Engineering of thermostable phytase–xylanase for hydrolysis of complex biopolymers

Abstract

Industrial processing of enzymes requires higher heating that affects the thermal stability of the enzyme and increases the production cost. In this study, xylanase–phytase (XP) fusion protein was generated via co-expression in a single vector with a cold-shock promoter, leading to improved activity at optimal pH, temperature and the thermal behaviour of the protein. Xylanase–phytase (XP) fusion and phytase proteins were characterized by differential scanning calorimetry (DSC) and thermogravimetric analysis (TGA). The XP fusion was thermally stable up to 124 °C, higher than phytase which was steady up to 113.5 °C. XP fusion exhibits higher stability at its thermal transition midpoint (T_m) 108 °C, higher than the T_m value of phytase which is 90 °C. Industrially efficient and environment-friendly proteins with low production cost and higher stability can be generated by ‘fusion protein’ technology.

Supplementary Information

The online version contains supplementary material available at 10.1007/s13205-021-02936-z.

Introduction

Enzymes like phytases and xylanases are efficiently used as feed supplements in veterinary applications (dos Santos et al. 2017). Industrial production and use of phytases (Enshasy et al. 2018) as well as xylanases are known to significantly improve the animal feed’s nutritive value (Mendes et al. 2013). However, considering the production cost of these enzymes, co-expressing them in a suitable host can aid in cost reduction (Roongsawang et al. 2010).

Xylanase has potential biotechnological and industrial applications in diverse industries such as paper and pulp industries (Bhardwaj et al. 2019), pharmaceutical industries, biofuel production, food, bakery industries (Deborah and Ramalingam 2010) as well as animal feed industries (Basit et al. 2020). Xylanases are used for the improvement of the digestibility of animal feed (Zhang et al. 2014). Animal feed supplemented with xylanase, phytase, or its combination improved their performance, which is credited to reduced weight and digesta viscosity with increased metabolizable energy (Lü et al. 2009).

In many of the industrial sectors, xylanase production at a low investment cost is necessary for xylan’s commercial consumption. Recombinant DNA technology plays a significant role in the large-scale expression of xylanases. Hence, engineering the most efficient microorganisms suitable for xylanase gene cloning calls for much attention (Deesukon et al. 2011). The majority of the relevant research articles are committed to the characterization and mining of xylanases considering their vast industrial application (Walia et al. 2017). Commercial applications necessitate higher enzyme expression levels, cost-effective enzymes, and xylanase’s competent secretion contributing to an economically feasible process.

Phytic acid (inositol hexakisphosphate) has a robust chelating ability, which chelates and binds with minerals like magnesium, iron, calcium, zinc, etc. (Abdulwaliyu et al. 2019). Under both alkaline and acidic environments, the highly negatively charged structure of phytate forms complexes with protein structure, which decreases the digestibility, resulting in reduced bioactivity of micronutrients (Vashishth et al. 2017). Since hydrolysis of phytate is of great concern, phytases have vast potential, it catalyses the hydrolysis of phytate–phosphate. This reaction, hydrolysis of the phytic acid into lower inositol phosphate releases the inorganic phosphate stored into phytate (Gupta et al. 2015). This conversion of phytic acid to inorganic phosphate in a biological system is of much significance in environmental biogeochemical cycling (Perera et al. 2018).

The animal feed contains a large amount of phytate, but non-ruminant animals such as fish, poultry, pigs are monogastric and cannot utilize phytate as they lack intestinal phytase activity (Singh and Satyanarayana 2015). The unconsumed phytate phosphorous runs off in the environment and leads to eutrophication and other ecological complications (Dave and Modi 2019).

