Activity Of Recombinant Xylanase Research Articles

Optimization of culture conditions for the production and activity of recombinant xylanase from microalgal platform

Xylanases are enzymes responsible for the hydrolysis of the heteropolymer xylan. They have wide applications ranging from the bakery, animal husbandry, and textile to pulp and paper industry and biofuel productions. Recombinant xylanase production has been previously reported from different hosts such as bacteria and yeast. Microalgae offer a safe and cost-effective photosynthetic platform for producing recombinant proteins, including therapeutics and industrial enzymes. In this study, we optimized the production of recombinant xylanase expressed and secreted from the green alga, Chlamydomonas reinhardtii. The growth of the culture was optimized using response surface methodology (RSM) based on central composite design (CCD), with two numeric (culture incubation time and agitation rate) and one categoric (light intensity) factors. The optimum biomass concentration was obtained as 0.71 mg/mL from the CCD values. In addition, bubble column photobioreactors were set and compared for the culture growth, the protein concentration, and the enzyme activity under different light intensities and air flows. Increasing the aeration rate from 1 vvm to 2 vvm resulted in improved enzyme activity from 5330.5 U/g to 6277.7 U/g under 3500 lux illumination on the 3rd day of the culture. This study may lead to the further large-scale production of xylanase with high enzyme activity and reveal the advantage of the microalgae as a sustainable platform.

ISOLATION AND STUDY OF A RECOMBINANT CARBOHYDRASE XYLANASE FROM BACILLUS LICHENIFORMIS

Xylanases are hydrolytic enzymes involved in the degradation of xylan, the main component of plant biomass. In the present study xylanase, XynA, from Kazakh strain Bacillus licheniformis was obtained in recombinant form in Escherichia coli cells. The xynA gene was amplified from the genomic DNA of the B. licheniformis and cloned in pET-28c (+) vector under the control of the promoter of bacteriophage T7. Recombinant XynA was produced in ArcticExpressRP(DE3) cells by plasmid gene expression, protein purification was carried out by metal affinity chromatography. During the study, the dependence of the enzymatic activity of recombinant xylanase on temperature and pH was observed, it was established that xylanase has the highest activity at +50°C and pH of 6.9. The activity units at these values of temperature and pH were1859 per ml of purified protein. The working temperature and pH ranges in which xylanase conserves more than 70% activity from maximum is 40-60°C and 5-8, respectively.
 The data obtained are essential for the use of carbohydrase xylanase from B.licheniformis in biotechnological processes in the processing of vegetable raw materials.

Biological synthesis of Au nanoparticles using liquefied mash of cassava starch and their functionalization for enhanced hydrolysis of xylan by recombinant xylanase.

Au nanoparticles (AuNPs) have shown the potential for a variety of applications due to their unique physical and chemical properties. In this study, a facile and affordable method for the synthesis of AuNPs via the liquefied mash of cassava starch has been described and the functionalized AuNPs by L-cysteine improved activity of recombinant xylanase was demonstrated. UV-Vis absorption spectroscopy, transmission electron microscopy, and zeta potential measurements were performed to characterize the AuNPs and monitor their synthesis. The presence of Au was confirmed by energy-dispersive X-ray spectroscopy (EDX) and the X-ray diffraction patterns showed that Au nanocrystals were face-centered cubic. The C=O stretching vibration in the Fourier transform infrared spectrum of AuNPs suggested that the hemiacetal C-OH of sugar molecules performed the reduction of Au³⁺ to Au⁰. The presence of C and O in the EDX spectrum and the negative zeta potential of AuNPs suggested that the biomolecules present in liquefied cassava mash were responsible for the stabilization of AuNPs. The surface of AuNPs was easily functionalized by L-cysteine, which improved the stability of AuNPs. Moreover, cysteine-functionalized AuNPs could significantly improve recombinant xylanase efficiency and stability.

Simplified feeding strategies for the fed-batch cultivation of Kluyveromyces lactis GG799 for enhanced recombinant xylanase production

A xylanase gene (xyn2) from Trichoderma reesei ATCC 58350 was previously cloned and expressed in Kluyveromyces lactis GG799. The production of the recombinant xylanase was conducted in a developed medium with an optimised batch and with fed-batches that were processed with glucose. The glucose served as a carbon source for cell growth and as an inducer for xylanase production. In a 1-L batch system, a glucose concentration of 20gL(-1) and 80% dissolved oxygen were found to provide the best conditions for the tested ranges. A xylanase activity of 75.53UmL(-1) was obtained. However, in the batch mode, glucose depletions reduced the synthesis of recombinant xylanase by K. lactis GG799. To maximise the production of xylanase, further optimisation was performed using exponential feeding. We investigated the effects of various nitrogen sources combined with the carbon to nitrogen (C/N) molar ratio on the production of xylanase. Of the various nitrogen sources, yeast extract was found to be the most useful for recombinant xylanase production. The highest xylanase production (110.13UmL(-1)) was measured at a C/N ratio of 50.08. These conditions led to a 45.8% increase in xylanase activity compared with the batch cultures. Interestingly, the further addition of 500gL(-1) glucose led to a 6.2-fold increase (465.07UmL(-1)) in recombinant xylanase activity. These findings, together with those of the exponential feeding strategy, indicate that the composition of the C/N molar ratio has a substantial impact on recombinant protein production in K. lactis.

