recombinant xylanase expanded its scope of application, and lays the foundation for its industrial applications.