Phytases have several beneficial effects on health and stabilize animal agriculture (Priyodip et al. 2017). Phytases can be produced from animals, plants, and especially from bacterial species (Jain et al. 2016). Bacterial phytases are engineered to improve phosphorus utilization (Reddy et al. 2017) in food industries, phytate reduction, environmental protection, medicine, and soil nutrients enhancement (Gupta et al. 2015; Dave and Modi 2018). Both xylan and phytate are significant antinutritive components and their use is inevitable in animal feed. Commercial-scale phytases are isolated from Escherichia coli and Aspergillus niger (Menezes-Blackburn et al. 2011). However, for animal feed application, Bacillus sp. is a potential source of phytases due to its unique characteristics such as optimum pH, substrate specificity, proteolysis resistance, and higher thermal stability (Borgi et al. 2015). Hence, several research groups focus on bacterial phytases from Bacillus sp. Tedious procedures such as recombinant host fermentation, enzyme purification, activity testing and raw materials are required for recombinant enzyme industrial production. Here, we propose the xylanase–phytase fusion for the possible application in feed processing industries. Moreover, the approach for cost and time reduction is the co-expression of the enzymes in a single host, where both the enzymes are functionally active and stable. Internal ribosome entry sites and usage of protease enzymes are two methods used for the co-expression of proteins in eukaryotes but they are not industrially efficient as the former requires another recombinant enzyme activity that is tough to control, leading to lower enzyme expression (Douin et al. 2004). Thus, the concept of fusion proteins is becoming more advantageous for balanced multiple proteins where co-expression can be achieved from a single promoter.

In the present study, we established a co-expression system for the fusion protein, i.e., xylanase from Bacillus amyloliquefaciens and phytase from Bacillus licheniformis (Fig. 1). Phytase and XP fusion were analysed to investigate the thermal behaviour through thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC).

Fig. 1.

Strategy employed for designing industrially valuable XP-pCold fusion protein

Materials and methods

NucleoSpin Gel and PCR Clean-up kit (TAKARA Bio, USA) were used for the purification of PCR products. pColdI, from TAKARA Bio, USA, was used as an expression vector. All the other reagents were of molecular biology grade and purchased from Sigma-Aldrich. The bacterial genomic sources were procured from the Microbial Type Culture Collection (MTCC) Chandigarh, India. The laboratory-maintained strain of Escherichia coli DH5α and Escherichia coli BL21 (DE3) was used for cloning and gene expression analysis. The antibiotic ampicillin was used as a selection marker at the concentration of 100 µg/ml.

Primers and gene amplification

The primers were designed based on the gene of interest from two different bacterial strains (Mamiatis et al. 1985); i.e., gene phyL from Bacillus licheniformis ATCC 14580 and xynA from Bacillus amyloliquefaciens ATCC 23350/DSM7. The primers were synthesized and obtained from Eurofins Genomics India Pvt, Ltd. Sequences of the primers are as shown in Table 1.

Table 1.

Primers used for gene amplification

	Primer name	Primers
A	B.lic_ PHY_ FP(Forward Primer)	5ʹ-ATT AGT AAA CAT ATG ATGAAC TTT TAC AAA ACG CTC-3ʹ
	B.lic_ PHY_RP (Reverse Primer)	5ʹ-TTA ATA AAA CTC GAG TTA TTT GGC TCG TTT CAG-3ʹ
B	B.Amy_ xyl_ FP	5ʹ- TTT AAA TAT TCT AGA AAA GGA TTT TCC GCT AAT AGT-3ʹ
	B.Amy_ xyl_RP	5ʹ-TTT AAA TAT GGA TCC ATG ACG GTA AGA CCT GAA TAC-3ʹ
C	pCold I _FP	5ʹ-CCA TAT CGC CGA AAG G- 3ʹ
	pCold I _RP	5ʹ-GGC AGG GAT CTT AGA TTC TG-3ʹ

Construction of the P-pCold and sequencing

The PCR amplified and subsequently purified gene fragment phyL and vector pCold I was double-digested with 0.5 U of restriction endonucleases (RE) Nde I and Xho I (Invitrogen USA). The post-digestion products were eluted using the gel extraction kit and electrophoresis, insert (1.14 kb) and the digested vector (4.4 kb) were kept overnight for ligation with T4 DNA ligase at 16 °C. Competent cells of Escherichia coli DH5α were transformed with the ligated samples. The transformants were positively selected using plasmid DNA isolation followed by its comparison with the control pColdI plasmid. The clones were further verified by performing allele-specific PCR of transformant. Subsequently, for further confirmation, the recombinant DNA was isolated from the selected clone, digested with the same RE pair and observed for insert release by gel electrophoresis.