Homologous constitutive expression of Xyn III in Trichoderma reesei QM9414 and its characterization

Xylanase III (Xyn III), a specific endoxylanase that belongs to family 10 of the glycoside hydrolases, was overexpressed in Trichoderma reesei QM9414 using a constitutive strong promoter of the gene encoding pyruvate decarboxylase (pdc). The maximum recombinant xylanase activity achieved was 817.2 ± 65.2 U/mL in the transformant fermentation liquid. The productivities of Xyn III accounted for approximately 53% of the total protein secreted by the recombinant. The enzyme was optimally active at 60 °C and pH 6. The recombinant Xyn III was stable at pH 5-8. This is the first report on the homologous expression of xyn3 in T. reesei QM9414. The properties of Xyn III make it promising in a variety of industrial use.

Cloning and expression of a xylanase xynB from Aspergillus nigerIA‐001 in Pichia pastoris

The high-level expression of the xylanase GH11 gene from Aspergillus niger IA-001 called xynB was successfully completed in Pichia pastoris. The xynB gene encoding a mature xylanase of 225 amino acid was subcloned into the pPICZαA vector and was transformed into P. pastoris X-33 under the control of the alcohol oxidase I (AOX1) promoter. The xynB gene was ligated with a sequence encoding modified α-factor signal peptide (pPICZαmA) and the recombinant xylanase activity, which was measured 1280 U ml(-1), was 1.5-fold higher than when it was inserted into pPICZαA and was 19.39-fold greater than the native xylanase in the original strain. In a 10 L fermenter, the recombinant xylanase activity measured 10,035 U ml(-1) after 114 h. The SDS-PAGE analysis revealed that the purified xynB protein migrated as a single band with an apparent molecular weight of 24 kDa. The specific activity, using beechwood xylan as a substrate, was 1916 U mg(-1). The xylanase activity was optimal at pH 5.0 and at 50 °C. In addition, the xynB was active over a pH range of 2.2 to 10.0. The apparent Km and Vmax values were 4.429 mg ml(-1) and 1429 U mg(-1), respectively.

Heterologous expression of a gene for thermostable xylanase from Chaetomium thermophilum in Pichia pastoris GS115

We studied heterologous expression of xylanase 11A gene of Chaetomium thermophilum in Pichia pastoris and characterized the thermostable nature of the purified gene product. For this purpose, the xylanase 11A gene of C. thermophilum was cloned in P. pastoris GS115 under the control of AOX1 promoter. The maximum extracellular activity of recombinant xylanase (xyn698: gene with intron) was 15.6 U ml(-1) while that of recombinant without intron (xyn669) was 1.26 U ml(-1) after 96 h growth. The gene product was purified apparently to homogeneity level. The optimum temperature of pure recombinant xylanase activity was 70°C and the enzyme retained its 40.57% activity after incubation at 80°C for 10 min. It exhibited quite lower demand of activation energy, enthalpy, Gibbs free energy, entropy, and xylan binding energy during substrate hydrolysis than that required by that of the donor, thus indicating its thermostable nature. pH-dependent catalysis showed that it was quite stable in a pH range of 5.5-8.5. This revealed that gene was successfully processed in P. pastoris and remained heat stable and may qualify for its potential use in paper and pulp and animal feed applications.

Extracellular recombinant protein production from an Escherichia coli lpp deletion mutant

E. coli is one of the most commonly used host strains for recombinant protein production. However, recombinant proteins are usually found intracellularly, in either cytoplasm or periplasmic space. Inadequate secretion to the extracellular environment is one of its limitations. This study addresses the outer membrane barrier for the translocation of recombinant protein directed to the periplasmic space. Specifically, using recombinant maltose binding protein (MalE), xylanase, and cellulase as model proteins, we investigated whether the lpp deletion could render the outer membrane permeable enough to allow extracellular protein production. In each case, significantly higher excretion of recombinant protein was observed with the lpp deletion mutant. Up to 90% of the recombinant xylanase activity and 70% of recombinant cellulase activity were found in the culture medium with the deletion mutant, whereas only 40-50% of the xylanase and cellulase activities were extracellular for the control strain. Despite the weakened outer membrane in the mutant strain, cell lysis did not occur, and increased excretion of periplasmic protein was not due to cell lysis. The lpp deletion is a simple method to generate an E. coli strain to effect significant extracellular protein production. The phenotype of extracellular protein production without cell lysis is useful in many biotechnological applications, such as bioremediation and plant biomass conversion.