The nucleotide sequence of the insert was also determined through automated sequencing (ABI 3730XL) by Eurofins Genomics India Pvt. Ltd. Clone verification was performed by BLAST homology search (Altschul 1997). ClustalW was used for sequence alignment of the PhyL amino acid sequences (Gouet et al. 1999; Thompson et al. 1994), and the construct clone was named as P-pCold.

Construction of the XP-pCold with xynA insertion in P-pCold

After confirming the presence of phyL in transformants, the xynA gene was inserted between Xba I and Bam H I restriction sites, adjacent to the phyL gene, the details are as depicted in Fig. 2. Subsequently, it was transformed into Escherichia coli DH5α. To verify the presence of xynA in XP-pCold, we randomly selected ten colonies and transferred them into a separate vial containing ampicillin along with the culture media from which the plasmid DNA was extracted, and subjected to PCR-based verification. The forward primer for phyL and reverse primer for xynA were used to confirm the amplification of both the genes present on the same fragment and the vector was digested for detection of insert release. The construct is further referred to as XP-pCold. Subsequently, the orientation of the insert was determined by DNA sequencing of XP-pCold.

Fig. 2.

Construct of XP-pCold fusion. Showing cloning and expression region having csp A promoter, the lac operator, cspA5ʹUTR- has a significant role low-temperature stimulation, the translation-enhancing element supports for gene expression regulation in pCold vector. phyL and xynA gene sequences were introduced at the NdeI–XhoI and BamHI–XbaI restriction sites of pColdI

Gene expression and purification of phyL and XP fusion gene

The XP-pCold clone contains two enzymes. These proteins were further purified to evaluate their in-vitro activity. For high-level expression of recombinant proteins, the XP-pCold clone was transformed into E. coli BL21 (DE3). The recombinant proteins were overexpressed in the host as described by Sudhir et al. (2014). The cells were suspended in lysis buffer (50 mM Tris-HCl, 300 mM NaCl and 20 mM Imidazole, pH 8.0) and cell lysis was done with sonication (QSonica, LLC USA), further the concentrated lysate was then centrifuged at 7800×g for 30 min at 4 °C. Finally, the lysate was injected into the column specially designed for Ni–NTA (Invitrogen, US) affinity chromatography. The His-tagged fusion proteins were eluted using the elution buffer, which contains 250 mM imidazole and the proteins were extracted towards the imidazole gradient. Subsequently, the concentrate of recombinant proteins and extra salt was removed by filtering the proteins through a 10 kD Millipore filter (Amicon).

Estimation and analysis of recombinant protein

The concentration of the total recombinant protein was measured by Bradford’s assay (Ernst and Zor 2010). Denaturing gel electrophoresis was performed with 12% sodium dodecyl sulphate-polyacrylamide gel electrophoresis for detecting the fusion proteins (Laemmli 1970). The recombinant protein from P-pCold was evaluated for comparative analysis using a premixed Protein Molecular weight Marker (Broad, TAKARA). Coomassie brilliant blue R-250 (Bio-Rad) was used for staining and visualization purposes.

Enzyme activity assays

The gradual release of inorganic phosphates from substrate sodium phytate was measured to estimate the Phytase activity. The specific reaction contains 1.5 mM sodium phytate, 0.6 mM CaCl₂, 50 μl of 1 mg/ml standard and expressed Phytase, and the reaction was performed at pH 7.0. The total system volume was adjusted to 1 ml with the sodium phosphate buffer, and the system was further incubated at 75 °C for 2 min 1 ml of trichloroacetic acid (10% w/v) was added to stop the reaction. Followed through the addition of 2 ml colour reagent that contains 1% (w/v) ammonium molybdate, 3.2% (v/v) sulfuric acid, and 7.2% of (w/v) ferrous sulphate and further centrifuged at 13,000×g. To analyse released inorganic phosphate, we measured the optical density at 700 nm (Borgi et al. 2014). The 1 unit of Phytase activity was defined as the amount of enzyme catalyzing the release of 1 μmol of inorganic phosphate per min under standard conditions.

Characterization and thermal behaviour analysis for P-pCold and XP-pCold fusion

The purified enzyme was incubated at varying temperatures from 30 to 90 °C to study the effect of temperature on the enzyme activity. The P-pCold and XP-pCold recombinant enzyme activity was assessed at acidic to alkaline range pH, and value ranges to 5.0–9.0. For pH 3.0 to 5.0 sodium acetate buffer was used, for pH 6.0 to 8.0 and pH 8.0 to 9.0 sodium phosphate buffer and Tris-HCl buffer was used, respectively. Phytase activity was quantitatively assayed using the method described in Borgi et al. (2014). Phytase activity was measured as described earlier.

Effect of metal ions on recombinant XP-pCold fusion activity

The recombinant proteins P-pCold and XP-pCold were purified by nickel affinity chromatography. To determine the effect of metal ions on the enzyme activity, enzyme assay was carried out following standard conditions with pre-incubation of the purified enzyme with metal ions such as FeCl₂, CaCl₂, MgCl₂, MnCl₂, CuCl₂, and ZnCl₂ for 20 min.

Thermal analysis

The commonly used techniques for the rapid evolution of protein are thermal stability, thermogravimetric analysis, and differential scanning analysis. The characterization and thermal behaviour studies of phytase and XP fusion were accomplished using a calorimetric cell (Mettler Toledo DSC3) and a thermobalance (Mettler Toledo TGA/DSC1), using the subsequent experimental procedure for DSC and TGA: (a) temperature from 25 to 250 °C; (b) N₂ atmosphere at a heating rate 10 °C/min; (c) sample mass of approximately 5 mg were sealed in aluminium DSC pans for the DSC analysis, and about 15 mg in crucibles for the TG analysis. The TGA and DSC thermogravimetric curves were obtained using the adapted protocols (Ricci et al. 2018).

Result and discussion

DNA isolation and amplification of xynA and phyL gene

Genomic DNA from Bacillus amyloliquefaciens (ATCC 23350/DSM7) and Bacillus licheniformis (ATCC 14580) were isolated and visualized on 0.8% agarose gel stained with ethidium bromide and observed under UV. Intense band on the agarose gel indicates good quality DNA extracted. The xynA gene from Bacillus amyloliquefaciens and phyL gene from Bacillus licheniformis were amplified using gene-specific primers at an annealing temperature of 58 °C for 35 cycles. The amplified gene products were checked on 0.8% agarose gel. A single PCR amplified band of 747 bp size (Fig. 3A) and 1.14 kb (Fig. 3B) were observed corresponding to xynA gene from Bacillus amyloliquefaciens and phyL gene from Bacillus licheniformis, respectively.

Fig. 3.

Amplification of xynA and phyL gene A L-M: 100 bp DNA ladder, L-1: xynA gene (747 bp), B L-M: 1 kb DNA ladder, L-1: phyL gene (1.14 kb)

Construction of the P-pCold and sequencing

phyL gene was cloned in the pColdI vector, which was subsequently transformed in E. coli DH5α and expressed in E. coli BL21 strain. The presence of cloned phyL gene was confirmed through insert release with size 1.14 kb which corresponds to the size of phyL form Bacillus licheniformis (Fig. 4A), PCR amplification with gene-specific primer (1.14 kb) (Fig. 4B) as well as size difference compared with control plasmid DNA (Fig. 4C), The presence of phyL in clone in desired orientation was also confirmed with DNA sequencing of the chimeric plasmid. The results of sequencing are presented in Supplementary Figure 1.

Fig. 4.

P-pCold clone confirmation A L-1: Insert release of phyL gene (1.14 kb) from P-pCold clone plasmid DNA, L-M: 1 kb DNA ladder, B L-1: phyL gene (1.14 kb) with gene-specific primers from P-pCold clone plasmid DNA, L-M: 1 kb DNA ladder, C L-1: control pCold plasmid, L-M: 1 kb DNA ladder, L-2–5: P-pCold clone plasmid DNA

Construction of the XP-pCold with xynA gene insertion in P-pCold and sequencing

Gene-specific primer combinations were used to amplify the phyL gene using the P-pCold clone plasmid as the template. Each primer combination for phyL and xynA was used to amplify the equivalent gene from Bacillus licheniformis and Bacillus amyloliquefaciens DNA, respectively. As expected, the phyL and xynA-specific primer and its combination amplified the identical fragment from the XP-pCold in pCold plasmid (Fig. 5A) (1.14 kb, 0.744 kb, and 1.88 kb respectively), which confirms the P-pCold and XP-pCold constructs in pCold plasmid. The XP-pCold was further cleaved with restriction enzymes Nde I–Xba I, used for the PCR amplicon cut during the cloning procedure. Figure 5B shows the agarose gel separation of an insert release with an expected size 1.88 kb. The clone was confirmed with DNA sequencing of XP-pCold (Supplementary Figure 2).

Fig. 5.

XP-pCold fusion clone confirmation, A L-1: xynA gene (747 bp) with genespecific primers, L-2: phyL gene (1.14 kb) with gene-specific primers both from XP-pCold clone plasmid DNA, L-M: 1 kb DNA ladder, L-3: XP fusion gene (1.88 kb) with phyL and xynA gene-specific reverse and forward primers from XP-pCold clone plasmid DNA, B L-1: XP fusion gene insert release [1.14 kb-phy + 0.74 kb-xynA = 1.88 kb] from XP-pCold clone plasmid DNA-Using restriction endonuclease (Nde I and Xba I), L-M: 1 kb DNA ladder

Effect of temperature and pH on recombinant P-pCold and XP-pCold fusion

Desirable enzyme properties such as high thermostability and pH stability are essential in animal feed industries. The optimum phytase enzyme activity was found at a pH of 6.0 (Fig. 6A). Activity at optimum pH was defined as 100%. The enzyme remains 80% active in the range of pH 5.0–6.0 and the activity gradually decreases as the pH increases. XP fusion enzyme activity was increased as pH rise from 4.0 to 6.0 (Fig. 6B). Moreover 80% active at pH 5.0 and about 90% functional at pH 7.0 which then gradually decreased as pH increase. The pH optima of both phytase (pH 5.0) and xylanase (pH 9.0) are considerably distinct. In this set-up, the XP fusion protein has achieved stability for a broad pH range that is often needed for various feed processing and hence proposing the fusion catalyst for feed processing can be a clever choice (Dave and Modi 2018).

Fig. 6.

pH, temperature and metal ion effect activity profiles for phytase (A, D, E) and XP fusion protein (B, C, F), respectively. Activities at optimal pH and temperature were defined as 100%. Error bars represent the standard deviation from three separate experiments

The phytase is susceptible to a higher temperature, and steep loss in enzyme activity is observed for temperatures above 60 °C (Borgi et al. 2015). In contrast, the selected xylanase was still partially active at a temperature above 60 °C (Prajapati et al. 2018). At the same time, XP fusion has extended this limit to 80 °C (Fig. 6C), higher than phytase temperature, which was 50 °C (Fig. 6D). The effect of various divalent metals on the phytase activity is depicted in Fig. 6E. Phytase enzyme showed maximum activity in the presence of Zn⁺², whereas the Mg⁺², Ca⁺², Mn⁺² reduces the phytase activity to a considerable extent. Fe⁺², Cu⁺² almost inhibit the enzyme action. A similar reaction with XP fusion protein also demonstrated maximum activity in the presence of Zn⁺² (Fig. 6F) followed by Ca⁺² and Mn⁺², whereas the lowest activity was noted in the presence of Fe⁺² and Cu⁺². Phytase being metal-dependent, showed some activity in the presence of Ca⁺² and Mn⁺². These results suggest their dependence on metal ions for optimal catalytic activity.

Thermal analysis

Protein denaturation is a thermally stimulated process that can be perceived through DSC analysis. The temperature at the maximum peak is noted as T_m. DSC analysis of phytase and XP fusion protein showed a denaturation peak between the temperature range of 62 to 99.33 °C and 106.33 to 118 °C, respectively (Fig. 7A). TGA was utilized to study the thermal stability of phytase (P) and XP fusion proteins (XP). In TG analysis, the temperature at which proteins start to degrade (T_d) was presumed as the onset temperature (noted as mass loss step’s thermal stability limit). It corresponds to the volatilization of proteins generated by thermally activated reactions. Preliminary phytase and XP fusion showed a 20% mass loss within the temperature range of 25–55 °C, which ended at 113.5 °C and 124 °C, respectively.

Fig. 7.

DSC (A) and TGA (B) plots of the thermograms for stability comparison among phytase and XP fusion proteins isolated from different organisms. Thermal transition midpoint (T_m), Thermal denaturation and mass loss (T_d) of the phytase and XP fusion

Further heating demonstrated 80% mass loss when the temperature rose from 55 to 113.5 °C and 55 to 124 °C. Thus ~ 100% mass loss is observed at 113.5 °C and 124 °C for phytase and XP fusion respectively, as shown in Fig. 7B. The temperature value of the thermal stability limit (T_d) was found to be 113.5 °C (P) and 124 °C (XP). Moreover, the higher the thermal transition midpoint T_m, more stable is the protein molecule. Analysing the protein stability through the DSC technique by measuring a heat change is associated with the thermal denaturation of the molecule when heated at a constant rate.

Many countries import commercial xylanase and phytase for animal feed production. The challenge of reducing the production cost with feed improvement can be achieved by exploring the recombinant fusion protein in a single cold expression cloning vector. To achieve desired characteristics such as increased apparent metabolizable energy, reduced digesta viscosity a combination of the different enzymes such as xylanase, phytase and β-glucanase is supplemented in the animal feed (Wu et al. 2004). XP fusion is an environment-friendly, economically cheap biotechnological intervention that can diminish environmental pollution by moderating the amount of phytase excreted in the environment.

Conclusion

This study demonstrates that xylanase and phytase are efficient enzymes for XP fusion protein generation in a single promoter and vector. It is very much evident that xylanase is suitable as an efficient alternative for XP fusion and an inducer of this expression system. In the future, XP-pCold expression system can be used to overexpress the phytase gene in pCold vector, which can be easily achieved in an industrial process and reduce the cost and time of animal feed processing. The presence of xylanase and its interaction with phytase promoted positive effects on the temperature and pH stability. XP fusion offered better properties such as thermal behaviour as compared to individually expressed phytase. These results also demonstrated the importance of thermo-analytical techniques (TGA and DSC) in thermal behaviour study and protein characterization. The precise and high-quality data obtained from DSC provides vital information on the stability of the protein. It can be concluded that the presence of xylanase has a potential effect on phytase. However, further detailed studies are required to investigate the suitability of this fusion protein for large-scale production.

Supplementary Information

Below is the link to the electronic supplementary material.

Authors' contributions

(1) Materials preparation, data collection, and analysis were performed. (2) The first draft of the manuscript was written by DP. The idea and design of work with critical revision the work were done by GD. Kiran Patel and Darshan Patel have provided scientific input for the comment of reviewer 1. They made considerable efforts to revise the scientific language of the manuscript.

Declarations

Conflict of interest

The authors declare that they have no conflict of interest.